Automated Sequential Sample Analysis for Some Amino Acids by Elution Chromatography Barry Dymond
Department of Health and Safety, United Kingdom Atomic Energy Authority, Chapelcross, Dumfriesshire, Scotland A new technique for automatically loading a ctiromatographic column is illustrated by its application to a single-buffer-elution sequential analysis for a number of amino acids. When using a Technicon AutoAnalyzer to monitor the column eluate, peak resolution and symmetry are comparable to manual loading, and the relative standard deviation on an overnight run of seven consecutive analyses is 3%. The technique is based on two chromatographic pumps of differing characteristics, one loading the sample, the other eluting it and regenerating the column. One of the main features of the method is that the additional chromatographic equipment, including sample loading apparatus, elution valves, and programmer, can be easily and inexpensively constructed. Sample cooling facilities are provided. The underlying principles, limitations, and possible further applications are briefly discussed.
THEBIOLOGICAL IRRADIATION PROGRAM at this laboratory requires a large number ofanalyses of physiological fluids for a limited range of amino acids. Accelerated methods, using single buffer elutions, were therefore adopted based on the continuous-gradient Technicon procedure initially used. Rapid separations of groups of amino acids have been very thoroughly dealt with by Shih, Efron, and Mechanic (1) and Benson, Cormick, and Patterson (2). In order to make use of the analytical system over a 24hour period, thereby increasing the number of analyses and also permitting some relaxation in the operators’ schedule, a fully automatic procedure was needed which could be based on inexpensive additions t o existing equipment. In any sequential chromatographic system, the crucial problem is that of automatically loading the samples onto a column while preserving an acceptable degree of resolution and precision of the substances of interest. Also, the conditions under which samples awaiting analysis are kept are an important consideration. Because the program at this laboratory involved tracer studies, and an extension of automation to the analysis of samples for other metabolites was envisaged, the provision of sample cooling facilities was clearly desirable. A review of published pertinent references revealed, for our purposes, certain limitations. Dus et ai. (3) employed a system of rotating valves for sample selection and elution programming, the samples being held in long lengths of narrow-bore tubing coupled between the valves. Eveleigh and Thomson (4) placed samples on miniature columns of resin which were clamped, in turn, to the analytical column. The loaded columns awaiting analysis were left open to the atmosphere. Neither of these (1) V. E. Shih, M. L. Efron, and G. L. Mechanic, Anal. Biochem., 20, 299 (1967). (2) . . J. V. Benson., Jr.., J. Cormick. and J. A. Patterson. ibid.. 18. 481 (1967). (3) K. Dus, S . Lindroth, R. Pabst, and R. M. Smith, ibid., 14, 41 (1966). (4) J. W. Eveleigh and A. R. Thomson, Biochem. J., 99,49 P (1966). ,
methods provided cooling facilities and it is not evident how such facilities could be easily incorporated. In both procedures, the samples were placed in the high-pressure part of their respective systems, and although this is desirable chromatographically, it was felt that the coupling and recoupling required would ultimately prove less reliable than placing samples on the low-pressure side of the pump. The latter course was adopted by Murdock, Grist, and Hirs (5) when they experienced leakage at pressures greater than 100 lb/inch2, using a system somewhat similar to that described in (3). Refrigerated water could be circulated around the samples but the general description of their system is insufficient. The prior filling of the long coils of tubing with samples did not appear to be a simple procedure and the same criticism could also be applied to Reference 3. This paper describes how the necessary requirements were met, indicates briefly the underlying concepts of the method, and discusses the limitations and possible further applications. Since this work was completed, a comprehensive system of analysis for amino acids based on Reference 5 has been demonstrated at the Technicon European Symposium (1967), and an interesting cooled loading device has been described (6). EXPERIMENTAL
Apparatus. PUMPS.The complete chromatographic system is illustrated in Figure 1. Two chromatographic micropumps feeding a column consecutively formed the basis of the method. One pump (12) loaded the sample, the other (11) eluted it and regenerated the column. The placing of a sample on the inlet side of a pump has previously been reported (3, with the same pump also being used for elution. However, the requirements for loading and elution appear to be mutually exclusive. The former demands a minimum pump-chamber volume for the smooth flowthrough of sample solution with minimum “back-mixing,” hence a short-stroke pump, while the latter requires longstroke positive displacement, therefore high chamber-volume, for precise elution in a high-pressure system. The two pumps were connected on their high-pressure sides, immediately below the ion exchange column (13). A fourway coupling was used, one side normally being blanked off during operation (not illustrated). This side could be opened when necessary to prime either pump line or to insert a pressure gauge. The pumps were carefully leveled, as proper seating of ball valves is essential to prevent leakage through the nonoperational unit. The pumps were serviced regularly. IONEXCHANGE COLUMN.The column (13) was constructed from 6-mm precision-bore borosilicate glass and was 60 cm long and mounted directly onto the four-way coupling. (Details of column fittings and water jacketing have been omitted from the diagram.)
