Accelerated Auto matic Chromatog ra phic Ana lysis of Amino Acids on a Spherical Resin JAMES V. BENSON, JR., and JAMES A. PATTERSON' Beckman Instruments, Inc., Palo Alto, Calif. Acceleration of analysis of amino acids by ion exchange chromatography has been facilitated in the Beckman amino acid analyzer by the use of a spherical particle resin. The acidic and neutral amino acids are analyzed on a 57- by 0.90-cm. resin column at a buffer volumetric input of 68 ml./hour (linear flow rate of 106 cm./hour). The basic amino acids are analyzed on a 5.0- by 0.90cm. resin column at the same buffer flow rate. This system reduces ihe total analysis time to 4 hours, 5 minutes; (3 hours, 10 minutes for the acidic and neutral amino acids and 55 minutes for the basic amino acids). With the proper analysis sequence, three analyses can b e completed in an 8-hour day.
the performance of a newly available Styrene-divinylbenzene fully sulfonated nominal 8% cross-linked spherical particle ion exchange resin suitable for the chromatography of amino acids in column procedures patt,erned after, or similar to, the method of Spackman, Stein, and Moore ( 7 ) . With this resin, and by modification of the accelerated system of Spackman (8),used in this work as a base line, the time of analysis of a protein hydrolysate was reduced to 4 hours, 5 minutes. With proper analysis sequence, three analyses can be completed in an 8-hour day. The decreased time of analysis was achieved by an increased flow rate through a 0.9- by 57-cm. resin column; a t a volumetric input of 68 ml./hour (equivalent to a linear flow rate of 106 cm./hour) the acidic and neutral amino E REPORT
1 Consultant to Beckman Instruments, Inc.
PH Sodium concentration Sodium citrate 2 H20 Concentrated HC1 Thiodiglycol (TG) Brij-35 solution (50 g./ 100 ml.) Caprylic acid Final volume
1108
acids were analyzed a t the same flow rate on a 0.9- by 5.0-cm. resin column in 55 minutes. The 69-cm. column was packed with Beckman custom research resin, mean diameter 22 =t 6 microns, and the 23-cm. column was packed with similar resin of 15 i 6 microns diameter. Column size employed and narrow particle diameter range helped keep pressures below 250 p.s.i., consistent with standard Beckman equipment, and resulted in improved resolution compared to our base line. EXPERIMENTAL
Apparatus. A standard Beckman Model 120B amino acid analyzer was used. Resin and Reagents. Beckman Custom Research Resin. For analysis of acidic and neutral amino acids, Type AA-15 with spherical particles having a mean diameter of 22 i 6 microns was used. For analysis of basic amino acids, Type AA-27 with spherical particles having a mean diameter of 15 6 microns was used. Buffers. For analysis of acidic and neutral amino acids, pH 3.28 (0.20N) and p H 4.25 (0.20N) sodium citrate buffers were used. For analysis of basic amino acids, pH 5.28 (0.35N) sodium citrate buffer was used. All buffers were prepared with reagent grade sodium citrate (2), as shown in Table I. Ninhydrin Reagent. Prepared according to the method of Spackman, Stein, and Moore ( 7 ) . Preparation of Ion Exchange Columns. A 69- by 0.9-cm. column was used for the analysis of the acidic and neutral amino acids and a 23- by 0.9-cm. column for the basic amino acids. The resin without pretreatment was slurried with 2 volumes of buffer. The p H 4.25, 0.20N buffer was used to
Table I. Sodium Citrate 3 28 f 0 01 0 20N 784 3 grams
*
200 ml.
Buffers 4 25 f 0 02 0 20,v 784 3 grams 335 ml. 200 ml.
80 ml. 4 ml. 40 liters
4 ml. 40 liters
493 ml.
ANALYTICAL CHEMISTRY
80 ml.
5 28 f 0 02 0 35ih' 1372 6 grams 260 ml.
