CN103869021B - Based on high-efficiency liquid chromatography method for detecting and the device of Surface enhanced raman spectroscopy - Google Patents
Based on high-efficiency liquid chromatography method for detecting and the device of Surface enhanced raman spectroscopy Download PDFInfo
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of high-efficiency liquid chromatography method for detecting based on Surface enhanced raman spectroscopy and device.Be connected with the entrance point of detector tube by pipeline in the sample outflow port of high performance liquid chromatograph, the endpiece of detector tube is connected with the sample inlet end of Raman spectrometer by pipeline; Described detector tube is the glass tube that golden nanometer particle is modified, and is applied to liquid chromatography-Surface enhanced raman spectroscopy coupling and detects.When detecting detected sample, the present invention adopts the detector tube prepared by self-assembly as a kind of high performance liquid chromatography detector means, effectively can be separated on the one hand by high performance liquid chromatography to multi-component material; The deficiency that high performance liquid chromatography cannot differentiate molecular structure can be overcome again on the other hand, Surface enhanced raman spectroscopy identification and concentration determination be carried out to isolated compound, a kind of novel detection device being applicable to high performance liquid chromatography is provided.
Description
Technical field
The invention belongs to the field of on-line analysis detection technique, be specifically related to a kind of high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy.
Background technology
Nineteen sixty, in order to the macromolecular substances that isolated protein, nucleic acid etc. not easily gasify, the Theories and methods of gas chromatography is reintroduced into classical liquid chromatography.1971, the people such as section's Crane (Kirkland) published " modern practice of liquid chromatography " book, indicate that high performance liquid chromatography (HPLC) is formally set up.
Through the development of decades, because high performance liquid chromatography has higher separation efficiency, well selectivity, higher detection sensitivity and wide range of application, chemistry and the indispensable analysis testing tool of association area are become.Compared with vapor-phase chromatography, high performance liquid chromatography is the volatility of test sample and thermal stability restriction not, applied range; Mobile phase kind is many, and the optimization by mobile phase reaches high separation efficiency; Generally at room temperature analyze, do not need high column temperature, thus be applicable to the compound analyzing higher boiling, large molecule, strong polarity, poor heat stability.
Although high performance liquid chromatography has above plurality of advantages, but inevitably still come with some shortcomings, as differentiated the design feature of different compound, multiple different compound may have identical retention time etc., cause scientists thus to study the expansion of high performance liquid chromatography detecting device, High Performance Liquid Chromatography/Mass Spectrometry (HPLC-MS) coupling technique such as generally applied at present, its core is for high performance liquid chromatography provides follow-up compound structure to differentiate means.
Surface enhanced raman spectroscopy (SERS), as one of the sensitiveest detection technique, receives publicity (see document: Nature Biotechnology 26,83 – 90 (2008)) in various fields.The key realizing SERS high-sensitivity detection is the surface reinforced Raman active substrate that preparation enhancing ability is strong, stability is good, sensitive, stable SERS signal can be obtained on the one hand, expand the range of application of SERS, the foundation (see document: Physical Review E 62,4318 – 4324 (2000)) of quantitative measurement can also be established on the other hand.And the key in strengthening as SERS, the pattern feature of nano particle and arrangement thereof determine the quality of strengthening the property.In the method for common structure nano-device, have a kind of efficient, fast and the nano particle assemble method of " from bottom to top " of low cost, i.e. the self assembly of nano particle.Its cardinal principle is by the effect with bifunctional molecules, makes its one end and substrate compact siro spinning technology, and the functional group of the other end can produce chemistry or electrostatic force with nanoparticle surface, thus forms one deck nano particle coverlay at substrate surface.By the two dimension of nano particle or the package assembly of three-dimensional, the even and stable SERS substrate on definite meaning can be obtained.
Therefore, research is needed to strengthen the preparation of substrate by special surface, thering is provided a kind of can realize the New-type detector of Surface enhanced raman spectroscopy as high performance liquid chromatography, thus under simple experiment condition, complete the recognition function to efficient liquid phase efflux Middle molecule, to meet high performance liquid chromatography, there is range of application widely.
