GB2250691A - Apparatus for low pressure liquid chromatography - Google Patents

Abstract

Apparatus for low pressure liquid chromatography which is particularly suitable for the testing of body fluids and in particular blood, comprising means for passing a sample through a chromatography column 4 containing a packing material so that a constituent of the sample is retained by the packing material and passing a buffer solution having a varying concentration through the column. When the concentration of the buffer solution reaches a particular level, the constituent will be disassociated from the packing material and flows out of the column through a detector. The detector measures the light absorption of the fluid and by relating the time varying light absorption of the fluid to the time varying concentration of the buffer solution it is possible to identify the constituent. The column is releasably held between two mountings 43, 45 at least one of which is movable between a position allow insertion and removal of the column and a position in which the column is held squeezed between the mountings. Mounting 45 is movable by means of cam 48. When the column is held tight, the mountings are fluid sealed to it by seats e.g. 46, 46A. <IMAGE>

Description

APPARATUS FOR LOW PRESSURE- LIOUlD CHROMATOGRAPHY The present invention relates to apparatus for low pressure liquid chromatography and in particular to an automated, chromatography apparatus designed-to give quantitative results.

Liquid chromatography is a technique in which a sample containing constituents to be neasured is passed into a chromatography column containing a packing material. The packing materiAl is chosen so that the constituents which are to be measured associate with or bind to it in such-a way that it can be disassociated by passing a certain concentration of a buffer solution through the column.

Different substances will bind to the packing material with different strengths and so different concentrations of buffer will be needed to disassociate them from the packing material. The presence of the constituents in the solution passing out of the column is detected by measuring its light absorption at a certain wavelength. --Thus by knowing the concentration of the buffer which caused the constituent to disassociate from the packing material, the identity of the constituent is known. In the established techniques the process is carried out at high pressure, i.e. several thousand psi, in steel chromatography columns about 30 cm long and 2 cm in diameter. Clearly the construction of the column and associated apparatus needs to be sufficiently strong to withstand the high pressures used.The apparatus tends, therefore, to be bulky, heavy and expensive.

Liquid chromatography, in particular, cation-exchange chromatography can be used to analyse haemoglobins as it can detect the presence of different types of haemoglobins in the blood. For instance, haemoglobins with an abnormal amino acid sequence can be detected, such as haemoglobin S which results in sickle-cell anaemia or glycated haemoglobin (i.e. haemoglobin which has reacted with glucose) which is indicative of a previous high concentration of blood glucose resulting from a diabetic condition. The concentration of glycated haemoglobin is used to monitor the treatment of diabetes and is a better indicator of a patient's condition than the instantaneous blood glucose level which tends to vary quite considerably over the short-term - for instance, it rises quickly after a meal.The concentration of glycated haemoglobin varies on a longer time scale than the blood glucose level because blood cells are replaced about every 120 days, and so is representative of an average of the blood glucose level over the preceding few months. However, since liquid chromatography has required the use of the high-pressure complex equipment described above, even if alternative tests were used, it has been necessary for many clinics and smaller hospitals to send blood samples away for testing which usually takes several days and is thus unsatisfactory.

Recently, the development of new packing materials for chromatography columns have made it possible to use a small column of the order of S to 20 am long and to perform the process at low pressure e.g. 10 to 30 psi. The samples are prepared manually and loaded manually on to the column, and then, one after the other, a number of different concentration buffers are individually passed through the column and the solution flowing out of the column monitored with the usual optical detector. Again, by knowing which buffer concentration caused which constituent to flow out of the column, the identity of the constituents is known. An example of this technique applied to blood testing is as follows. Blood collected into tubes containing EDTA anti-coagulent is centrifuged and the plasma and white cells removed.The red cells (1 vol) are washed by mixing with isotonic saline (4 vol) and then centrifuged at 2000xg for 5 min. After repeating this washing process, the cells (1 vol) are haemolysed by vigourously mixing with 0.01 M potassium cyanide solution (1.5 vol). They are then allowed to stand for 5 min to ensure all the haemoglobin was in the reduced form. Lipid material is removed by shaking with carbon tetrachloride (1 vol) for 30 s. After centrifuging, a portion of the clear supernatant (SOrL) is removed and mixed with the loading buffer (950awl) appropriate to the analysis system under investigation. Portions of this solution (S0;#L-1.5yL of red cells) are loaded into the column.Various column packing materials can be used including CM cellulose, BioRex 70, Glycogel B and polyaspartic acid silicas. Columns of CM cellulose and plyaspartic acid silica are eluted with Bis-tris (bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane) buffer. Two stock buffers can be used : one, stock buffer (a) containing 40 infl Bis-tris and 4ex potassium cyanide adjusted to pH 6.2 with concentrated hydrochloric acid, and a second stock buffer (b) comprising buffer (a) to which sodium chloride was added to a concentration of 200 mM before ps Adjustment. These buffers can be mixed in various proportions to obtain eluents of intermediate sodium chloride concentration.