80 ml. 4 ml. 40 liters
pack the long column and the pH 5.28, 0.35N buffer was used for the short column. Brij-35 and thiodiglycol were not present in these column packing buffers. Before pouring the column, about 10 ml. of buffer was forced. with air pressure, through the disk in the bottom of the column to remove any trapped air. About 1 cm. of liquid was left above the disk. The bottom of the column was capped. The resin slurry was stirred just before pouring and the column was filled. After 5 minutes the bottom cap was removed, and the column effluent line was connected. A buffer flow rate of 68 ml./ hour was used to pack the column. When the resin bed had packed, excess buffer was aspirated and the column was again filled with the resin slurry, taking care that the previously packed section was not disturbed. Several additional packings were necessa.ry to fill the long column. The long column was packed to a height of 57 cm. The short column was packed to a height of 5.0 cm. Each column was regenerated with 0.2W NaOH after which they were equilibrated with the appropriate buffer. ANALYSIS OF A M I N O ACIDS AND RESULTS
Figure 1 illustrates the resolution of a standard calibration mixture of basic, acidic, and neutral amino acids obtained at a buffer flow rate of 68 ml./ hour. Both analyses were carried out a t 55' C. The back pressure on the long column was 130 p.s.i. and on the short column, 50 p.s.i. The sample (0.5 ml.) placed on each column contained 0.25 pmole of each amino acid. Recorder chart speed was 6 inches per hour Kith 1 dot/2 seconds printing speed. To prevent the recorder dots from cluttering on the chromatogram, the alternate 570-mp Helipot potentiometer was adjusted so that the pen printed on the left side of the chart. For the analysis on the short column (basic amino acids) a t the flow rates indicated above, the total analysis time was 55 minutes. The total analysis time for t,he long column (acidic and neutral amino acids) was 3 hours, 10 minutes. The buffer change was made a t 85 minutes to take effect just before valine. The hexosamines, glucosamine, and galactosamine were present in the sample to indicate their lack of interference with the amino acid elution
a 0. 0.
Figure 1.
Chromatogram of a n amino acid
Basic amino acids and ammonia (shorter curve at far
pattern; they are resolved well in this system. Cysteic acid, if present, is eluted at approximately 20 minutes and methionine sulfone emerges between aspartic acid and threonine. Citrulline, if present in the sample, would be eluted Table 11. Time short Col. snaysia 8:30 a.m. 8:38 a.m.
8:43 a.m.
9:25 a.m. Long col. analysis 8:55 a.m.' 9:25 a.m.
Typical Daily Schedule Lspsed time (min.) Operational changes 0 Start buffer pump (pH 5.28, 0.35N)
and ninhydrin pump 8 Turn on recorder 13 Lysine peak emerges 55 Analysis is complete
0 Start buffer pump
12:oO Naon
~WNUS-Z
lah) sepd. en 5.0-cm. COI.
rv19.ulmtmg 0.25 fimole of each amino acid
Acidic and neutral amino odds (longer curve.) sepd. on 57-cm. col.
between glutamic acid and proline. Alphsramino adipic acid is eluted just ahead of glycine and h o m o c i t d i n e just after cystine. Glucosamine, if present in the sample, would be eluted off the short column just before lysine. Chlorotyrosine and an acid degradsr tion product of tryptophan are also eluted early in the analysis. If the resin column is increased in height from 5 to 10 cm., the distance between these two peaks will then increase from 5 minutes to 11 minutes, with other separations increasing proportionally. Total analysis time will then he 1 hour, 35 minutes through the elution of arginine. A typical daily schedule for the complete analysis of amino acid analyses is summarized in Table 11. To obtain the same linear Bow rate in a 0.9-cm.
column as in the 0.6-em. column employed by Spackman (8) a t a volumetric input of 30 ml./hour, it was necessary to incresse the volumetric input t o the former column to 68 ml./hour. With these rates, a reaction coil was used which was twice the standard length (22.50-ml. volume instead of 11.25 ml.). Recovery determinations on various sample concentrations indicated that Beer's law was obeyed ( 1 ) . A standard length reaction coil was also tried (11.25ml. volume), and recovery values o b tained over a sample concentration range of 0.1 t o 0.5 pmole indicated that Beer's law was still obeyed. T o determine statistical accuracy and precision, 11 analyses were performed at the 0.25-pmole level t o he used as a calibration point for the analyzer. The results at the 0.5- and 0.l-Mmole load levels are shown in Table 111, using a
(DH 3.28, (pH 3.28. 0.20N)
to drain tb
30 Turn column effluent t.n . coil and ninhydrin to coil 85 Buffer change takes place to pH (4.25, ~~~~
10:20 a.m.
