GB2132349A

GB2132349A - Reaction detector apparatus

Info

Publication number: GB2132349A
Application number: GB08334055A
Authority: GB
Inventors: Timothy D Schlablach
Original assignee: Varian Associates Inc
Current assignee: Varian Medical Systems Inc
Priority date: 1982-12-22
Filing date: 1983-12-21
Publication date: 1984-07-04
Also published as: GB8334055D0; DE3344649A1

Abstract

A post-column reaction apparatus and technique for use in liquid column chromatography systems is disclosed. This apparatus comprises a reaction vessel whose interior is traversed by a tubular semipermeable membrane whose lumen defines a flow path for the column effluent through the vessel. The space between the exterior of the membrane and the interior surface of the vessel wall contains a solution of a reagent that is capable of diffusing through the membrane and reacting with a desired component of the effluent. This apparatus and technique may be used to convert analytes in the effluent to detectable reaction products or to convert components in the effluent that interfere with detecting analyte into noninterfering reaction products. <IMAGE>

Description

SPECIFICATION Reaction detector apparatus Technical field The invention is in the field of chromatography.

More specifically, it is in the field of post-column reaction detectors that are used in liquid column chromatography systems.

Background art Chemicai reaction detection in liquid column chromatography is a well known technique for making samples more suitable for resolution or analysis. Post-column reactions chemically convert substances eluting from the column to improve the detectability of the analyte. The reaction occurs immediately after the separated sample leaves the column and prior to detection.

The object of post-column reactions is typically to increase the detection sensitivity of the chromatographically separated sample components or to enhance their sensitivity with respect to interfering bands that overlap bands of interest.

The reaction cells that are commonly used to carry out post-column reactions have been simple devices that consist basically of a segment of conduit that carries the column effluent and is intersected by another conduit that carries the reagent and is connected to one or more reagent reservoirs. The amount of reagent that is introduced into the column effluent is usually regulated by the delivery rate of a pump that pumps the reagent from the reservoir(s) into the effluent. One of the major disadvantages of such post-column reaction celis is the additional hardware in the form of pumps and valving that is required to introduce the reagent into the effluent at a suitable pressure and rate.

A principal object of the present invention is to provide a post-column reaction apparatus and technique that avoids the need for post-column pumps and intersecting conduits. Another object is to provide a post-column reaction cell that is easily replaced and in which reagent may be stored dry until just prior to use. These and other objects will be apparent from the following description of the invention.

Disclosure of the invention One aspect of the invention is an apparatus for used in analyzing the effluent from a liquid chromatography column for the presence of an analyte comprising: (a) a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semipermeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of the effluent through the vessel and the second compartment being defined by the space between the membrane and the wall of the vessel; and (b) a reagent contained in the second compartment that is capable of reacting with the analyte to form a detectable reaction product which, when in solution, will permeate through the semipermeable membrane into the first compartment and therein react with any analyte in the effluent flowing through the vessel to form said detectable reaction product.

A second aspect of this invention is a process for detecting an analyte in the effluent from a liquid chromatogrphy column comprising: (a) providing a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semipermeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of effluent through the vessel and the second compartment being defined by the space between the membrane and the vessel wall; (b) forming a solution of reagent in the second compartment in contact with the semipermeable membrane which reagent is capable of diffusing through the semi permeable membrane and reacting with the analyte to form a detectable reaction product;; (c) passing the effluent through the first compartment in contact with the semi permeable membrane whereby the reagent diffuses through the membrane into the first compartment and reacts with analyte in the effluent to form the detectable reaction product; and (d) detecing the detectable reaction product.

Other aspects of the invention are an apparatus and process that are used to reduce or eliminate components in the effluent that interfere with detecting analyte in the effluent. This apparatus and process are similar to those used to produce detectable reaction products except that the reagent does not react with analyte but instead reacts with the interfering component(s) to produce noninterfering reaction products. One embodiment of this aspect of the invention is a process for reducing background conductance in the effluent from an ion exchange column in which the reagent constitutes a first ionic species displacably bound to an insoluble matrix in the second compartment. This first ionic species exchanges with a second ionic species that is associated with the component(s) responsible for the background conductance to form nonconducting reaction products.