Columns of Biorex 70 are eluted with a modification of a known phosphate buffer system (e.g. described by Xynoch P.A.M. and Lehman s. (1977) Lancet ii, 16). Again two buffers are used: buffer (a) was 4lmM sodium, pH 6.4, and extra sodium chloride was added to the original buffer (b) (136 mn sodium, pH 6.8) to a final concentration of 200 mM.

Columns of Glycogel B are equilibrated with ammonium acetate buffer (250 n#r#+SOmM MgSO4, 3mM NaN3, pH 8.5) as described in, for example, Gould et al. (1982) Clin. Chin.

Acta 125, 41.

All buffers were passed through a millipore filter (0.45 m) under reduced pressure, before use. This process also helped to degas the solutions. The techniques are however, still slow, and are too complex to be used by unskilled technicians in individual clinics as the sample preparation requires several pieces of equipment and several preparation chemicals and the operation of the chromatography apparatus requires some skill.

It is an object of the inventi#on to provide a simplified liquid chromatography apparatus which is suitable for use by relatively unskilled technicians in individual clinics.

It is a further object of the invention to provide a liquid chromatography apparatus which is easy to use, provides easily understandable results and in which the parts which have a short useful life are easily renewable.

According to the present invention there is provided an apparatus for low pressure liquid chromatography comprising: a chromatography column containing a packing material; sample induction means for drawing a sample into the apparatus and passing it through the column whereby a constituent of the sample is retained in the packing material; means for producing a buffer solution having a concentration which varies as a predetermined continuous, function of time during an interval of time and for passing the varying concentration buffer solution through the column; and a detector for measuring the concentration of said constituent in the fluid flowing out of the column as a function of time; and wherein the column is removably mounted between first and second mountings in the apparatus, including an inlet and an outlet for supplying fluid to and receiving fluid from the column, at least one of the mountings being moveable between a first position in which the column may be inserted between the mountings and a second position in which the column is retained between the mountings and is fluid sealed to each of them.

Preferably the apparatus further include display means for displaying the measured concentration as a function of time on a printed graph or a video display or both. Conveniently the concentration of the buffer solution can be varied in the required manner by varying the mixing ratio of two solutions of different concentrations. This may be done by using a gradient valve connected to two reservoirs of the solutions combining the outputs of two syringes driven at separately controlled rates each containing a buffer solution having a different concentration. Preferably the column is less than 10 mm long and uses polyaspartic acid-silica as a packing material. The process may be carried out between 10 and 30 psi and preferably at about 15 psi.

Preferably the chromatography column is removably mounted in the apparatus conveniently by being squeezed between mountings including respectively an inlet and outlet for supplying fluid to and receiving fluid from the column.

This allows the colum to be easily removed and replaced.

Preferably the apparatus and method is controlled by a microprocessor operating according to a stored program so that when the sample has been mounted in the apparatus it can simply be commanded to proceed and will then draw the sample into the apparatus, perform the analysis and display the results without any further action required by the operator.

The invention will be further described by way of non-limitative example with reference to the accompanying drawings in which: Figure 1 is schematic diagram of the apparatus according to the present invention; Figure 2 shows the sample loading part of the apparatus of Figure 1; Figure 3 shows a chromatography column for use with the apparatus of Fig 1; Figure 4 is a side view of the column mounting in apparatus according to the present invention; Figure 5 is a back view of the column mounting shown in Figure 4; and Figure 6 shows the output results of using the apparatus of Figure 1.

