JP2017215215A - Sample introduction device and sample introduction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce external effects not only when the volume of a sample or the amount of a solute is too large for a column but also when a higher column performance is to be presented for a sample which appears to have an appropriate volume or amount for the column in view of existing techniques.SOLUTION: The sample introduction method divides a sample band in a flow path from a sample injection device to before a column and causes at least a part of the divided sample band to enter the column in liquid chromatography.SELECTED DRAWING: Figure 1

Description

  The present invention relates to liquid chromatography and, more particularly, to high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) operating methods under conditions that provide high performance and ultra high performance, in particular The present invention relates to a sample introduction method, and further relates to a sample introduction apparatus necessary for these methods.

Here, “high performance liquid chromatography (HPLC)” means liquid chromatography performed under a pressure of up to 40 MPa. Also, “ultra-high performance liquid chromatography (UHPLC)” means liquid chromatography that is performed under a pressure of greater than 40 MPa and up to 140 MPa.

In HPLC or UHPLC, it is necessary to use a column packed with a small packing material in order to speed up the separation, that is, to generate a large number of theoretical plates in a short time and to shorten the separation time. However, a small column packing material reduces the packing gap (flow path through which the mobile phase passes), and therefore requires a high pressure for liquid feeding. Therefore, a short column is used to lower the pressure.

Further, when the liquid is fed at high speed to the gap between the small fillers, heat is generated due to friction between the liquid and the particle wall. The generated heat is dissipated from the column tube wall to the outside of the column, but the temperature at the center of the column becomes high, resulting in a temperature distribution in the column. Therefore, even if the same solute is used, the moving speed becomes non-uniform depending on the position in the column. This widens the solute bandwidth and degrades the separation performance of the column. Thus, in order to quickly achieve temperature equilibrium in the column and maintain the separation performance of the column, it is effective to make the column thinner.

For the above reasons, a small packing material is packed into a short and narrow column in order to exhibit the high performance of the column. And the solute band eluted from a small high-performance column has a very small volume and dispersion (Non-patent Document 8).

  Further, as a solvent for preparing a sample by dissolving a solute, a solvent having a strong dissolution power, a solvent having a high dissolving power, or a solvent having the same composition as the mobile phase is often used. When the sample is injected into the column, the solute advances in the column by the solvent itself in the sample, so that the sample bandwidth is widened and the number of theoretical plates is reduced. Therefore, it is very important to reduce the volume of the sample to be injected. However, when using a column having an inner diameter of 1.0 to 4.6 mm based on the ease of sample handling and the sensitivity of the detector, A 0.1-20 μL sample is injected. When the sample flows through a flow path from the sample injection device such as an injector to the column, for example, a connecting pipe, a laminar flow (in the form of a parabolic flow profile that is fast at the center of the pipe and slow at the wall) The presence of unswept volume (residence effect) widens the sample bandwidth.

The peak at which each sample component (solute) separated by the column appears on the chromatogram is approximated by a Gaussian distribution. Dispersion of Gaussian distribution (σ ² : square of standard deviation σ) is additive, and dispersion of solute bands separated using a certain HPLC apparatus and column (dispersion of the entire solute band, σ _total ² ) is As shown in Equation 1, the solute band dispersion σ _col ² provided by the column main body and the solute band dispersion σ _extra ^{2 in} the channel outside the column are represented as the sum (Non-patent Document 1).

(Formula 1)
σ _total ² = σ _col ² + σ _extra ²

The dispersion σ _extra ² of the solute band outside the column is expressed as follows: (1) The dispersion of the solute band (σ _inj ) in the sample injection device (inj) and the pipe (tube 1) from the sample injection device to the column inlet. ² + σ _tube1 ² ), (2) Dispersion of solute band (σ _tube2 ² + σ _det ² ) in the pipe (tube2) and detector cell (det) from the column outlet to the detector, and (3) from the detector It consists of the signal averaging and the dispersion of the solute band σ _data ² resulting from the time interval at which the data processor takes the signal.

(Formula 2)
σ _extra ² = σ _inj ² + σ _tube1 ² + σ _tube2 ² + σ _det ² + σ _data ²

The peak dispersion (total solute band dispersion, σ _total ² ) actually obtained in the HPLC apparatus or UHPLC apparatus is calculated by adding the solute band dispersion (σ _col ² ) in the column to the right side of Expression 2 As shown.

(Formula 3)
σ _total ² = σ _inj ² + σ _tube1 ² + σ _col ² + σ _tube2 ² + σ _det ² + σ _data ²

The theoretical plate number (N) of the column is calculated using the retention time (t _R ) (or retention volume (V _R )) of the solute and the dispersion of the solute band, as shown in Equation 4. Here, σ _total-t ² is the variance calculated as the observed time, and σ _total-v ² is the variance calculated as the volume. Hereinafter, the number of theoretical plates was calculated assuming that the peak half-width (peak width at ½ of the peak height) = 2.35σ.

(Formula 4) N = (t _R ² / σ _total-t ² ) = (V _R ² / σ _total-v ² )

Compared with the theoretical plate number (N _col ) that the column should show under ideal conditions without σ _extra ² shown in Equation 5, it is obtained by actually connecting the column to an HPLC device or UHPLC device shown in Equation 4. The number of theoretical plates (N) that can be achieved is generally small. This is based on the contribution of the dispersion σ _extra ² of the solute band outside the column shown in Equations 1 and 2.

(Formula 5) N _col = (t _R ² / σ _col-t ² ) = (V _R ² / σ _col-v ² )

Here, the reason why σ _data ² of (3) is included in σ _extra ² will be described below. The chromatogram plots the detector signal against time. When the chromatogram is displayed on the data processor, the detector output slowness and the data processor slow data acquisition also give the peak spread (dispersion) on a time scale. When the mobile phase liquid is flowing at a constant flow rate, the dispersion of the solute band with the time as a scale can be converted into a dispersion with the volume as a scale by using the flow speed.

In order to obtain high performance in HPLC separation using a small column or UHPLC separation, it is necessary to minimize the dispersion σ _extra ² of the solute band outside the column. Among σ _extra ² , σ _data ^{2 in} (3) above is minimized by shortening the response time of the detector and shortening the data acquisition interval of the data processing device.

In addition, σ _tube2 ² + σ _det ^{2 in} (2) above is minimized by reducing the inner diameter and capacity of the detector cell flow path by reducing the length and length of the connection pipe between the column outlet and the detector cell. It becomes.

When injecting a liquid sample into an HPLC apparatus or UHPLC apparatus, a sample band is a flow path from a sample injection apparatus such as an autosampler or injector through a pipe to a separation column, laminar flow (parabolic flow profile), and Diffusion occurs due to the retention effect due to the unswept volume caused by the corners of the connecting parts. Σ _inj ² + σ _tube1 ^{2 in} (1) above also reduces the inner diameter of the pipe from the sample injector to the column, shortens the length, and decreases the inner diameter of the sample loop and sample needle of the sample injector. Is minimized by reducing the orifice of the valve through which the sample passes, but the sample loop of the sample injection device, the sample needle, and the orifice of the valve need to pass the liquid sample with good reproducibility under suction or atmospheric pressure. A practical inner diameter that is difficult to resist is required.

As described above, the dispersion σ _extra ² of the solute band outside the column becomes a relatively large contribution as the column size becomes smaller, and the smaller the column, the less likely the column's inherent performance is exhibited. In a generally used HPLC apparatus or UHPLC apparatus, when the column tube has a volume of less than 200 μL (for example, a column having an inner diameter of 2.1 mm and a length of 5 cm (column tube capacity of about 173 μL), an inner diameter of 1.0 mm and a length of 25 cm ( This tendency becomes remarkable in the case of a column having a column tube capacity of about 196 μL) or a column having a smaller capacity than these.

Specifically, in a UHPLC apparatus, a column having an inner diameter of 2.1 mm and a length of 5 cm is often used, and in many cases, a sample of 0.1 to 2.0 μL is injected. In many UHPLC apparatuses, due to the dispersion σ _extra ² of the solute band outside the column, about 30% in the column having an inner diameter of 2.1 mm and a length of 5 cm and an inner diameter of 1 for a solute giving a retention coefficient k = about 5. A decrease in the number of theoretical plates of about 70% or more has been reported for a column of 0.0 mm and a length of 5 cm (column tube capacity: about 39 μL) (Non-patent Document 2).

In addition, the smaller the column, the larger the proportion of the solute band dispersion σ _extra ² occupies outside the column relative to the _total solute band dispersion σ _total ² . When the retention coefficient is small, it becomes more remarkable. In the UHPLC apparatus, about a solute that gives a retention coefficient k = about 1, a reduction in the number of theoretical plates of about 50% has been reported even in a column having an inner diameter of 2.1 mm and a length of 5 cm (Non-patent Document 3). For a column with an inner diameter of 1.0 mm and a length of 5 cm, σ _extra ² causes a further significant performance degradation (Non-Patent Document 8).

In addition, in order to reduce the dispersion σ _extra ² of the solute band outside the column of the UHPLC apparatus, the pipe that was originally provided in the apparatus from the sample injection device to the column (inner diameter 0.127 mm, length 55 cm) To improve the performance of the 2.1 mm inner diameter, 5 cm long column, 2.1 mm inner diameter, 10 cm long column by changing to a shorter pipe (inner diameter 0.127 mm, length 25 cm) An example has been reported (Non-Patent Document 5).

The sample injection device of the HPLC apparatus or UHPLC apparatus is installed between the liquid feed pump and the column, and usually has an injection port for storing the sample and a sample loop for temporarily storing the sample, and is inserted from the injection port. The sample is incorporated into a flow path between the liquid feed pump and the column and introduced into the column. Since the sample incorporated in the flow path moves while diffusing the flow path to the column inlet, the capacity of the flow path greatly affects the dispersion of the sample band. An autosampler, which is a kind of sample injection device, is equipped with a flow path switching valve connected to a sample loop with a volume of 5.0 to 50 μL. The connection from the outlet of the flow path switching valve to the column inlet has an outer diameter. A pipe having a length of 1.6 mm, an inner diameter of 0.1 to 0.25 mm, and a length of 20 to 30 cm is often used. Among stainless steel tubes having an outer diameter of 1.6 mm that has a pressure resistance and is not easily damaged even when the column is repeatedly replaced, the inner diameter is 0.1 mm, which is the smallest. In addition, when the autosampler and the column oven are separate units, a certain length of piping is required from the flow path switching valve to the column inlet.

The dispersion σ _extra ² of the solute band outside the column becomes a relatively large contribution when the column size is reduced, and lowers the column performance. It is necessary to minimize σ _extra ^{2 in} order to obtain the performance (theoretical plate number) inherent to the column using a column with a small inner diameter and a short length. As for the piping from the injection device to the column inlet, there is an actual limitation as described above for minimization. So, for the sample band that has been injected by the sample injection device and then diffused in the flow path to the column inlet, There is an effective solution other than the above method or a method of concentrating the sample by adsorbing the sample as a narrow band on the stationary phase at the column inlet using a solvent having a small dissolution power (Non-patent Documents 6 and 7). It was not found.

On the other hand, in capillary gas chromatography columns and capillary liquid chromatography columns with a small amount of packing material (stationary phase), if the volume of the sample or the weight of the solute contained in the sample is overloaded to the column In addition, column performance is maintained by split injection to reduce the volume of a sample or the weight of a solute contained in the sample to such an extent that the column can be processed (Patent Document 1, Non-Patent Document 4).

This split injection is performed in such a way that a portion is taken from an amount of sample that is too large for the column, i.e. a portion of the flowing sample band is taken from the column inlet and the majority is discharged and discarded. In this split injection method, many samples are inevitably discarded. This split injection method is performed when the volume of the sample or the weight of the solute contained in the sample is excessive with respect to the column, and is not performed when the sample is in an appropriate amount with respect to the column. .

In addition, since split injection is performed in the form of branching the flow path in the middle of the pipe from the sample injection apparatus to the column inlet in the HPLC apparatus or UHPLC apparatus, dispersion of the solute band cannot be reduced.

On the other hand, in the HPLC apparatus, a second liquid feeding port is provided at the column inlet separately from the first liquid feeding port, and the method of distinguishing the liquid feeding to the column center and the liquid feeding to the column peripheral part is as follows. It has been proposed for the purpose of controlling the laminar flow (a flow profile that draws a parabolic shape) that occurs in the inside (Non-Patent Document 9 and Non-Patent Document 10).

US Pat. No. 3,513,636

A. Prus, C. Kempter, J. Gysler, T. Jira, Journal of Chromatography A, 1016, 129-141 (2003). Extracolumn band broadening in capillary liquid chromatography. N. Wu, A. C. Bradley, Journal of Chromatography A, 1261, 113-120 (2012). Effect of column dimension on observed column efficiency in very high pressure liquid chromatography F. Gritti, C. A. Sanchez, T. Farkas, G. Guiochon, Journal of Chromatography A, 1217, 3000-3012 (2010). Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments. N. Wu, J. A. Lippert, M. L Lee, Journal of Chromatography A, 911, 1-12 (2001). Practical aspects of ultrahigh pressure capillary liquid chromatography. F. Gritti, CA Sanchez, T. Farkas, G. Guiochon, Journal of Chromatography A, 1217, 7677-7689 (2010). On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1mmI. D. columns packed with sub-2 μm particles. J. P. C. Vissers, A. H. de Ru, M. Ursem, J.-P. Chervet, Journal of Chromatography A, 746, 1-7 (1996). Optimised injection techniques for micro and capillary liquid chromatography. A. C. Sanchez, J. A. Anspach, T. Farkas, Journal of Chromatography A, 1228, 338-348 (2012) Performance optimizing injection sequence for minimizing injection band broadening contributions in high efficiency liquid chromatographic separations. Y. Ma, AW Chassy, S. Miyazaki, M. Motokawa, K. Morisato, H. Uzu, M. Ohira, M. Furuno, K. Nakanishi, H. Minakuchi, K. Mriziq, T. Farkas, O. Fiehn , N. Tanaka, Journal of Chromatography A, 1383, 47-57 (2015). Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions. T. J. N. Webber, E. H. McKerrell, Journal of Chromatography, 122, 243-258 (1976). Optimization of liquid chromatographic performance on columns packed with microparticulate silicas. R. A. Shalliker, M. Camenzuli, L. Pereira, H. J. Ritchie, Journal of Chromatography A, 1262, 64-69 (2012). Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a ‘virtual’ narrow bore column.

As described above, the sample injected into the HPLC apparatus or the UHPLC apparatus causes a large dispersion in the piping from the sample injection apparatus to the column. When using a small column (small inner diameter and short length), it is necessary to minimize dispersion of the solute band outside the column in order to obtain the performance (theoretical plate number) that the column originally has. For the sample band that diffuses in the flow path from the sample injection device to the column inlet while having a volume and solute weight appropriate for the above, the above-mentioned conventional technique, in particular, a solvent having a low solute power has been used. No effective solution was found other than the method of adsorbing and concentrating the sample as a narrow band on this stationary phase.

There is also a method in which a valve with a concentration column is provided between the sample injection device and the column, and the sample is once adsorbed to the concentration column as a narrow band and then introduced into the column. Concentration according to the solute is necessary, and cannot be applied under conditions where concentration is difficult.

  The split injection described in Non-Patent Document 4 is performed when the sample volume or the solute weight in the sample is excessive with respect to the column. This is not done when the sample is already in the correct amount for the column. Further, since the flow path is branched in the middle of the pipe from the sample injection device to the column inlet, the dispersion of the solute band cannot be reduced.

Furthermore, there is a method in which the sample attached to the sample injection apparatus is repeatedly switched to divide the sample and cut into narrow bands, but thereafter, the sample band causes a large dispersion in the pipes leading to the column.

In addition, in the examples of Non-Patent Document 9 and Non-Patent Document 10, a second liquid feeding port is provided at the column inlet separately from the first liquid feeding port, and the liquid feeding to the center of the column and the feeding to the periphery of the inner wall of the column tube are performed. The purpose is to distinguish the liquids, keep the speed ratio between the liquid delivery ports constant, and reduce the effect of the mobile phase solvent speed difference between the center of the column and the periphery of the inner wall of the column tube over the entire length of the column. The liquid feeding from the two liquid feeding ports was not a technique used for dividing the sample band introduced from the first liquid feeding port or reducing the dispersion of the solute outside the column.

Therefore, the present invention expresses even higher column performance not only when the volume of the sample and the solute weight in the sample are excessive with respect to the column, but also when the sample is considered to be an appropriate amount for the column. Therefore, an object is to reduce dispersion of the solute band outside the column.

The present invention as a means for solving the above-mentioned problem is that in liquid chromatography, when a sample is introduced into a separation column, the sample band is cut and divided in a flow path between the sample injection device and the column, The invention improves the separation performance of the column by reducing the dispersion of the sample bands entering the column at once.

When a liquid sample is injected into a chromatographic apparatus, particularly a liquid chromatography apparatus, the sample band diffuses in the process from the sample injection apparatus (autosampler or injector, etc.) through the flow path to the column, thereby spreading the sample band. Wake up. Sample bands that extend to the column inlet reduce column performance. The effect of this expansion of the band outside the column (dispersion of the solute band outside the column) on the column performance (out-column effect) becomes a relatively large contribution when the column size is reduced, and high performance liquid chromatography can be applied to small columns. When used in an (HPLC) apparatus or an ultra high pressure liquid chromatography (or ultra high performance liquid chromatography, UHPLC) apparatus, the performance inherent in the column cannot be expressed. In the present invention, a sample band injected by a sample injection device and diffused in a pipe is cut at a column inlet in a direction crossing the axial direction (flow direction) of the sample band, and is introduced into the column at a time. By reducing the volume and dispersion of the sample band, the separation performance (theoretical plate number) of the column is improved. In the HPLC apparatus and UHPLC apparatus, the dispersion of the solute band outside the column is important. It solves the problem.

  More specifically, it is a device provided between a sample injection device of a sample introduction device and a column, and is a sample band dividing device for dividing a sample band.

  The sample band splitting device is a device provided between the sample injection device and the column of the sample introduction device for introducing the sample into the column in the liquid chromatography device, for sending the sample band to the column A sample band dividing device for dividing a sample band, comprising a first liquid feeding port and a dividing port for dividing a sample band sent from the first liquid feeding port.

  In the sample band dividing device, the sample band dividing device is a device for dividing the sample band in a direction crossing the axial direction.

In the sample band splitting device, the split port is configured to include a second liquid feeding port for sending the mobile phase to the column and / or a first discharge port for discharging a part of the sample band. Is a sample band dividing device characterized by the following.

  In the sample band dividing device, the volume of the flow path from the flow path switching point between the first liquid feed port and the second liquid feed port or the first discharge port to the column inlet is 1 of the column void volume. /13.9 or less is a sample band splitting device.

  In the sample band dividing device, the volume of the flow path from the flow path switching point between the first liquid feed port and the second liquid feed port or the first discharge port to the column inlet is 1 of the column void volume. /21.1 or less is a sample band splitting device.

  In the sample band dividing device, the volume of the flow path from the flow path switching point between the first liquid feed port and the second liquid feed port or the first discharge port to the column inlet is 1 of the column void volume. This is a sample band dividing device characterized by having a ratio of 21.9 or less.

  In the sample band dividing device, the volume of the flow path from the flow path switching point between the first liquid feed port and the second liquid feed port or the first discharge port to the column inlet is 1 of the column void volume. This is a sample band dividing device characterized by being / 259 or less.

The sample band splitting device is characterized in that a flow path switching valve is provided.

  In the sample band dividing device, the flow path switching valve is a three-way valve, a four-way valve, a six-way valve, an eight-way valve, or a ten-way valve.

The sample band splitting device is characterized in that a three-way or four-way joint is provided.

  Furthermore, the present invention is a sample introduction device provided with the sample band dividing device.

In the sample introduction apparatus, the second introduction port for discharging a part of the sample band upstream from the sample injection apparatus is provided.

  Furthermore, a liquid chromatography apparatus comprising the sample introduction apparatus.

In the liquid chromatography apparatus, a column having an inner diameter of 4.6 mm or less is provided.

In the above liquid chromatography apparatus, the peak elution volume is 10.2 mL or less.

  In liquid chromatography, the sample introduction is characterized in that the sample band is divided in the flow channel before the column from the sample injection device by switching the flow channel, and at least a part of the divided sample band is introduced into the column. Is the method.

In the sample introduction method, the sample introduction is characterized in that when the sample band crosses the column inlet or the channel switching point closest to the column inlet, the channel is switched and the sample band is divided. Is the method.

Further, in the sample introduction method, the sample introduction method is characterized in that the volume of a part of the divided sample bands to be introduced into the column at a time is 1 / 13.9 or less of the column void volume. It is.

In the sample introduction method, the sample band is divided in a direction crossing the axial direction of the sample band.

Further, in the sample introduction method, the sample introduction method is characterized in that all of the portions of the divided sample bands are introduced into the column.

In the sample introduction method, a part of the divided sample band is introduced into a column.

In the sample introduction method, the sample introduction method is characterized in that the front portion of the divided sample band is introduced into the column.

In the sample introduction method, the sample introduction method is characterized in that an intermediate portion of the divided sample band is introduced into a column.

In the sample introduction method, the sample introduction method is characterized in that a rear portion of the divided sample band is introduced into a column.

In the sample introduction method, the sample introduction method is characterized in that a portion of the divided sample band other than the portion introduced into the column is discharged.

In the sample introduction method, the rear part including the middle part of the sample band is not introduced into the column, but is left in the flow path, and the front part of the divided sample band is introduced into the column or discharged from the flow path. Then, the sample introduction method is characterized in that a rear portion of the sample band placed in the flow path is divided and an intermediate portion of the sample band is introduced into the column.

Further, in the sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and the front part of the sample band injected from the sample injection device enters the column, and the sample When the band straddles the column inlet or when the sample band straddles the flow path switching point closest to the column inlet on the flow path, the liquid feeding from the first liquid feeding port that sends the sample band to the column is stopped. , The liquid feeding from the second liquid feeding port provided near the column inlet is started, the sample band is divided, the front part of the sample band is introduced into the column, and the rear part of the sample band is removed from the sample injection device. The sample introduction method is characterized in that it is retained in the connection pipe.

Further, in the sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and the front part of the sample band injected from the sample injection device enters the column, and the sample When the band straddles the column inlet or when the sample band straddles the flow path switching point closest to the column inlet on the flow path, the liquid feeding from the first liquid feeding port that sends the sample band to the column is stopped. Alternatively, the liquid feeding is maintained, the liquid feeding from the second liquid feeding port provided near the column inlet is started, the sample band is divided, the front part of the sample band is introduced into the column, and the vent valve Is opened, the liquid feeding from the first liquid feeding port is resumed or maintained, and the rear part of the sample band is discharged from the first discharge port provided in the flow path near the column inlet. Sample introduction It is the law.

Further, in the sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and a first discharge port is created between the sample injection device and the column to inject the sample. When the front part of the sample band injected from the apparatus enters the column and the sample band straddles the column inlet, or when the sample band straddles the channel switching point closest to the column inlet on the channel, A sample introduction method characterized by opening a first discharge port and discharging a rear portion of a sample band.

Further, in the above sample introduction method, the injected sample band is divided by the liquid supply from the second liquid supply port, and the liquid is discharged between the pump and the sample injection device via the joint and the valve. A second discharge port is provided so that liquid can be discharged at any time by opening and closing the valve. After the sample band is divided, the rear part of the sample band placed in the first liquid supply port is supplied from the second liquid supply port. Thus, the sample introduction method is characterized in that the sample is pushed back through the sample injection device and discharged from a second discharge port provided upstream from the sample injection device.

In the above sample introduction method, the injected sample band is divided by the liquid feed from the second liquid feed port, the rear part of the sample band is left in the first liquid feed port, and the sample band introduced into the column After the sample components contained in the front part are eluted from the column, the part after the sample band placed in the first liquid feeding port is introduced into the column by restarting the liquid feeding from the first liquid feeding port. This is a characteristic sample introduction method.

In the above sample introduction method, the rear part of the sample band placed in the first liquid feeding port is further divided, and at least a part of the part is reintroduced from the first liquid feeding port and introduced into the column. A sample introduction method characterized by the following.