Brief description of the drawings In the drawings: Fig. 1 is a schematic block diagram showing a 'liquid chromatography system that includes a post-column reaction apparatus (optional components of the system are shown in phantom); Fig. 2 is an enlarged sectional view of one embodiment of the post-column reaction apparatus of the invention; and Fig. 3 is an enlarged sectional view of another embodiment of the post-column reaction apparatus of the invention.

Modes for carrying out the invention Fig. 1 shows a complete liquid chromatography system in which the postcolumn reaction apparatus is combined with conventional liquid chromatography (LC) separation and detecting equipment. The system that is illustrated is a high performance liquid chromatography (HPLC) system.It includes: mobile phase reservoirs 11 that contain an adequate supply of mobile phase for proportioning or gradient elution; a mobile phase delivery system 12 into which the reservoirs feed that pumps the mobile phase through the remainder of the system; a monitoring means 13 for delivering a variable amount of each reservoir simultaneously; a column 14 which is typically a stainless steel tube packed with a stationary phase; a sample injector 1 5 that is typically a rotary valve but could be an on-column syringe device; a reaction cell 16 in which the column effluent is reacted with a reagent; temperature control means 17, eg a bath, for controlling the reaction temperature; a detector 18 that detects one or more of the reaction products formed in cell 16; and a recorder or data system 19 that records or stores the output of the detector.

The mobile phase is a liquid that is a solvent for the sample to be separated. The chemical composition of the mobile phase will thus depend primarily on the nature of the sample. It must also be compatible with the stationary phase, with the reagent(s) for the reaction and the product(s), and inert with respect to the tubular membrane.

Mobile phases that inhibit the post-column reaction or lead to reagent or product precipitation should of course be avoided, as well as those phases that obscure detection of the reaction product. Most of the currently used postcolumn reactions require a single phase aqueous solvent system. Thus, water immiscible solvents such as heptane and ether should be avoided in such systems. In such systems the solvent may consist of just water or buffered solution or it may be a mixture of the above with water miscible organic solvents such as alcohols and acetonitrile.

Flow of the mobile phase through the system is effected by means of a mobile phase delivery system, commonly called a "pump". In HPLC systems considerabie pressure, typically up to 40 Mum~2, are required to overcome column resistance to flow. Constant mobile phase flow at such high pressures can be achieved with either reciprocating pumps or motirized syringe pumps.

The mobile phase delivery system introduces the mobile phase into the column at a suitable operating pressure. The sample is introduced simultaneously into the column by means of an injector, typically a rotary valve. Once injected onto the column, the sample mixes with the mobile phase and is carried into contact and interaction with the column packing. The packing is chemically derivitized with a bonded phase and is referred to as the stationary phase. The stationary phase interacts with sample components as they migrate through the column.

Differential migration of the sample components is achieved through ionic, hydrophobic, or exclusion mechanisms depending on the nature of the stationary phase. Sample components are thus physically separated and enter the reaction cell as individual bands separated by mobile phase.

The reaction cell provides for continuous addition of the reagent by steady state diffusion, followed by mixing and incubation under conditions that favor reaction between the reagent and any analyte(s) in the effluent. Its geometry is such as to substantially maintain the band separation in the effluent as the effluent passes through cell. Referring to Fig. 2 the reaction cell comprises a cylindrical vessel 22 whose interior is divided longitudinally by a tubular semipermeable electrically neutral membrane 23 into an inner compartment 24 defined by the lumen of membrane 23 and an outer concentric compartment 25 defined by the space between the inside of the vessel wall and the outside of the tubular semipermeable membrane. Although not shown in Fig. 2, compartment 25 could be subdivided into two or more compartments.The joints between the ends of the tubular membrane and the end walls of the vessel are fluid tight. The vessel has an inlet 26 in its one end wall that connects to a line 27 that leads to the column outlet and an outlet 28 in its other end wall that connects to a line 29 that leads to the detector. The inlet and outlet open into the lumen of the tubular membrane, providing a flow path for the column effluent through the vessel.