The chromatography apparatus of the present invention is shown schematically in Figure 1. The main parts of the apparatus are two syringes 1 and 3 which are used to control the flow of fluids around the system, a chromatography column 4 attached to a detector 13, a sampling induction tube 5, two hold-up loops 7 and 9 which hold a fixed, predetermined volume of liquid and two reservoirs 15 and 17. The apparatus also includes a microprocessor which is used to control the syringes 1 and 3 via stepper motors and to control the various valves connecting the fluid parts together and to process and output the results from the detector 13.

The reservoirs for buffer solution can be either rigid plastic bottles or flexible aseptic containers similar to the well known wine boxes which have an outer box and and inner collapsible bay.

In use, the apparatus operates as follows. First, a sample is drawn up into the apparatus via the induction line 5. (This part of the apparatus is also shown in Figures 4 and 5). The sample is drawn up by syringe 1 via the two hold-up loops 7 and 9. Then valve 8 is closed to isolate the sample in hold-up loop 7 from the syringe while the sample in hold-up loop 9 is ejected as waste. This is done so that the sample in hold-up loop 7 which will actually be tested does not include the first fluid drawn from the sample which can be unsatisfactory for a number of reasons e.g. because of contamination or air bubbles.

Subsequently, valve 8 is opened and syringe 1 pumps the sample onto the column 4. Various constituents of the sample will bind with different strengths to the packing material in the column.

The two buffer reservoirs 15 and 17 contain buffers of different concentrations and the two syringes 1 and 3 are filled respectively from the two reservoirs. Subsequently, under control of the microprocessor, the two syringes are driven so as to pump buffer solution to the mixing point 16 and from there through the column 4. The concentration of the mixed solution is clearly dependent on the respective rates of the two syringes and the concentrations of the buffers and so it is possible for the concentration of the mixed solution to be varied by the microprocessor according to a pre-stored program. Note that in the illustrated embodiment no mixing chamber is necessary, the solutions mix at the junction of the three tubes. A mixing chamber can be used if desired.Alternatively a gradient valve connected directly to pressurized reservoirs, e.g. syringes driven at the same rate, can be used to control the concentration of solution supplied to the column. When the concentration of the buffer reaches a sufficient strength to disassociate a particular sample constituent from the column packing material, then that constituent will pass out of the column and through the detector 13 where it can be detected. The microprocessor is conveniently programmed to increase the concentration of buffer passing through the column gradually in a series of defined steps or in a continuous gradient.

Thus it will be known at any time exactly what concentration of buffer is passing through the column and thus which of these sample constituents is being disassociated from the column packing material and appearing in the detector. If this is done then by integrating the output of the detector the amount of that constituent in the sample can be calculated and thus a quantitative result obtained.

The detector used is similar to that used in previous low pressure liquid chromatography apparatus and simply measures the light absorption at a certain wavelength of the fluid passing through it. The output is supplied to the microprocessor controlling the apparatus which also processes and outputs the results. The results are displayed in graphical form both as a hard copy via printer 23 and on a video display 21. An example of the results produced is shown in Figure 6.

The control apparatus is designed so that when the apparatus is switched on, a number of blank runs are performed, e.g. five, that is runs without a sample on the column so that air bubbles and contaminants are removed from the system. The system is preferably arranged to continue performing blank runs until the column is ready for use.

The same procedure is also used when the machine is shut down. The circuitry also counts the number of times the column is used so that a limit can be set on the life of the column. In the present case the limit is 100 samples.

The chromatography column is shown in more detail in Figure 3. It comprises a two-part plastics body consisting of a lower part 31 and an upper part 33. The two parts are formed so that they push-fit together in an interference fit by virtue of the wide, open-end of part 31 fitting around a portion 34 a reduced external diameter. An inlet 36 for fluid is provided in the end wall of the lower part 31 and an outlet 35 in the end wall of the upper part 33. The inlet and outlet are covered by permeable membranes 37 made from polyurethane or P.T.F.E. and the upper part 33 is filled with column packing material 38, the remainder of the volume the column being made up by inert permeable material 39.