I
( 5 ) A. L. Murdock, K. L. Grist, and C. H . W. Hirs, Arch. Biochem. Biophys., 114, 375 (1966). (6) E. Monch, Technicon European Symposium, Brighton, England, (1967). VOL. 40, NO. 6, MAY 1968
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6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 22a.
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Figure 1. Diagram of apparatus for automated sequential chromatography (not to scale) 23. Upper needle stop (clamp omitted) Elutant reservoirs 24. Sample plate support (clamp omitted) Soda-lime tubes 25. Sample plate 15-cm diameter Solenoids 26. Cups holding "wash" solution Solenoid plunger 27. Trip pins 13-mm rubber bung 28. Sample plate pivot with 12-point locating mechanism Valve support frame 29. Needle guide (clamp omitted) Valve plunger 30. 18-gauge sample needle Valve body 31. Glass sleeve 6-mm O-ring Side inlet to valve 32. 6-mm bore PVC connecting sleeve 33. Sample tube 3-mm bore flexible PVC tubing 34. Sample solution Elution pump 35. Polythene rod insert Sample loading pump Ion exchange column (water jacket omitted) 36. 15-cm Dewar ice bowl 37. Crushed ice Eluate for analysis Plunger stop (glass funnel) 38. Programmer drive unit Nipple inserted in bung 39. Microswitch bank (stand omitted) 40. Cardboard drum of 30-cm diameter 2-mm-bore flexible PVC tubing 41. Microswitch trips Pivot 6 B.A. nut with soldered nipple 42. Turntable of 60-cm diameter 6 B.A. brass rod 43. Pegs 44. 50-point locating mechanism 5-mm polythene rod end fitting 1-mm-bore flexible PVC tubing 45. Trip for sample plate pins 3-mm-bore PVC tubing a
ANALYTICAL CHEMISTRY
The resin used was the 8 % DVB, sulfonated polystyrene Chromobeads A supplied by Technicon. Column temperature for this analysis was 68' C and the operating pressure at this temperature was 150 to 200 lb/inch2. Upward flow elution of the column was used in order to simplify the siting of the loading pump (12) and also to obtain a further reduction in dead space between sample and resin. A manual comparison showed no difference between upward and downward flow analyses. To pack the column, a small cotton wool plug was placed in the bottom of the column and a slurry of resin in buffer was poured in and allowed to settle overnight. The elution pump (11) was connected to the column top and buffer was pumped through for several hours. The column was toppedup with more slurry and repumped, repeating until contraction ceased. The pump was then reconnected to its normal operating position at the column base. A second cotton wool plug and outlet fitting was inserted in the column top. This fitting was connected to an unmodified amino acid AutoAnalyzer for the analysis of the column eluate (14). Periodically, as further contraction of the column occurred in use, the topping-up was repeated. An adjustable column fitting such as that described by Dus et al. (4) would be a useful accessory. After 100 analyses, the column was lifted from the coupling and the dirt-laden lower plug of cotton wool was replaced. ELUTIONSYSTEM,The normal solenoid-operated metal valves used in chromatography are not entirely satisfactory because they heat the solutions passing through and produce air bubbles (7). They also tend to be rather expensive. The valves illustrated in Figure 1 were therefore designed and built and have operated satisfactorily for the past 12 months. The solenoid (3) was adapted from a continuously rated gas valve (Type A.V.G. 2, Black Automatic Controls, Corsham, England) by removing the brass valve body. For simplicity, the plunge. sleevs is omitted from the figure. The rubber sealing disk in the plunger was replaced by a tight-fitting rubber bung (4) into which was fitted a 6-mm perspex rod (6) which was pointed and grooved to hold the valve seal (8). The valve body (7) was made from a 5-ml polystyrene syringe, with a n additional syringe and piece glued on the side (9). Four valves were built into a perspex frame (5) which is shown unsupported and incomplete for simplicity. Each valve formed a n integrated level system with its corresponding reservoir (1). To avoid any cross-contamination of solutions, switching was arranged to close one valve fractionally before another opened. The flexible polyvinyl chloride (PVC) tubing to the pump accommodated any temporary contraction. The pump used for elution was a MiltonRoy positive displacement minipump. SAMPLE LOADING SYSTEM.The loading