Summary of the invention
Problem to be solved by this invention overcomes existing high performance liquid chromatography to detect in the deficiency to existence in compound structure discriminating, a kind of eigen vibration signal that can obtain molecule contained by some is provided, reaches the high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy differentiating molecular structure function.
To achieve the above object of the invention, the technical solution used in the present invention is to provide a kind of high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy, and it comprises high performance liquid chromatograph, Raman spectrometer; Be connected with the entrance point of detector tube by pipeline in the sample outflow port of high performance liquid chromatograph, the endpiece of detector tube is connected with the sample inlet end of Raman spectrometer by pipeline; Described detector tube is the glass tube that golden nanometer particle is modified, and preparation method is: the glass tube after washing is placed in H
20, NH
4oH and H
2o
2mixed solution in, by volume, H
20:NH
4oH:H
2o
2for 5:1:1, be activation process 0.5 ~ 1 hour under the water bath condition of 60 ~ 80 DEG C in temperature, obtain the glass tube of coupling aminopropyl trimethoxysilane molecule; Glass tube is placed in aurosol solution to soak 10 ~ 12 hours, golden nanometer particle is fixed on glass tube inwall again, obtains Surface enhanced raman spectroscopy detector tube through cleaning; The mean diameter of described aurosol GOLD FROM PLATING SOLUTION particle is 45 ~ 65nm.
A preferred version of the present invention is the internal diameter of described glass tube is 0.91 ~ 1mm, and wall thickness is 0.1 ~ 0.15mm, and pipe range is 80 ~ 120mm.
Technical solution of the present invention also comprises carries out with said apparatus the method that detects, comprises the steps:
1, detected sample enters chromatographic column through the injector of high performance liquid chromatography, and mobile phase is continued through chromatographic column by the adjustment through pump, and wash-out is adsorbed in the solute molecule in chromatographic column;
2, the eluent obtained enters the UV-detector that high performance liquid chromatography is equipped with, and detects the uv absorption signal of efflux in real time;
3, the measuring samples that high performance liquid chromatograph outflow port flows out is entered Raman spectrometer after the detector tube that inwall fixes golden nanometer particle, strengthening Raman shift and the intensity of vibration peak in spectrogram by detecting compound surface to be checked, obtaining structure and the concentration of compound to be detected.
The present invention adopts H by volume
20:NH
4oH:H
2o
2the mixed solution of=5:1:1 carries out activation process to detector tube, detector tube inwall coupling aminopropyl trimethoxysilane (APTMS) molecule, makes the exposed a large amount of hydroxyl of inside pipe wall, utilizes the chemical interactions of silicon in hydroxyl and APTMS, fixing one deck APTMS molecule; The acting force that golden nanometer particle is fixed is chemical bond power, by effect that is amino in APTMS and gold, golden nanometer particle is fixed on glass detector tube inwall, surface reinforced Raman active substrate detector tube is connected with high performance liquid chromatograph, and the Raman spectrometer that is coupled, chromatographic resolution can be realized and spectral detection is carried out in real time.
Principle of the present invention is: utilize the detector tube being assembled with golden nanometer particle, can by the efflux after being separated in high performance liquid chromatography by this detector tube, the efflux being controlled different retention time by valving flows through the detector tube being assembled with golden nanometer particle separately, if the substance be separated is adsorbed on golden nanometer particle surface and reaches certain concentration, just can be obtained some chemical bond oscillations spectrum peak of this molecule by Surface enhanced raman spectroscopy, thus the Structure identification of compound is played a role; Read according to the change of spectral intensity the concentration flowing out species simultaneously.The surface reinforced Raman active substrate that the present invention is prepared by self-assembly is as a kind of high performance liquid chromatography detecting device, solve the deficiency that high performance liquid chromatography cannot differentiate molecular structure, realize identifying to some characteristic oscillation modes of institute's separating compound, thus make high performance liquid chromatography and these two kinds of technology of Surface enhanced raman spectroscopy complement each other, effectively combine.