The part of the chromatography apparatus which holds the column is shown in Figures 4 and 5. It comprises a lower mounting 43 which includes a fluid path 44 through which fluid is supplied to the column and an upper mounting AS including a fluid path 47. The upper mounting includes an outer seat 46 which abuts the top corners of the column and an inner seat 46A which abuts and seals to the end of the column and surrounds the outlet from the column. Fluid from the outlet of the column passes through the region within the inner seat 46A and via an outlet 47 to the main.

part of the apparatus. The upper mounting 45 is moveable between the illustrated lower position and an upper position in which the seats 46 and 46A are raised above the column so that the column can be lifted out of the apparatus through the opening 40 in the front. The mounting is moved by a cam arrangement 48 operated by handles 49 (see Figure 5) and the operating surface 50 of the cam arrangement is configured so that the mounting will remain stationary in either of its positions.

It will be recalled that the illustrated column is formed of two parts which pushed together, and it will therefore be appreciated that the pressure exerted on the column by the upper mounting when it is lowered tends to hold the column together. Figure 5 shows a rear view of the mounting part of the apparatus and more clearly shows the handles 49 of the cam arrangement.

The sample induction part of the apparatus is shown in detail in Figure 2. It is designed to take a sample from a standard medical vial 25.The induction part includes a moveable lower platform 27 which is movable between a raised position shown in full and a lowered position shown dotted and is urged towards its upper position by a spring 24. It also includes an upper seat 28 through which the sample induction tube 5 extends. The vial is placed over the induction tube 5 with its upper edge near the seat 28 while the platform is held in its lowered position. The moveable platform 27 is allowed to rise to its upper position to support the lower part of the vial and hold the vial in position with its open end against the seat 28.In the present embodiment, which is intended to be-used with a method of collecting the samples which will be described below, the sample vial 25 will contain a capillary tube 26 (originally used to collect the sample). While the illustration shows the platform arranged to hold the vial vertically, in an alternative embodiment in order that this capillary tube does not impede the sample induction tube 5, the platform is arranged so as to tilt the sample vial so that the capillary tube falls to one side and clears the sample inlet tube 5.

The present invention is particularly intended for use in analysing blood samples and is designed to be suitable for use by operators with relatively little training. One of the main problems with prior art techniques as mentioned above, is the skill required to take a blood sample and prepare it so that it is suitable for analysis. The present invention, however, is designed to work with the blood sample taken in a particularly simple way and processed in a single solution process.

With the present invention a blood sample can be taken not by a syringe, but instead simply by pricking the patient's finger and applying a capillary tube to the droplet of blood which emerges. The capillary tube in this embodiment has a volume of 20 microlitres. The capillary tube is then placed in a standard sample vial containing a solution which dilutes and haemolises the blood. In this embodiment the sample vials are pre-filled with lmb of the processing solution. Different amounts can be used. No filtration or centrifuging of the resultant mixture is necessary, the sample is simply agitated, e.g. shaken by hand, for a recommended time e.g. not less than five minutes, and then placed in the sample induction part of the apparatus as described above.

For testing, the haemoglobins in blood samples, and particularly to test for glycated haemoglobins, the processing solution is one of the buffer solutions used in the elution process, in this case buffer A described below, and the vial needs to be agitated for not less than five minutes before being placed in the apparatus.

The testing apparatus is programmed with a number of tests which it can carry out automatically on command of the operator. Thus it will be possible for a number of tests to be available each selected by only one or two button operation. After the type of test has been entered and a .'start" switch operated the machine automatically executes the induction of the sample, the testing and processing the results and displays the output in the required form.

Figure 6 shows a typical ouput of a blood analysis for glycated haemoglobin. This was carried out using the apparatus described earlier and using the following two buffer solutions: Buffer A Bis-Tris 40mM:- 41.84g./5L.

KCN 4mM:- 1.30g./5L.

NaN3 (azide) 3mM:- l.OOg./5L.

Buffer B NaC# 200mM:- 58,44g./5L.

Bis-Tris AOmM:- 41.84g./5L.

XCN 4mM:- 1.30g./5L.

NaN3(azide) 3mM:- l.00g./5L.

The first large peak, labelled 2, is glycated haemoglobins passing through the detector and the second peak 3 represents non-glycated haemoglobins passing through the detector. As can be seen from the table below the graph the processing circuitry automatically works out the area of beneath of the peaks (allowing for their overlap) i.e.

integrates the curve, and this represents the total amount of glycated haemoglobins in the sample. The processing circuitry in this embodiment is also programmed in this to look out for certain other peaks in the sample which might be representative of other haemoglobin abnormalities.