Compartment 25 contains a predetermined amount of a reagent 32 that is shown in dry, solid form in Fig. 2. In alternative embodiments the reagent may be in solution or displacably bound to an insoluble matrix or support. In the event that more than a single reagent is required the additional reagents, if compatible, could be present in compartment 25, or the additional reagents could be housed in separate subcompartments within compartment 25. The reagent becomes activated by the passage of mobile phase through the reactor cell. Mobile phase permeates through the semipermeable membrane and enters compartment 25. The composition of the mobile phase is such as to promote dissolution of the solid reagent and prepare it for use in the post-column reaction. If the reagent solubilization by the mobile phase is slow, it can be enhanced by injecting a more suitable solvent 33 into compartment 25 via a solvent injector 34 inserted through septum 35.

Alternatively, the reagent may be predissolved in an appropriate solvent and added to compartment 25 through the same solvent injector 34. The reagent is capable of reacting with the analyte or analytes in the column effluent to form a reaction product that is (are) detectable by the detector.

It also must be capable of diffusing through the semipermeable membrane in controlled amounts when in solution.

The rate, R, of reagent diffusion into the column effluent flowing through the tubular membrane can be approximated by using Fick's Law of diffusion and making the steady-state assumption resulting in the following equation: R=-DxC/L; where D is the diffusion coefficient of the reagent molecule, C is the reagent concentration, and L is the thickness of the semipermeable membrane. The amount, Q, of reagent entering a fluid element as it passes through the tubular membrane can be determined by the equation: Q=RxAxT; where A is the inner surface area of the membrane, and T is the residence time of a fluid element passing through the membrane.

By applying these equations to a typical set of reaction cell design parameters one may determine the typical mean concentration of reagent in the reaction chamber that may be achieved with the invention apparatus. With the following set of design parameters: L=0.05 cm, C=1 millimol/cc, D=1 x 10-5 cm2/sec, A=1 5.7 cm2, and F=1 5 sec Q is 4.71 x 10-2 millimol. The resulting mean concentration in such a reactor is then about 60 millimolar. Accordingly, the invention apparatus is useful for carrying out post-column reactions that may be done at reagent concentrations of this order.

Reactions requiring a substantial change in the pH of the fluid flowing through the reactor will not be readily feasible because high buffer concentrations are typically required to effect such a change. Often buffer concentrations of 0.5M or higher must be introduced to effect the appropriate pH change. These high concentrations are outside the normal operating range of this invention.

Assuming the reagent in compartment 25 is completely dissolved and that the reagent is in equilibrium with the fluid flowing through the cell and that the volume of the reagent compartment is 100 cc, then 50% of the reagent would be depleted after about 5 hours of operation, which would be adequate for at least several analyses.

The operation time can be directly increased by adding to the volume of the reagent compartment.

Although the mean reagent concentration inside the tubular reactor gradually decreases with time, the amount of product formed will not be affected as long as the reaction reaches completion. This requires at least a stoichiometric amount of reagent to be present. For reactions that do not achieve complete conversion of analyte, the amount of product formed may still be nearly constant, provided that the reagent concentration is several times the stoichiometric requirement. This can be readily deduced from the law of mass action. Because analyte concentrations are usually in the micromolar range when they elute from the chromatographic column, reagent concentrations in the millimolar range will generally satisfy the mass action requirement for a reproducible response.

For reactions that do require a fairly constant reagent concentration, then another aspect of this invention must be employed. A constant rate of reagent delivery to the membrane lumen requires a constant reagent concentration in the external environment (ie, in compartment 25). This can be achieved with a saturated solution of reagent that is in dynamic equilibrium with the effluent. The reagent solution in compartment 25 will remain saturated provided that dissolution of undissolved reagent in the compartment is fast relative to the rate of diffusion. This aspect of the invention will also be important in those applications where the reagent contributes significantly to the baseline offset of the detector. Thus, a constant baseline is dependent on a constant reagent concentration reaching the detector.

There are chromatographic limitations to efforts to maximize reagent flux by increasing the surface of the membrane or the residence time of the effluent in the reaction cell. As the chromatographically separated bands move through the cell they tend to merge. This phenomenon is called "bandbroadening" and is directly proportional to the tube radius and proportional to the square root of the tube length. In most embodiments the tubular radius will be limited to less than 0.03 cm, thus limiting the maximum surface area of the membrane. Surface area can be increased with minimal bandbroadening by increasing the tube length.