Due to the employing of technique scheme, compared with prior art, tool of the present invention has the following advantages:
1, adopt the method for Chemical self-assembly, golden nanometer particle is assembled into glass detector tube inwall, obtains enhancing substrate in a big way, and the effect of on-line checkingi can be realized.
2, high performance liquid chromatography is effectively combined with Surface enhanced raman spectroscopy by the present invention, high performance liquid chromatography is first adopted to be separated potpourri, recycling Surface enhanced raman spectroscopy identifies the eigen vibration of compound in liquid phase stream fluid, realize the mensuration of structure and concentration, be compared to UV-detector simultaneously, use Surface enhanced raman spectroscopy can realize the detection of the different molecular with identical retention time, and the detectability of Surface enhanced raman spectroscopy is lower than other detection techniques, thus actual meaning is played to the analysis of some low concentration compound.
3, pick-up unit provided by the invention can utilize the existing checkout equipment in laboratory, and detection method is simple, mild condition, is conducive to applying.
Accompanying drawing explanation
The structural representation of the pick-up unit that Fig. 1 provides for the embodiment of the present invention;
The mean grain size that the reduction of sodium citrate method that Fig. 2 provides for the embodiment of the present invention prepares is about the TEM figure of the golden nanometer particle of 55 nm;
Fig. 3 assembles the variation diagram in kind of glass detector tube before and after golden nanometer particle in the embodiment of the present invention; Wherein, figure a is the glass tube before assembling Au nano particle, and figure b is the glass detector tube after assembling Au nano particle;
The SEM figure that be assembled with golden nanometer particle glass detector tube of Fig. 4 prepared by the embodiment of the present invention;
Fig. 5 is assembled with the surface increasing Raman spectrum figure of golden nanometer particle glass detector tube for different probe molecule prepared by the embodiment of the present invention; Wherein, a is to sulfydryl benzenethiol, and b is 4,4-dipyridine;
Fig. 6 is that the embodiment of the present invention adopts high performance liquid chromatography to be separated 4,4-dipyridine and the uv absorption result collection of illustrative plates to sulfydryl benzenethiol;
Fig. 7 is that the glass detector tube being assembled with golden nanometer particle prepared by the embodiment of the present invention applies to high performance liquid chromatography separation 4,4-dipyridine and detects collection of illustrative plates to the SERS of sulfydryl benzenethiol efflux;
Fig. 8 is that the embodiment of the present invention adopts high performance liquid chromatography to be separated the uv absorption result collection of illustrative plates of 4,4-dipyridine and p-aminophenyl thiophenol;
The glass detector tube that be assembled with golden nanometer particle of Fig. 9 prepared by the embodiment of the present invention applies to the SERS detection collection of illustrative plates that high performance liquid chromatography is separated 4,4-dipyridine and p-aminophenyl thiophenol efflux.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
See accompanying drawing 1, it is the structural representation of a kind of high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy provided by the invention.The sample introduction solution of high performance liquid chromatography enters chromatographic column via the injector of high performance liquid chromatography, afterwards, mobile phase continue through chromatographic column by the adjustment of pump and the solute molecule making to be adsorbed in chromatographic column along with the flowing of mobile phase by wash-out, afterwards, eluent enters the UV-detector that high performance liquid chromatography is equipped with, the uv absorption signal of real-time detection efflux, enter the glass detector tube be positioned in Raman detection platform again via polytetrafluoro connecting pipe, detect the surface-enhanced Raman signals of solute molecule in real time.