The detector 13 can be provided with scans for measuring at several different wavelengths e.g. by using a unable laser or by having several different fixed frequency light sources in it, or an achromatic light source with a number of interchangeable filters. Thus tests at several different wavelengths are available. Thus by using different buffer solutions and (if necessary) different column packing materials (easily used by virtue of the easily replaceable column) the apparatus can provide a wide variety of tests all of which are relatively simple for the operator to select.

The syringes, column, fluid paths detector and displays and the electronic processing circuitry all form a single unit of such a size that it can be conveniently mounted on a desk top. The apparatus is therefore very suitable to use in individual clinics. A clinic would be provided with a supply of suitable buffer solutions, pre-filled sample vials and capillary tubes of the correct length foretaking samples and a number of replacement columns. The supply of buffer solutions and columns vials and capillary tubes could be replenished on demand or at regular intervals and all of the equipment is tough enough to withstand rough handling and light and small enough so they can be despatched by mail etc.This means that individual clinics can make tests, e.g. blood tests, instantly on their own premises and thus have a blood test result within minutes of a sample being taken. Further, the staff do not need specialist training in order to use the apparatus or replace the column etc. The equipment supplied to a clinic and the set of replacement materials can also include a standard sample (in a marked vial) which can be passed through the apparatus for quality control purposes.

It would also be possible to run a blind quality control system in which samples whose composition is known only to the quality inspector are despatched to the clinics and the results returned to the inspector.

The present invention therefore provides a relatively small apparatus which is easy to use and could be used on an individual basis by clinics, hospitals and surgeries around the world. Further, all of the parts which are used up during testing can be replenished easily e.g. by dispatching a pack of columns, buffer solutions, pre-filled vials and capillary tubes to the user. The apparatus is also suitable for programming and performing a wide variety of tests for haemoglobin abnormalities.

This application describes matter claimed in copending application number 8813761.2 from which this application was diVided and also copending application number which was also divided from that application.

Claims (14)

1. Apparatus for low pressure liquid chromatography comprising: a chromatography column containing a packing material; sample induction means for drawing a sample into the apparatus and passing it through the column whereby a constituent of the sample is retained in the packing material; means for producing a buffer solution having a concentration which varies as a predetermined continuous, function of time during an interval of time and for passing the varying concentration buffer solution through the column; and a detector for measuring the concentration of said constituent in the fluid flowing out of the column as a function of time; and wherein the column is removably mounted between first and second mountings in the apparatus, including an inlet and an outlet for supplying fluid to and receiving fluid from the column, at least one of the mountings being moveable between a first position in which the column may be inserted between the mountings and a second position in which the column is retained between the mountings and is fluid sealed to each of them.

2. Apparatus according to claim 1 wherein at least one mounting is reciprocally movable between the two positions, the column being held between the mountings by pressure exerted axially of the column by contact with the mountings.

3. Apparatus according to claim 1 or 2 further comprising display means for displaying the measured concentration as a function of time on at least one of a printed graph and a video display.

4. Apparatus according to claim 1, 2 or 3 wherein column packing material is polyaspartic acid-silica.

5. Apparatus according to claim 1, 2, 3 or 4 wherein said column is less than 100 mm long.

6. Apparatus according to claim 4 wherein said column is 5 mm long.

7. Apparatus according to any one owf claims 1 to 6 further comprising a holder for receiving a container containing the sample; and wherein said sample induction means are adapted to draw the sample from the container when it is in position on the holder.

8. Apparatus according to any one of claims 1 to 7 wherein said means for producing the buffer solution comprise mixing means for mixing two solutions of different concentrations at a mixing ratio which is a controlled function of time.

9. Apparatus according to claim 8 wherein the mixing means comprises two syringes connected to a common outlet and including means for driving the syringes at independently controllable rates.

10. Apparatus according to claim 8 wherein said mixing means comprises a gradient valve.

11. Apparatus according to any one of claims 1 to 10 including a mounting for receiving two reservoirs for said two buffer solutions.

12. Apparatus according to claim 11 wherein the mounting is a mounting for a flexible plastics bag of fluid.

13. Apparatus according to any one of claims 1 to 12 including a microprocessor for controlling the sample induction means, buffer solution producing means and the detector according to a stored program and for monitoring the output of the detector and processing it according to a stored program.

14. Apparatus for low pressure liquid chromatography substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.