Practically the lengths are limited to less than 50 feet by the reactor design. For common mobile phase and reagent flow rates, the reactor residence time is typically less than 120 sec, and more often than not in the 20 to 50 sec range..

Thus, the mean concentration achievable with this device cannot be expected to exceed 250 millimolar in normal operation. Because the membrane will offer some resistance to diffusion, values will be somewhat lower in practice.

Because the membrane is permeable to small reagent molecules, it will also be permeable to small analytes passing through the tubular membrane. The diffusion can also be estimated from Fick's law assuming that steady state is obtained and that diffusion is everywhere radial so that the following equation may be used: Q=--2nx DxTx C/log(b/a) where b is the outer radius and a is the inner radius of the tube and the other parameters are as defined previously. Given the same typical parameter values as before except that C=10 nanomol/cc and b/a=2, then Q=-3 13 x 10-5 nanomol/cm. Assuming that the band width of a peak passing through the tubular membrane is 10 cm in the axial direction, then there will be about a 4% loss in analyte, assuming constant diffusion in one direction.Because the analyte will also diffuse back into the flowing stream, then analyte loss will be around 2% under conditions where the reagent concentration will be about 60 millimolar. If conditions are chosen to reduce the diffusing reagent concentration to 10 millimolar then the analyte loss will be around 0.3%, which is much more tolerable. When the molecular size of the analyte is substantially greater than the molecular size of the reagent it is preferable to select a membrane that is substantially impermeable to the analyze.

Fig. 3 illustrates an embodiment of the invention in which the reagent is displacably bound to an insoluble matrix, such as an ionic species bound to an ion exchange resin. This embodiment may be used to deionize the effluent from an ion chromatography column, The reaction cell shown in Fig. 3 comprises a vessel 36, one end of which has a removeable end cap 37. The interior of the vessel is traversed axialiy by a tubular semipermeable membrane 38 that divides the interior into two compartments 42, 43 as in the embodiment of Fig. 2. Compartment 42 is defined by the lumen of the membrane and provides a flow path for the column effluent.

Compartment 43 is packed with an ion exchange resin 44 (the matrix) that can readily exchange hydrogen ions (the reagent) for sodium or other cations. The resin may be present as a suspension or as a dry filtrate. The resin compartment can be opened for filling with fresh resin by removing the fitting 45 which holds it in place and unscrewing it from the end of the vessel. Such construction eliminates the need for a reagent refilling port.

In operation the resin is put into suspension in an appropriate fluid either by adding same to compartment 43 or by diffusion of mobile phase into compartment 43. Cations from the effluent diffuse through the semipermeable membrane and displace protons from the resin. The displaced protons diffuse from compartment 43 into compartment 42 and promote association of weak acids or bases in the mobile phase into nonconducting chemical species, thus lower the background conductance of the mobile phase.

Anion (analyte) exchange can be inhibited by Donnan exclusion by using an ion exchange resin of like charge to the anion.

The following are examples of post column reactions that may be carried out with the invention. These examples are not intended to limit the invention in any manner.

1. Indicator post-column reactions When acidic or basic compounds elute from a column in an unbuffered or weakly buffered solution, then the local pH will be strongly altered by the presence of the acidic or basic solute.

Carboxylic acids, for example, can be readily separated by HPLC. Conventional uv detection is not very sensitive owing to the poor molar absorbtivity of these compounds. Post-column detection with a pH indicator can be accomplished by adding a low concentration of indicator dye with a post-column reagent pump.

Such an indicator could be readily introduced with the invention by diffusion across the semi permeable membrane as previously described.

2. Dye-binding reactions.

Proteins are large polymers of amino acids that are generally greater than 10,000 in molecular weight. Semipermeable membranes are available that restrict the oufflow of proteins but permit the free diffusion of smaller molecules. Thus, proteins would not be lost with this invention but reagent could be added conveniently. One analytically useful property of proteins is their ability to selectively bond dyes. This complexation alters the absorbance spectrum of the dye and can be used to quantitate the amount of protein present.