Utilize above-mentioned detection device to detect, its step is as follows:
(1) in 100 ml three-neck flasks, add the chlorauric acid solution that 100 ml concentration are 25 mM, solution is faint yellow; This solution is heated to boil, and under vigorous stirring, adds the sodium citrate solution that 0.75ml concentration is 0.01 g/ml.Now solution changes black into by faint yellow gradually, then becomes the aubergine of the slightly colour of loess from black, and whole color change process is 1 ~ 3 min.Become aubergine with solution and start timing, keep system fluidized state 15 min under stirring condition, naturally cool to room temperature subsequently.See accompanying drawing 2, it is the TEM figure of the golden nanometer particle that the present embodiment adopts reduction of sodium citrate legal system standby, adopts Dutch FEI Co. TEcnaiG20 type high resolution transmission electron microscopy, as seen from Figure 2, golden nanometer particle is spherical, uniform particle sizes, and monodispersity is good.
(2) get simple glass pipe, in ultrapure water after cleaning in ultrasonic ten minutes, use H
20:NH
4oH:H
2o
2=5:1:1 mixed solution, 60 ~ 80 DEG C of water-bath activation 0.5 ~ 1 hour, is immersed in after washing in APTMS solution and keeps spending the night, and cleans rear 120 DEG C of oven 0.5 ~ 1 hour.
(3) get step (1) in the 55nm aurosol centrifugal concentrating of preparation, remove supernatant liquor, by step (2) in be full of in prepared glass tube concentrated after aurosol, keep about 10 ~ 12 hours.
By step (3) in liquid sucking-off in the glass tube for preparing, ultrapure water cleaning glass tube 2 ~ 3 times, just obtains the glass tube substrate that inwall is assembled with Au nano particle.The glass tube prepared is kept in ultrapure water.See accompanying drawing 3, it changes pictorial diagram before and after glass tube assembling golden nanometer particle in this step, and wherein, a figure is the glass tube before assembling, and in water white transparency, b figure is the glass tube after assembling, in claret, as detector tube.
(5) utilize high performance liquid chromatography to be separated 4,4-dipyridine and to sulfydryl benzenethiol mixed solution, mobile phase adopts 50% acetonitrile and 50% water, flow velocity 1mL/min.
the glass detector tube adopting step (4) to prepare receives the efflux in high performance liquid chromatography, the efflux of different time sections is carried out to the sign of Surface enhanced raman spectroscopy, when in UV detect result 4, after 4-dipyridine flows out completely from high performance liquid chromatography, valve is converted to another interface, the efflux after making it flows through another glass detector tube being assembled with golden nanometer particle and detects.
Characterize the high-efficient liquid phase color spectrum detection device character based on Surface enhanced raman spectroscopy of above-mentioned preparation, result is as follows:
See accompanying drawing 4, it is the SEM figure being assembled with the glass detector tube of golden nanometer particle prepared by the present embodiment; Adopt the Hitachi S-4700N scanning electron microscope that Japanese Hitachi company produces, as shown in Figure 4, glass detector tube inwall gold nanometer particle grain size about 55 nm, favorable dispersibility, there is multi-Layer Phenomenon in subregion.
See accompanying drawing 5, it is the surface enhanced performance study being assembled with the glass detector tube of golden nanometer particle prepared by the present embodiment; Adopt the HR800 type Raman spectrometer of French Jobin Yvon company; The enhancing signal of test different probe molecule.As shown in Figure 5, signal is strengthened good.Wherein, a figure is 10
-3mol/L to sulfydryl benzenethiol, b figure is 10
-34, the 4-dipyridines of mol/L.
See accompanying drawing 6, it is 4,4-dipyridines and the high performance liquid chromatography Separation Research to sulfydryl benzenethiol mixed solution in the present embodiment; Adopt 1200 type high performance liquid chromatographs of Agilent company of the U.S.; Detecting device adopts UV-detector, and mobile phase is 50% acetonitrile and 50% water, be separated same concentrations 4,4-dipyridines and to sulfydryl benzenethiol solution, as shown in Figure 6, the corresponding two kinds of different materials in the absorption peak position of uv absorption spectrogram.