  In the sample introduction method, the range of the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) from the time of sample injection to the sample band division is 10/0 to 1/5. A sample introduction method characterized by the following.

According to the present invention, in liquid chromatography, a sample band spread in a flow path from a sample injection device to a separation column is divided at a column inlet in a direction crossing the axial direction of the sample band and introduced into the column. By reducing the dispersion of the sample band, the separation performance of the column, that is, the number of theoretical plates can be improved.

Overview of sample introduction device Sample band division method The figure which shows embodiment of a sample band division | segmentation apparatus The figure which shows embodiment of a sample band division | segmentation apparatus, and a division | segmentation process The figure which shows the Example of a sample band division | segmentation apparatus The figure which shows the Example of a sample band division | segmentation apparatus The figure which shows the Example of a sample band division | segmentation apparatus The figure which shows the Example of a sample band division | segmentation apparatus The figure which shows the Example of a sample band division | segmentation apparatus The figure which shows the Example of the sample introduction apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure The figure which shows the Example of the sample band division | segmentation apparatus which has a vent structure Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example Chromatogram of analysis results using the sample band dividing method of the present invention and the injection method of the comparative example The graph which shows the correlation with the liquid feeding volume from the 1st liquid feeding port until sample band division | segmentation, and the introduction rate to a column

In the liquid chromatography including HPLC and UHPLC, the present invention includes a sample injection device and a sample injection device from the sample injection device to the column inlet. The sample band that has spread in the flow path including the pipe is cut in the direction that intersects the axial direction of the sample band in the sample band splitting device provided before the column entrance, specifically, between the sample injection device of the sample introduction device and the column. By dividing and introducing a part of the sample band into the column, dispersion of the solute band in the sample injection device (inj) and the pipe (tube1) from the sample injection device to the column inlet of Equation 2, that is, (σ _{inj ²} + ^{σ tube1 2)} a with a smaller decrease the variance (σ _extra ²⁾ of the solute band in outer column, equations 1 and 4 Definitive (sigma _total ^2), i.e., by subtracting the dispersion of the solute band in the whole separation system, column performance (theoretical plate number N, Equation 4) to increase, i.e., the sample improves the separation performance actually column brings the band A dividing device, a sample band introducing device, and a sample band introducing method for dividing a sample band and introducing it into a column.

Liquid chromatography includes analysis and fractionation. The amount of sample injected from the sample injection device is not particularly limited, but in the case of analytical HPLC, a small volume of 0.01 to 5 μL, and in preparative HPLC, it is appropriate depending on the size of the device and the column. Can be injected. The chromatography apparatus using the method of the present invention or installing the sample introduction apparatus of the present invention is a known apparatus constructed by connecting a pump, a sample injection device, a column, and a detector in this order.

In addition, the present invention is more specifically described when the sample band reaches the column inlet, specifically, when the front part of the sample band that has been injected and expanded further enters the column, that is, the sample band straddles the column inlet. Or when the sample band straddles the flow path switching point closest to the column inlet, the sample band is cut by switching the flow path, and part of the sample band, for example, the front part and the rear part (tail Separation of the column by reducing the volume and dispersion of the sample band entering the column by allowing only the front part of the sample band to enter the column and not allowing the rear part to enter the column at least temporarily. A method and apparatus for improving performance. In addition, the part ahead of the advancing direction among the parts which the sample band was cut | disconnected and divided | segmented is called a front part, the part behind the advancing direction is called a rear part, and parts other than a front part and a rear part are called intermediate parts.

The sample injection device includes, for example, an injector using a 6-way switching valve or a similar valve, an autoinjector, and an autosampler, and a known device generally used in HPLC or UHPLC can be used.

The method of dividing the sample band by cutting the sample band in the vicinity of the column inlet and introducing the divided sample band into the column, the sample band dividing device for dividing the sample band and introducing it into the column, and the sample introduction device are shown below. Methods and apparatus can be employed.

(1) Sample band splitting-Sample introduction method and apparatus with rear partial detention When a sample is entering the column inlet from the pipe (first liquid feeding port) from the sample injection apparatus, the joint between the pipe and the column (ie At the column inlet), the sample band is divided into two or three or more by starting the supply of the mobile phase solvent from a route (second liquid feeding port) different from the first liquid feeding port (FIGS. 1 and 2). . At this time, if the supply of the mobile phase solvent from the first liquid feeding port is stopped, only the front part of the sample band enters the column, and the rear part of the sample band remains in the first liquid feeding port (FIG. 2 (4)). To (6)). Thereafter, the components in the front portion of the sample band introduced into the column can be separated by liquid feeding from the second liquid feeding port. FIG. 1 shows an outline of a sample introduction device provided with a sample band dividing device having a second liquid feeding port, and FIGS. 3, 4 and 5 show embodiments and examples of the sample band dividing device. . As will be described later, when separation analysis of the rear part of the sample band is not necessary, a discharge port (vent) may be provided at an appropriate position upstream or downstream of the sample injection device to discharge the rear part. Yes (FIGS. 6 and 7).

(2) Sample band splitting-Sample introduction method and apparatus with rear partial discharge At the connection between the pipe from the sample injection device and the column inlet, a second liquid feeding port for supplying the mobile phase solvent by another route, and A third port (first discharge port or second discharge port) that can discharge the liquid from the flow path by valve operation is provided, and when the front part of the sample band is entering the column, the liquid is fed from the second liquid feed port. By starting the liquid and discharging from the third port, the sample band can be cut, the rear part of the sample band can be discharged from the flow path, and only the front part of the sample band can be introduced into the column (see FIG. 6 and FIG. 7).

(3) Sample introduction method and apparatus with sample division by rear part discharge from the sample band Port at which the liquid can be discharged from the flow path by valve operation at the connection part between the pipe from the sample injection device and the column inlet (first discharge) Port) and when the front part of the sample band enters the column and the rear part (tail part) of the sample band remains in the pipe, it is discharged from the first discharge port. The part can be discharged from the flow path and only the front part of the sample band can be introduced into the column (FIG. 8).

In these methods, the sample band dividing unit in the sample band dividing apparatus uses a division port constituted by the second liquid feeding port or / and the first discharge port for discharging a part of the sample band, Dividing the sample band injected from the sample injection device in a direction crossing the axial direction (traveling direction) of the sample band, introducing the front part of the sample band into the column and discharging the rear part of the sample band, the front part of the sample band To discharge only the rear part of the sample band into the column, to discharge the front part and the rear part of the sample band and to introduce only the middle part into the column, or to continuously or partially part of the sample band By dividing, the divided parts can be continuously introduced into the column at a predetermined interval. That is, the sample band can be divided into two or more, one or more divided portions can be introduced into the column, and other portions can be discharged.

By the method as described above, a part of the sample band, for example, the rear part (tail part) is discharged, the other part of the sample band, for example, only the front part is introduced into the column, and the dispersion of the sample band is reduced to improve the column performance. It is possible to improve. By reducing the dispersion of the sample band entering the column, the peak width of the chromatogram can be reduced.

Next, a sample introduction method involving the division of the sample band and the partial indwelling will be described in detail. In carrying out this method, as shown in FIG. 1, the liquid chromatograph is a sample provided with a sample band dividing device 10 having a sample band dividing unit 11 for dividing a sample band in the vicinity of the inlet of the column 1. A liquid chromatograph apparatus provided with an introduction apparatus 91 is used. The sample introduction device 91 includes the sample band dividing device 10 and the column 1 from the liquid feeding pump 21 through the sample injecting device 22 and the connection pipe 23. The sample band dividing device 10 includes the first liquid feeding port 20 and the like. In addition, a second liquid feeding port 29 that can separately supply a mobile phase solvent and a flow path 28 to the column 1 inlet can be provided. Further, the sample band dividing device may be provided in the sample introduction device together with the second discharge port 601 as shown in FIG. 6, or may be provided with the first discharge port 60 as shown in FIG. .

As shown in FIG. 1, the sample band dividing unit 11 includes a flow path switching point 12 for switching the flow path between the first liquid feeding port and the second liquid feeding port, and the first liquid feeding port in the vicinity of the flow path switching point 12. 20, a tip portion of the second liquid feeding port 29, and a flow path 28 to the column 1 inlet. The cutting of the sample band is performed by switching the liquid feeding at the flow path switching point 12 that is the outlet of the first liquid feeding port. In FIG. 1, the mobile phase is supplied to the second liquid feeding port 29 through a route different from that of the first liquid feeding port 20 or from another liquid feeding pump (not shown). A filter 25 may be installed at the tip of the column 1.

As shown in FIG. 1, the sample is supplied from the sample injection device 22 to the column 1 through the first liquid feeding port 20. The first liquid feeding port 20 may be disposed between the connection pipe 23 connected to the sample injection device 22 and the column 1, or may be disposed between the sample injection device 22 and the column 1. The mobile phase solvent and the sample are sent to the column 1 through the connection pipe 23 and the first liquid feeding port 20 or only through the first liquid feeding port 20.

FIG. 2 shows the progress of the sample band 3 in the first liquid feeding port 20 from the sample injection device 22 to the column 1 and the operating state of the pump (+ = on, 0 = in the sample introduction device 91 and the sample band dividing device 10. FIG. 5 is a liquid feeding process diagram showing a liquid feeding pump 21 for feeding off from the first liquid feeding port 20 and liquid feeding pump 291 for feeding liquid from the second liquid feeding port 29 by B. As shown in FIGS. 2 (1) to (3), the sample band 3 advances in the first liquid supply port 20 by the operation of the liquid feed pump 21, and as shown in FIG. 2 (4), the sample band When 3 crosses the flow path switching point 12 of the sample band dividing section 11, the liquid feeding pump 21 is stopped and the liquid feeding from the first liquid feeding port 20 is stopped, and at the same time, the liquid feeding pump 291 is operated, When liquid feeding is started from the second liquid feeding port 29, the sample band 3 is divided in the sample band dividing unit 11 as shown in FIGS. 2 (5) to (6), and the front portion 31 of the divided sample band 3 is formed. It is fed into the column 1 from the column inlet, and the rear portion 32 of the sample band 3 is retained in the first liquid feeding port 20.

Instead of switching the liquid feeding state of the two pumps of the liquid feeding pump 21 and the liquid feeding pump 291, a flow path switching valve is installed at the outlet of the liquid feeding pump 21, and the liquid flow of the pump is continued and the flow path is maintained. Even by switching, the liquid feeding from the first liquid feeding port 20 can be stopped and the liquid feeding from the second liquid feeding port 29 can be started at the same time.

The sample band 3 is divided when the front part of the sample band 3 injected from the sample injection device enters the column, the liquid supply from the first liquid supply port for sending the sample band 3 to the column 1 is stopped, and the column It is good also as starting liquid feeding from the 2nd liquid feeding port provided in the entrance vicinity. In addition, liquid feeding from the second liquid feeding port 29 is continuously performed in advance simultaneously with liquid feeding from the first liquid feeding port 20 before sample injection, and liquid feeding from the first liquid feeding port 20 is performed at the time of sample band division. In the method of stopping and continuing the liquid feeding from the second liquid feeding port 29, the liquid feeding from the second liquid feeding port 29 is performed in advance simultaneously with the liquid feeding from the first liquid feeding port 20 before the sample injection. It is also possible to adopt a method that is continuously performed and stops the liquid feeding from the first liquid feeding port 20 at the time of dividing the sample band and increases the liquid feeding amount (flow velocity) from the second liquid feeding port 29.

Thereafter, when the liquid feeding from the second liquid feeding port 29 is continued, the front portion 31 of the sample band 3 introduced into the column 1 is separated and eluted through a normal chromatographic separation process. According to this introduction method, the sample band is divided into a front portion and a rear portion (tail portion) near the inlet of the column 1 by dividing the sample band 3 in the intersecting direction including the direction orthogonal to the axial direction (flow direction). In this case, only the front portion is introduced into the column 1, and the band broadening (dispersion) of the sample entering the column 1 can be made extremely small.

After the separation and elution of the sample band front portion 31 introduced into the column 1 is completed, if the liquid feeding pump 21 restarts the liquid feeding from the first liquid feeding port 20, the liquid is kept in the first liquid feeding port 20. A rear portion 32 of the sample band is introduced into the column 1 for a second chromatographic separation. By repeating this operation, the entire sample band 3 injected at a time can be divided into two or more times and introduced into the column 1, and each part can be separated and eluted or separated and analyzed.

After separation of the sample band front portion 31 into the column 1, when separation analysis is not necessary for the sample band rear portion 32, the second portion is provided between the liquid feed pump 21 and the sample injection device 22 as shown in FIG. A sample band is provided by providing a discharge port (vent) 601 for discharge or by providing a first discharge port 60 between the sample injection device 22 and the column 1 for discharge as shown in FIG. It is possible to discharge the rear part of the sample band from the discharge port before the start of separation elution of the front part of the sample band, during separation elution, or after the end of separation elution. These structures and operating methods will be described later.

As an example of a sample band splitting device for carrying out such a method of splitting a sample band by liquid feeding from the second liquid feeding port and introducing it into a column, as shown in FIG. ) A chromatographic apparatus having a sample band dividing device 10 using 41 and the double pipe 42 and having the inner pipe 421 of the double pipe 42 as the first liquid feeding port and the outer pipe 422 as the second liquid feeding port. Can be used. This structure of the sample band splitting device makes it possible to split the sample band immediately before the solvent filter (frit) at the column inlet for a commonly used column. By configuring the first liquid feeding port and the second liquid feeding port using a double tube, the sample introduction of the present invention is applied to a column having a general column end joint (column end joint having one connecting pipe port). The method can be carried out, and there is an advantage that a column end joint having a special structure having a plurality of liquid feeding passages is not required.

In FIG. 3, the T-shaped connector 41 can connect a general 1/16 inch (outer diameter 1.6 mm) outer diameter tube. The inner pipe 421 of the double pipe, that is, the first liquid feeding port 20 is a stainless steel pipe (length: 10 to 25 cm) having an inner diameter of 0.05 to 0.25 mm and an outer diameter of 0.1 to 0.4 mm. The double tube outer tube 422 and the second liquid feeding port 29 connected to the column 1 inlet are stainless steel tubes having an inner diameter of 0.4 to 1.0 mm and an outer diameter of 1.6 mm. The flow path switching point 12 for switching the flow path between the first liquid feeding port 20 and the second liquid feeding port 29 is formed in a double pipe outer pipe 422. Further, the branch pipe for feeding liquid to the second liquid feeding port is a stainless steel pipe having an inner diameter of 0.1 to 0.5 mm and an outer diameter of 1.6 mm. In addition, the size of the connecting pipe is appropriately adjusted without being bound by the above range depending on the scale and flow rate range of the liquid chromatograph apparatus. The connection between each tube and the T-shaped connector or column end joint is made using an appropriate ferrule and oscine or nut.

As shown in FIG. 3A, the ends of the inner tube 421 and the outer tube 422 of the double tube are structured on the same plane as the orifice inlet of the column end joint 17. Further, as long as the inner tube of the double tube does not damage the filter 25 and the packing layer of the column 1, the inner tube may be longer than the outer tube (FIGS. 3B and 3C) or slightly shorter. . Further, a configuration in which the sample can be directly introduced into the filler layer 18 by removing the intra-column filter 25 (FIG. 3D) may be employed.

As shown in FIG. 3, when a double pipe inner pipe 421 is used as the first liquid feed port 20 and a double pipe outer pipe 422 is used as the second liquid feed port 29, a T-shaped connector is used. 421 penetrates the T-shaped connector, and the outer tube 422 is located outside one inner tube 421 and connected to one port of the T-shaped connector. When the sample band is moving from the liquid port to the column, the sample band is divided by stopping the liquid supply from the first liquid supply port and switching to the liquid supply from the second liquid supply port. It is possible to adopt a sample introduction method in which only the front portion (tip portion) of the sample band is introduced into the column while keeping the rear portion in the first liquid feeding port.

Further, as shown in FIG. 4, the sample injection device 22 and the column 1 are switched using a flow path switching valve for switching the liquid flow path from the pump, for example, a three-way, four-way, six-way, eight-way, or ten-way switching valve. The second liquid feeding port 52 is formed on the flow path close to the column 1 inlet between the two, and the sample band is cut at the flow path switching point 50 by using the second liquid feeding port 52, and the sample band is divided into the front part and the rear part. Even with a structure having a function of being placed in the ring 53 or discharged from the loop cleaning pipes 57 and 58 by using a separate liquid feeding pump, the sample introduction method of the present invention involving the division of the sample band and the partial placement of the sample band can be carried out.

As shown in these examples, by dividing the sample band at the flow path switching point near the column 1 inlet, the dispersion of the sample diffused in the sample injection device and the pipe from the sample injection device to the column inlet is greatly reduced. Can be made small. If the capacity of the flow path from the flow path switching point to the column inlet is large, the divided sample band diffuses before being introduced into the column, so the capacity of the flow path from the flow path switching point to the column inlet If the other factors affecting the solute dispersion are constant, the sample of the present invention should have a smaller ratio of the channel volume from the channel switching point to the column inlet / column void volume. The effectiveness of the introduction method is high.

  In the following, three examples of the ratio of the volume from the flow path switching point to the column inlet / the column void volume are shown from the examples where the effectiveness of the sample introduction method of the present invention was confirmed.

  In Examples 1 to 9, 12, and 13 (FIGS. 10 to 18, 21, and 22), since the same sample dividing apparatus is used, the capacity from the flow path switching point to the column inlet is the same. This volume is from the elution volume of naphthalene 0.65 μL (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.013 min) in the second chromatogram of Example 4, FIG. It is calculated as 0.07 μL by subtracting the zero dead volume union (ZDU, capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 0.55 μL), and the detector cell capacity 6 nL. Non-patent document 2 is referred to for the method of measuring the capacity of the apparatus flow path based on the measured value. As for the void volume of the column, the void volume of Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, column tube capacity about 39.3 μL) was measured with the uracil in Example 3 and FIG. 12 (A). Value (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.40 min) and the measured value of naphthalene in Example 4, FIG. 13B (measured value at a flow rate of 0.05 mL / min) , 0.05 mL / min × 0.037 min), calculated as 20.0−1.85 = 18.15 μL, and the capacity of ZDU used in Example 4 and FIG. 13B is 0.02 μL. Is calculated as 18.13 μL, and the porosity is about 46.1%.

In these calculations, when there is no column, the response of the detector is 0.01 seconds, the data acquisition interval is 5 mSec, and the peak top value of the chromatogram is 0. When the column is present, the response of the detector is 0. The peak value of the chromatogram at 50 mSec is used for 05 seconds and the data acquisition interval is assumed to contribute little to the original peak shape and retention time, so the detector response and data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / column void capacity = 1/259. As will be described later, in Example 3, the number of theoretical plates is 1.2 to 2.8 times higher than that of the conventional injection method by the sample introduction method of the present invention. The increase in the number of theoretical plates is higher in the peak with the smaller retention capacity, and in particular, the number of theoretical plates 2.8 times as large as that of the non-retention (k = 0) uracil peak. Since the uracil peak passes through the column, the volume necessary for the uracil peak to elute from the column alone is the column void volume (18.13 μL).

On the other hand, in Examples 14 and 15 (FIGS. 23 and 24), the sample dividing apparatus using the micro 6-way flow switching valve shown in FIG. Since the inner pipe has an inner diameter of 0.13 mm and a length of 6.5 cm, the capacity from the flow path switching point to the column inlet is at least 0.86 μL, and the flow path inside the micro 6-way flow path switching valve This is a value to which a capacity (a valve having a flow path diameter of 0.25 mm, the length of the groove of the rotor seal is 5 mm and about 0.25 μL) is added. The average value of the elution volume of uracil obtained from Example 15 and the second to sixth chromatograms of FIG. 24 (F) was 2.45 μL (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.049 min), ZDU (capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 1.12 μL) and the detector cell capacity 6 nL are subtracted and calculated to 1.304 μL. Non-patent document 2 is referred to for the method of measuring the capacity of the apparatus flow path based on the measured value. Regarding the void volume of the column, the void volume of Inert Sustain C18 (filler diameter: 2 μm, inner diameter: 1.0 mm, length: 5 cm, column tube capacity: about 39.3 μL) is measured by measuring uracil in Example 14 (D). Values (measured values at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.53 min) and measured values of the uracil of Example 15 (D) (measured values at a flow rate of 0.05 mL / min) , 0.05 mL / min × 0.167 min), calculated as 26.5-8.35 = 18.15 μL, and the ZDU capacity used in Example 15, FIG. 24D is 0.02 μL. Is calculated to be 18.13 μL, and the porosity is about 46.1%.

In these calculations, when there is no column, the response of the detector is 0.01 seconds, the data acquisition interval is 5 mSec, and the peak top value of the chromatogram is 0. When the column is present, the response of the detector is 0. The peak value of the chromatogram at 50 mSec is used for 05 seconds and the data acquisition interval is assumed to contribute little to the original peak shape and retention time, so the detector response and data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / column void capacity = 1 / 13.9. Further, at least 0.86 μL / 18.15 μL = 1 / 21.1 or more. As will be described later, when Example 14 and FIGS. 23D and 23E are compared, the number of theoretical plates is 1.03 to 3.06 times greater by the sample introduction method of the present invention than the conventional injection method. doing. The increase rate of the number of theoretical plates is higher at the peak with the smaller storage capacity.

On the other hand, in Examples 16 and 17 (FIGS. 25 and 26), the sample band splitting device using the cloth shown in FIG. 7B is used, and the pipe from the cloth to the column inlet has an inner diameter of 0. Since 03 mm and a length of 6 cm are used, the capacity from the flow path switching point to the column inlet is at least 0.042 μL or more. As for the void volume of the column, the void volume of MonoCap C18 (silica monolith column, inner diameter 0.1 mm, length 15 cm, column tube volume about 1.18 μL) was measured with the thiourea in Example 16, FIG. 25 (A). Value (measured value at a flow rate of 0.002 mL / min, 0.002 mL / min × 0.77 min) and the measured value of naphthalene in Example 17 (A) of FIG. 26 (measured value at a flow rate of 0.003 mL / min) , 0.003 mL / min × 0.18 min) is calculated as 1.54-0.54 = 1.00 μL, and the fused silica capillary tube used in Example 17 and FIG. Insert the volume of 0.042 μL and the volume of the zero dead volume union (0.02 μL × 2) to connect the inlet and outlet of the fused silica capillary tube. By subtracting 0.918 μL, the porosity is about 77.8%.

In these calculations, when there is no column, the response of the detector is 0.01 seconds, the data acquisition interval is 5 mSec, and the peak top value of the chromatogram is 0. When the column is present, the response of the detector is 0. The peak value of the chromatogram at 50 mSec is used for 05 seconds and the data acquisition interval is assumed to contribute little to the original peak shape and retention time, so the detector response and data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / the void volume of the column = 1 / 21.9. As will be described later, in Example 16, the number of theoretical plates is 2.5 to 5.0 times higher than that of the conventional injection method by the sample introduction method of the present invention. The increase rate of the number of theoretical plates is higher at the peak with the smaller storage capacity.

  As described above, the largest value of the ratio of the capacity from the flow path switching point to the column inlet / column void capacity is 1 / 13.9, which confirms the effectiveness of the sample introduction method of the present invention. there were. If the ratio is smaller than this, the effectiveness of the sample introduction method of the present invention will be exhibited.

As an example of a sample band dividing device, a sample band introducing device, and a sample band dividing and introducing method, FIG. The structure of the apparatus 93 and the process of dividing the sample band using the apparatus 93 and introducing it into the column are shown. 4A shows a state in which the sample band 3 has started to enter the flow path of the flow path switching valve 5 from the sample injection device 22, and FIG. 4B shows that the sample band 3 has reached the column 1 inlet, The state which straddles the adjacent flow-path switching point 50 (liquid-feeding port switching point) is shown. FIG. 4C shows a state in which the sample band 3 is divided into two parts by switching the flow path from FIG. 4B, and from this state, as shown in FIG. The sample band front part 31 enters the column 1 by the liquid feeding, and the components contained in the sample band front part 31 are separated and eluted. In the state of FIG. 4D, it is possible to discharge the sample band rear portion 32 placed in the first liquid feeding port 51 and the loop 53, but after the elution of the sample band front portion 31 is completed, FIG. As shown in (E), by switching to the liquid feeding from the first liquid feeding port 51, the retained sample band rear portion 32 can be introduced into the column 1 and separated and eluted.

