DE19718652A1 - Liquid chromatography apparatus with improved separation - Google Patents

Abstract

This original apparatus improves separation in liquid chromatography. A novel feature is the continuous taper in the direction of flow, over the lower section of the separation column. Flow is accelerated here, compressing and stabilising the packing (matrix MX). There are connections at either end of the column, each with two openings, but which differ in construction. That on the inlet and distribution side has a large dead volume, expanding conically. In this, a sieve plate is mounted to allow movement, this being held against gravity by retaining springs during operation. The connection at the outlet side has a fixed sieve plate with funnel collecting eluate. This has an outlet, with at least one further opening at the upper edge, which can be closed.

Description

In chromatography, the separation efficiency is over defines the number of dividers. This is from the depending on various factors. she will for example by finer, more porous and Separation matrix particles of more homogeneous size improved.

The ratio of flow velocity to parti size and the binding strength of the separating matrix also influences the separation efficiency. High flow speeds lead to strong pressure waste in the separating tube, what happens with compressible matrices can lead to complete blockage.

Are all matrix-related parameters can be optimized it still comes to that before, during or after fractionation due to interference a renewed mixing comes that the achieved separation again nullifies. causes a channel formation in the matrix can be used for this ("chanelling") or dead spaces at the ends of the Separating tube, in the drain and in detectors be. Even those in a pipe are always closed observing curvature of the flow front has the same effect of material carryover, which is makes noticeable in the elution profile as "tailing".

To reduce these sources of error there was the most varied of approaches. So was to avoid it of "chanelling" the sizes of the Chromato graphical columns bounded and by an outer Compression stabilizes the matrix packing. A Overview of the various compression options columns as well as something about the curvature of the Flow front can be found in M. Perrut: J.Chrom. Ser. A 658 (1994) 293-313: Advances in supercritical fluid chromatographic processes.

A device to reduce "tailing" at curved elution front can be found in: DE G 93 17 551.5 (11/16/93). The background to this is in DE OS 44 40 805.6-41 (11/16/93).

So far, the approaches that attempted to compensate for the different flow through the edge zone and center have remained unmentioned in the case of separating pipes with large diameters:
DE OS 19 34 015 (15.11.70), DE OS 39 39 854 (13.06.90), EP OS 01 08 242 (29.09.83), DE 41 18 50 102 (03.06.91), DE OS 17 73 578 ( 06.06.68), DE OS 21 57 594 (20.11.72). The last-mentioned laid-open specification uses tube pieces of the same length as supply and discharge to the periphery and center. (This corresponds to part of the first approach.)

In addition to a uniform material flow distribution is the change in volume of the matrix during separation another major problem. To fix this For example, the order zone was disrupted as Stamp designed: DE OS 30 21 366 A1 (06.06.81).

Another compression column will be the packing of the separation matrix by back and forth Pushing a mandrel held constant.

For such compression columns is a complex one Regulation and mechanics necessary. Also kick with the high pressures on the moving parts Sealing problems.

We see the inventive task in this To fix disadvantages - or at least to mitigate.

These disadvantages are summarized again:

• - the uneven cross-section of the separating tube Flow,
• - the risk of channel formation in the matrix packing with volume changes,
• - the sealing that occurs with compression columns problems.

We want two solutions to this problem to introduce. Theirs is the compression by one accelerated material flow within the matrix pack together. The two solutions differ On the other hand, however, in sample application and in the Tracking of the sieve plate in the application zone.

In the first case, the same material flow branching system used and the sieve plate becomes the matrix surface pneumatically tracked.

This is a compression column, the forces of which are brought up within the steadily narrowing separating tube by the accelerated flow of material. The actual compression work is done by the liquid pump. The known type of punch compression is not used here - as is usually the case in compression columns - for compressing the matrix, but only for tracking and superficial pressing of the sieve plate in the short, cylindrical separating tube area of the application zone. This is to prevent the formation of a gap between the sieve plate and the matrix. Pneumatically, therefore, only a slightly higher pressure has to be applied than from the liquid pump. This is an essential difference to the compression columns described, in which the pressure must be sufficient to compress the matrix packing. The curvature of the elution front associated with the narrowing of the tube is negligible for most applications up to a diameter ratio of one to three. However, if the elution front is still to be equalized, there are again two solutions:

• 1. The use of domed chromatography floors as described in DE G 93 17 551.5 or
• 2. the use of closed cones or truncated cones to laminate the material flows as shown in FIGS . 7B and C. FIG.