See accompanying drawing 7, it is the Surface enhanced raman spectroscopy research of glass detector tube for high performance liquid chromatography efflux being assembled with golden nanometer particle prepared by the present embodiment; Adopt the HR800 type Raman spectrometer of French Jobin Yvon company; As shown in Figure 7, corresponding to the retention time of ultraviolet absorption peak, the surface enhanced Raman spectroscopic signals of corresponding molecule can be detected.
Embodiment 2
The present embodiment carries out detecting based on the high performance liquid chromatography of Surface enhanced raman spectroscopy to sample, and as described in Example 1, detecting step is as follows for pick-up unit:
(1) in 100 ml three-neck flasks, add the chlorauric acid solution that 100 ml concentration are 25 mM, solution is faint yellow; This solution is heated to boil, and under vigorous stirring, adds the sodium citrate solution that 0.75ml concentration is 0.01 g/ml.Now solution changes black into by faint yellow gradually, then becomes the aubergine of the slightly colour of loess from black, and whole color change process is 1 ~ 3 min.Become aubergine with solution and start timing, keep system fluidized state 15 min under stirring condition, naturally cool to room temperature subsequently.
(2) get simple glass pipe, in ultrapure water after cleaning in ultrasonic ten minutes, use H
20:NH
4oH:H
2o
2=5:1:1 mixed solution, 60 ~ 80 DEG C of water-bath activation 0.5 ~ 1 hour, is immersed in after washing in APTMS solution and keeps spending the night, and cleans rear 120 DEG C of oven 0.5 ~ 1 hour.
(3) get step (1) in the 55nm aurosol centrifugal concentrating of preparation, remove supernatant liquor, utilize capillarity by step (2) in be full of in prepared glass tube concentrated after aurosol, keep about 10 ~ 12 hours.
By step (3) in liquid sucking-off in the glass tube for preparing, ultrapure water cleaning glass tube 2 ~ 3 times, just obtains the glass detector tube substrate that inwall is assembled with Au nano particle.The glass detector tube prepared is kept in ultrapure water.
(5) utilize high performance liquid chromatography to be separated 4,4-dipyridine and p-aminophenyl thiophenol mixed solution, mobile phase adopts 30% acetonitrile and 70% water, flow velocity 1mL/min.
the glass detector tube adopting step (4) to prepare receives the efflux in high performance liquid chromatography, the efflux of different time sections is carried out to the sign of Surface enhanced raman spectroscopy, when in UV detect result 4, after 4-dipyridine flows out completely from high performance liquid chromatography, threeway is converted to another interface, the efflux after making it flows through another glass detector tube being assembled with golden nanometer particle and detects.
See accompanying drawing 8, it is the high performance liquid chromatography separating resulting of 4,4-dipyridines and p-aminophenyl thiophenol mixed solution in the present embodiment; Adopt 1200 type high performance liquid chromatographs of Agilent company of the U.S.; Detecting device adopts UV-detector, and mobile phase is 30% acetonitrile and 70% water, is separated 4,4-dipyridines and the p-aminophenyl thiophenol solution of same concentrations, as shown in Figure 8, and the corresponding two kinds of different materials in absorption peak position of uv absorption spectrogram.
See accompanying drawing 9, it is the Surface enhanced raman spectroscopy research of glass detector tube for high performance liquid chromatography efflux being assembled with golden nanometer particle prepared by this example; Adopt the HR800 type Raman spectrometer of French Jobin Yvon company; As shown in Figure 9, corresponding to the retention time of ultraviolet absorption peak, the surface enhanced Raman spectroscopic signals of corresponding molecule can be detected.