4D, when the sample band rear portion 32 is large, after the elution of the sample band front portion 31 is completed, the flow path is switched and liquid is fed from the first liquid feed port 51, whereby the sample band rear portion. A part of the solution is introduced into the column (FIG. 4 (E)), returned to FIG. 4 (D) again, and can be eluted by feeding from the second solution feeding port 52. Thereafter, by switching the flow paths in FIGS. 4 (E) to 4 (D), the sample injected at once from the sample injection device can be introduced into the column in multiple times and separated and eluted. .

In the sample introduction method of the present invention, by controlling the liquid feed rate (flow rate) from the first liquid feed port and the time from sample injection to sample band division and column introduction, The ratio of the portion introduced into the column, that is, the sample introduction rate can be adjusted. The timing of dividing the sample band and introducing into the column is from the time when the sample band tip reaches the column inlet or the channel switching point until the sample band rear end reaches the column inlet or channel switching point. While the sample band crosses the column inlet or the flow path switching point. The sample introduction rate can be 96 to 4%, and 93 to 10% is preferable.

In addition, in the sample introduction method of the present invention, at the time of sample injection, not only the liquid is fed only from the first liquid feeding port, but also the mobile phase solvent is partially fed from the second liquid feeding port. Control of the rate of introduction of the sample band into the column can also be achieved by stopping the liquid feeding from the first liquid feeding port and increasing the liquid feeding speed from the second liquid feeding port when the sample is divided and introduced into the column. Is possible. In addition, the range of the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) from the time of sample injection to sample division can be 10/0 to 1/9.

In addition, when the sample is injected, the mobile phase solvent is partially fed from the second liquid feeding port, and the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) is adjusted. The sample introduction rate into the column can be easily adjusted. In addition, the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from the time of sample injection to the sample division that maximizes the column performance without degrading the column performance, and the sample to the column You can choose the rate of introduction. The ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from the time of sample injection to sample division is preferably 10/0 to 3/7.

As an example of the sample band splitting device, when the column is directly connected to the injector, the first liquid feeding port is provided and the second liquid feeding is performed using a T-shaped connector and a double tube as shown in FIG. 5A. As shown in FIG. 5B, the column is directly connected to the connection port of the sample injection device (injector) and the second liquid feeding port 52 is provided at the column inlet, as shown in FIG. As shown, by arranging a T-shaped connector between the sample injection device and the column 1 and providing the second liquid feeding port 52 with a branch pipe, the same principle and operation as described above and FIG. It is possible to carry out the sample introduction method involving the division of the sample band and the partial partial placement of the invention.

As an example of the sample band splitting device, as shown in FIG. 5D, the sample band can be split at the inlet of the column 1 and introduced into the column by creating the second liquid feeding port 52 in the column end joint 19. In that case, a column having a specially structured end joint with the second liquid feeding port 52 is required.

As an example of the sample introduction device, when the analysis of the rear portion 32 of the sample band 3 after the division of the sample band is unnecessary, a second discharge port 601 is provided upstream of the sample injection device 22 as shown in FIG. By operating the vent valve 62, the liquid is supplied from the second liquid supply port 29 through the resistance pipe 63 or without the resistance pipe before the separation elution of the front portion 31 of the sample band 3, during the separation elution, or after the separation elution. It is possible to discharge the rear part 32 of the sample band 3.

Next, a sample introduction method involving sample band division and rear partial discharge will be described in detail. In carrying out this method, a liquid chromatograph apparatus, as shown in FIG. 6, in addition to the first liquid feeding port 20, a second liquid feeding port 29 capable of supplying a mobile phase solvent separately, and the column 1 A sample band splitting device 10 having a flow path 28 to the inlet for cutting and splitting the sample band, and a liquid feed pump 21 to a sample injection device 22 and a first liquid feed port 20 in a normal liquid chromatograph. The sample introduction device 95 provided with the connection pipe 23 connected to can be used. A junction of the first liquid feeding port 20 and the second liquid feeding port 29 becomes the flow path switching point 12. In FIG. 6, the mobile phase is supplied to the second liquid feeding port 29 through a route different from that of the first liquid feeding port 20 or from another liquid feeding device (not shown). Further, as described above, the second discharge port 601 is provided upstream of the sample injection device 22.

Then, as shown in FIG. 7A, when a sample band injected from a sample injection device (not shown) is entering the column 1 inlet (or when straddling the flow path switching point 121), the first feeding is performed. The liquid supply from the liquid port 20 is stopped or maintained, the sample band is cut by the liquid supply from the second liquid supply port 29, the sample band is divided, and the sample band from the first discharge port 60 is divided. A sample band dividing apparatus 101 having a sample band dividing portion for discharging (venting) the rear portion (tail portion) can be used.

FIG. 7B shows that a four-way joint 7 is arranged between a sample injection device (not shown) and the column 1, and in addition to the first liquid feeding port 20 and the connection port to the column 1, the second liquid feeding port. 29 is a sample band splitting device 101 having a structure in which 29 and a first discharge port (vent port) 60 are incorporated. In the case of this configuration, the connection portion of the first liquid supply port 20, the second liquid supply port 29, and the first discharge port 60 becomes the flow path switching point 121.

When using the apparatus and method having such a configuration, when the sample band passes through the central portion where the flow paths of the four-way joint (cross) 7 intersect, that is, the flow path switching point 121, in other words, Alternatively, when the sample band straddles the column entrance, the sample band is stopped by stopping the liquid feeding from the first liquid feeding port 20 or maintaining the liquid feeding and starting the liquid feeding from the second liquid feeding port 29. The sample band front part is introduced into the column 1, and the vent valve is opened before starting the separation elution of the sample band front part, during the separation elution, or after the separation elution is completed. It is possible to improve the column performance by restarting or maintaining the liquid feeding and discharging (venting) the rear part of the sample band from the first discharge port 60 to reduce the dispersion of the sample band entering the column.The first discharge port 60 may be provided with a storage pipe (not shown), and the rear portion of the sample band sent to the first discharge port 60 may be placed in the storage pipe instead of being discharged.

In particular, in the case of a sample band that spreads forward and backward (front part and rear part), after the front part of the sample band is introduced into the column or discharged out of the system, it is placed in the connecting pipe from the sample injection device to the column. The rear part of the sample band is divided and placed, or by repeating this operation, the rear part of the sample band is divided, and only the middle part of the sample band is introduced into the column. Thus, the dispersion of the sample band entering the column can be reduced, and the separation performance of the column can be improved.

In this way, the first discharge port 60 is provided together with the second liquid supply port 29 between the sample injection device and the column 1 to start the liquid supply from the second liquid supply port 29 and discharge the rear part of the sample band. As an example of dividing the sample band and introducing the sample band front part into the column, the injector and the column 1 are connected to the four-way joint 7 and the other two ports are connected to the second liquid feeding port 29 and the second one. Used as one discharge port 60. In this case, when the sample band straddles the center of the four-way joint, liquid feeding from the second liquid feeding port 29 is started, and the sample band is divided and introduced into the column in the front part of the sample band, It is possible to discharge the rear part of the sample band from the first discharge port before the start of separation and elution of the solute contained in the front part of the sample band, during separation or elution, or after the end of separation and elution. By installing the second liquid supply port 29 and the first discharge port 60 in another positional relationship between the sample injection device and the column, the start of liquid supply from the second liquid supply port and the first discharge are performed in the same manner as described above. It is possible to discharge the rear part of the sample band from the port and divide the sample band and introduce the part in front of the sample band into the column. In any case, by arranging a storage pipe (not shown) between the first discharge port and the vent valve 62, the rear portion of the sample band discharged from the flow path connecting the sample injection device and the column is temporarily stored. It is also possible to have a structure for storing the target.

In addition, using the first liquid feeding port and the second liquid feeding port, when the sample band straddles the center of the four-way joint, the vent valve is opened without stopping the liquid feeding from the first liquid feeding port, By starting the liquid feeding from the second liquid feeding port, the sample band may be divided and the rear part of the sample band may be discharged. Further, the rear portion of the sample band can be discharged from a discharge port upstream of the sample injection apparatus as shown in FIG. In this case, not only a four-way joint but also a sample band dividing unit using a T-shaped connector and a double tube as shown in FIG. 3 can be used.

  FIG. 5E shows an embodiment of the sample band dividing device in which the first liquid feeding port 51, the second liquid feeding port 52, and the first discharge port 60 are connected to the flow path switching point provided in the column end joint. It can be used in the same manner as the sample band dividing apparatus shown in FIGS. 7 (A) and 7 (B).

Next, an example of a sample introduction method involving sample division by discharging the rear portion (tail portion) from the sample band will be described in detail. In carrying out this method, as shown in FIG. 8, when the sample band is entering the column 1 inlet, it is opened and closed by operating a valve 62 provided immediately before the column 1 inlet. A liquid chromatograph apparatus including a sample band dividing device 101 having a sample band dividing unit 11 for discharging (venting) a rear portion (tail portion) of the sample band from the first discharge port 60 is used. I can do it. The sample band splitting device 101 is configured so that the rear portion (tail portion) of the sample band from the first discharge port 60 is discharged at a flow path switching point 13 that is a connection portion between the first liquid supply port 20 and the first discharge port 60. Split the sample band.

FIG. 8A shows a sample band dividing apparatus 101 having a structure in which the column 1 is connected to the column port 77 communicating with the first liquid feeding port 20 and the first discharge port 60 is provided. The sample band splitting device 101 is not provided with the second liquid feeding port, but includes the first liquid feeding port 20 and the first discharge port 60. 8B shows a structure in which the outlet of the sample injection device (not shown), the sample band dividing section 11 and the column 1 inlet are integrated using the T-shaped connector 41 and the double tube 42. FIG. A first discharge port 60 is provided by a T-shaped connector 41 and a double tube 42 outside the liquid feeding port 20, and the sample band dividing portion 11 is arranged at a position very close to the column inlet in both cases. In FIG. 8D, a T-shaped connector 41 is disposed between the sample injection device and the column 1, and the branch pipe is used as the discharge port 60.

In principle, in any of FIGS. 8A to 8D, a T-connector (three-way joint) is disposed between the sample injection device and the column 1, and the first liquid feeding port 20 and the connection port to the column 1 are provided. In addition, the first discharge port (vent port) 60 is incorporated. Further, as shown in FIG. 8E, the sample band dividing device 101 is provided with the sample dividing portion 11 in the column end joint 28, that is, the tip portions of the first liquid feeding port 20 and the first discharge port 60 are connected to the column end joint. 28 may be provided.

8A to 8E, the front part of the sample band passes through the connection part of the first discharge port 60, that is, the flow path switching point 13, and the rear part of the sample band (tail part) is the flow path switching point 13. At a later time, the vent valve 62 is opened, and the rear part of the sample band is discharged from the first discharge port 60, whereby the sample band is cut and divided, and the rear part of the sample band is not introduced into the column. It is possible to improve column performance by reducing the dispersion of sample bands entering the column.

Incidentally, when the injected sample band enters the column inlet or when the sample band straddles the flow path switching point 13, the liquid feeding speed (flow rate) to the column 1 is temporarily reduced, or By not changing the flow rate or increasing the flow rate, the vent valve 62 is opened and the flow to the first discharge port 60 is started. It is possible to place or discharge in a storage pipe (not shown) provided between the two. In order to introduce the rear portion of the indwelled sample band into the column, a liquid feed pump or the like that can feed liquid from the first discharge port 60 toward the column is separately required.

As described above, an injector is provided as an example of dividing the sample band and introducing it into the column by providing the first discharge port 60 between the sample injection device and the column and discharging the rear portion of the sample band. And the column are connected to a three-way joint, and a branch pipe is used as the discharge port 60. 8A and 8D, the sample band is divided by operating the vent valve 62 when the sample band crosses the flow path branch point 13, and in the case of FIGS. 8B, 8C, and 8E, the sample band is divided. Divides the sample band by operating the vent valve 62 when it crosses the inlet of the column 1, discharges the rear part of the sample band from the first discharge port 60, and then closes the vent valve to feed the liquid to the column By restarting, it is possible to introduce the sample band in front of the column and to perform separation elution and analysis. In any case, by arranging a storage pipe (not shown) between the first discharge port 60 and the vent valve 62, the rear portion of the sample band discharged from the flow path connecting the sample injection device and the column is temporarily stored. It is also possible to have a structure for storing.

As shown in FIG. 9, when the T-shaped connector 73 or the four-way joint 74 is used for the sample band dividing portion 11, FIGS. 9A and 9B corresponding to FIGS. 7B and 8D. In order to prevent the diffusion of the front part of the sample band to be analyzed and the rear part of the discharged sample band from re-entering the flow path to the column 1 due to diffusion, the sample band is divided and introduced into the column. It is preferable that the gap between the pipes on the flow path connecting the sample injection device and the column that enter the inside of the joint for carrying out is narrowed to 0.5 mm or less. A similar structure is possible in the connection form of the other sample band dividing sections (FIG. 5C).

Next, the timing of the start of feeding from the second feeding port and the ratio of (first feeding port flow velocity) / (second feeding port flow velocity) will be described. In the method of dividing the sample band accompanied with the liquid feeding from the second liquid feeding port, by stopping the liquid feeding from the first liquid feeding port and starting the liquid feeding from the second liquid feeding port at the same time, Separation and introduction into the column can be performed, but before the sample injection, the liquid feed from the second liquid feed port is within the range of 1 to 90% of the flow rate from the first liquid feed port. You can start it. Performing liquid feeding from the second liquid feeding port in advance at the time of sample injection facilitates the division of the sample band and acceleration of the second liquid feeding port liquid pump at the time of introduction into the column, so that the column performance is maximized. It is effective to enable the introduction of a sample to be expressed in the cell and to adjust the introduction rate of the sample band into the column.

As described above, the present invention differs from the split injection method conventionally used in capillary liquid chromatography and gas chromatography in the following points.
1. The present invention further divides samples of sample volume (0.01-5 μL) that are standard in normal HPLC or UHPLC and are considered sufficiently small in the state of the art.
2. Rather than venting a large portion of a sample solution flow and taking a portion of it into the column, the present invention divides a standard amount of sample in a direction that intersects its axial direction. The part (front part) is sent to the column for separation elution and analysis, and at the same time, part of the divided sample (rear part) is placed in the flow path between the sample injection device and the column or discharged out of the system .
3. In the present invention, the rear part of the sample placed in the flow path between the sample injection device and the column is separated and eluted after the separation and elution of the front part of the sample is completed, or released from the discharge port before and after the separation and elution. Is done.
4). In split injection used in conventional gas chromatography, the difference in behavior between solutes becomes a problem. In the method of the present invention, the difference in behavior between solutes occurs due to the division of the sample band. Since it is possible to continuously analyze the front part and the rear part of the divided sample band, it is possible to examine it by one injection of the sample.
5. In the conventional sample injection method, even when the solute band provided by the minimum sample volume giving the best performance flows from the sample injection device to the column inlet, the band spread is increased. Can be further divided and introduced into the column.

Hereinafter, a method for embodying the apparatus and operation method of the present invention will be described as an example together with a comparative example. The following examples relate to sample band division-sample introduction method with rear partial placement, sample band division-sample introduction method with rear partial discharge, and sample introduction method with sample division by rear partial discharge from the sample band. Each example is shown. In each of these methods, the column size, the sample injection volume, the detector cell volume, and the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from the time of sample injection to sample division, etc. In some combinations, the sample introduction method of the present invention has an effect of increasing the number of theoretical plates of the column as compared with the conventional sample injection method. How the injected sample band spreads in several combinations of vessel cell volume, (first liquid delivery port flow rate) / (second liquid delivery port flow rate) ratio from the time of sample injection to sample division In this sample introduction method, it is directly visualized how the sample is divided and introduced into the column. In the following examples, the conventional sample injection method (incorporating the sample band in the sample loop into the flow path of the mobile phase by operating the sample injection valve) is referred to as “conventional sample injection method” or “conventional sample injection”. "Method" or "injection".

For sample introduction method with sample band splitting and rear partial placement, sample injection volume 0.1-5.0 μL, column inner diameter 1.0-4.6 mm, column length 5-15 cm, detector cell volume 1 μL Examples include 6 nL, as well as several combinations of ((first liquid feed port flow rate) / (second liquid feed port flow rate) ratio from sample injection to sample splitting. The numerical value attached to the peak is the half width of the peak (unit: min), and corresponds to 2.35σ (σ: standard deviation) of the peak in the case of Gaussian distribution, from this numerical value and the retention time (t _R ). The number of theoretical plates of the column can be calculated (Equation 4), and it can be directly understood that the bandwidth, that is, the peak dispersion (σ ² ) can be reduced by the sample introduction method according to the present invention. Table 1 shows the actual results Example, a method of dividing the sample band in the comparative example, the column size, detector cell volume, the sample injection volume, showing the relevance of each example another.

In order to confirm the improvement in column performance by the sample introduction method with sample band splitting and rear partial placement, the conventional sample injection method (FIG. 10A) (comparative example) and the sample introduction method of the present invention (FIG. 10B) )) Column performance was compared.

Among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band-rear partial placement, the sample band injected with 0.4 μL is divided into two and introduced into the column, and the first sample from the front part of the sample band is introduced. It is an example in which an improvement in the number of theoretical plates in the chromatogram and a second chromatogram given by the rear part of the sample band held at the first liquid feeding port are shown.

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm). And a mobile phase (acetonitrile / water = 60/40) at 0.05 mL / min. The temperature condition was room temperature. The room temperature is 24.5 ± 0.5 ° C. (hereinafter the same). For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. As a connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. Samples include: Sample I: uracil (U, 0.13 mg / mL), methyl benzoate (MB, 1.8 mg / mL), toluene (T, 2.6 mg / mL), naphthalene (N, 0.7 mg / mL) mL) A mixture of acetonitrile / water = 60/40 was used.

The sample band splitting unit of the sample band splitting apparatus used in this example has the structure shown in FIG. 3A, and uses a T-shaped connector U-428 (inner diameter 0.5 mm, IDEX). , A stainless steel tube (capacity: about 0.92 μL) having an outer diameter of 0.236 mm, an inner diameter of 0.108 mm, and a length of 10 cm, connected to a connection pipe from the sample injection valve to the column inlet (inner pipe for feeding the first liquid feeding port) And, the outer pipe for feeding the second liquid feeding port was constituted by a stainless steel pipe having an outer diameter of 1.6 mm, an inner diameter of 0.5 mm, and a length of 5 cm. A stainless steel tube having an outer diameter of 1.6 mm and an inner diameter of 0.25 mm was used as a connection pipe (branch pipe) for feeding liquid to the second liquid feeding port of the T-shaped connector.

FIG. 10A is a chromatogram obtained by injecting 0.4 μL of sample I using a conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feeding port at a flow rate of 0.05 mL / min.

In addition, in FIGS. 10-28, the numerical value on each peak shows the band width (half value width, unit: min) in 50% peak height. This half-value width corresponds to 2.35σ (σ: standard deviation) in the case of a Gaussian distribution, and from this value, the dispersion of the peak and the theoretical plate number (N, Formula 4) can be calculated. When the peak dispersion (σ ² , Formula 1) is expressed in the volume dimension, σ is converted into the volume dimension using the flow rate (mL / min).

FIG. 10 (B) shows that 0.4 μL of sample I was injected under the same mobile phase conditions as FIG. 10 (A), and the first liquid supply port liquid supply was stopped 0.03 minutes after the injection, and the second liquid supply port 3. Liquid feeding (acetonitrile / water = 60/40, 0.05 mL / min) was started to divide the sample band, and the front part of the divided sample band was introduced into the column for elution separation, and 4. After 0 minute, the second liquid supply port liquid supply was stopped, and the liquid supply from the first liquid supply port (acetonitrile / water = 60/40, 0.05 mL / min) was returned to the first liquid supply port. It is the chromatogram which introduce | transduced the separated part of the sample band into the column and performed separation elution.

In FIG. 10A (comparative example), the theoretical plate numbers 450, 2540, 4400, and 5110 are obtained for U, MB, T, and N, respectively. On the other hand, about 60% of the injected sample is left in the first liquid feeding port by dividing the sample band and about 40% is introduced into the column in the first half of FIG. In the chromatogram, the band width of each peak is narrowed by dividing the sample band and introducing it into the column in the front part of the sample band, and the number of theoretical plates 960, 4640, 6120 and 6740 for U, MB, T and N, respectively. The number of theoretical plates about 1.2 to 2.1 times that of FIG. 10A can be generated.

As shown in the latter half of the chromatogram (second chromatogram) in FIG. 10 (B), 0.03 from the sample injection by returning to the liquid supply from the first liquid supply port after 4.0 minutes from the sample injection. The portion after the sample band that has been left in the first liquid-feeding port tube after the division of the sample band after the separation is introduced into the column and separated and eluted. The four portions of the second chromatogram in FIG. Each peak shows a smaller band width and dispersion than the elution peak in FIG. 10A (comparative example) eluted without dividing the sample band. That is, the sample bands divided into two in the direction crossing the axial direction and introduced into the column are separated with high performance as compared with the case where the sample bands are injected without being divided.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, compared to the conventional sample injection method (FIG. 11A) (comparative example), the division of the sample band of the present invention-after A decrease in sample bandwidth by the sample introduction method with partial placement (FIG. 11B) was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving the division of the sample band-rear partial placement, the 0.4 μL-injected sample band is divided into two and introduced into the zero dead volume union. The first chromatogram and the second chromatogram given by the rear part of the sample band held in the first liquid feeding port are shown. Each peak on the chromatogram includes band broadening in the connection tube and the detection cell. 11A and 11B can be considered to represent the state of the sample band at the column entrance in FIGS. 10A and 10B, respectively.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). The same sample band dividing part as that in Example 1 having the structure shown in FIG. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. A PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used as a connecting tube from the ZDU outlet to the detector cell inlet. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 11 (A) shows a case in which 0.4 μL of sample naphthalene is injected using a conventional sample injection method, and liquid is supplied from the first liquid supply port (acetonitrile / water = 60/40, 0.05 mL / min). This is an eluted chromatogram. FIG. 11 (B) is a chromatogram in which 0.4 μL of sample naphthalene injected is divided into two using the sample introduction method of the present invention, introduced into ZDU and eluted. After 0.03 minutes from the sample injection, liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min) was stopped, and liquid feeding from the second liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min), the sample band is divided, the front part is introduced into the ZDU and eluted (first chromatogram), 0.2 minutes later, the second liquid feeding port is fed. Stop the liquid, return to the liquid feed from the first liquid feed port (acetonitrile / water = 60/40, 0.05 mL / min), and return to the ZDU at the rear part of the sample band held in the first liquid feed port Is introduced and eluted (second chromatogram).

In the second chromatogram in FIG. 11 (B), the sample remaining in the first liquid feeding port at the time of dividing the sample band 0.03 minutes after the sample injection is eluted, and the sample band is near the ZDU inlet. It is directly shown that is divided into two in the direction crossing the axial direction and introduced into each ZDU. Compared with the peak in FIG. 11A (comparative example), the bandwidth of each peak in the two chromatograms in FIG. 11B is reduced.

In both Examples 1 and 2, after the sample injection, each peak passes through the UV detector cell (capacity 1 μL) regardless of whether the sample band is divided or not. Each peak includes a connecting tube from the column outlet or the ZDU outlet to the detector cell, and a dispersion of solute bands in the detector cell.

In order to confirm the column performance improvement by the sample introduction method involving the division of the sample band and the partial partial placement, the conventional sample injection method (FIGS. 12A and 12B) (comparative example) and the sample introduction method of the present invention ( The column performance in FIG. 12 (C), (D), (E)) was compared.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. It is an example in which the second chromatogram given by the rear part of the sample band is shown.

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm). And a mobile phase (acetonitrile / water = 60/40) at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm connected to ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected for use. As the sample band dividing apparatus, the sample band dividing unit having the structure shown in FIG. Samples include: Sample I: uracil (U, 0.13 mg / mL), methyl benzoate (MB, 1.8 mg / mL), toluene (T, 2.6 mg / mL), naphthalene (N, 0.7 mg / mL) mL) A mixture of acetonitrile / water = 60/40 was used.