pump (12) was a short-stroke D.C.L. Micropump Series I1 (F. A. Hughes & Co., Epsom, England). The entry and exit ports were sleeved with tightly fitting 2-mm-bore polythene tubing to reduce dead-space volume, similar to the Teflon inserts of Murdock, Grist, and Hirs (5). Because this type of pump was not considered sufficiently accurate to meter a sample to the column, a pipetted aliquot was necessary. This posed difficulties of either air entrainment or prior diffusion of sample into the following wash solution. The sample-loading tube illustrated (26, 33) was designed to overcome these difficulties. The tubes were constructed primarily from polystyrene syringes. The lower section (33) was 40 mm long with a 4-mm bore. The polythene plug (35) served to cushion the fall of the needle (30). The upper section (26) was of 25-mm diameter and 25 mm high. Its syringe end fitting was shortened and shaped to act as a needle guide. The two sections were held together by a PVC sleeve (32) which fitted snugly into the sample plate (25). (7) P. B. Hamilton, ANAL.CHEM.. 35,2055 (1963).
Table I. Schedule of Operations Peg No.
Time, min
50
0
Start of NaOH regeneration
5
15
Buffer regeneration
10
30 33
11
12 13
14 50
36 39 42 150
Sample loading Amino acid elution NaOH regeneration
Operations NaOH valve opensbuffer valve closes Buffer valve opens/NaOH valve closes Sample needle lifts Mechanical trip to next sample Sample needle drops M.R. stops/DCL starts M.R. starts/DCL stops Cycle repeats
The purpose of the constriction was to retain an air gap between the sample and the wash solution both prior to and during sample loading. The sample needle (30) was made from 18-gauge stainless steel hypodermic tubing, 80 mm long, and partially sleeved with glass capillary tubing. The function of the glass was to ensure, by its favorable contact angle with water, that nonturbulent replacement of sample by wash solution occurred during sample loading and that the air space stayed reasonably constant and immobile. (The efficiency of various designs was investigated by connecting the needle directly to the colorimeter and monitoring the pick-up of a suitably colored solution.) The tubing which connected needle to pump (22) was of the minimum length consistent with free needle movement, and it passed through the rod end fitting (21) as a tight slip fit. In order to change over to the next sample in line, it was necessary to lift the needle 75 mm. This was achieved by a IO-mm movement of the solenoid plunger by simple lever arrangement (see Figure 1 and Table I). The approximate distance between pivot (18) and nut (19) was 15 mm and from pivot to rod end (21) was 110 mm. Counterweights could be incorporated, if necessary, and fine adjustment of needle movement was readily obtainable. The sample plate (25) was made from 6-mm perspex sheet, with steel trip pins (27) inserted near the edge; IO-mm holes, to accommodate the sample tubes, were drilled 20 mm from the edge. The supporting rod (24) was clamped above the plate to allow for the insertion of the plate into the rim of the ice bowl (36). When the latter was filled with crushed ice (37) to a level just below the sample tubes it kept the samples awaiting analysis at an overnight temperature of 5" to 7" C. SYSTEM PROGRAMMER. The master module was an adjustable-speed Kymograph drive unit (38) with drum removed. The pointer which normally operated electrical contacts on the unit was used to trip mechanically the pegs (43) inserted into the outermost holes of an obsolete fraction collector turntable (42). The turntable was 60 cm in diameter and held 50 pegs with a corresponding locating mechanism (44). The drive unit was set at 1 revolution in 3 minutes. A complete cycle, corresponding to one analysis and regeneration, was thus 150 minutes. Mounted on the turntable was a stout cardboard drum (40) of 30-cm diameter and covered with linear graph paper to facilitate the mounting of the microswitch trips (41). The trips were cut from 6-mm polythene sheet, drilled and bolted to the drum. For this analysis, five microswitches (39) were used to control the two pumps and the solenoids of the sample needle, the sodium hydroxide valve, and the eluting buffer valve. On the underside periphery of the turntable a single metal pointer was inserted (45) which, at every revolution of the VOL. 40, NO. 6, MAY 1968
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..