Both equalization methods are also used if necessary applied to the following second solution.

In the second case, it is again one Compression column based on the principle of flow-related internal compression works. Here sample and elution buffer are over two different piping systems into one big one Dead space, for the layer structure outside the matrix, guided. In addition, one becomes the matrix surface stabilizing perforated and filter plate through their Intrinsically heavy, or with the reverse arrangement mechanical springs, pressed on. The feathers are like that heavily designed to take the weight of the matrix in Can hold position when the separating tube is turned over becomes. Unlike the first solution, they have to here not also the pressure of Counteract liquid pump because they are are together with the matrix in the separating tube. This eliminates the sealing problems of a conventional compression column.

The buffer feed ends at the top of one conical inner lumen behind a porous diffuser plate, which allows the mixing in the Counteracts dead space. Contrary to common chromato graphic practice is here in the order zone aimed for a large dead volume. In this However, it becomes similar to a trap Separating funnel used to build up layers. (All dings shows the dead volume compared to last rem a reverse arrangement.) Contrary to more common Opinion of chromatographic practice can so despite large dead volume an excellent separation can be achieved when it is guaranteed that in him mixing is prevented. Through the Layer build-up outside the separation matrix is over the entire cross-section has a uniform fabric ver division achieved. This makes a branched Zulei management system superfluous.

Usually the specifically heavier sample, the buffer already in the dead space, when the column outlet is initially closed and open overflow, layered. The order must be specifically heavier than the buffer for this - or it must be specifically made heavier. If this is not possible or not desired, the separating tube is turned over so that the order floats up and is below the separation matrix can collect.

If he has extremely sharply delimited application zones the zone of the first may be more dilute Order to be removed via the equalizing valve. This possibility exists with the first approach Not.

Both solutions described improve the separation in chromatographic processes and allow the operation of separation tubes with larger diameters than before. This has further advantages:

• 1. The separation matrix is used more intensively and one comparatively uses less matrix.
• 2. The construction of plants with larger diameters leads to lower production costs.
• 3. Larger diameters with the same filling volume and the same separation performance lead to less construction heights. This has further advantages:
• - at the same operating pressure and at the same Separation capacity shorten the separation times,
• - This will be used in automatic production more cutting cycles per time unit possible,
• - the separations can take place at lower pressure be performed. This represents lesser Requirements for the equipment and the pressure insensitivity of the separation matrix.

1st example Device for the preparative separation of ingredients from a solution or suspension that is specifically heavier than the elution buffer

Particularly advantageous applications for this example are the desalination of a protein solution and fractional crystallization. Fig. 2 shows the upper part, Fig. 6 shows the lower part of the separating tube. (A curved partition plate is not required up to a diameter ratio of one to three.)

Fig. 1 shows the flow scheme for its application.

2nd example

Device for the preparative separation of Ingredients from a compared to Elution buffer specifically lighter solution or Emulsion.

Compared to Example 1, the device must be modified. The direction of separation is now from bottom to top (direction differs from the illustration in FIG. 1). Correspondingly, the order end piece shown in FIG. 3 must also be used. Since gravity counteracts the direction of separation, another eluate drainage must be selected that no longer depends on gravity. A suitable end piece is shown in Fig. 5 and Fig. 11.

3rd example

For the same task as in example 1 or 2 you can also use preparative columns with a conical displacement body in the arrangement B or select C. These pillars then have a level Ground.

For this purpose, the upper part in FIG. 8 is combined with the lower part from FIG. 11 to form a separating tube. In the arrangement C, FIG. 11 shows the cross section through one side of the separating tube which is formed at the bottom.

4th example Device for analytical separation ( Fig. 4)

For the analytical separation is compared to the previous examples a much slimmer one Separating tube used. The elution front is through the inserted cone largely straightened.