12A and 12B are chromatograms obtained by injecting 0.2 μL of sample I and 0.1 μL of (B), respectively, using a conventional sample injection method, and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feeding port at a flow rate of 0.05 mL / min.

12 (C), (D), and (E) show the first liquid feeding port liquid (acetonitrile / water) 0.03 minutes after injection of 0.4 μL, 0.2 μL, and 0.1 μL of sample I, respectively. = 60/40, flow rate 0.05 mL / min) stopped, second liquid feed port liquid feed (acetonitrile / water = 60/40, flow rate 0.05 mL / min) was started to divide the sample band, and the sample band The first part is introduced into the column for separation and elution, and the first liquid feeding port liquid (acetonitrile / water = 60/40, flow rate 0.05 mL / min) is restarted 4.0 minutes after the sample injection, This is a chromatogram in which the sample I was divided into two parts and introduced into the column by separating the liquid feeding port liquid feeding and separated.

In FIG. 12A (comparative example), theoretical plate numbers 1240, 4870, 6370, and 6900 are obtained for U, MB, T, and N, respectively.

On the other hand, 0.4 μL of sample I was injected, and about 40% of the structure shown in FIG. 3A was introduced into the column using the same sample band dividing unit as in Examples 1 and 2. 12 (C) In the first chromatogram, theoretical plate numbers 3330, 6840, 7480 and 7800 are obtained for U 1, MB, T and N, respectively. In the first chromatogram of FIG. 12 (D), in which 0.2 μL of sample I was injected and about 47% was introduced into the column using the same sample band divider as in Examples 1 and 2, U 1, MB, T And N, theoretical plate numbers 3530, 8560, 8140, and 8410 are obtained, respectively. In the first chromatogram of FIG. 12 (E) in which 0.1 μL of sample I was injected and about 48% was introduced into the column using the same sample band divider as in Examples 1 and 2, U 1, MB, T And N, theoretical plate numbers 3330, 8720, 8160, and 8610 are obtained, respectively.

In Example 3, as compared with the conventional injection method, the peak width of the chromatogram is narrowed by dividing the sample band in the direction intersecting the axial direction of the sample band and introducing it into the column. The generation of about 2.8 times the number of theoretical plates is possible. Note that when the injection volume is small, the sample band width is also small. Therefore, in the division of the sample band at the same time after the sample injection, the ratio of the sample remaining in the first liquid feeding port is reduced. However, as seen in FIGS. 12D and 12E, at a low flow rate (0.05 mL / min), which is a condition in which diffusion of the sample in the flow channel hardly occurs, 0.2 μL or 0.1 μL. When the injection volume is reduced to a certain extent as described above, the sample band width after diffusion does not substantially change, so that the ratio at which the front portion of the divided sample band is introduced into the column does not change.

In Example 3, when a small-capacity capillary UV cell (6 nL) was used, a larger number of theoretical plates was obtained than in the case where a 1 μL cell was used in Example 1. In particular, for solutes (U, MB) having a small retention, a large increase in the number of theoretical plates was observed compared to the conventional injection method by dividing the sample band and introducing it into the column. These results show that the capacity of the piping from the column outlet to the detector cell inlet (about 0.48 μL in Example 1, about 0.55 μL in Example 3) and the detector cell capacity (1 μL in Example 1) In Example 3, 6 nL), the condition that each solute peak eluted from the column is more difficult to disperse is that a larger number of theoretical plates can be obtained, and the number of theoretical plates increases more greatly by the sample introduction method of the present invention. Is shown. In addition, the effect of solute dispersion outside the column was reduced by the sample introduction method of the present invention, and the performance of solute peaks (theoretical plate number) close to the original performance was obtained even for solute peaks that appear in the early chromatogram. It is shown that.

Compared with the conventional sample injection method (FIGS. 13A, 13B, and 13C) (comparative example), the present sample injection method and the sample band expansion and shape in the sample introduction method of the present invention Sample band splitting of the invention—Decrease in sample band width by the sample introduction method (FIGS. 13 (D), (E), (F)) with rear partial placement was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving the division of the sample band and the subsequent partial placement, the sample bands injected with 0.4 μL, 0.2 μL, and 0.1 μL are divided and introduced into the ZDU, It is an example in which the first chromatogram from the front part of the band and the second chromatogram given by the rear part of the sample band held at the first liquid feeding port are shown. FIGS. 13B and 13C, FIGS. 13D and 13E are respectively shown in FIGS. 12A and 12B (conventional sample injection method), and FIGS. It can be considered to represent the state of the sample band at the column entrance in (D) and (E) (sample introduction method of the present invention). However, these chromatograms also include a connecting tube from the ZDU outlet to the detector cell, and dispersion of solute bands in the detector cell.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was used. As the sample band dividing device, the sample band dividing unit having the structure shown in FIG. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIGS. 13A, 13B, and 13C show a conventional sample injection method using a valve, injecting 0.4 μL, 0.2 μL, and 0.1 μL of sample naphthalene, respectively, and acetonitrile / water as a mobile phase. = 60/40 was sent from the first liquid feed port at 0.05 mL / min and eluted chromatogram.

FIGS. 13D, 13E, and 13F show that sample naphthalene is injected by 0.4 μL, 0.2 μL, and 0.1 μL, respectively, and the sample band is divided into ZDUs by the sample introduction method of the present invention. It is the chromatogram which was introduced and eluted. After 0.03 minutes from the sample injection, liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min) was stopped, and liquid feeding from the second liquid feeding port (acetonitrile / water = (60/40, 0.05 mL / min) was started, the sample band was divided, the front part was introduced into the ZDU and eluted (first chromatogram), 0.2 minutes after the sample injection, the second liquid feed Stop feeding the port, return to the first feeding port (acetonitrile / water = 60/40, 0.05 mL / min), and introduce and elute the rear part of the divided sample band into the ZDU. Yes (second chromatogram).

13 (A), (B), (C) (comparative example), the naphthalene peak shows large tailing, whereas in FIGS. 13 (D), (E), (F), The peak of the first chromatogram has a small width, and the tailing portion is cut off from the peaks of FIGS. 13 (A), (B), and (C). Even an apparatus using a small capacity pipe having an inner diameter of 0.108 mm and a length of 10 cm (capacity of about 0.92 μL) in the pipe from the sample injection apparatus to the column inlet, the injection volume was reduced by the conventional sample injection method. It is shown that a narrow sample band that cannot be obtained by itself is obtained by splitting in the direction intersecting the axial direction of the sample band in the vicinity of the column inlet according to the present invention (in this case ZDU). The half-value width (50% peak height) of naphthalene in the first chromatograms of FIGS. 13D, 13E and 13F is the peak of naphthalene in FIGS. 13A, 13B and 13C, respectively. Compared to the width, it is reduced to about ½. In addition, as a result of using a sample band splitting device having a sample band splitting unit having the structure shown in FIG. It shows that the effect of dispersing and introducing solutes outside the column can be reduced by dividing and introducing them.

  The measured value of the elution volume of naphthalene in Example 4 and FIG. 13B (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.037 min) was 1.85 μL. Subtracting the ZDU (capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 0.55 μL) and the detector cell capacity 6 nL from this value yielded 1.274 μL. From this, the timing for dividing the sample at a flow rate of 0.05 mL / min was set to 0.03 min after sample injection (0.05 mL / min × 0.03 min = after 1.5 μL of liquid was fed). As a result, 50% peak height of naphthalene peak injected and eluted at 0.4 μL, 0.2 μL, and 0.1 μL, respectively, in FIGS. 13A, 13B, and 13C (comparative example). The symmetric coefficients in were 2.5, 2.4 and 2.1, but in FIGS. 13 (D), (E) and (F), 0.4 μL, 0.2 μL and 0.1 μL, respectively. Injected, divided by the sample introduction method of the present invention, introduced into ZDU (sample introduction rates of about 29%, about 41% and about 40%, respectively) and eluted at 50% peak height of the naphthalene peak The symmetry coefficients were 0.99, 0.93, and 1.04, and peaks with good symmetry.

In FIGS. 13 (A), (B) and (C) (comparative example), the eluted peak is tailed, and the peak with a larger injection volume is behind the whole peak (the retention time is longer). It has spread to. For this reason, when the sample is divided after a certain time of 0.03 min after the sample injection, the ratio of the most divided sample (100% -29%) when 0.4 μL is injected (FIG. 13D). = 71%) became large.

Figure 13 (A), (B) in and (C) (Comparative Example), the variance of the peak of naphthalene, the moment method (BEM), 0.34MyuL ^2, and 0.42MyuL ² and 0.41MyuL ² calculated. On the other hand, in FIGS. 13D, 13E, and 13F, it is calculated as 0.04 μL ² , 0.05 μL ^2, and 0.07 μL ^2. 0.4 μL ² (about 80% or more) also resulted in small solute dispersion. Under the condition that the solute is dispersed outside the column (outside the ZDU) by reducing the capacity of the flow path from the ZDU to the detector cell using a small 6nL capillary cell for UV detection. It was shown that the sample introduction method of the present invention has a very large effect of reducing the dispersion of the sample band by 80% or more as compared with the conventional sample injection method. The fact that these results were obtained under the same conditions except for the column in Example 3 and FIGS. 12 (C), (D) and (E) is that in FIGS. 12 (C), (D) and (E). The factor that made it possible to develop a performance close to the performance originally possessed by a small column having an inner diameter of 1.0 mm and a length of 5 cm is considered to be the effect of reducing the dispersion of the sample band in the vicinity of the column inlet. When calculating the variance of peaks by the moment method, the half width given to each peak is not used. The reason why the dispersion of the peak is calculated by the moment method is that the dispersion reflecting the peak shape can be calculated more for a peak with poor symmetry. For a peak with good symmetry, multiply the full width at half maximum (unit: min) by the flow velocity and change it to a volume unit, then calculate σ using this value as 2.35σ and the square (σ ² ) as variance The variance and value calculated by the method of moments are close.

In Example 3 and FIG. 12 (A), a 0.2 μL sample was injected by the conventional sample injection method, and the dispersions for the solutes U, MB, T, and N separated and eluted by the column were respectively peaks. Calculated from the full width at half maximum (unit: min) of 0.31 μL ² , 0.93 μL ² , 2.04 μL ² and 2.96 μL ² (the half width (unit: min) multiplied by the flow rate of 0.05 mL / min) Then, after changing to the unit of volume, this value was set to 2.35σ, and σ was calculated, and the square (σ ² ) was regarded as variance). For these, in the FIG. 12 (D) first chromatogram, 0.11μL ^2, 0.55μL ^2, calculated to 1.66MyuL ² and 2.53MyuL ^2, the sample introduction method of the present invention, the solute peak The dispersion was reduced by 0.3 to 0.4 μL ^{2 and} the theoretical plate number of the solute peak could be increased by 1.2 to 2.8 times compared to the conventional sample injection method.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. FIG. 14B shows an example in which the second chromatogram given by the rear part of the sample band is shown (FIG. 14B). Further, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved, and the rear part of the sample band retained in the first liquid feeding port is not introduced into the column but discharged from the discharge port shown in FIG. (FIG. 14C). Also, the sample band is divided, the rear part of the sample band is placed in the first liquid feeding port, the front part of the sample band is separated and eluted, and then the first liquid feeding port liquid and the second liquid feeding port liquid are alternately supplied. This is an example in which the rear part of the sample band is introduced into the column and separated and eluted by dividing the sample band into five parts at regular intervals, and the separation performance is improved in each chromatogram (FIG. 14D). For comparison of column performance, an example of a conventional sample injection method using a valve is also shown (FIG. 14A) (comparative example).

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm). , 5 cm in length, GL Sciences Inc.), the mobile phase (acetonitrile / water = 60/40) from both the first and second liquid feeding ports, the first liquid feeding port flow rate (0.09 mL / min), The liquid was fed at a second liquid feeding port flow rate (0.01 mL / min) for a total of 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm connected to ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected for use. As the sample band dividing apparatus, the sample band dividing unit having the structure shown in FIG. Further, as shown in FIG. 6, a sample introduction device having a vent discharge function in which a second discharge port 601 is provided upstream of the sample injection device 22 was used. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 14A (comparative example) is a chromatogram obtained by injecting 1 μL of sample II using a conventional sample injection method using a valve. The mobile phase (acetonitrile / water = 60/40) is supplied from both the first and second liquid-feeding ports from the first liquid-feeding port flow rate (0.09 mL / min) and the second liquid-feeding port flow rate (0.01 mL / min). The solution was fed at a total of 0.1 mL / min.

FIG. 14B shows a case where 1 μL of sample II was injected under the same mobile phase conditions as in FIG. 09 mL / min) is stopped, the sample band is divided by increasing the flow rate of the second liquid feeding port liquid (acetonitrile / water = 60/40, 0.01 mL / min) to 0.1 mL / min, Introduced into the column for separation and elution, 6.0 minutes after sample injection, the first liquid feeding port flow rate (0.09 mL / min), the second liquid feeding port flow rate (0.01 mL / min), 0.1 mL / min in total It is the chromatogram which returned to min liquid feeding, introduce | transduced the back part of the sample band to the column, and isolate | separated and eluted.

14C, 1 μL of sample II was injected under the same mobile phase conditions as in FIG. 14A, and 0.03 minutes later, the first liquid feeding port liquid feeding (acetonitrile / water = 60/40,. 09mL / min) is stopped and the sample band is divided by increasing the flow rate of the second liquid feeding port liquid (acetonitrile / water = 60/40, 0.01mL / min) to 0.187mL / min. The front portion is introduced into the column to start separation and elution, and the rear portion of the sample band is discharged from the second discharge port 601 by opening the vent valve 62 in FIG. Is closed, and the first liquid feed port flow rate (0.09 mL / min), the second liquid feed port flow rate (0.01 mL / min), and the total of 0.1 mL / min are returned to separate the front part of the sample band. Eluted chromatogram A.

For details of the sample band rear portion discharge, a second discharge port 601 is provided upstream of the sample injection device 22 of the sample band dividing device shown in FIG. The vent valve 62 installed was opened, and the rear part of the sample band was pushed back to the upstream side of the sample injection device through the resistance of the resistance tube 63 (fused silica tube having an inner diameter of 0.03 mm and a length of 30 cm). Discharge). During the period from 0.03 to 1.0 minute after sample injection, the flow rate required for the separation elution from the column in the front part of the sample band is added to the flow rate necessary for the discharge of the rear part of the sample band from the discharge port, The flow rate of the second liquid supply port liquid supply was 0.187 mL / min. A fused silica tube having an inner diameter of 0.03 mm and a length of 30 cm functions as a liquid feeding resistance and prevents a rapid pressure drop caused by opening the vent valve. In addition, by adjusting the flow rate of the second liquid feeding port, it is possible to obtain a separation chromatogram equivalent to the first chromatogram when there is no discharge operation (FIG. 14B). Moreover, separation and elution can be performed in the same time as the separation elution by the conventional valve injection method (Fig. 14 (A)) by dividing the sample band accompanied by the discharge from the discharge port and introducing it into the column. It was shown that can be improved.

FIG. 14 (D) shows the same mobile phase conditions as in FIGS. 14 (A), (B), and (C), that is, the mobile phase (acetonitrile / water = 60/40) is both the first and second liquid feeding ports. Then, 3 μL of sample II was injected while feeding at a first liquid feed port flow rate (0.09 mL / min), a second liquid feed port flow rate (0.01 mL / min), and a total of 0.1 mL / min. 0.02 minutes later, the first liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0.09 mL / min) was stopped, the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0.01 mL / min). ) Is increased to 0.1 mL / min, the sample band is divided, introduced into the column and separated and eluted (FIG. 14 (D) first chromatogram), and after 6.0 minutes from sample injection, 0.01 minutes Between (6.00 to 6.01 minutes), the first feeding port flow rate (0.0. 9 mL / min), the second liquid feeding port flow rate (0.01 mL / min), and a total of 0.1 mL / min for 0.01 minute from the sample band held in the first liquid feeding port The first liquid feeding port liquid feeding is stopped and the flow rate of the second liquid feeding port liquid feeding is set at 0. 0 to 12.00 minutes after the sample injection. The sample introduced into the column was separated and eluted at 1 mL / min (FIG. 14 (D) second chromatogram).

Between 12.00 minutes and 12.01 minutes after sample injection, the first liquid feeding port flow rate (0.09 mL / min), the second liquid feeding port flow rate (0.01 mL / min), a total of 0.1 mL / min 1 min from the sample band held in the first liquid feeding port, and only the liquid feeding for 0.01 min is introduced into the column, and from 12.01 min to 18.00 min after sample injection During this time, the first liquid feeding port liquid feeding is stopped, the flow rate of the second liquid feeding port liquid feeding is 0.1 mL / min, and the sample introduced into the column is separated and eluted (FIG. 14 (D) third chromatogram). In the same manner, the fourth sample band was introduced between 18.00 minutes and 18.01 minutes after sample injection, and between 18.01 minutes and 24.00 minutes after sample injection. The sample introduced into the column is separated and eluted (FIG. 14 (D) fourth chromatogram) The fifth sample band was introduced between 24.00 minutes and 24.01 minutes later, and the sample introduced into the column was separated and eluted between 24.01 minutes and 30.00 minutes later ( FIG. 14 (D) fifth chromatogram), after 30.00 minutes from the same time, the first liquid feeding port flow rate (0.09 mL / min), the second liquid feeding port flow rate (0.01 mL / min), a total of 0. The solution was fed at 1 mL / min, and the entire amount of the sample band remaining in the first solution feeding port was introduced into the column for separation and elution (FIG. 14D, sixth chromatogram).

In this way, after the elution of the front part of the sample band injected into the column was completed, the liquid feeding pump resumed the liquid feeding from the first liquid feeding port and was left in the first liquid feeding port 20. The middle part and the rear part 32 of the sample band are introduced into the column 1, and the next chromatographic separation and elution is performed. By repeating this operation, the sample band is retained in the first liquid feeding port every 6 minutes. By separating and eluting by repeatedly dividing the sample band in the direction crossing the axial direction and introducing it into the column, a large amount of sample is injected into the first liquid feeding port and the loop of the Rheodyne 8125 valve as a large tail. The possibility of continuous analysis was demonstrated for the rear part of the clamped sample band.

In addition, the result of FIG. 14D shows that the theoretical plate number of the column can be reduced as compared with the conventional sample injection method by discharging the front part and the rear part of the sample band and introducing only the middle part of the sample band into the column. It shows that it is possible to increase. For example, in FIG. 13, the sample band injected by the conventional sample injection method is tailed, but the peak width of the sample band divided and introduced into the ZDU is small in both the front part and the rear part. In FIG. 10B, the peak widths of the first chromatogram and the second chromatogram in which the front part and the rear part of the sample band divided and introduced into the column are separated are shown in FIG. Since it is smaller than the peak width derived from the same solute in (A), using the sample introduction method of the present invention, the front part of the sample band is discharged without being introduced into the column, and only the rear part of the sample band. It is self-evident that column performance can be improved by reducing the dispersion of the entire sample band by introducing the column into the column.

As shown in FIG. 8, if a sample dividing part with a T-shaped connector with a discharge port or a four-way joint is used in front of the column inlet, the front part or rear part of the sample band is discharged, and only the middle part is discharged. It can also be introduced into the column.

In FIG. 14 (A) (comparative example), 1 μL of sample II was injected by the conventional sample injection method, and the number of theoretical plates for the separated and eluted TU, N and OcPh (for the first, fourth and seventh peaks), respectively. 500, 6690 and 7990 stages are obtained. On the other hand, in the first chromatogram of FIG. 14 (B) in which the sample II injected with 1 μL is divided, the front portion (about 65% of the total injected sample) is introduced into the column and separated and eluted, The theoretical plate numbers 1910, 9240, and 8620 are obtained for TU, N, and OcPh, respectively. In the first chromatogram of FIG. 14C in which the sample II injected with 1 μL is divided and the front part is introduced into the column and separated and eluted, the number of theoretical plates 2130, 9140 and TU, N and OcPh are respectively shown. 8870 stages were obtained. Regardless of whether or not the sample band is discharged, the sample band is divided in the direction crossing the axial direction and introduced into the column, so that the theoretical plate number is about 1.1 to 4 times that of the conventional sample injection method. Is possible.

In addition, liquid feeding from the first liquid feeding port is stopped and the liquid feeding speed from the second liquid feeding port is reduced even under conditions where liquid is fed from both the first liquid feeding port and the second liquid feeding port at the time of sample injection. By increasing the number, the sample band can be divided and introduced into the column, and the flow rate ratio between the first liquid feed port and the second liquid feed port (first liquid feed port flow rate / second liquid feed port flow rate). It was shown that the column performance can be improved by the sample introduction method of the present invention without degrading the column performance even when the value is changed from 9/1 to 0/10 and then again 9/1. In FIG. 14B, the division of the sample band and the introduction rate into the column were about 65% when the liquid feeding volume from the first liquid feeding port was 2.7 μL from the time of sample injection.

In order to confirm the performance improvement by the sample introduction method involving the division of the sample band and the partial partial detention, the conventional sample injection method using a valve (FIG. 15A) (comparative example) and the sample introduction method of the present invention (FIG. 15). The column performance in (B) and (C)) was compared.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved, and the sample is kept in the first liquid feeding port. FIG. 15B shows an example in which the second chromatogram given by the rear part of the sample band is shown (FIGS. 15B and 15C).

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and column Kinex C18 (filler diameter 2.6 μm, inner diameter 2. 1 mm, length 5 cm, Phenomenex), the mobile phase (acetonitrile / water = 60/40) was fed at a total of 0.2 mL / min from both the first and second liquid feeding ports. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. As a connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. As the sample band dividing device, the sample band dividing unit having the structure shown in FIG. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 15A is a chromatogram obtained by injecting 1 μL of sample II by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feeding port at 0.2 mL / min.

FIG. 15 (B) shows that the mobile phase (acetonitrile / water = 60/40) is fed at 0.2 mL / min from the first liquid feeding port, and 1 μL of sample II is injected. Stops liquid transfer port liquid supply, starts the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.2 mL / min), divides the sample band, and introduces the front part to the column for separation and elution. After 6.0 minutes from the sample injection, the liquid feeding of the second liquid feeding port was stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.2 mL / min) was returned, It is the chromatogram which introduce | transduced and isolate | separated to the column after the sample band.

FIG. 15C shows a mobile phase (acetonitrile / water = 60/40) from both the first and second liquid feeding ports, the first liquid feeding port flow rate (0.199 mL / min) and the second liquid feeding port flow rate. (0.001 mL / min), a total of 0.2 mL / min, and 1 μL of sample II was injected. After 0.02 minutes, the first liquid supply port liquid supply was stopped, and the second liquid supply port liquid flow rate (0.2 mL / min) to divide the sample band, introduce the front part into the column for separation and elution, and 6.0 minutes after sample injection, send from both the first and second liquid delivery ports. It is the chromatogram which returned to the liquid and isolate | separated and eluted at the 1st liquid feeding port flow rate (0.199 mL / min) and the 2nd liquid feeding port flow rate (0.001 mL / min), and a total of 0.2 mL / min.

In FIG. 15A (comparative example), TU (non-retention), BuPh (retention coefficient k = 1.8) and N (k = 2.8), which are the first, third and fourth peaks, The number of stages 3030, 8520 and 10200 are obtained. In contrast, in the first chromatogram of FIG. 15B, theoretical plate numbers 3760, 9340, and 10960 were obtained for TU, BuPh, and N, respectively. The sample introduction method according to the present invention, which is divided in the direction crossing the axial direction and introduced into the column, enables generation of the theoretical plate number about 1.07 to 1.25 times as compared with the conventional sample injection method. . The effect on the peak eluting early in the chromatogram is large.