Some dyes only bind to a very limited number of proteins. Bromocresol green is an example of a dye that binds selectively to serum albumin but not other proteins. This reagent could readily be added to the column effluent according to the present invention. Other dyes bind to a wide variety of proteins and can be used to quantitate many proteins. Coomassie blue is used to determined protein concentrations in batch assay.

The reaction does require that the effluent be acidified but this can be accomplished with stoichiometric quantities of acid for this reaction and not large excess. Typical buffer concentrations for protein separations are around 20 millimolar which are within the limits of the invention. Furthermore, because most dyes are not readily soluble in aqueous systems, precipitates form that must be filtered away to prevent clogging, but the natural filtering action of the semipermeable membrane easily prevents such filtrates from entering the post-column reaction system.

3. Enzyme reactions Enzymes are proteins that catalyze chemical reactions. They efficiently convert substrate(s) to product(s), and the rate of this reaction is the means used to quantitate the amount of enzyme present. Some enzymes have a number of structurally related forms that ali catalyze the same reaction. They are known as isoenzymes and are important in clinical diagnosis and genetic linkage. Such isoenzymes can be separated by HPLC and quantitated with an in-line post-column reaction. The substrate concentrations used in the post-column reaction are typically less than 100 millimolar. The substrates are also unstable and often must be stored in a powder form, and the reagent prepared fresh daily. This invention accommodates both the storage requirements and the substrate requirements for post-column enzyme assays. Its use will make post-columnreaction detection of enzymes more convenient because it eliminates the post-column reagent pump(s) and the tedium of reagent preparation.

4. Deionization The deionization of the column effluent is particularly important in ion-chromatography where the sample ions separated by the chromatographic column must be measured against the background of mobile phase ions. The mobile phase counter ions are typically weak acids or bases, whose association into the unionized form can be promoted by a change in pH. This can be achieved with a column packed with an ion-exchange resin that readily exchanges protons for sodium or other cations. The protons promote association of weak acids into nonconducting species, and thus lower the background. Donnan exclusion allows the resin to be placed on the other side of a semipermeable membrane. The counter cations diffuse freely across the membrane and displace resin bound hydrogen ions.Sample anions of interest such as chloride or nitratc tend not to diffuse into the resin compartment because of the fixed negative charge of the resin. Thus, the appropriate ionexchange resin can be simply placed in the reagent compartment for use in ionchromatography. The use of this invention eliminates direct contact of the sample with ion exchange resin.

Although not shown in Figs. 2 and 3 temperature and temperature control play an important part in many post-column reactions.

Temperature increases the reaction rate and thus the amount of product formed during the residence time in the reactor. Not only does temperature increase the reaction rate but it also lowers viscosity and thus enhances the diffusion coefficient and improves mixing. Thus, temperature elevation is the simplest means for improving the performance of the invention apparatus. For consistent performance the temperature should be held constant. A regulated bath may be used to accurately control the temperature.

Once the reaction is complete the effluent passes from the reaction cell into the detector.

The nature of the detector will depend upon the nature of the product of the post-column reaction or the analyte. Most products can be adequately detected with a photometric detector, either absorbance, fluorescence or luminescence.

Electroactive products can best be detected with an electrochemical detector. When the invention is used in ion chromatography a conductivity detector will be used most often although a refractive index detector could be used in some applications.

Modifications of the above described modes for carrying out the invention that are obvious to those of ordinary skill in the field of chromatography, analytical chemistry, or related fields are intended to be within the scope of the following claims.

Claims

1. An apparatus for use in analyzing an effluent from a liquid chromatography column for the presence of an analyte comprising: (a) a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semi permeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of the effluent through the vessel and the second compartment being defined by the space between the membrane and the wall of the vessel; and (b) a reagent contained in the second compartment that is capable of reacting with the analyte to form a detectable reaction product and which, when in solution, will diffuse through the semipermeable membrane into the first compartment and therein react with any analyte in the effluent flowing through the vessel to form said detectable product.

2. The apparatus of claim 1 wherein the membrane is substantially impermeable to the analyte.

3. The apparatus of claim 1 wherein the reagent contained in the second compartment is in the form of a solid that is soluble in the mobile phase of the effluent.