--. eu
L
:*
..
.
. . . .
. .
Figure 2. Recorder trace illustrating automatic sequence
turntable, made contact with one of the pins (27), tripping the sample plate 30" and bringing the next sample into line for analysis. Reagent Solutions. The regenerating solution was 0.4N aqueous sodium hydroxide, prepared as follows. An approximately 20N solution was centrifuged to remove insoluble carbonate. The clear supernate was diluted to 0.4N with boiled demineralized water. A soda-lime tube was inserted in the reservoir stopper to prevent subsequent carbonate formation during operation. These precautions were taken because it was found that the resultant liberation of COZduring column regeneration upset the analytical system and caused pen oscillation. Similar precautions were taken to minimize carbonate formation in the eluting buffer, which had the following aqueous composition: citrate, 0.05M; Na+, 0.40N; Brij 35, 0.3%; n-caprylic acid, 0.1 %. After adjustment to pH 3.80 with 6N HC1, 10% by volume of ethanol was added. Procedure. The sequence of operations determined by the system programmer is summarized in Table I. Before starting, the turntable was rotated manually to peg 50. The water-circulating bath for the column was switched on and the column was brought up to temperature. The analytical system was started and checked for eficient working. External standards were run. The blanking-off stud of the four-way coupling was removed and both pump lines were primed-the D.C.L. with water, and the MiltonRoy with buffer. The pressure gauge was connected and the flow rates of the two pumps were individually adjusted to 1 ml per minute, inserting temporary trips under the microswitches as necessary. The stud was replaced, and all units were then switched on. The drive unit initiated the start of
Consecutive detn
NaOH regeneration and all subsequent operations. The dela of 36 minutes before sample pick-up was used to prepar the first samples for loading. Twenty per cent by volume of 2M sulfosalicylic acid was incorporated into each sample, and the resultant solution was centrifuged and filtered as necessary to remove any traces of insoluble matter (plasma and tissue homogenate samples were first deproteinated by ultrafiltration). A microsyringe was used to transfer a suitable aliquot to the clean dry sample tube (33). Care was taken to avoid both contamination of the tube above the sample surface and air bubbles beneath it. The normal aliquot was usually within the range of 0.1 to 0.5 ml. Approximately 4 ml of the wash solution (in this case demineralized water) was poured into the upper cup (26), this volume being in slight excess of pumping requirements. The loaded tubes were inserted in sequence into the sample plate. For the purposes of this paper, an aliquot of 0.2 ml of a synthetic mixture containing between 0.05 and 0.2 hmole of each amino acid was used. An overnight sequence of seven analyses, total time 17.5 hours, was run. RESULTS AND DISCUSSION
The precision data obtained from the consecutive sqies of determinations on the synthetic amino acid mixture is given in Table 11. Part of the actual recorder trace of the same series is illustrated in Figure 2. Because passing a sample solution through a pump introduces some tailing, and therefore locates the sample as an undesirable broad band on the column, chromatographic conditions were employed to minimize this.
Table 11. Amino Acid Recoveries as a Percentage of Their Mean y-Amino-isoP- Amino-isobutyric acid Phenylalanine butyric acid Tyrosine
1 2 3 4 5 6 7
102.3 103.8 105.2 96.5 99.4 99.4 95.4
101.1 101.4 99.4 95.2 101.7 101.7 101.7
101.7 97.5 103.3 95.0 102.1 104.1 98.4
Re1 std dev
3.6
2.5
3.5
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0
ANALYTICAL CHEMISTRY
104.6 103.4 100 95.4 98.9 98.9 96.6 3.4
Tryptophan 100 97.3 104.7 97.3 98.0 102.7 98.6 2.9
The rate of travel of a substance on a column is proportional t o the reciprocal of its distribution coefficient-that is, its relative binding to the resin. Therefore, an overall increase in the distribution coefficients of the sample amino acids will result in a narrower band being loaded onto the column. Two known factors which d o this are acidification (3
[creating the protonated form RCH(NH3)COOH] and a n initial eluent of low ionic strength. The conditions employed here were chosen for operational simplicity and may not be ideal, but their effectiveness is illustrated by the symmetry and resolution of the prescribed amino acids in Figure 2. Cysteic acid and taurine were included in the sample to demonstrate the degree of tailing of those substances which have little affinity for this resin, and whose distribution coefficients cannot be appreciably altered by the technique employed. This is a limitation of the met hod. A further limitation is the introduction of artifacts by this loading technique. Pump changeover produces “blips” on the recorder trace, one being just visible on the trailing edge of cysteic acid. The sulfosalicylic acid incorporated in the sample is eluted after taurine and produces a base line depression. Also, the hump occurring after phenylalanine appears to be an artifact associated with the water-wash.