At the time of sample injection, liquid feeding is performed from both the first and second liquid feeding ports at a ratio of first liquid feeding port flow velocity / second liquid feeding port flow velocity = 199/1, and 0.02 as in FIG. 15B. In the first chromatogram of FIG. 15C in which the sample band is divided after a minute and the front part is introduced into the column, TU (non-retention), BuPh (retention coefficient k = 1.8) and N (k = 2) 8), the number of theoretical plates is 3830, 9070, and 10970, respectively, and the sample introduction method of the present invention that divides in the direction crossing the axial direction and introduces it to the column is compared with the conventional sample injection method. The generation of the theoretical plate number about 1.07-1.26 times is enabled. Performing liquid feeding from the second liquid feeding port at a flow rate in an appropriate range between the time of sample injection and the division of the sample band is one form in which the effect of the sample introduction method of the present invention can be obtained. Thus, there is an effect of facilitating the increase in the second liquid feeding port liquid feeding speed when the sample band is divided and introduced into the front column.

In order to confirm the improvement in column performance by the sample introduction method with sample band division and rear partial placement, the conventional sample injection method using a valve (FIG. 16A) (comparative example) and the sample introduction method of the present invention (FIG. 16 (B)) was compared.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. FIG. 16B shows an example of the second chromatogram given by the rear part of the sample band (FIG. 16B).

As a sample injection device, a UHPLC apparatus LC800 (GL Sciences Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Kinex C18 (filler diameter 2.6 μm, inner diameter 2. The mobile phase (acetonitrile / water = 60/40) was fed at 0.3 mL / min using 1 mm, length 5 cm, Phenomenex). The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm connected to ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected for use. As the sample band dividing device, the sample band dividing unit having the structure shown in FIG. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 16A is a chromatogram obtained by injecting 1 μL of sample II by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.3 mL / min from the first liquid feeding port.

FIG. 16 (B) shows that the mobile phase (acetonitrile / water = 60/40) was fed at 0.3 mL / min from the first liquid feeding port, and 0.01 μm after the injection of 1 μL of sample II. Stops liquid transfer port liquid supply, starts the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.3 mL / min), divides the sample band, introduces the front part to the column and separates and elutes it. After 5.0 minutes from the sample injection, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.3 mL / min) is returned, It is the chromatogram which introduce | transduced into the column after a sample band, and performed separation elution.

In FIG. 16A (comparative example), TU (non-holding), BuPh (holding coefficient k = 1.8), N (k = 2.8) and the first, third, fourth and seventh peaks. The theoretical plate numbers 3330, 9490, 11220 and 10890 are obtained for OcPh (k = 10.9), respectively. On the other hand, in the first chromatogram of FIG. 16B in which the sample band is divided in the direction crossing the axial direction and the front portion is introduced into the column, TU (non-retention), BuPh (retention coefficient k = 1.8), N (k = 2.8), and OcPh (k = 10.9), theoretical plate numbers 6130, 11530, 12320, and 11570 were obtained, respectively. The sample introduction method of the present invention enables generation of the theoretical plate number about 1.06-1.84 times that of the conventional sample injection method. The effect on the peak eluting early in the chromatogram is large.

Here, with respect to FIG. 16B, Table 3 shows the results of the sample introduction rate in which the peak area of the first chromatogram / (the peak area of the first + second chromatogram) was calculated. For each peak (FIG. 16B, in order from the left of the first chromatogram, TU, AcPh, BuPh, N, HxPh, HpPh, OcPh), the ratio of the divided sample bands introduced into the column is 65.2 ± 0. It is considered that no particular solute has been screened by the sample introduction method of the present invention and introduced into the column more than other solutes (difference in behavior between solutes). , Discrimination is not considered to have occurred).

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, the sample band of the present invention is compared with the conventional sample injection method (FIGS. 17A and 17B) (comparative example). The reduction in sample bandwidth by the sample introduction method (FIG. 17C) with partial separation and rear partial placement was demonstrated without a column.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. FIG. 17C shows an example in which the second chromatogram given by the rear part of the sample band is shown (FIG. 17C). FIGS. 17A and 17C respectively show the state of the sample band at the column entrance in FIG. 16A (conventional sample injection method) and FIG. 16B (sample introduction method of the present invention). .

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). The mobile phase (acetonitrile / water = 60/40) was fed at 0.3 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was used. For the sample band dividing device, the sample band dividing unit having the structure shown in FIG. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

17 (A) and 17 (B) use a conventional sample injection method, inject 1.0 μL and 0.5 μL of sample naphthalene, respectively, and use acetonitrile / water = 60/40 as a mobile phase from the first liquid supply port. It is the chromatogram which pumped and eluted at 0.3 mL / min.

FIG. 17C is a chromatogram obtained by injecting 1.0 μL of sample naphthalene, dividing the sample band into two, introducing it into ZDU, and eluting it. After 0.01 minute from the sample injection, liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.3 mL / min) was stopped, and liquid feeding from the second liquid feeding port (acetonitrile / water = (60/40, 0.3 mL / min) was started, the sample band was divided, the front part was introduced into the ZDU and eluted (first chromatogram), 0.2 minutes after the sample injection, the second liquid feed Stop feeding the liquid to the port and return to the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.3 mL / min) to elute the rear part of the divided sample band (first 2 chromatograms).

In FIGS. 17A and 17B (comparative example), the peak of naphthalene shows large tailing, whereas in FIG. 17C, the peak width of the first chromatogram is small. The tailing portion is cut off from the peak in FIG. A direction in which a narrow sample band that cannot be obtained only by reducing the injection volume with the conventional sample injection method (FIG. 17B) intersects the axial direction of the sample near the column (in this case, ZDU) inlet according to the present invention. It is shown that it was obtained by splitting in and introducing into ZDU. In FIG. 17C, about 69% of the sample injected with 1 μL is introduced into the ZDU and appears in the first chromatogram. In FIG. 17B, the 0.5 μL sample is obtained by the conventional sample injection method. Compared to the peak that appears as a result of the injection of, the sample volume introduced into the ZDU is large, but a narrow peak is given. FIG. 17D is an overwriting of the first chromatogram of FIGS. 17A, 17B, and 17C, and the peak width of the first chromatogram of FIG. 17C is the narrowest. You can see clearly.

In FIG. 17 (A), (B) ( Comparative Example), the variance of the peak of naphthalene, the moment method, 1.44MyuL ^2, was calculated to 1.33μL ^2. On the other hand, in FIG. 17C, it was calculated as 0.43 μL ^2, and by the sample introduction method of the present invention, the dispersion was reduced by about 70% compared to the conventional sample injection method. This result was obtained under the same conditions as in Example 7 and FIG. 16 (B) except that the column was originally a small column with an inner diameter of 2.1 mm and a length of 5 cm in FIG. 16 (B). The factor that made it possible to achieve performance close to the performance is considered to be the effect of reducing the dispersion of the sample band near the column inlet. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

Sample band splitting-conventional sample injection method using a valve (FIGS. 18A and 18G) (comparative example) and sample of the present invention for confirmation of column performance improvement by sample introduction method with partial partial placement Comparison of column performance in the introduction method (FIGS. 18B, 18C, 18D, 18E, 18F, 11H) was performed. For FIGS. 18B and 18C, the peak area of the first chromatogram / (the peak area of the first + second chromatogram) was calculated, and reproducibility was confirmed. Also, the effect of the discharge timing of the rear portion of the sample band and the time from the sample injection to the division of the sample band applied to the sample introduction method of the present invention (first liquid supply port liquid supply speed) / (second (Liquid feed port liquid feed speed) ratio and the liquid feed time from sample injection to sample band division were confirmed to have an effect on column performance.

18 (B) and 18 (C) show the improvement in the number of theoretical plates in the first chromatogram from the front part of the sample band by the sample introduction method of the present invention, in which the sample band is divided and the partial partial placement is performed. And a second chromatogram provided by the rear part of the sample band held in the first liquid feeding port. Reproduction of the sample introduction method of the present invention at the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) = 0.6 / 1.2 mL / min from the time of sample injection until the sample band splitting Sex was confirmed.

18D, 18 </ b> E, and 18 </ b> F, in the sample introduction method of the present invention, in which the sample band is divided and the rear partial placement is performed, the discharge time of the rear part of the sample band and the sample band are shown. It was confirmed that the (first liquid feed port flow rate) / (second liquid feed port flow rate) ratio after discharge of the rear portion and the effect of the liquid feed time on the column performance were confirmed. The ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) from the time of sample injection to the time when the sample band was divided was set to 0.6 / 1.2 mL / min. In FIG. 18 (G), in the conventional sample injection method using a valve, the effect of the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from the time of sample injection to the end of separation elution (FIG. 18). 18 (A) compared). The ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) from the time of sample injection to the end of separation and elution was set to 0.3 / 1.5 mL / min.

18 (H) and (I), in the sample introduction method according to the present invention, which is a sample introduction method involving the division of the sample band and the partial indwelling, from the time of sample injection until the division of the sample band (first transmission). The effect of the (liquid port flow velocity) / (second liquid feed port flow velocity) ratio (comparison with FIGS. 18B and 18C) could be confirmed. The ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from sample injection to sample band splitting was set to 0.3 / 1.5 mL / min.

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 4.6 mm). The mobile phase (acetonitrile / water = 60/40) was fed at a total rate of 1.8 mL / min from both the first and second liquid feeding ports. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. As a connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. For the sample band dividing device, the sample band dividing unit having the structure shown in FIG. Further, as shown in FIG. 6, a sample introduction device having a vent discharge function in which a second discharge port 601 is provided upstream of the sample injection device 22 was used. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 18A shows a chromatogram obtained by injecting 2 μL of sample II by the conventional sample injection method and separating and eluting it, which is a comparative example. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding port flow rate) ratio = 0.6 / 1. 2 mL / min, total 1.8 mL / min). 18 (B) and 18 (C), the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feeding ports ((first liquid feeding port flow velocity) / (second liquid feeding). Liquid port flow rate) ratio = 0.6 / 1.2 mL / min, total 1.8 mL / min), 2 μL of sample II was injected, and the first liquid supply port liquid solution (acetonitrile / water 0.01 minutes after sample injection) = 60/40, 0.6 mL / min) is stopped, and the flow rate of the second liquid feeding port (acetonitrile / water = 60/40, 1.2 mL / min) is increased to 1.8 mL / min The band was divided and the front part was introduced into the column and separated and eluted. 9.0 minutes after sample injection, the ratio of liquid feeding from both the first and second liquid feeding ports ((first liquid feeding port flow velocity) / (second liquid feeding port flow velocity) ratio = 0.6 / 1.2 mL / min, a total of 1.8 mL / min), and a chromatogram obtained by separating and eluting the rear part of the sample band held in the first liquid feeding port.

In FIG. 18A (comparative example), theoretical plate numbers 4540, 9510, 10070, and 11290 are obtained for TU, AcPh, BuPh, and N, which are the first, second, third, and fourth peaks, respectively. Compared to this, in FIGS. 18B and 18C, the average values of the theoretical plate numbers of 5980, 10930, 11480, and 11490 were obtained for TU, AcPh, BuPh, and N, respectively. Compared with the conventional sample injection method, the sample introduction method of dividing the sample in the direction crossing the axial direction of the present invention and introducing it into the column is 1.02 to 1.30 for the peak eluting at the initial chromatogram. It is possible to generate twice as many theoretical plates.

Table 4 shows the results of the sample introduction rates obtained by calculating the peak area of the first chromatogram / (peak area of the first + second chromatogram) for FIGS. 18 (B) and (C). For each peak (FIG. 18B, in order from the left in the first chromatogram, TU, AcPh, BuPh, N, HxPh, HpPh, OcPh), the ratio of the divided sample bands introduced into the column was 90.0 ± Within the range of 0.8%, the reproducibility of the sample introduction method of the present invention was shown. In addition, a specific solute is not selected by the sample introduction method of the present invention and introduced into the column more than other solutes (discrimination does not occur).

18D, 18E, and 18F, the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feeding ports ((first liquid feeding port flow velocity) / (Second liquid feeding port flow rate) ratio = 0.6 / 1.2 mL / min, total 1.8 mL / min), 2 μL of sample II was injected, and the first liquid feeding port was fed 0.01 minutes after sample injection. (Acetonitrile / water = 60/40, 0.6 mL / min) is stopped, and the flow rate of the second liquid feeding port liquid (acetonitrile / water = 60/40, 1.2 mL / min) is increased to 1.9 mL / min. By dividing the sample band, the front part of the sample band is introduced into the column to start separation and elution, and the rear part of the sample band is opened by opening the vent valve of the discharge port shown in FIG. Between 01 minute and 1.0 minute later, the inner diameter is 0.03 mm Through resistance of fused silica capillary tube of length 30 cm, was discharged from the upstream sample injector (back discharge). The operation of the vent valve of the first liquid supply port liquid supply, the second liquid supply port liquid supply, and the discharge port after 1.0 minute from the sample injection is shown in FIGS. 18 (D), 18 (E) and They are different in (F). The reason why the flow rate from the second liquid feeding port was set to 1.9 mL / min from 0.01 minute to 1.0 minute after the sample injection was to separate and elute the front part of the sample band from the column. This is because the flow rate necessary for discharging the rear part of the sample band from the discharge port is added to the required flow rate.

In FIGS. 18D, 18E, and 18F, the first liquid feeding port liquid feeding, the second liquid feeding port liquid feeding, and the discharge port are performed from 1.0 minute to 9.0 minutes after sample injection. Regarding the operation of the vent valve, the following operations are performed. In FIG. 18D, the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) = 0 / 1.9 mL / min was continued, and the vent valve was kept open. In FIG. 18 (E), the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) = 0.6 / 1.2 mL / min, and the vent valve was closed. In FIG. 18F, the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) was set to 1.8 / 0 mL / min, and the vent valve was closed.

The presence or absence of the sample introduction method of the present invention, and from the time of sample injection to the time of sample band division, after dividing the sample and introducing the front portion into the column (first liquid feed port flow rate) / (second liquid feed) Table 5 shows the effect of the port flow rate ratio and the presence or absence of the discharge operation on the number of theoretical column columns.

18 (D), (E), and (F), TU, BuPh, and N, which are the first, third, and fourth peaks, are obtained by the conventional sample injection method in FIG. A theoretical plate number 1-20% larger than the obtained theoretical plate number was obtained. These results also show that the sample introduction method of dividing the sample in the direction crossing the axial direction and introducing the sample into the column is effective for increasing the number of theoretical columns of the column of 4.6 mm inner diameter. The ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) after the divided sample band front part is introduced into the column and the rear part of the sample band is discharged is the sum of the flow amount flowing through the column. If the flow rate is the same, the column performance is not significantly affected (the difference in the number of theoretical column columns is 4.2% or less).

In this example, the fused silica capillary tube having an inner diameter of 0.03 mm and a length of 30 cm functions as a liquid feeding resistance, and prevents a sudden pressure drop caused by a discharge operation that opens the vent valve. The adjustment made it possible to obtain a separation chromatogram equivalent to that obtained when there was no discharge operation. Also, by performing the sample introduction method of the present invention involving the use of a discharge port, the column performance is improved as compared with the conventional sample injection method, and separation and elution can be performed in the same time as the analysis by the conventional sample injection method. It was shown that.

FIG. 18G is a comparative example, and is a chromatogram obtained by injecting 2 μL of sample II by the conventional sample injection method and separating and eluting it. The mobile phase (acetonitrile / water = 60/40) was sent from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding port flow rate) ratio = 0.3 / 1. .5 mL / min, total 1.8 mL / min).

18 (H) and (I), the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding). Liquid port flow rate) ratio = 0.3 / 1.5 mL / min, total 1.8 mL / min), 2 μL of sample II was injected by the conventional sample injection method, and the first liquid supply port 0.02 minutes after sample injection Stop feeding the liquid (acetonitrile / water = 60/40, 0.3 mL / min) and set the flow rate of the second liquid feeding port (acetonitrile / water = 60/40, 1.5 mL / min) to 1.8 mL / min. The sample band was divided by increasing to min, the front part was introduced into the column and separated and eluted. 9.0 minutes after the sample injection, the ratio of liquid feeding from both the first and second liquid feeding ports ((first liquid feeding port flow velocity) / (second liquid feeding port flow velocity) ratio = 0.3 / 1.5 mL / min, a total of 1.8 mL / min), and a chromatogram obtained by separating and eluting the rear part of the sample band held in the first liquid feeding port.

In FIG. 18G (comparative example), theoretical plate numbers 1850, 6300, 8690, and 9140 were obtained for TU, AcPh, BuPh, and N, which are the first, second, third, and fourth peaks, respectively. Compared to this, in the first chromatogram in FIG. 18 (H) and the first chromatogram in FIG. 18 (I), the average values 2840, 7700, 9450, and 9760 of the theoretical plate numbers for TU, AcPh, BuPh, and N are shown. Obtained. Compared with the conventional sample injection method, the sample introduction method of dividing the sample in the direction crossing the axial direction of the present invention and introducing the sample into the column is 1.02 to 1.50 with respect to the peak eluting at the initial chromatogram. It is possible to generate twice as many theoretical plates. However, as shown in Table 5, the mobile phase liquid feeding condition ((first liquid feeding port flow velocity) / (second liquid feeding port flow velocity) ratio of FIG. 18 (B) and (C) = 0.6 / 1. 2 mL / min, a total number of 1.8 mL / min) lower than the theoretical plate number is obtained, and from this result (sample liquid flow velocity) / (second liquid feed) from sample injection to sample band splitting It has been shown that proper selection of the port flow rate ratio results in a higher number of theoretical plates.

Table 6 shows the results of the sample introduction rates for calculating the peak area of the first chromatogram / (peak area of the first + second chromatogram) for FIGS. 18 (H) and (I). For each peak (FIG. 18 (H), from the left of the first chromatogram, TU, AcPh, BuPh, N, HxPh, HpPh, OcPh), the ratio of the divided sample bands introduced into the column was 92.4 ±. Within the range of 0.8%, the reproducibility of the sample introduction method of the present invention was shown. In addition, a specific solute is not selected by the sample introduction method of the present invention and introduced into the column more than other solutes (discrimination does not occur).

When the first liquid feeding port flow rate was 0.3 mL / min, when the sample band was divided at 0.02 minutes and introduced into the column, about 92% of the injected sample was analyzed as the first chromatogram. Reproducibility was seen in the ratio of the peak areas of the first chromatogram and the second chromatogram. As shown in Table 4, in FIGS. 18B and 18C, when the first liquid feeding port flow rate is 0.6 mL / min, the sample band is divided in 0.01 minutes and introduced into the column. About 90% of the injected sample was analyzed as the first chromatogram. Reproducibility was seen in the ratio of the peak areas of the first chromatogram and the second chromatogram. The results of these examples show that the sample introduction method of the present invention is compared with the case of using a conventional sample injection method for a large column with an inner diameter of 4.6 mm and a length of 5 cm, particularly for the peak in the initial chromatogram Thus, a large theoretical plate number of up to about 50% can be obtained, a difference in behavior between solutes is not caused, and the reproducibility of the sample introduction method of the present invention is shown.

The results of Examples 1 to 9 are
1. Dividing the sample band, feeding volume from the first feeding port until the sample front part is introduced into the column, that is, the flow rate of the first feeding port, dividing the sample band from sample injection, and the front part of the sample The ratio (sample introduction rate) that the front part of the sample band used for the first separation analysis occupies in the entire sample can be controlled based on the time until introduction into the column.
2. Even if a large flow rate is required for a large column, if the flow rate of the first liquid feed port is reduced and the liquid feed from the second liquid feed port is partially used, the flow rate of the whole and the first liquid feed port is adjusted, By considering the timing, the sample band can be divided and introduced into the column in front of the sample appropriately.
3. If the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from sample injection to sample band splitting becomes too small, the theoretical plate number of the column decreases, so a better theoretical plate number (higher It is appropriate to select a flow rate ratio that gives performance.
And
4). The effect of the (first liquid feed port flow rate) / (second liquid feed port flow rate) ratio on the column performance is limited to the time when the sample band is divided from the time of sample injection. After introducing into the column and discharging the rear part of the sample band from the discharge port, the (first liquid feed port flow rate) / (second liquid feed port flow rate) ratio = 100/0 to 0/100 The flow rate ratio in the range should give an equivalent result.
And so on.

Among the sample introduction methods of the present invention, the conventional sample injection method using a valve (FIG. 19 (A)) and the present invention are used for confirmation of column performance improvement by the sample introduction method involving sample band splitting and partial partial placement. The column performance in the sample introduction method (FIG. 19B) was compared. Among the sample introduction methods of the present invention, the improvement in the number of theoretical plates in the first chromatogram given by the front part of the sample band was confirmed by the sample introduction method involving the division of the sample band and the partial indwelling.

As a sample injection device, a UHPLC apparatus LC800 (GL Sciences Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used. 4.6 mm, length 10 cm, Merck KGaA, Darmstadt, Germany) using a mobile phase (acetonitrile / water = 60/40) from both the first and second liquid feeding ports at a total of 1.0 mL / min. Liquid was sent. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. As a connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. The sample band splitting device uses a sample band splitting unit having the structure shown in FIG. 3A, and is connected to an injection valve-column inlet connecting pipe (inner pipe of a double pipe for feeding a first liquid feeding port). Was a stainless steel tube having an outer diameter of 0.363 mm, an inner diameter of 0.185 mm, and a length of 10 cm (capacity of about 2.7 μL). Further, as shown in FIG. 6, a sample introduction device having a vent discharge function in which a second discharge port 601 is provided upstream of the sample injection device 22 was used. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 19A (comparative example) is a chromatogram obtained by injecting 5 μL of sample II by the conventional sample injection method, and separating and eluting it. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding port flow rate) ratio = 0.3 / 0. 0.7 mL / min, total 1.0 mL / min).

In FIG. 19B, the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding port flow rate). Ratio = 0.3 / 0.7 mL / min, total 1.0 mL / min), 5 μL of sample II was injected by the conventional sample injection method, and the first liquid supply port solution (acetonitrile 0.03 minutes after sample injection) / Water = 60/40, 0.3 mL / min) is stopped, and the flow rate of the second liquid feeding port liquid (acetonitrile / water = 60/40, 0.7 mL / min) is increased to 1.0 mL / min. To divide the sample band, introduce the front part of the sample band into the column for separation and elution, and 12 minutes after the sample injection, the second feed port flow rate is kept at 1.0 mL / min and the vent valve is turned on. Open the test that was left in the first liquid delivery port He was discharged the band after partial.

In FIG. 19A (comparative example), TU (non-holding), BuPh (holding coefficient k = 1.1), N (k = 1.7) and OcPh which are the first, third, fourth and seventh peaks. The theoretical plate numbers 7850, 12450, 13280 and 11930 were obtained for (k = 6.5), respectively. Compared to this, in FIG. 19B, TU (non-holding), BuPh (holding coefficient k = 1.1), N (k = 1.7) and OcPh (k = 6.5) are respectively shown. The theoretical plate numbers 9550, 13900, 13620 and 11940 were obtained. Compared with the conventional sample injection method, the sample introduction method of dividing the sample in the direction crossing the axial direction of the present invention and introducing it into the column is TU (non-retained) and BuPh (retained) in the initial chromatogram. It is possible to generate 1.03 to 1.22 times the number of theoretical plates for the three peaks of coefficient k = 1.1) and N (k = 1.7).

In this example, the mobile phase was fed from both the first and second liquid feeding ports ((first liquid feeding port flow velocity) / (second liquid feeding port flow velocity) ratio = 0.3 / 0.7 mL / min. , Total 1.0 mL / min). It is easy to adjust the rate of introduction of the sample band into the column by feeding the liquid at a flow rate in an appropriate range from the second liquid feeding port between the sample injection and the division of the sample band. There is an effect of facilitating an increase in the second liquid feeding port flow velocity at the time of division and introduction into the column.

19 (A) and 19 (B), the elution volume of OcPh of the seventh peak is 10.2 mL. Among the elution volumes of the peaks for which the effect of the sample introduction method of the present invention was confirmed in the examples. It was the largest value.