4. The apparatus of claim 3 wherein the rate of dissolution of the reagent in the mobile phase is fast relative to the rate of diffusion of the reagent through the membrane.

5. The apparatus of claim 1 wherein the reagent contained in the second compartment is displaceably bound to an insoluble matrix.

6. The apparatus of claim 1 wherein the reagent contained in the second compartment is in both a dissolved and undissolved form constituting a saturated solution that is in dynamic equilibrium with the effluent flowing through the lumen.

7. The apparatus of claim 1 wherein (i) the analyte is a carboxylic acid and the reagent is a dye indicator; or (ii) the analyte is a protein and the reagent is a dye that binds to the protein; or (iii) the analyte is an enzyme and the reagent is a substrate on which the enzyme acts.

8. A method of detecting an analyte in an effluent from a liquid chromatography column: (a) providing a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semipermeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of effluent through the vessel and the second compartment being defined by the space between the membrane and the wall of the vessel; and (b) forming a solution of a reagent in the second compartment in contact with the semipermeabie membrane which reagent is capable of diffusing through the semi permeable membrane and reacting with the analyte to form a detectable reaction product; (c) passing the effluent through the first compartment in contact with the semi permeable membrane whereby the reagent diffuses through the membrane into the first compartment and reacts with analyte in the effluent to form the detectable reaction product; and (d) detecting the detectable reaction product.

9. The method of claim 8 wherein the solution of reagent is formed by the mobile phase of the effluent diffusing through the membrane into the second compartment and dissolving the reagent.

10. The method of claim 9 wherein the rate of dissolution of the reagent is fast relative to the rate of diffusion of the reagent through the membrane.

11. The method of claim 8 wherein the reagent is an ionic species that is displacably bound to an ion exchange material.

1 2. The method of claim 8 wherein (i) the analyte is a carboxylic acid and the reagent is a dye indicator; or (ii) the analyte is a protein and the reagent is a dye that binds to the protein; or (iii) the analyte is an enzyme and the reagent is a substrate on which the enzyme acts.

13. An apparatus for use in eliminating or reducing the amount of a component of an effluent from a liquid chromatography column that interferes with detecting the presence of an analyte in the effluent comprising: (a) a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semipermeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of the effluent through the vessel and the second compartment being defined by the space between the membrane and the wall of the vessel; and (b) a reagent contained in the second compartment that is capable of reacting with the component to thereby convert the component into a noninterfering product and which, when in solution, will diffuse through the semipermeable membrane into the first compartment and therein react with said component.

14. The apparatus of claim 13 wherein the reagent is an ionic species that is displacably bound to an ion exchange resin.

1 5. A method of eliminating or reducing the amount of a mobile phase component of an effluent from a liquid chromatography column that interferes with detecting the presence of an analyte in the effluent comprising: (a) providing a reaction vessel, the interior of which is divided into a first compartment and a second separate compartment by a tubular semipermeable membrane that traverses the vessel's interior, the first compartment being defined by the lumen of the membrane and providing a passageway for the flow of effluent through the vessel and which, when in solution, will diffuse through the semi permeable membrane into the first compartment and therein react with the component; and (b) forming a solution of a reagent in the second compartment in contact with the semipermeable membrane which reagent is capable of diffusing through the semi permeable membrane and reacting with the component to convert the component to a non interfering reaction product; and (c) passing the effluent through the first compartment in contact with the semi permeable membrane whereby the reagent diffuses through the membrane into the first compartment and reacts with the component to form the noninterfering product.

1 6. The method of claim 1 5 wherein the reagent is an ionic species that is displacably bound to an ion exchange material and said solution is formed by another ionic species diffusing from the effluent through the semipermeable membrane into the second compartment and displacing said ionic species.

1 7. The method of claim 1 6 wherein the ion exchange material and the analyte have like charges whereby the diffusion of analyte into the second compartment is inhibited by Donnan exclusion.

1 8. Liquid chromatography apparatus substantially as hereinbefore described with reference to and as illustrated in Figure 2 or Figure 3 of the accompanying drawings.

19. A liquid chromatography method substantially as hereinbefore described with reference to the accompanying drawings.