If more usual loading conditions are employed-for example, a minimum acidification of sample followed by a “wash” of the eluting buffer-then artifacts are reduced, but so is peak resolution. In any application of this system, the relative requirements must therefore be considered. The dotted lines in Figure 1 indicate the additional reservoirs, valves, and controlling microswitches necessary to extend the system to multidiscrete-buffer analysis. “Wash” solution volume can be increased by using a larger cup (26) and forms a convenient additional parameter. Some of the apparatus and techniques described may be applicable to other fields of column chromatography. ACKNOWLEDGMENT
The author expresses his appreciation to H. Smith for helpful discussion, to S. Macaulay for photography, and to the Electrical, Instrument, and Typing Sections of Chapelcross for their able technical assistance. RECEIVED for review August 31, 1967. Accepted January 8, 1968. This work was done under the auspices of J. H. Martin as part of the program at Chapelcross on the biological indicators of radiation effects, financed by the United Kingdom Atomic Energy Authority who have also given permission to publish.
A High-Resolution Liquid Chromatograph R. E. Jentoft and T. H . Gouw Cheuron Research Co., Richmond, Calif. 94802 A description is given of an apparatus for high resolution liquid chromatography which can be used for the analysis of high molecular weight substances, thermally labile compounds, and reactive products which cannot be analyzed by gas chromatography. A pulseless high pressure pump capable of generating pressures up to 1000 psi is used. The sample is introduced into the stream using a specially designed injector system. After separation on the chromatographic column, the eluted components are detected by determining the ultraviolet absorption of the stream in a 2 0 4 flow-through cell with a 1-cm path len th. Chromatograms are shown of the separation o coronene, benzo[a]pyrene, and phenanthrene on two different columns.
9
THELITERATURE of the past few years shows a resurgent interest in liquid chromatography. Motivated in part by the great successes in thin-layer chromatography (TLC) and building on the experience gained in gas chromatography, many workers are exploring for improvements in the separations possible by liquid chromatography. Theoretical investigations are contributing especially strongly to the understanding and to the prediction of the possibilities and limitations of this technique (1-4). Liquid chromatography is particularly applicable to the separation of high molecular weight substances, thermally (1) J. C. Giddings, ANAL.CHEM., 37, 61 (1965). (2) J. C. Giddings, ibid., 35, 2215 (1963). (3) D. C. Locke, ibid., 39, 921 (1967). (4) L. R. Snyder, ibid., 39, 698, 705 (1967).
labile compounds, and reactive products which cannot be analyzed by gas chromatography. The liquid chromatographic systems currently available commercially are not sufficiently versatile for research purposes, and for specific applications it is still necessary to construct sections of the unit oneself. Descriptions of liquid chromatographs have been presented by Lambert (5) and Stouffer, Oakes, and Schlatter (6). Factors affecting the design of these instruments have been discussed by Scott, Blackburn, and Wilkins (7). The chromatographic system described in this paper was specifically developed for the analyses of polynuclear aromatic hydrocarbons; the system can, of course, be used in a much wider area of application. Separation of polynuclear aromatic hydrocarbons is generally carried out by TLC (8, 9). However, extensive work may be necessary to remove background material; and the amounts involved are too small for easy handling when additional information is required. . Liquid chromatography can, to some extent, obviate the problems observed in TLC. In addition, advantages observed in using liquid-liquid over liquid-solid systems also favor the (5) S. M. Lambert, ANAL.CHEM., 37, 959 (1965). (6) J. E. Stouffer, P. L. Oakes, and J. E. Schlatter, J . Gas. Chromatog., 4, 89 (1966). (7) R. P. W. Scott, D. W. J. Blackburn, and T. Wilkins, ibid., 5, 183 (1967). (8) E. Sawicki, T. W. Stanley, W. C. Elbert, and J. D. Pfaff, ANAL.CHEM., 36, 497 (1964). (9) R. H. White and J. W. Howard, J . Chromatog., 29, 108 (1967). VOL. 40, NO. 6, MAY 1968
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