The present invention relates to the conventional sample injection method using a valve and the spread and shape of the sample band in the sample introduction method of the present invention, compared with the conventional sample injection method (FIGS. 20A and 20B) (comparative example). The reduction of the sample bandwidth by the sample introduction method (FIGS. 20 (C) and (D)) involving the splitting of the sample band and the rear partial placement was demonstrated without a column.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. FIG. 20C shows an example in which the second chromatogram given by the rear part of the sample band is shown (FIGS. 20C and 20D). 20B and 20D show the state of the sample band at the column entrance in FIG. 19A (conventional sample injection method) and FIG. 19B (sample introduction method of the present invention), respectively. It can be thought of as representing.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). The mobile phase (acetonitrile / water = 60/40) was fed at a total of 1.0 mL / min from both the first and second liquid feeding ports. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. As a connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. The sample band splitting device uses a sample band splitting unit having the structure shown in FIG. 3A, and is connected to an injection valve-column inlet connecting pipe (inner pipe of a double pipe for feeding a first liquid feeding port). Was a stainless steel tube having an outer diameter of 0.363 mm, an inner diameter of 0.185 mm, and a length of 10 cm (capacity of about 2.7 μL). In addition, as shown in FIG. 6, a vent discharge function provided with a second discharge port 601 upstream of the sample injection device 22 was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

20 (A) and 20 (B) are chromatograms obtained by injecting 2 μL and 5 μL of sample II, respectively, and separating and eluting them by the conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feeding ports ((first liquid feeding port flow rate) / (second liquid feeding port flow rate) ratio = 0.3 / 0. 0.7 mL / min, total 1.0 mL / min). 20 (C) and (D) show that the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow rate) / (second feed). Liquid port flow rate) ratio = 0.3 / 0.7 mL / min, total 1.0 mL / min), sample II was injected 2 μL and 5 μL, respectively, and the first liquid transfer port was sent 0.02 minutes after sample injection (Acetonitrile / water = 60/40, 0.3 mL / min) was stopped, and the flow rate of the second liquid feeding port liquid (acetonitrile / water = 60/40, 0.7 mL / min) was increased to 1.0 mL / min. By dividing the sample band, the sample band front part is introduced into the ZDU for elution, and 0.2 minutes after the sample injection, (first liquid feed port flow rate) / (second liquid feed port flow rate) Ratio = 0.3 / 0.7 mL / min, total 1.0 mL / min Returned. 20E is an overwriting of FIGS. 20A and 20C, and similarly, FIG. 20F is an overwriting of FIGS. 20B and 20D.

20 (A) and 20 (B) (comparative example), the naphthalene peak shows a large tailing, whereas in FIGS. 20 (C) and 20 (D), the peak width of the first chromatogram is small. The tailing portions are cut off from the peaks of FIGS. 20A and 20B, respectively. It is shown that the rear part (tail part) of the sample band was removed by dividing the sample band in the direction crossing the axial direction of the sample band immediately before the column inlet and introducing the sample band into the ZDU.

In FIG. 20D and FIG. 19B, the liquid feeding volume from the first liquid feeding port from the sample injection to the sample band splitting is different between 9 μL and 6 μL. However, this point and the fact that the results were obtained under the same conditions except for the column indicate that the chromatogram in FIG. 19B is particularly in the initial chromatogram even for a large column with an inner diameter of 4.6 mm and a length of 10 cm. It is considered that the reason why the peak can exhibit the performance close to the original performance of the column is the effect of reducing the dispersion of the sample band in the vicinity of the column inlet.

20 In (A), (B) (Comparative Example), the variance of the peak of naphthalene, the moment method, 22.6MyuL ^2, was calculated to 60.1μL ^2. In contrast, in the first chromatogram of FIGS. 20 (C) and (D), 14.7 μL ² and 23.1 μL ² are calculated, and in FIG. 20 (D) the first chromatogram, FIG. ), The dispersion of the solute band was reduced by 60% or more. In contrast, in the first chromatogram in FIG. 20C, the solute band dispersion decreased only about 35% compared to FIG. 20A. These results are shown in FIG. 20 under the condition that the volume of the mobile phase fed from the first liquid feeding port from sample injection to sample division is 0.3 mL / min × 0.02 min = 0.006 mL = 6 μL. In (C), the sample injection volume is 2 μL, and only a small part was placed in the first liquid feeding port when the sample band was divided, whereas in FIG. 20 (D), the sample injection volume was 5 μL, This is based on the fact that a larger part was placed in the first liquid feeding port when the sample band was divided. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, compared to the conventional sample injection method (FIG. 21 (A)) (comparative example), the division of the sample band of the present invention-after A decrease in sample bandwidth by the sample introduction method with partial placement (FIGS. 21B and 21C) was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving the division of the sample band and the partial indwelling, the state where the sample band injected by 1 μL is divided into two at different timings and introduced into the ZDU and eluted. Examples shown as a first chromatogram given by the front part of the band, a second chromatogram given by the rear part of the sample band held in the first liquid feeding port (FIGS. 21B and 21C), and the sample This is an example (FIG. 21D) in which the sample band does not reach the ZDU inlet and the first chromatogram cannot be obtained at the timing of the division and introduction into the ZDU. 21 (A) and 21 (C) respectively show Example 13, FIG. 22 (A) (conventional sample injection method using a valve) and FIG. 22 (B) (sample introduction method of the present invention) described later. This is considered to represent the state of the sample band at the column entrance in FIG.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was used. As the sample band dividing device, the sample band dividing unit having the structure shown in FIG. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 21A is a chromatogram obtained by injecting 1 μL of sample naphthalene by the conventional sample injection method and eluting it. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min from the first liquid feeding port. 21 (B), (C), and (D), the mobile phase (acetonitrile / water = 60/40) is fed from the first liquid feeding port at 0.1 mL / min, and 1 μL of sample naphthalene is injected. After the sample injection, 0.03 minutes, 0.02 minutes, and 0.01 minutes later, the first liquid supply port liquid supply was stopped, and the second liquid supply port liquid supply (acetonitrile / water = 60/40,. 1 mL / min) is started, the sample is divided, introduced into the ZDU, and eluted. After 0.2 minutes from the sample injection, the liquid supply from the second liquid supply port is stopped and the liquid supply from the first liquid supply port is stopped. It is the chromatogram which separated and returned to the liquid (acetonitrile / water = 60/40, 0.1 mL / min).

In FIG. 21A (comparative example), the peak of naphthalene shows large tailing, whereas in FIGS. 21B and 21C, the peak of the first chromatogram is shown in FIG. The tailing part was cut off from the peak of the sample, and it was shown that the rear part of the sample band was placed in the first liquid feeding port by dividing the sample band and introducing it into the ZDU.

In FIG. 21A (comparative example), the dispersion of the naphthalene peak was calculated to be 0.95 μL ² by the moment method. On the other hand, in the first chromatogram of FIGS. 21B and 21C, it is calculated as 0.27 μL ² and 0.17 μL ^2, and the sample of the present invention is divided in the direction crossing the axial direction, The sample introduction method introduced into the column (in this case ZDU) resulted in a reduction of 0.7 to 0.8 μL ² . Under the condition that the solute is dispersed outside the column (outside the ZDU) by reducing the capacity of the flow path from the ZDU to the detector cell using a small 6nL capillary cell for UV detection. It has been shown that the sample introduction method of the present invention has a very large effect of reducing the dispersion of the sample band by 80% compared to the conventional sample injection method. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

The measured value of the elution volume of naphthalene in Example 12 and FIG. 21A (measured value at a flow rate of 0.1 mL / min, 0.1 mL / min × 0.025 min) was 2.5 μL. From this value, the ZDU (capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 0.55 μL), and the detector cell capacity 6 nL were subtracted to be 1.924 μL. In FIG. 21 (D), the sample is divided into 0.01 minutes after the sample injection (the liquid supply volume from the first liquid supply port is 0.1 mL / min × 0.01 min = 0.001 mL = 1 μL). When introduction into ZDU is attempted, the tip of the sample band does not reach the outlet of the first liquid feed port (inner pipe of the double pipe), that is, the flow path switching point, and the entire sample band is the first liquid feed. No peak appeared in the first chromatogram because it was trapped in the loop of the port and Rheodyne 8125 valve (injector). It shows that the entire sample band was eluted as the peak of the second chromatogram with the resumption of liquid feeding from the first liquid feeding port.

In FIGS. 21B and 21C, 0.1 mL / min × 0.03 min = 0.003 mL = 3 μL and 0.1 mL / min × 0.02 min = 0.002 mL = 2 μL are sent from the sample injection, respectively. When the sample is divided and introduced into the ZDU at the time when it is liquid, about 78% of the sample in FIG. 21 (B) and about 40% of the sample in FIG. 21 (C) are introduced into the ZDU. Eluted. The symmetry coefficient at the peak height of 50% of the peak of FIG. 21A is 2.0, the peak of FIG. 21B is the first chromatogram, 1.32, and the peak of FIG. 21C is the first chromatogram. The peak was 1.0. From these results, by controlling the liquid feeding capacity from the first liquid feeding port from the sample injection to the division of the sample band, the sample band having a different volume can be changed from the constant volume sample band injected by the conventional method. It was shown that can be introduced into the column.

In order to confirm the column performance improvement by the sample introduction method involving the division of the sample band and the rear partial placement, the conventional sample injection method using a valve (FIG. 22A) (comparative example) and the sample introduction method of the present invention (FIG. 22 (B)) was compared.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved and the sample is kept in the first liquid feeding port. FIG. 22B shows an example in which the second chromatogram given by the rear part of the sample band is shown (FIG. 22B).

As a sample injection device, a UHPLC apparatus LC800 (GL Sciences Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and column Monobis C18 (C18-modified monolithic silica gel, inner diameter 1.0 mm). , 15 cm in length, Kyoto Monotech Co., Ltd.), and a mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm connected to ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected for use. As the sample band dividing apparatus, the same sample band dividing unit as that of Examples 1 to 8 and 12 having the structure shown in FIG. Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 22A is a chromatogram obtained by injecting 1 μL of Sample II by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min from the first liquid feeding port.

FIG. 22 (B) shows that the mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min from the first feeding port, and 1 μL of sample II was injected, and 0.02 minutes later, Stops liquid transfer port liquid supply, starts the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.1 mL / min), divides the sample band, introduces the front part to the column and separates and elutes it. 10 minutes after the sample injection, the liquid feeding from the second liquid feeding port was stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) was returned to the sample band. It is the chromatogram which introduce | transduced into the column of a rear part, and performed separation elution.

In FIG. 22A (comparative example), the first, third, fourth, and seventh peaks are TU (non-holding), BuPh (holding coefficient k = 1.4), N (k = 2.3) and The theoretical plate numbers 6060, 11970, 15360 and 15960 are obtained for OcPh (k = 9.4), respectively. On the other hand, in the first chromatogram of FIG. 22B in which the sample band is divided and the front part is introduced into the column, TU (non-retention), BuPh (retention coefficient k = 1.4), N ( For k = 2.3) and OcPh (k = 9.4), theoretical plate numbers 13330, 17120, 18890 and 17350 were obtained, respectively. The sample introduction method according to the present invention, which divides a sample in a direction crossing the axial direction and introduces it into a column, can generate about 1.09 to 2.20 times as many theoretical plates as the conventional sample injection method. It is said.

In order to confirm the column performance improvement by the sample introduction method involving the separation of the sample band and the partial partial placement, the conventional sample injection method using a valve (FIG. 23A) (comparative example) and the sample introduction method of the present invention (FIG. 23 (B), (C)) column performance was compared. Compared with the conventional sample injection method (FIG. 23 (D)), the sample band width according to the example (FIG. 23 (E)) in which the sample introduction method involving the division of the sample band and the partial indwelling of the present invention is continuously performed. Confirmed the decrease.

Among the sample introduction methods of the present invention, the method of dividing the sample band and placing it in the rear part allows the number of theoretical plates in the first chromatogram from the front part of the sample band to be improved, the inside of the first liquid feeding port, and the micro six-way switching valve. This is an example in which the improvement in the number of theoretical plates in the second chromatogram given by the rear portion of the sample band held in the loop is shown (FIGS. 23B and 23C). For comparison of the column performance with these examples, an example by a conventional sample injection method is also shown (FIG. 23A).

In addition, the sample band is divided, the rear part of the sample band is placed in the first liquid feed port and the loop of the micro 6-way flow switching valve, the first part of the sample band is separated and eluted, and then the first liquid feed port is fed. In this example, the portion after the sample band is introduced into the column, separated and eluted, and the separation performance is improved in each chromatogram. Yes (FIG. 23E). For comparison of the column performance with this example, an example by a conventional sample injection method is also shown (FIG. 23D) (comparative example).

UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (2 μL loop, inner diameter 0.13 mm, length 15.0 cm and length 5 μL loop, inner diameter 0.20 mm, length 15.9 cm) as a sample injection device And using a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, GL Sciences Inc.), the mobile phase (acetonitrile / water = 60/40) was 0.1 mL / min, and 0. The solution was fed at 05 mL / min. The temperature condition was 40 ° C. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 240 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a stainless steel tube (capacity approx. 0.86 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm, connected to ZDU (capacity 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was used. As the sample band dividing device, a sample band dividing unit having the structure shown in FIG. 4 was used.

A micro 6-way flow path switching valve (C72X-1696EH, VALCO) for switching the flow path between the Rheodyne 8125 valve and the column is shown in FIG. 4 (first liquid feed port 51, second liquid feed port) 52, the flow path switching point 50, the loop 53, and the column 1), and this valve was operated by the LC 800 to divide the sample band and introduce it into the column.

The pipe from the Rheodyne 8125 valve to the C72X-1696EH valve uses a stainless steel tube (capacity: 0.79 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.1 mm, and a length of 10 cm, and the loop 53 has a capacity of 2 μL (see FIG. 23 (A), (B), (C)) and 5 μL (used in FIGS. 23 (D), (E)). For the piping from the flow path switching point 50 to the column inlet, a stainless steel tube (capacity: about 0.86 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm was used. The other pipes connected to the C72X-1696EH valve were stainless steel pipes having an outer diameter of 1.6 mm and an inner diameter of 0.1 mm.
Samples in FIGS. 23A, 23B, and 23C are Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0. 3 mg / mL), naphthalene (N, 0.3 mg / mL), hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0. 6 mg / mL) A mixture of acetonitrile / water = 60/40 was used.

23D and 23E, sample III: uracil (0.005 mg / mL), acetanilide (AcAn, 0.05 mg / mL), acetophenone (AcPh, 0.05 mg / mL), propiophenone (PrPh, 0.05 mg / mL), butyrophenone (BuPh, 0.05 mg / mL), benzophenone (BePh, 0.05 mg / mL), valerophenone (VaPh, 0.05 mg / mL), hexanophenone (HxPh, 0 0.05 mg / mL), heptanophenone (HpPh, 0.05 mg / mL), octanophenone (OcPh, 0.05 mg / mL) mixture in acetonitrile / water = 65/35 solution was used.

In FIGS. 23A, 23B, and 23C, the loop capacity of the Rheodyne 8125 valve and the loop capacity of the C72X-1696EH valve were 2 μL. In FIGS. 23D and 23E, the loop capacity of the Rheodyne 8125 valve and the C72X-1696EH valve was 5 μL. The numerical value given to each peak on the chromatogram indicates the peak half-value width.

FIG. 23A is a chromatogram obtained by injecting 0.2 μL of Sample II by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min from the first liquid feeding port.

FIG. 23 (B) shows a mobile phase (acetonitrile / water = 60/40) delivered at 0.1 mL / min from the first delivery port, and 0.04 minutes after 0.2 μL of sample II was injected. The micro six-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 is operated to switch, the first liquid feeding port liquid feeding is stopped, and the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0 .1 mL / min) to start, divide the sample band, introduce the front part into the column to separate and elute, and after 5.0 minutes from sample injection, operate the micro 6-way channel switching valve to By switching to the flow path, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first liquid feeding port. Liquid feed port and micro 6 way flow path switching valve And introduction into the column of a sample band after the portion has been placed inside the loop of a chromatogram was isolated elution.

FIG. 23 (C) shows a mobile phase (acetonitrile / water = 60/40) fed at 0.1 mL / min from the first liquid feeding port and 0.05 minutes after 0.2 μL of sample II was injected. The micro six-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 is operated to switch, the first liquid feeding port liquid feeding is stopped, and the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0 .1 mL / min) to start, divide the sample band, introduce the front part into the column to separate and elute, and after 5.0 minutes from sample injection, operate the micro 6-way channel switching valve to By switching to the flow path, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first liquid feeding port. Liquid feed port and micro 6 way flow path switching valve And introduction into the column of a sample band after the portion has been placed inside the loop of a chromatogram was isolated elution.

FIG. 23D is a chromatogram obtained by injecting 0.5 μL of Sample III by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min from the first liquid feeding port.

FIG. 23 (E) shows that the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feeding port at a flow rate of 0.05 mL / min, and 5 μL of sample III was injected. By operating the one-way switching valve, the first liquid supply port liquid supply is stopped and the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.05 mL / min) is switched to divide the sample band. And introduced into the column for separation and elution (first chromatogram in FIG. 23 (E)), 0.02 minutes after 10.00 minutes from sample injection (between 10.00 and 10.02 minutes), microscopic By operating the 6-way flow switching valve, the second liquid supply port liquid supply is stopped, the liquid supply from the first liquid supply port (acetonitrile / water = 60/40, 0.05 mL / min) is performed, and the first liquid supply port liquid supply is stopped. Liquid port and micro 6 way flow path switching valve By introducing 0.02 minutes of liquid feeding volume (volume of 1 μL) from the sample band placed in the loop, and operating the micro 6-way flow switching valve 10.02 minutes after sample injection. Then, the first liquid feeding port liquid feeding was stopped, and the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0.05 mL / min) was switched to separate and eluted (FIG. 23 (E) second chromatogram). . Further, every 10 minutes thereafter, that is, 20.00, 30.00, 40.00 and 50.00 minutes after the sample injection, respectively 0.01 minutes each, the volume is determined by operating the micro 6-way flow switching valve as described above. Sample bands of 0.5 μL each were introduced into the column, separated and eluted, and the third to sixth chromatograms of FIG. 23 (E) were obtained. Finally, from 60.00 minutes after sample injection, the micro six-way flow path switching valve is operated to switch to the liquid feed from the first liquid feed port (acetonitrile / water = 60/40, 0.05 mL / min), The sample band remaining in the first liquid feeding port and the loop of the micro 6-way flow path switching valve was introduced into the column and separated and eluted to obtain a seventh chromatogram of FIG.

In FIG. 23A (comparative example), theoretical plate numbers 440, 3520, 5220, and 7880 were obtained for TU, BuPh, N, and OcPh, which are the first, third, fourth, and seventh peaks, respectively. On the other hand, in the first chromatogram of FIG. 23 (B) in which the sample band is divided by switching the liquid feeding port using a valve and the front part is introduced into the column, each of TU, BuPh, N, and OcPh The theoretical plate numbers 900, 5660, 7140 and 8540 were obtained. The sample introduction method of dividing the sample in the direction crossing the axial direction and introducing it into the column of the present invention can generate a theoretical plate number about 1.08 to 2.00 times that of the conventional sample injection method. It is said. The effect on the peak in the initial chromatogram is large.

In the first chromatogram of FIG. 23 (C) in which the sample band is divided by switching the liquid feeding port using the micro 6-way flow path switching valve and the front part is introduced into the column, TU, BuPh, N and OcPh are shown. Respective theoretical plate numbers of 680, 4900, 6410 and 8640 were obtained. The sample introduction method of the present invention enables generation of the number of theoretical plates about 1.07 to 1.50 times that of the conventional sample injection method. In FIG. 23B, a sample introduction rate of 41% was obtained by dividing the sample 0.04 minutes after the sample injection and introducing it into the column, whereas in FIG. A sample introduction rate of 73% was obtained by dividing the sample after 0.05 minutes and introducing it into the column. When the sample introduction rate is large, the sample band introduced into the column includes a larger rear portion (tail portion). Therefore, in the first chromatogram in FIG. 23B and the first chromatogram in FIG. FIG. 23 (B) shows a higher theoretical plate number in the first chromatogram.

In FIGS. 23B and 23C, the theoretical plate number of the peak of the second chromatogram given by the rear part of the divided sample band is larger than that in FIG. In FIG. 23 (A), theoretical plate numbers 440, 3520, 5220, and 7880 were obtained for TU, BuPh, N, and OcPh, respectively, whereas in the second chromatogram of FIG. 23 (B), TU, The theoretical plate numbers 640, 4790, 6320 and 8330 were obtained for BuPh, N and OcPh, respectively. In the second chromatogram in FIG. 23C, theoretical plate numbers 750, 5280, 6770, and 8460 were obtained for TU, BuPh, N, and OcPh, respectively. The sample introduction method of dividing the sample in the direction crossing the axial direction and introducing it into the column according to the present invention enables generation of a higher number of theoretical plates than the conventional sample injection method.

In FIG. 23D (comparative example), theoretical plate numbers 930, 3910, 5110 and 6640 were obtained for AcAn, BuPh, VaPh and HpPh, which are the second, fifth, seventh and ninth peaks, respectively. On the other hand, in the third to sixth chromatograms of FIG. 23 (E), the average number of theoretical plates of 2840, 6950, 7270, and 6860 was obtained for AcAn, BuPh, VaPh, and HpPh, respectively. The sample introduction method of dividing the sample in the direction crossing the axial direction and introducing it into the column according to the present invention can generate 1.03-3.06 times the theoretical plate number as compared with the conventional sample injection method. It is said. When compared with the band width (half-value width) at 50% height of the peak, the third to sixth chromatograms in FIG. 23 (E) are 1.7 to 43% smaller than those in FIG. 23 (D).

Incidentally, a very small part of the sample band intermediate portion existing in the rotor channel of the valve immediately before the flow path switching point 50 of the first liquid feeding port shown in FIG. 4B is the micro 6-way flow path switching valve. In accordance with the switching, the sample band moves along with the front part or the rear part of the sample band depending on the rotation direction of the rotor. Further, as shown in FIG. 4D, after the sample band is divided by switching the flow path, the loop cleaning pipe 58 of the switching valve is used in the loop during or after the separation and elution of the front part of the sample band. It is possible to discharge the rear part of the placed sample band.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, compared to the conventional sample injection method (FIG. 24 (A)) (comparative example), the division of the sample band of the present invention-after A decrease in sample bandwidth by the sample introduction method with partial placement (FIGS. 24B and 24C) was demonstrated without a column. Further, as compared with the conventional sample injection method (FIGS. 24D and 24E), the sample introduction method involving the division of the sample band and the rear partial placement of the present invention is continuously performed (FIG. 24F). The decrease in sample bandwidth due to was demonstrated without a column.

Among the sample introduction methods of the present invention, by the sample introduction method involving the division of the sample band and the partial indwelling, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved, the inside of the first liquid supply port, and the switching valve This is an example in which the improvement in the number of theoretical plates in the second chromatogram given by the rear portion of the sample band held in the loop is shown (FIGS. 24B and 24C). For comparison with these examples, an example by a conventional sample injection method is also shown (FIG. 24A). In addition, the sample band is divided, the rear part of the sample band is placed in the first liquid feed port and the loop of the micro 6-way flow switching valve, the first part of the sample band is separated and eluted, and then the first liquid feed port is fed. In this example, the portion after the sample band is introduced into and eluted from the ZDU by dividing the portion after the sample band into six times at regular intervals by alternately repeating the second liquid feeding port and the second liquid feeding port liquid feeding. (FIG. 24 (F)). For comparison with this example, an example by a conventional sample injection method is also shown (FIGS. 24D and 24E). 24 (A), (B), and (C) show Example 14, FIG. 23 (A) (conventional sample injection method), and FIGS. 23 (B) and (C) (sample introduction method of the present invention), respectively. ) Represents the state of the sample band at the column entrance. 24 (E) and 24 (F) show the results at the column entrance in Example 14, FIG. 23 (D) (conventional sample injection method) and FIG. 23 (E) (sample introduction method of the present invention), respectively. Represents the state of the sample band.

UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (2 μL loop, inner diameter 0.13 mm, length 15.0 cm and length 5 μL loop, inner diameter 0.20 mm, length 15.9 cm) as a sample injection device And a zero dead volume union (U-435, inner diameter 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used instead of the column. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min and 0.05 mL / min. The temperature condition was 40 ° C. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 240 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a stainless steel tube (capacity of about 0.86 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of another ZDU (capacity of 0.02 μL). And a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, a sample band dividing unit having the structure shown in FIG. 4 was used.