Claims (3)
1. the high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy, it comprises high performance liquid chromatograph, Raman spectrometer, it is characterized in that: the sample outflow port of high performance liquid chromatograph is connected with the entrance point of detector tube by pipeline, and detector tube is positioned in the detection platform of Raman spectrometer; Described detector tube is the glass tube that golden nanometer particle is modified, and preparation method is: the glass tube after washing is placed in H
20, NH
4oH and H
2o
2mixed solution in, by volume, H
20:NH
4oH:H
2o
2for 5:1:1, be activation process 0.5 ~ 1 hour under the water bath condition of 60 ~ 80 DEG C in temperature, be immersed in after washing in APTMS solution and keep spending the night, obtain the glass tube of coupling aminopropyl trimethoxysilane molecule; Glass tube is placed in aurosol solution to soak 10 ~ 12 hours, golden nanometer particle is fixed on glass tube inwall again, obtains Surface enhanced raman spectroscopy detector tube through cleaning; The mean diameter of described aurosol GOLD FROM PLATING SOLUTION particle is 45 ~ 65nm.
2. a kind of high performance liquid chromatography detecting device based on Surface enhanced raman spectroscopy according to claim 1, is characterized in that: the internal diameter of described glass tube is 0.91 ~ 1mm, and wall thickness is 0.1 ~ 0.15mm, and pipe range is 80 ~ 120mm.
3. carry out the method detected with the high-efficient liquid phase color spectrum detection device based on Surface enhanced raman spectroscopy according to claim 1, it is characterized in that comprising the steps:
(1) detected sample enters chromatographic column through the injector of high performance liquid chromatography, and mobile phase is continued through chromatographic column by the adjustment through pump, and wash-out is adsorbed in the solute molecule in chromatographic column;
(2) eluent obtained enters the UV-detector that high performance liquid chromatography is equipped with, and detects the uv absorption signal of efflux in real time;
(3) enter via connecting pipe the detector tube that the inwall be positioned on Raman spectrometer monitor station fixes golden nanometer particle again, strengthening Raman shift and the intensity of vibration peak in spectrogram by detecting compound surface to be checked, obtaining structure and the concentration of compound to be detected.
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| CN107655873A (en) * | 2016-07-25 | 2018-02-02 | 岛津分析技术研发（上海）有限公司 | Raman spectrum analysis device and method |
| CN109342602B (en) * | 2018-11-21 | 2024-07-16 | 中国计量大学 | Aflatoxin detection device in liquid |
| CN114923996B (en) * | 2022-04-27 | 2024-08-20 | 苏州大学 | Automatic HPLC-SERS (high performance liquid chromatography-surface enhanced Raman scattering) combined device and detection method based on paper substrate |
| CN116165188B (en) * | 2022-12-12 | 2024-02-09 | 苏州大学 | Automatic HPLC-SERS combined system based on liquid phase substrate and application thereof |
| CN117589687B (en) * | 2024-01-18 | 2024-04-09 | 成都艾立本科技有限公司 | Optical cavity container based on air-wrapped liquid, application and spectrum detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128908A (en) * | 2010-01-19 | 2011-07-20 | 华东理工大学 | On-site quick detection method for organic pollutants in water |
| CN103398995A (en) * | 2012-09-07 | 2013-11-20 | 欧普图斯(苏州)光学纳米科技有限公司 | Integrated equipment capable of realizing chemical separation and light dispersion |
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2014
- 2014-03-28 CN CN201410121804.7A patent/CN103869021B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128908A (en) * | 2010-01-19 | 2011-07-20 | 华东理工大学 | On-site quick detection method for organic pollutants in water |
| CN103398995A (en) * | 2012-09-07 | 2013-11-20 | 欧普图斯(苏州)光学纳米科技有限公司 | Integrated equipment capable of realizing chemical separation and light dispersion |
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| Identification of illicit drugs by a combination of liquid chromatography and surface-enhanced Raman scattering spectroscopy;Bernd Sagmuller et al.;《Journal of Molecular Structure》;20061216;第661-662卷;279-290 * |
| Quantification of DNT isomers by capillary liquid chromatography using at-line SERS detection or multivariate analysis of SERS spectra of DNT isomer mixtures;Bernhard Zachhuber et al.;《J. Raman Spectrosc.》;20120803;第43卷;998-1002 * |
| 干净、均一的表面增强拉曼基底的制备和表征;徐燕慧 等;《光散射学报》;20091231;第21卷(第4期);295-299 * |
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