5th example Separation column with external compression

According to the principle shown in FIG. 10 and FIG. 11, the division of the exit area into smaller areas, from which one can supply or discharge individually, not only preparative columns (as shown here) but also analytical columns (not shown here) can be constructed become. In such columns, because of the smaller diameter, you get by with a smaller number of material flows. A suitable arrangement is the application via an annular channel (according to Fig. 10-5) with the supply of 6 partial flows and the discharge via only one central funnel-shaped bore (according to Fig. 11-5).

The material flow diagram from FIG. 12 is used for this example.

6th example Laminarization of the material flow in conical Separation pipes by using conical Displacement bodies

Fig. 7 shows an overview of the way in which the flow of material can be laminarized within a conically narrowing separating tube (production of a parallel flow). In the diagram shown, the cross-section of such separation devices is shown to scale, the application areas of which relate to the elution area as 3 (F3) to 1 (F1). The easiest way to obtain the separating device shown in B is to work from the outset with a separating device that opens in an annular shape.

Of course, the same result is also obtained if the application zone shown in FIG. 2 with the outlet of FIG. 11 is used. However, one then has to replace the distributor plate with a plate with an integrated cone, as shown in FIG . As can be seen from the schematic representation of FIG. 7B, one cannot expect complete parallelization of the material flow with this separating device which tapers downward in the form of a ring. In the illustrated diameter ratios, however, the deviations are so small that they can be tolerated.

For other, more extreme diameter ratios, one can go two ways for a complete correction. In the first solution, a second downwardly conically pointed ring is inserted into the separating device. This means that the corrections shown in Fig. 7B and Fig. 7C used simultaneously in the same device (a cone comes from below and the other from above).

The second approach is to use ge curved dividing trays showing the elution front equalize. So you can use any Combinations of inserted cones and curved Use partitions. There are procedures for lead to the same goal.

List of reference symbols

Fig.

1
Flow chart for separate sample and buffer application

1

Valve to choose between sample (PE, a) and buffer (BR, b)

2

Pump for sample and buffer application

3

Valve: normal operation (a) or backwashing (b)

4th

Valve for selecting the application site in the separation column, separation matrix surface (a) or elution cone (b)

5

Valve to choose between buffer application (a) and opening of the pressure equalization / overflow (b)

7th

Shut-off valve at the end of the separating pipe, to (a), to (b)

8th

Valve for selecting the container for product (EL, b) or waste (WA, b)

Fig.

2
Application side for normal separation direction and separate application of sample and buffer

1

Separation matrix

2

Perforated distributor plate with high dead weight

3

conical separating tube

4th

conical lid

5

Sieve plate

6th

Diffuser: sieve plate to distribute the buffer

7th

Sample feed

8th

Ring seal with triangular cross-section

12th

Clamp seal

13th

cover

14th

Overflow or buffer feed

15th

Closure / stopper

16

O-ring

Fig.

3
Application side for any direction of separation with separate feed lines for sample and buffer

1

to

4th

;

6, 7

and

12th

to

16

are identical to

Fig.

2

5

Fiber fleece serves as a sieve and as a side seal

8th

Stainless steel compression spring

9

Stainless steel plates for positioning the springs

10

Perforated plate to absorb the spring pressure

11

Flange seal for pressure plate and cover

Fig.

4th
Compression column for analytical separations

1

Separation matrix

2

Perforated plate with integrated displacement body

3

Separation pipe

4th

Screw cap

5

Sieve plate

6th

Diffuser

7th

resilient cannula as sample feed line

8th

Pressure spring made of stainless steel

9

slightly curved sieve plate (curvature not essential)

10

Collecting funnel with thread

11

O-rings

12th

Ring seals with triangular cross-section

13th

Guide grooves that serve as a buffer feed line

14th

Buffer supply line

Fig.

5
Derivation from the curved perforated plate through a closed hose system for any direction of separation
(The funnel as a housing for building up a pneumatic counter pressure has been omitted for the sake of simplicity.)

1

curved perforated plate with conical bores and nipples for draining the eluate

2

Sieve plate made of non-rusting fiber fleece

3

conical separating tube

4th

Tube lumens of the leads are the same size

5

Conical bore

6th

Merging of the hose lines

Fig.