A micro 6-way flow path switching valve (C72X-1696EH, VALCO) for switching the flow path between the Rheodyne 8125 valve and the column is shown in FIG. 4 (first liquid feed port 51, second liquid feed port) 52, channel switching point 50, loop 53, column 1), and this valve was operated by LC800 to divide the sample band and introduce it into the ZDU. The piping from the Rheodyne 8125 valve to the C72X-1696EH valve uses a stainless steel tube (capacity: about 0.79 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.1 mm, and a length of 10 cm, and the loop 53 has a capacity of 2 μL (see FIG. 24 (A), (B), (C)) and 5 μL (used in FIGS. 24 (D), (E), (F)). For the piping from the flow path switching point 50 to the ZDU inlet, a stainless steel tube (capacity: about 0.86 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm was used. Other piping connected to the C72X-1696EH valve was a stainless steel tube having an outer diameter of 1.6 mm and an inner diameter of 0.1 mm. Sample uracil: A solution of uracil (0.07 mg / mL) in acetonitrile / water = 60/40 was used. The numerical value attached to each peak on the chromatogram indicates tR: retention time and tW: peak half-value width.

FIG. 24A is a chromatogram obtained by injecting 0.2 μL of sample uracil by the conventional sample injection method and eluting it. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min from the first liquid feeding port.

FIG. 24 (B) shows a mobile phase (acetonitrile / water = 60/40) delivered at 0.1 mL / min from the first delivery port, and 0.04 minutes after 0.2 μL of sample uracil was injected. The micro six-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 is operated to switch, the first liquid feeding port liquid feeding is stopped, and the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0 .1 mL / min) to start, divide the sample band, introduce the front part into the ZDU, separate and elute, and 0.5 minute after sample injection, operate the micro 6-way flow path switching valve By switching to the flow path, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first liquid feeding port. Inside the liquid feed port and the flow path switching valve And introduction into ZDU sample band after the portion has been placed in the flop, a chromatogram was isolated elution.

FIG. 24 (C) shows a mobile phase (acetonitrile / water = 60/40) fed from the first liquid feeding port at 0.1 mL / min and 0.05 minutes after injecting 0.2 μL of sample uracil. The micro six-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 is operated to switch, the first liquid feeding port liquid feeding is stopped, and the second liquid feeding port liquid feeding (acetonitrile / water = 60/40, 0 .1 mL / min) to start, divide the sample band, introduce the front part into the ZDU, separate and elute, and 0.5 minute after sample injection, operate the micro 6-way flow path switching valve By switching to the flow path, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first liquid feeding port. Switching between liquid feed port and micro 6-way flow path And introduction into ZDU sample band after the portion has been placed in the loop of the lube, a chromatogram was isolated elution.

FIG. 24D is a chromatogram obtained by injecting 0.5 μL of sample uracil by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min from the first liquid feeding port. FIG. 24E is a chromatogram obtained by injecting 5.0 μL of sample uracil by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min from the first liquid feeding port.

FIG. 24F shows that the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feeding port at a flow rate of 0.05 mL / min, 5 μL of sample uracil was injected, and after 0.09 minutes, micro 6 By operating the one-way switching valve, the first liquid supply port liquid supply is stopped and the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.05 mL / min) is switched to divide the sample band. , Introduced into ZDU and separated and eluted (FIG. 24F, first chromatogram), 0.50 to 0.02 minutes after sample injection (between 0.50 and 0.52 minutes) ), By operating the micro six-way flow path switching valve, the second liquid feeding port liquid feeding is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min) is returned, In the first liquid supply port and micro 6 way flow path switching bar Introduce 0.02 minute liquid supply (capacity 1 μL) from the sample band placed in the loop of the tube into the ZDU, and operate the micro 6-way flow path switching valve 0.52 minutes after sample injection. Thus, the first liquid supply port liquid supply was stopped, and the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.05 mL / min) was switched to separate and eluted (FIG. 24 (F) second chromatograph). Grams). After that, every 0.5 minutes, ie, 1.0, 1.5, 2.0, and 2.5 minutes after sample injection, each 0.01 minute each, the micro 6-way flow switching valve is operated as described above. The sample bands with a volume of 0.5 μL were introduced into and eluted from the ZDU to obtain the third to sixth chromatograms of FIG. Finally, from 3.0 minutes after sample injection, the micro six-way flow path switching valve is operated to switch to liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min), The sample band remaining in the first liquid feeding port and the loop of the micro 6-way flow path switching valve was eluted to obtain a seventh chromatogram of FIG.

As shown in FIG. 24 (B), when the liquid supply volume from the first liquid supply port from the sample injection to the division in the direction crossing the axial direction of the sample band and the introduction to the ZDU is 4 μL, About 41% of the total injected sample was introduced and appeared as the peak of the first chromatogram. In the peak of FIG. 24A (comparative example), the dispersion of the sample band calculated by the moment method was 1.68 μL ² , whereas in the peak of FIG. The dispersion was 0.68 μL ² , which was reduced by about 60% compared to the peak in FIG. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

As shown in FIG. 24 (C), when the liquid feeding volume from the first liquid feeding port from the sample injection to the division in the direction crossing the axial direction of the sample band and the introduction to the ZDU is 5 μL, About 73% of the total injected sample was introduced and appeared as the peak of the first chromatogram. In the peak of the first chromatogram in FIG. 24C, the dispersion of the sample band calculated by the moment method was 0.97 μL ² , which is about 40% lower than the peak in FIG. In both cases of FIGS. 24 (B) and (C), the peak of the second chromatogram shows a decrease in the sample bandwidth as compared with the peak of FIG. 24 (A). Compared with the peak in FIG. 23A, the peak width in the second chromatogram peak in FIGS. 23B and 23C is similar to the tendency to decrease. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

24B and 24C, the elution volume of the peak of the second chromatogram was 0.1 mL / min × 0.035 min = 0.535 mL = 3.5 μL. Subtracting the ZDU capacity (0.02 μL), the pipe capacity from the ZDU to the detector inlet (1.12 μL), and the detector cell capacity (6 nL) from this value gives 2.354 μL, and the flow shown in FIG. The measured value of the capacity from the path switching point 50 to the ZDU inlet at 0.1 mL / min was 2.354 μL. In FIG. 24A, the peak elution volume was 0.1 mL / min × 0.068 min = 0.068 mL = 6.8 μL. Subtracting the ZDU capacity (0.02 μL), the pipe capacity from the ZDU to the detector inlet (1.12 μL), and the detector cell capacity (6 nL) from this value gives 5.654 μL, and the above measured value 2 Subtracting .354 μL yielded 3.3 μL. Therefore, under the condition where 0.2 μL of sample uracil is eluted at a flow rate of 0.1 mL / min, the capacity from the injector to the flow path switching point is 3.3 μL, and the capacity from the flow path switching point to the ZDU inlet is 2.354 μL. It became. Under these conditions, in FIGS. 24B and 24C, when the liquid feeding volume from the first liquid feeding port until the sample band splitting is 4 μL and 5 μL, respectively, the sample introduction rate is 41%, 73 %. These sample introduction rates coincide with the sample introduction rates in FIGS. 23 (B) and 23 (C).

In FIG. 24D, the peak elution volume was 0.05 mL / min × 0.167 min = 0.835 mL = 8.35 μL. Subtracting the ZDU capacity of 0.02 μL from this value yields 8.33 μL. When a 0.5 μL sample is injected using the conventional sample injection method with a 5 μL loop injector, the column is calculated from the peak elution time (in this experiment, not the column but the ZDU). The volume of was 8.33 μL. On the other hand, in the third to sixth chromatograms of FIG. 24F introduced into the ZDU by 0.5 μL each from the sample band placed in the loop of the flow path switching valve of the sample band dividing device, Since sample uracil was eluted after an average of 0.049 minutes from the introduction, the peak elution volume was 0.05 mL / min × 0.049 min = 0.245 mL = 2.45 μL. Subtracting the ZDU capacity of 0.02 μL from this value yields 2.43 μL. By the sample introduction method of the present invention, it was confirmed that the volume outside the column (in this experiment, outside the ZDU) was reduced by 5.9 μL in the measured value. Also, subtracting the pipe capacity (1.12 μL) from the ZDU to the detector inlet from 1.43 μL and the capacity of the detector cell (6 nL) to 1.304 μL, from the flow path switching point 50 shown in FIG. The measured value of the capacity up to the ZDU inlet at 0.05 mL / min was 1.304 μL.

As disclosed in Non-Patent Document 3, in a normal UHPLC instrument, the volume outside the column measured by the same method as in Non-Patent Document 2 is 12 to 18 μL at a flow rate of 0.1 to 2.0 mL / min. There is big. If the sample introduction method of the present invention is also applied to these apparatuses, the capacity outside the column can be reduced and the dispersion of the solute can be made very small.

Figure 24 (D), (E) ( Comparative Example) each dispersion band peak uracil calculated by the moment method in, 1.00 L ^2, whereas was 1.16μL ^2, FIG. 24 (F The dispersion of the uracil peak band in the 3rd to 6th chromatograms) was 0.26 μL ² on average. In each of the third to sixth chromatograms of FIGS. 24 (D) and 24 (F), a sample having a volume of 0.5 μL is injected or introduced into the ZDU. The dispersion of the eluted peaks is very different. This is the effect of dividing the sample in the direction crossing the axial direction of the sample and introducing it into a column (here ZDU). When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

In Example 14 and Example 15, according to the example shown in FIGS. 23 (E) and 24 (F), the sample introduction method of the present invention can originally analyze one sample band by dividing it into many. As shown in FIG. 8, the sample band splitting section including the four-way joint shown in FIG. 7 and the T-shaped connector shown in FIG. If the sample band dividing part is provided, the front part of the sample band is discharged without being introduced into the column, and the intermediate part of the sample band can be analyzed quickly.

  In Examples 14 and 15, the sample introduction method of the present invention using the micro 6-way flow switching valve was shown. However, using the 3-way flow switching valve, the liquid feeding from the second liquid feeding port was performed separately. If a liquid feed pump is used, the sample introduction method of the present invention can be carried out. In addition, even if a four-way flow switching valve, an eight-way flow switching valve, or a ten-way flow switching valve is used, the sample introduction method of the present invention is achieved by adopting the same flow path configuration as the six-way flow switching valve. Can be implemented.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, compared to the conventional sample injection method (FIG. 25 (A)) (comparative example), the sample band of the present invention is divided after The improvement of the theoretical plate number of the column was confirmed by the sample introduction method with partial placement (FIG. 25 (B)) and the sample introduction method (FIG. 25 (C)) by dividing the sample band of the present invention after the partial discharge.

FIG. 25 (B) shows a sample introduction method according to the present invention, in which a sample band is divided and a partial partial placement is performed. This is an example in which the number of theoretical plates in the chromatogram is improved and the portion after the sample band is discharged. For discharging the rear part of the sample band, a discharge port upstream of the sample injection device (injector) as shown in FIG. 6 was used.

FIG. 25C shows the sample introduction method of the present invention, in which the sample band is divided and the partial discharge is performed after the sample band is divided and the front portion is introduced into the column. This is an example in which the number of theoretical plates in the chromatogram is improved and the portion after the sample band is discharged. For discharge of the rear part of the sample band, a discharge port connected to a cloth placed between the injector and the column shown in FIG. 7B was used.

A sample Monocap C18 (C18-modified monolithic silica gel, inner diameter 0.1 mm, long length) was used as a sample injection device using a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Valco injector valve (VICI C4-0004-01, 10 nL inner loop). 15 cm, Kyoto Monotech Co., Ltd.) was used. The mobile phase (acetonitrile / water = 60/40) was fed at 0.002 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet was used by connecting a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, a sample band dividing unit having a structure shown in FIG. 7B was used. Further, as shown in FIG. 6, a discharge port was separately installed upstream of the sample injection device.

The details of the sample band dividing section shown in FIG. 7B are as follows. The Valco injector valve and the cross (four-way joint, U-430, inner diameter 0.50 mm, inner capacity 0.72 μL, IDEX) have an outer diameter of 0. Connected by a fused silica capillary tube (capacity: about 0.035 μL) with a length of 375 mm, an inner diameter of 0.03 mm, and a length of 5 cm. The cloth and the column inlet are 0.375 mm outer diameter, 0.03 mm inner diameter, 6 cm long fused silica. A capillary tube (capacity: about 0.042 μL) was used for connection. As shown in FIG. 9A, the fused silica capillary connecting pipes connected to the cloth were connected by being inserted into the cloth, and the gap between the connecting pipes was set to 0.2 mm. In addition, a second liquid feeding port and a discharge port (a vent valve and a resistance tube (inner diameter 0.03 mm, length 30 cm) connected) were provided as separate ports on the cross.

Samples include: Sample II: Thiourea (TU, 0.1 mg / mL), Acetophenone (AcPh, 0.16 mg / mL), Butyrophenone (BuPh, 0.3 mg / mL), Naphthalene (N, 0.3 mg / mL) , Hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 25A is a chromatogram obtained by injecting 0.01 μL of sample II by the conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.002 mL / min from the first liquid feeding port.

FIG. 25 (B) shows that the mobile phase (acetonitrile / water = 60/40) was fed at 0.002 mL / min from the first liquid feeding port, and 0.15 minutes after 0.01 μL of sample II was injected. Stop the first liquid feeding port, start the second liquid feeding port (acetonitrile / water = 60/40, 0.02 mL / min) to divide the sample band, and introduce the front part into the column for separation. Elution, and after 5.0 minutes from the sample injection, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.002 mL / min) It is the returned chromatogram.

From 0.15 to 5.0 minutes after sample injection, the vent valve connected to the discharge port upstream of the sample injection device as shown in FIG. 6 is opened, and the inner diameter is 0.03 mm and the length is 30 cm. The rear part of the sample band was discharged through the resistance of a fused silica capillary tube. The flow rate from the second liquid feeding port was set to 0.02 mL / min from 0.15 to 5.0 minutes after sample injection, which is necessary to separate and elute the front part of the sample band with a column. This is because the flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the appropriate flow rate. In addition, the vent valve ahead of the discharge port connected to the cloth shown in FIG. 7B was kept closed.

FIG. 25 (C) shows a mobile phase (acetonitrile / water = 60/40) fed at 0.002 mL / min from the first liquid feeding port and 0.15 minutes after 0.01 μL of sample II was injected. Stop the first liquid feeding port, start the second liquid feeding port (acetonitrile / water = 60/40, 0.02 mL / min) to divide the sample band, and introduce the front part into the column for separation. Elution, and after 5.0 minutes from the sample injection, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.002 mL / min) It is the returned chromatogram.

From 0.15 minutes to 5.0 minutes after sample injection, the vent valve at the end of the discharge port connected to the cloth shown in FIG. 7B is opened, and the fuse has an inner diameter of 0.03 mm and a length of 30 cm. The rear part of the sample band was discharged through the resistance of the dosilica capillary tube. The flow rate from the second liquid feeding port was set to 0.02 mL / min from 0.15 to 5.0 minutes after sample injection, which is necessary to separate and elute the front part of the sample band with a column. This is because the flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the appropriate flow rate. Note that the vent valve at the end of the discharge port upstream of the sample injection apparatus as shown in FIG. 6 was kept closed.

As shown in FIG. 25 (A) (comparative example), when a very small capillary column (inner diameter: 0.1 mm, length: 15 cm) is used, the effect of dispersing the solute outside the column appears very greatly. The theoretical plate numbers 410, 1140, and 1960 were obtained for the fourth, sixth, and seventh peaks, N, HpPh, and OcPh, respectively. In contrast, in FIG. 25 (B) obtained from the sample band front portion obtained by the sample introduction method of the present invention, 2190, 4360, and 5430 theoretical plate numbers were obtained for N, HpPh, and OcPh, respectively. . In FIG. 25C, the theoretical plate numbers 2380, 4300, and 5440 are obtained for N, HpPh, and OcPh, respectively. Compared to the conventional sample injection method, the sample of the present invention is divided in the direction crossing the axial direction. In the sample introduction method for introducing into the column, the number of theoretical plates was 2.5 to 5.0 times.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, compared to the conventional sample injection method (FIG. 26 (A)) (comparative example), the sample band of the present invention is divided after Reduction of the sample band width by the sample introduction method with partial placement (FIG. 26 (B)) and the sample introduction method with division of the sample band of the present invention-after partial discharge (FIG. 26 (C)) without a column. Demonstrated.

26 (A), (B), and (C) show the state of the sample band at the column entrance in FIGS. 25 (A), (B), and (C), respectively.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Valco injector valve (VICI C4-0004-01, 10 nL inner loop) was used as a sample injection apparatus, and the column was replaced with an outer diameter of 0.375 mm, an inner diameter of 0.03 mm, Use a zero-dead volume union (U-435, inner diameter 0.25 mm, internal capacity 0.02 μL, IDEX) connected to the inlet and outlet of a 6 cm long fused silica capillary tube (capacity 0.042 μL) It was. The mobile phase (acetonitrile / water = 60/40) was fed at 0.003 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. A fused silica capillary tube (capacity: about 0.24 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm is connected from the outlet of the fused silica capillary tube instead of the column to the detector cell inlet. Used. As the sample band dividing device, a sample band dividing unit having a structure shown in FIG. 7B was used. Further, as shown in FIG. 6, a discharge port was separately installed upstream of the sample injection device.

The details of the sample band dividing portion shown in FIG. 7B are as follows. Valco injector valve and cross (4-way joint, U-430, inner diameter 0.50 mm, internal capacity 0.72 μL, IDEX), outer diameter 0.375 mm Connected with a fused silica capillary tube (capacity: about 0.035 μL) having an inner diameter of 0.03 mm and a length of 5 cm. The fused silica capillary tube instead of the cloth and column has an outer diameter of 0.375 mm, an inner diameter of 0.03 mm, and a length A 6-cm fused silica capillary tube (capacity: about 0.042 μL) was connected. As shown in FIG. 9A, the connecting pipes connected to the cross were connected by being inserted into the cross, and the gap between the connecting pipes was 0.2 mm. In addition, a second liquid feeding port and a discharge port (a vent valve and a resistance tube (inner diameter 0.03 mm, length 30 cm) connected) were provided as separate ports on the cross. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 26A is a chromatogram obtained by injecting 0.01 μL of sample naphthalene by the conventional sample injection method and elution. The mobile phase (acetonitrile / water = 60/40) was fed at 0.003 mL / min from the first liquid feeding port.

FIG. 26 (B) shows that mobile phase (acetonitrile / water = 60/40) was fed at 0.003 mL / min from the first liquid feeding port, and 0.1 μL after 0.1 μL of sample naphthalene was injected. Stop the first liquid supply port liquid supply, start the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.004 mL / min), divide the sample band, and replace the front part with a fused instead of a column. Introduced into the silica capillary tube and eluted, 1.0 minute after the sample injection, the liquid feeding from the second liquid feeding port was stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, It is a chromatogram returned to 0.003 mL / min.

From 0.1 minute to 1.0 minute after the sample injection, the vent valve connected to the upstream discharge port of the sample injection device as shown in FIG. 6 is opened, and the inner diameter is 0.03 mm and the length is 30 cm. The rear part of the sample band was discharged through the resistance of a fused silica capillary tube. The flow rate from the second liquid feeding port was set to 0.004 mL / min from 0.1 minute to 1.0 minute after sample injection because the front part of the sample band was a fused silica capillary instead of the column. This is because the flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the flow rate required for elution from the tube. In addition, the vent valve ahead of the discharge port connected to the cloth shown in FIG. 7B was kept closed.

FIG. 26 (C) shows that mobile phase (acetonitrile / water = 60/40) is fed at 0.003 mL / min from the first liquid feeding port, and 0.01 μL of sample naphthalene is injected by the conventional sample injection method. 0.1 minute after the first liquid supply port liquid supply is stopped, the second liquid supply port liquid supply (acetonitrile / water = 60/40, 0.004 mL / min) is started, and the sample band is divided. Is introduced into a fused silica capillary tube instead of the column and eluted. After 1.0 minute from the sample injection, liquid feeding from the second liquid feeding port is stopped, and liquid feeding from the first liquid feeding port (acetonitrile) / Water = 60/40, 0.003 mL / min).

From 0.1 minute to 1.0 minute after sample injection, the vent valve at the end of the discharge port connected to the cross shown in FIG. 7B is opened, and the fuse has an inner diameter of 0.03 mm and a length of 30 cm. The rear part of the sample band was discharged through the resistance of the dosilica capillary tube. The flow rate from the second liquid feeding port was set to 0.004 mL / min from 0.1 minute to 1.0 minute after sample injection because the front part of the sample band was a fused silica capillary instead of the column. This is because the flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the flow rate required for elution from the tube. Note that the vent valve at the end of the discharge port upstream of the sample injection apparatus as shown in FIG. 6 was kept closed.

In FIG. 26A (comparative example), the naphthalene peak shows large tailing, whereas in FIGS. 26B and 26C, the peak width of the chromatogram is reduced. The tailing part is cut off from the peak of (A). It was shown that the rear part (tail part) of the sample band was removed by splitting the sample band in the direction crossing the axial direction just before the column inlet and introducing it into the fused silica capillary tube instead of the column. Yes.

In FIG. 26A (comparative example), the dispersion of the naphthalene peak was calculated to be 0.092 μL ² by the moment method. In contrast, FIG. 26 (B), the variance of the peak of naphthalene (C), respectively, 0.023MyuL ^2, was calculated to 0.030μL ^2. By dividing the sample in the direction crossing the axial direction of the present invention and introducing the sample into the column (here, fused silica capillary tube), 0.06-0.07 μL ² (about 60-70%) is obtained. ) Dispersion became smaller. In FIGS. 26 (B) and (C), these results were obtained under the same conditions as in FIGS. 25 (B) and (C) except for the column and the flow rate. ) In the column having an inner diameter of 0.1 mm and a length of 15 cm, the factor that resulted in a higher theoretical plate number than the chromatogram obtained by the conventional sample injection method (FIG. 25A) is the column inlet. This is the effect of reducing the dispersion of the sample band in the vicinity. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

Compared with the conventional sample injection method (FIG. 27A) (comparative example), the sample at the column inlet of the present invention relates to the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention. The column performance was compared by the sample introduction method (FIG. 27 (B)) involving the sample division by discharging the back band part.

Among the sample introduction methods of the present invention, the column performance improvement, that is, the number of theoretical plates is improved by the sample introduction method using the sample band dividing unit shown in FIG. This is a chromatogram shown in FIG. 27 (B).

As a sample injection device, a UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used, and a column Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm, Length 5 cm, GL Sciences Inc.) was used. A mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.05 seconds and the data capture interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity approx. 0.88 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 45 cm connected to ZDU (capacity 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was used. As the sample band dividing device, a sample band dividing unit having a structure shown in FIG. 8C was used.

As for the details of the sample band dividing section shown in FIG. 8 (C), U-428 (inner diameter: 0.5 mm, IDEX) is used for the T-shaped connector (three-way joint). Stainless steel tube (capacity: about 0.92 μL) having an outer diameter of 0.236 mm, an inner diameter of 0.108 mm, and a length of 10 cm, and an outer diameter of the outer tube A stainless steel tube having a diameter of 1.6 mm, an inner diameter of 0.5 mm, and a length of 5 cm was used. A stainless steel pipe having an outer diameter of 1.6 mm and an inner diameter of 0.25 mm was used as a branch pipe of the T-shaped connector. A 6-way switching valve (Rheodyne 7000) was used as the vent valve at the end of the branch pipe. Samples include: Sample IV: (thiourea (TU), acetanilide (AcAn), acetophenone (AcPh), propiophenone (PrPh), butyrophenone (BuPh), naphthalene (N), hexanophenone (HxPh), heptanophenone A solution of (HpPh), octanophenone (OcPh) mixture in acetonitrile / water = 60/40 was used.

FIG. 27A is a chromatogram obtained by injecting 0.2 μL of sample IV by a conventional sample injection method and performing separation. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min from the first liquid feeding port.

FIG. 27 (B) shows a mobile phase (acetonitrile / water = 60/40) delivered at 0.05 mL / min from the first delivery port, and 0.03 minutes after 0.2 μL of sample IV was injected. The vent valve was opened until ˜0.04 minutes later, and the rear part of the sample band was discharged. The flow rate of the first liquid delivery port from 0.03 minutes to 0.04 minutes after the sample injection was 0.199 mL / min, and after 0.04 minutes after the sample injection, the flow rate was 0.05 mL / min. The flow rate from the first liquid feeding port was set to 0.199 mL / min from 0.03 minutes to 0.04 minutes after the sample injection, which is necessary to elute the front part of the sample band from the column. This is because a flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the flow rate.

In FIG. 27A (comparative example), theoretical plate numbers 1090, 5800, 6490, and 6820 were obtained for TU, BuPh, N, and OcPh, which are the first, fifth, sixth, and ninth peaks, respectively. On the other hand, the theoretical plate number in the chromatogram from the front part of the sample band obtained by the sample introduction method of dividing the sample in the direction crossing the axial direction and introducing it into the column of the present invention is shown in FIG. ), TU, BuPh, N, and OcPh were 4580, 8690, 8780, and 7310 stages, respectively, indicating a column performance improvement of 1.07 to 4 times. This is a result obtained by comparing the conventional sample injection method with the sample introduction method of the present invention using the same column in Example 3 (N in FIG. 12A is 6900 stages, FIG. N is a performance improvement close to 8410).

Compared with the conventional sample injection method (FIG. 28A) (comparative example), the sample at the column inlet of the present invention relates to the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention. The effect of reducing the sample band width by the sample introduction method (FIG. 28 (B)) accompanied by the sample division by discharging the rear part (tail part) of the band was demonstrated without a column.

A UHPLC apparatus LC800 (GL Science Co., Ltd.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection apparatus, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU). A mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The detector response was 0.01 seconds and the data capture interval was 5 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.88 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 45 cm, and another ZDU (capacity: 0.02 μL). And a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, the sample band dividing unit used in Example 18 having the structure shown in FIG. 8C was used. As a sample, a solution of sample uracil: uracil (U, 0.07 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 28A is a chromatogram obtained by injecting 0.2 μL of sample uracil by the conventional sample injection method and eluting it. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min from the first liquid feeding port.

FIG. 28 (B) shows a mobile phase (acetonitrile / water = 60/40) fed from the first feed port at 0.05 mL / min, and 0.2 μL of sample uracil was injected by the conventional sample injection method. The vent valve was opened from 0.03 minutes to 0.04 minutes later, and the rear part of the sample band was discharged. The flow rate of the first liquid delivery port from 0.03 minutes to 0.04 minutes after the sample injection was 0.199 mL / min, and after 0.04 minutes after the sample injection, the flow rate was 0.05 mL / min. The flow rate from the first liquid feeding port was set to 0.199 mL / min from 0.03 minutes to 0.04 minutes after the sample injection, which is necessary to elute the front part of the sample band from the ZDU. This is because a flow rate necessary for discharging the rear portion of the sample band from the discharge port is added to the flow rate. 28A and 28B show the spread and shape of the sample band at the column entrance in FIGS. 27A and 27B, respectively.

In FIG. 28 (A) (comparative example), the uracil band shows a large tailing, whereas as shown in FIG. 28 (B), the liquid supply capacity from the first liquid supply port is 1.5 μL ( The uracil band in which about 71% of the injected sample band was introduced into ZDU due to the division in the direction crossing the axial direction of the sample at a flow rate of 0.05 mL / min × 0.03 min) shows a large tailing. Not. In FIG. 28A, the dispersion of the sample band calculated by the moment method was 0.95 μL ² , whereas in FIG. 28B, it was 0.22 μL ² , and the dispersion of the sample band was 70% or more. I was able to decrease. In FIG. 28 (B), this result was obtained under the same conditions as in FIG. 27 (B) except that the column had an inner diameter of 1.0 mm and a length of 5 cm in the chromatogram of FIG. The factor that resulted in a higher theoretical plate number in the column than in the chromatogram obtained by the conventional sample injection method (FIG. 27A) is the effect of reducing the dispersion of the sample band near the column inlet. When calculating the variance of peaks by the moment method, the half width given to each peak is not used.

Conventional sample injection under the conditions discussed in Examples 1, 3, 5, 6, 7, 9, 10, 13, 16 (FIGS. 10, 12, 14, 15, 16, 18, 19, 22, 25) Table 7 shows the relationship between the increase in the number of theoretical column columns, the column size, and the detector cell capacity according to the sample introduction method of the present invention compared to the method.

As the column size increases, the effect of increasing the number of theoretical plates by dividing and introducing the sample band decreases, but when using a small column, the column has a high potential, especially for the peak at the beginning of the chromatogram. In order to achieve performance (large number of theoretical plates), it is effective to cut and divide the sample band in the direction intersecting the axial direction at the column entrance and introduce only the front part of the sample band into the column. It has been shown that the structure and operation of the sample band splitting device according to the present invention enables an increase in the number of theoretical columns of the column.

Table 8 shows the result of calculating the dispersion σ _extra ² of the solute band outside the column from an example measured by connecting ZDU or fused silica capillary piping instead of the column. Peaks with good symmetry were calculated from the half-width, and peaks with poor symmetry were calculated by the moment method. It has been shown that the sample introduction method of the present invention can reduce σ _extra ² by 30 to 80% compared to the conventional sample injection method. σ _extra ² increases as the injected sample volume increases, but the method of the present invention makes it possible to introduce the sample into the column as a very narrow band.

Sample band dispersion is smallest when the time to sample split is reduced (at low sample introduction rates) at low flow rates. From this, the peak appearing at the beginning of the chromatogram with a small dispersion (σ _col ² , Formula 1) of the original solute band of the column is reduced by 1 as compared with the conventional sample injection method by reducing σ _extra ² . It became possible to obtain 1 to 5 times the number of theoretical plates (Table 8).

In order to make the most effective use of the effects of the sample introduction method of the present invention, the dispersion of solute bands (σ _tube ² + σ _det ) in the pipe ( _tube ² ) from the column outlet to the detector and the detector cell (det). ² ) It is appropriate to use pipes and detector cells with small inner diameters and small capacities that can keep low.

The results of Examples 1, 3, 5, 6, 7, 9, 10, 13, and 14 (FIGS. 10, 12, 14, 15, 16, 18, 19, 22, and 23) are obtained by dividing the sample band from the time of sample injection. The ratio of (first liquid feeding port flow velocity / second liquid feeding port flow velocity) is not only 100/0, but also various ratios of 90/10, 199/1, 1/2, 1/5, 3/7 Even the values indicate that column performance is improved by the sample introduction method of the present invention as compared to the conventional sample injection method.

Further, the results of Example 5 (FIG. 14) and Example 9 (FIG. 18) show that the flow rate at the rear part of the sample band is increased by opening the vent valve at the time of dividing the sample band and increasing the second liquid feeding port flow velocity. It is shown that the discharge and separation elution on the column in front of the sample band can be performed simultaneously. In addition, after discharging the rear part of the sample band from the flow path, the vent valve is closed to separate and elute the column in the front part of the sample band at various ratios (first liquid feeding port flow rate / second liquid feeding port flow rate). Indicates that you can continue.

The result of Example 9 (FIG. 18) shows that the column exhibits high performance in the ratio of (first liquid feeding port flow rate / second liquid feeding port flow rate) from the time of sample injection to the sample band splitting. A ratio is present.

In addition, the sample introduction method of the present invention, as shown in Examples 2, 4, 8, 11, and 12 (FIGS. 11, 13, 17, 20, and 21) performed using ZDU instead of the column, sample injection The sample band injected from the apparatus is clearly divided in the direction intersecting the axial direction (traveling direction) of the sample band, and only the front part of the sample band is introduced into the column, so that the high performance of the column can be exhibited. Also, if a sample band splitting device having a vent port between the sample injection device and the column is used, the front part of the sample band can be discharged from the flow path and only the rear part of the sample band can be introduced into the column. As suggested by 5, 14, 15 (FIGS. 14, 23, 24), it is clear that the front and rear portions of the sample band can be discharged from the flow path and only the middle portion can be introduced into the column.

From the embodiments described above, the present invention can be used when the sample band injected from the sample injection device (injector or the like) is entering the column from the first liquid feeding port through the connecting pipe, that is, the column inlet or the flow path switching point. When the sample band is straddling, the sample band that has entered the column is divided by feeding from the second liquid feeding port connected to the column inlet, and the sample band is separated and eluted at the same time. The rear part (tail part) of the remaining sample band is retained in the first liquid feeding port tube, or pushed back to be discharged from the flow path, or discharged from the flow path, thereby reducing the spread of the sample band introduced into the column. It is clear that the column performance can be improved.

In particular, as a method for improving the column performance of a sample band provided by a sample injection volume of 0.01 to 5 μL, which is usually suitable for liquid chromatography, the structure of the disclosed sample band dividing device and the operation of the liquid feeding flow path It is clear that the method is useful. It is clear that the column performance improvement (increase in the number of theoretical plates) by dividing the sample band and introducing it into the column has been achieved over a sample introduction rate of 93 to 29%.

From the results of Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 18, and 19 (FIGS. 10 to 18, 21, 22, 27, and 28), the sample injection device to the column When the volume to the inlet is 1.274 μL (measured at a flow rate of 0.05 mL / min and a sample volume of 0.2 μL in Example 4, FIG. 13B), the sample injection volume is 0.01 to 5 μL. In this case, it is apparent that 1.5 to 6 μL is appropriate as the first liquid supply port liquid supply volume until the sample band is divided. Further, as shown in Table 8, in the example using ZDU or fused silica capillary piping instead of the column, the dispersion of the sample band introduced into the ZDU or the like (outside the column) at a sample introduction rate of 29 to 93%. In this case, the sample introduction method of the present invention can be reduced by 30 to 80% compared to the conventional sample injection method. 14, 16, 18 (FIGS. 10, 12, 16, 19, 22, 23, 25, 27), the number of theoretical plates of the column can be improved by 1.1 to 5 times.

In addition, it is apparent that the present invention can achieve improved column performance (theoretical plate number) by introducing a very small injected sample volume into the column. In the present invention, it is clear that a sample having a volume of 0.01 to 0.1 μL (10 to 100 nL), which is considered to be sufficiently small in the current technical level, can be further divided and introduced into the column at the column inlet. It is.

29, when the volume of the sample injected by the injector is 1 μL (Examples 5, 6, 7, 8, 12, 13 (FIGS. 14, 15, 16, 17, 21, 22)), the sample is divided. The relationship between the volume of the mobile phase fed from the first liquid feed port and the introduction rate into the column or ZDU in the front part of the sample band is shown on the horizontal axis. “Liquid feeding volume from one liquid feeding port (μL)”, and the vertical axis is “Sample introduction rate to the column.” However, when the liquid feeding volume until the division of the sample band is 6 μL, the volume of the injected sample Is 2 μL (Example 9, FIG. 18), and the liquid feeding volume until the sample band is divided is 1.5 μL, the injected sample volume is 0.4 μL (Examples 1, 2, 3, 4 (FIG. 10). , 11, 12, 13)) are plotted.

By creating a calibration curve illustrated in FIG. 29 for a sample band dividing device having a fixed configuration, the selection of the liquid supply capacity from the first liquid supply port from the sample injection to the division of the sample band, and the sample band in that setting The rate of introduction into the column can be predicted.

Table 9 shows the dispersion of the solute band (σ _col ² , equation 5) that gives a theoretical plate number of 10,000 on a column with a length of 5 cm and a porosity of 50%, and several retention factors between k = 0-10. The simulated value for the peak of (k value) is shown. If the column size to be compared and the column void volume are the same, it can be determined by comparing with Table 9 whether or not the peaks of the chromatograms collected at various flow rates have approached 10,000 theoretical plates. In order to obtain a theoretical plate number of 10,000 with a column having a length of 5 cm, it is generally necessary to pack a packing material of 2 μm or less. In particular, Inert Sustain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, GL Sciences) Is used in a conventional liquid chromatography device, the sample loop volume of the autosampler is 0.2 μL, and the connection tube from the sample injection valve of the autosampler to the column inlet has an inner diameter of 0.025 mm and a length of 10 cm (volume) From the column outlet to the detector cell inlet, the connecting tube has an inner diameter of 0.025 mm and a length of 10 cm (capacity of about 0.049 μL), and the detector cell has an inner diameter of about 0.15 mm and a length of 5 cm (capacity of about 0). .09 μL), a microcapillary HPLC device (Eksig) with a very small volume of the flow path through which the sample passes ent ExpressLC-Ultra, AB SCIEX) at a flow rate of 0.05 mL / min, the number of theoretical plates is 1790 at k = 0, 7130 at k = 2.2, 8550 at k = 4.0, k550 In 9.0, it was the limit to obtain 8720 (Non-patent Document 8). The porosity of the column varies depending on the type of filler, the way of packing, the type of a single substance in the column, etc., but Table 9 lists general porosity values.

Further, when the value of the solute dispersion decreased by the sample introduction method of the present invention as compared with the conventional injection method shown in Table 8 is compared with the dispersion value shown in Table 9, the sample introduction method of the present invention. Thus, the column size and peak retention coefficient that can obtain a higher effect can be confirmed. For example, in Example 4 of Table 8 and FIG. 13D, the dispersion is reduced by 0.3 μL ² . Even if the retention coefficient k is 8 in a column having an inner diameter of 1.0 mm and a length of 5 cm in Table 9, the dispersion is 3.11 μL ^2, so if the dispersion is reduced by about 10%, the column performance is greatly affected. Further, an inner diameter of 2.1 mm, so 2.99MyuL ² in retention factor k is 1, a column of length 5 cm, although larger impact on column performance When 0.3 [mu] L ² decrease, k is large, 6.73MyuL ² In 2 Therefore, even if 0.3 μL ^{2 is} reduced, the effect on the column performance is not expected to be very large.

For example, in the case of the column of Example 3, FIG. 12 (A) having an inner diameter of 1.0 mm and a length of 5 cm, the peak dispersion calculated from the half width for the solutes U, MB, T and N is 0.31 μL ² , 0.93 μL ² , 2.04 μL ² and 2.96 μL ² . In FIG. 12D obtained by dividing the sample band and introducing it into the column from the same sample injection volume of 0.2 μL, the peak dispersion for the solutes U, MB, T, and N is 0.11 μL ² , They were 0.55 μL ² , 1.66 μL ² and 2.53 μL ² . As shown in Example 4 and FIGS. 13B and 13E, the decrease in dispersion of these peaks is reduced by 0.3 to 0.4 μL ² by dividing the sample band and introducing it into ZDU. The theoretical plate number of each peak in FIG. 12D (3530 when k (U) = 0, 8560 when k (MB) = 2.5, 8140 when k (T) = 4.9. , K (N) = 6.4 and 8410) is the number of theoretical plates of each peak in FIG. 12A (1240 when k (U) = 0, 4870 when k (MB) = 2.5, k (T) = The result increased 1.2 to 2.8 times compared to 6370 at 4.9 and 6900 at k (N) = 6.4 (Table 2). In the peak dispersion (σ _col ² ) by the column (N = 10000) shown in Table 9, for example, the dispersion of the peak of k = 6 in a column having an inner diameter of 1.0 mm and a length of 5 cm is 1.88 μL ² . For N in (D) (k = 6.4 and peak dispersion 2.53 μL ² ), an HPLC apparatus having a larger flow path capacity than the microcapillary HPLC apparatus is used according to the sample introduction method of the present invention. However, the number of theoretical plates can be increased from 6900 to 8410, and the column performance close to the column performance obtained with the microcapillary HPLC apparatus (k = 4.0, theoretical plate number 8550, k = 9.0, 8720) is exhibited. It was shown that 12 (D), U (k = 0, peak dispersion 0.11 μL ² ) and MB (k = 2.5, peak dispersion 0.55 μL ² ) are analyzed by the UHPLC apparatus according to the sample introduction method of the present invention. Can be used to increase the number of theoretical plates from 1240 to 3530 and from 4870 to 8560, respectively, and the column performance obtained with the microcapillary HPLC apparatus (k = 0, theoretical plate number 1790, k = 2.2, 7130) It was shown that column performance exceeding) was demonstrated.

That is, with the sample introduction method of the present invention, an increase in the number of theoretical plates is expected for a column having an inner diameter of 1.0 mm and a length of 5 cm in a wide range of the retention coefficient k. Even in a column having an inner diameter of 2.1 mm and a length of 5 cm, or a column having an inner diameter of 3.0 mm and a length of 5 cm, an improvement in column performance is expected particularly with respect to a peak appearing in the initial chromatogram.

Table 10 shows an inner diameter of 1.0 mm with a porosity of 46%, a column with a length of 5 cm, an inner diameter of 2.1 mm with a porosity of 50%, a column with a length of 5 cm, and an inner diameter of 3.0 mm with a porosity of 50% and a length. The half-value width (unit: min) of a solute band that can obtain a theoretical plate number of 10,000 with a 5 cm column is a value obtained by simulating several retention coefficients (k values) between k = 0 and 10. The full width at half maximum is affected by the flow rate, but if the column size to be compared, the column void volume, and the flow rate are the same, whether the peak of the collected chromatogram has approached the theoretical plate number of 10,000 plates (or has high performance been achieved)? Whether or not) can be determined by comparing with Table 10. A full width at half maximum (unit: min) is assigned to each peak in the example. Moreover, the half value width is described in Tables 11-13 also about the peak which has not provided the half value width.

In Tables 11-13, the half width of each peak is shown about each Example.

According to the sample introduction method of the present invention, in the column for liquid chromatography having various inner diameters, dispersion of solutes outside the column can be reduced, and columns having inner diameters of 0.1 mm, 1.0 mm, 2.1 mm, and 4.6 mm can be reduced. , The effect of increasing the number of theoretical plate columns was observed. The present invention has made it possible to improve column performance for thin, short, and small columns required by UHPLC, particularly for peaks that elute early in the chromatogram. The operations of the pumps and flow path switching valves used in the examples are controlled in units of 1/100 minutes, but if the controllable time interval can be shortened to units of 1/1000 minutes to 1/10000 minutes, for example, Obviously, better control can be achieved to improve column performance.

  According to the present invention, the separation performance of a column, that is, the number of theoretical plates can be improved in liquid chromatography, so that the analysis and fractionation efficiency of substances are improved and used in research and applications in various fields. Is possible.

DESCRIPTION OF SYMBOLS 1 Column 10 Sample band dividing device 11 Sample band dividing part 12 Flow path switching point 20 1st liquid feeding port 21 Liquid feeding pump 22 Sample injection apparatus 29 2nd liquid feeding port 3 Sample band 31 The front part 32 of a sample band Rear part 5 6-way valve 50 Flow path switching point 51 First liquid feeding port 52 Second liquid feeding port 60 First discharge port 7 Four-way joint 91 Sample introduction device

Claims (26)

  In a liquid chromatography apparatus, a device provided between a sample injection device and a column of a sample introduction device for introducing a sample into a column, the first liquid supply port for sending a sample band to the column, and the first liquid supply A sample band dividing device for dividing a sample band, comprising a dividing port for dividing the sample band sent from the port. The sample band dividing device according to claim 1, wherein the sample band dividing device is a device for dividing the sample band in a direction crossing the axial direction. The said division | segmentation port is comprised with the 1st discharge port for discharging the 2nd liquid feeding port for sending a mobile phase to a column, and / or a part of sample band, It is characterized by the above-mentioned. Or the sample band splitting device according to 2. The volume of the flow path from the flow path switching point between the first liquid feed port and the second liquid feed port or the first discharge port to the column inlet is 1 / 13.9 or less of the column void volume. The sample band dividing device according to claim 3, wherein The sample band dividing device according to any one of claims 1 to 4, further comprising a flow path switching valve. 6. The sample band dividing device according to claim 5, wherein the flow path switching valve is a three-way valve, a four-way valve, a six-way valve, an eight-way valve, or a ten-way valve. The sample band dividing device according to any one of claims 1 to 6, further comprising a three-way or four-way joint.   A sample introduction device comprising the sample band dividing device according to any one of claims 1 to 7.   The sample introduction device according to claim 8, further comprising a second discharge port for discharging a part of the sample band upstream from the sample injection device.   A liquid chromatography apparatus comprising the sample introduction apparatus according to claim 8. The liquid chromatography apparatus according to claim 10, further comprising a column having an inner diameter of 4.6 mm or less. The liquid chromatography apparatus according to claim 10 or 11, wherein the peak elution volume is 10.2 mL or less. In liquid chromatography, a sample introduction method comprising: dividing a sample band in a flow channel before a column from a sample injection device by switching the flow channel, and introducing at least a part of the divided sample band into the column. 14. The sample introduction method according to claim 13, wherein when the sample band crosses the column inlet or the channel switching point closest to the column inlet, the sample band is divided by switching the channel. The sample introduction method according to any one of claims 13 to 14, wherein the sample band is divided in a direction intersecting an axial direction of the sample band. The sample introduction method according to any one of claims 13 to 15, wherein all parts of the divided sample bands are introduced into the column. 16. The sample introduction method according to any one of claims 13 to 15, wherein a part of the divided sample band is introduced into a column. The sample introduction method according to claim 17, wherein a portion other than the portion introduced into the column among the divided sample bands is discharged. The rear part including the middle part of the sample band is left in the flow path without being introduced into the column, and the front part of the divided sample band is introduced into the column or discharged from the flow path, and then placed in the flow path. 14. The sample introduction method according to claim 13, wherein a rear part of the sample band is divided and an intermediate part of the sample band is introduced into the column. Using a chromatograph that consists of a pump, sample injection device, column, and detector connected in this order, the front part of the sample band injected from the sample injection device enters the column, and the sample band straddles the column entrance Or when the sample band straddles the flow path switching point closest to the column inlet on the flow path, the liquid feeding from the first liquid feeding port that sends the sample band to the column is stopped, and the 2 starts liquid feeding from the liquid feeding port, divides the sample band, introduces the front part of the sample band into the column, and keeps the rear part of the sample band in the connection pipe from the sample injection device. The sample introduction method according to any one of claims 13 to 15, wherein the sample introduction method is characterized in that: Using a chromatograph that consists of a pump, sample injection device, column, and detector connected in this order, the front part of the sample band injected from the sample injection device enters the column, and the sample band straddles the column entrance When the sample band straddles the flow path switching point closest to the column inlet on the flow path, stop the liquid supply from the first liquid supply port that sends the sample band to the column or maintain the liquid supply. The liquid feeding from the second liquid feeding port provided near the column inlet is started, the sample band is divided, the front part of the sample band is introduced into the column, the vent valve is opened, and the first liquid feeding port 16. The method of any one of claims 13 to 15, characterized in that the liquid feeding from the pipe is restarted or maintained, and the rear part of the sample band is discharged from a first discharge port provided in the flow path near the column inlet. Or 1 Sample introduction methods of the placement. Using a chromatograph device constructed by connecting a pump, a sample injection device, a column, and a detector in this order, a first discharge port is created between the sample injection device and the column, and the sample band injected from the sample injection device When the front part enters the column and the sample band straddles the column inlet, or when the sample band straddles the channel switching point closest to the column inlet on the channel, the first discharge port is opened, and the sample is opened. The sample introduction method according to any one of claims 13 to 15, wherein a rear portion of the band is discharged. The injected sample band is divided by liquid feeding from the second liquid feeding port, and a second discharge port is provided between the pump and the sample injection device for discharging the liquid via a joint and a valve. After the sample band has been divided, the rear part of the sample band placed in the first liquid feeding port is pushed back through the sample injection device by the liquid feeding from the second liquid feeding port. The sample introduction method according to any one of claims 13 to 15, wherein the sample is discharged from a second discharge port provided upstream of the sample injection device. The injected sample band is divided by the liquid feeding from the second liquid feeding port, the rear part of the sample band is left in the first liquid feeding port, and the sample components contained in the front part of the sample band introduced into the column are 16. The portion after the sample band that has been placed in the first liquid feeding port after being eluted from the column is introduced into the column by restarting the liquid feeding from the first liquid feeding port. The sample introduction method according to any one of the above. 14. The rear part of the sample band placed in the first liquid feeding port is further divided, and at least a part thereof is introduced into the column by restarting the liquid feeding from the first liquid feeding port. The sample introduction method according to any one of 15. 14. The range of the ratio of (first liquid feeding port flow rate) / (second liquid feeding port flow rate) from the time of sample injection to sample band division is 10/0 to 1/5. The sample introduction method according to any one of 15.

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