6th
Optional open material flow discharge on the arched chromatography base

1

curved perforated plate

2

wire mesh reinforced fiber fleece

3

strange separating pipe

4th

Drain wires

5

Collecting funnel

6th

Outlet

7th

lockable ventilation / washing opening

8th

Additional opening for buffer or eluate

9

Socket for probe

Fig.

7th
Schematic representation of streamlines
A in the conical separating tube
B in the conical separating tube after inserting a sinker with the tip pointing upwards
C in the conical separating tube after inserting a sinker with the tip pointing downwards
F1, F3 denote 1 and 3 surface units, respectively

Fig.

8th
Application side with separate supply of sample and buffer with integrated displacement cone
The names are identical to

Fig.

2, only the displacement cone (

2

) is also integrated into the perforated plate. In the normal direction of separation it is solid and in the reverse direction of separation it is hollow so that it floats.

Fig.

9
Order page for normal and reverse operation with branched inlet and outlet
The names are besides those of

7th

and

14th

With those in

Fig.

2 identical

7th

Pressure supply line

14th

Buffer and sample feed

Fig.

10
Order page for normal and reverse operation with branched inlet and outlet as well as displacement bodies

Fig.

11
The names are identical to

Fig.

9
Flat separating pipe end plate for use in any direction of flow (the funnel as a housing for building up a pneumatic counterpressure has been omitted for simplification.)

1

Perforated plate with conical holes

2

Displacement cone (optional)

3

conical separating tube

4th

Nipple for eluate drainage

5

Sieve plate

6th

Hose system for drainage

Fig.

12th
Flow diagram of the material flow in compression columns with a branched pipe system

1

Valve to choose between sample (PE, a) and buffer (BR, b)

2

Liquid transport pump

3

Valve for pneumatic compression (a) or relief (b)

4th

Pump for pneumatic pressure build-up

7th

Shut-off valve of the separating pipe, to (a), to (b)

8th

Valve to choose between product (EL, b) and waste (WA, a)

Claims (5)

1. A device for improving the separation in liquid chromatography is characterized in that the separation tube narrows steadily in the flow direction in the lower part. This accelerates the flow of material, which compresses and stabilizes the matrix packing.
At the ends of the separating tube there are two differently designed end pieces, each with at least two openings.
The one on the order side has a large dead volume, preferably ending in a cone. Inside there is a movably mounted sieve plate and, when operating against gravity, there are also retaining springs.
The one on the outlet side has a fixed sieve plate and a funnel for collecting the eluate with at least one outlet and at least one further closable opening on the upper edge of the funnel.
The separating matrix is held in position between the two sieve plates, the sieve plate on the inlet side being pressed against the matrix via a heavy perforated plate or, if the arrangement is reversed, additionally by springs.
The sample feed lines lead directly to the perforated plate. The buffer feed line opens into the dead space behind a diffuser screen plate at the outermost end of the application cone. 2. A device for improving the separation in liquid chromatography is characterized in that the separation tube narrows steadily in the flow direction in the lower part. This accelerates the flow of material, which compresses and stabilizes the matrix packing.
At the ends of the separating tube there are two differently designed end pieces, each with at least two openings.
On the application side there is a movably mounted sieve plate with an overlying perforated plate that seals in the periphery. Hose pieces of equal length lead to the holes in the plate as a supply line. These are combined to form a line before it leaves the end piece.
The outlet side has an immovable sieve plate and a discharge line designed like the supply line. It leaves the funnel through its lower outlet.
The separating matrix is held in position between the two sieve plates by applying the same external gas pressure to the second, not yet occupied, openings in the end pieces. 3. The apparatus of claims 1 and 2 is characterized in that a closed conical displacement body in the narrowing area of the separating tube used becomes to the curvature caused by the constriction to reduce the elution front. 4. The apparatus of claims 1 and 2 is characterized in that a curved screen plate for equalizing the elution front on the Aus aisle side is used. 5. The apparatus of claims 3 and 4 is characterized in that a combination of Displacement body and curved sieve bottom for Equalization of the elution front is used.