CUIIYIUIIVII
0.2ON)
~~~~~
~
Start second short column analysis 12:05 p.m. 250 Analysis is complete 12:20 p.m.a Start second long column analysis 12:50 p.m. End second short column analysis 3:25 p.m. Stsrt third short column analysis 3:30 p m . End second long column analysis 3:50p.m." Start third lonc column analysis" 4:20 p.m. End third short column anal sis 7:OO p.m. End third Ibntg coly n n analys1,s usmg aut
Table Ill. Recoveries from 0.50- a n d 0.10-pmale Samples (One standard length reaction coil, 11.25-mi. volume, and buffer flow rate of 68 ml./hour) 0.10 pmoles (6 runs) 0.5 pmoles (5 runs) Range Av. Range Av. 99-ino 99 100 ion Lysine 100 ion 100 Histidine 100 101-102 in2 98 98 Ammonia 1oc-101 101 98 98 Arginine 101-102 in2 102 102 Asnsrtic acid 100 100 101 100-101 Thonine in2 99-103 103 101-102 Serine 102 99-103 103 101-102 Glutamic acid 96-115 ion Proline 100 10C-104 9&102 102 Glycine 100 100 98-102 102 100-102 Alanine 100 Half cystine 1oc-101 92-ino 100 100 Valine ioo-inz 94-103 98 102 Methionine ioi-ioZ 97-101 101 101 Isoleucine 102-103 102 99-103 100 100 Leucine 100-101 96100 100 T rosine romne 100-102 100 98-102 102 in1 gs-inz . QR d e n y lalanine 101-102 100 101 Av. Std. dev. 1.49 1.37 2 X Std. dev. 3.0 3.0
VOL 37, NO. 9, AUGUST 1965
1 109
standard length reaction coil (1 1.25-ml. volume). The results are expressed in terms of two standard deviations (95% probability). Thus, the average accuracy and precision in the 0.1- to 0.5-pmole sample range is 100 + 3% and 100 3%, respectively. To determine the resolution at higher flow rates, the 57.0-cm. column was operated (using the same buffer and ninhydrin system previously mentioned) at 120 ml./hour for the buffer and 60 ml./hour for the ninhydrin reagent. The operating back pressure was 210 p.s.i. A sample load of a synthetic amino acid mixture containing 0.25 pmole (G.50-ml. volume) of each amino acid was tested. The analysis was conducted a t 55’ C. and a buffer change from the p H 3.28, 0.20N to the p H 4.25, 0.20N sodium citrate buffer occurred a t 41 minutes. A slight loss of resolution was experienced. However, it is possible to quantitate accurately (height-width method) those peaks affected; namely, the threonine-serine and tyrosine-phenylalanine doublets. If the standard length rwction coil ( 7 ) is used, reproducibility of recoveries will be 100 f 6%. With a doublelength reaction coil, normal r+ producibility of recoveries will be 100 f 3%. An analysis time of 1 hour, 35 minutes for the analysis of the acidic and neutral amino acids is possible.
DISCUSSION
The goal of this work has been the optimization of resin, column length, column diameter, and flow rate to achieve the complete analysis of a protein hydrolysate in the minimum time. Limiting conditions imposed were; maintenance of precision and accuracy, operation at reasonable pressures, with maximal resolution and a minimum of change of existing equipment. We found no commercially available ion exchange resin that would allow us to meet these specifications using the conditions described above. I n the accomplishment of these aims, the geometry and chemical composition of the resin column packing is crucial. The resin was designed specifically to meet the above goals. T h a t these aims have been met adequately is demonstrated by the results obtained. The results with this resin are also in agreement with expectation indicated for the hydraulic characteristics of narrowly graded spherical column packing material (3, 6). The good resolution with fine particles that has been previously obtained with other systems (4, 6) is also confirmed for this system. Because of rigid control in the manufacture of the resin to ensure constancy of cross-linking, spherical shape, and predetermined particle diameter range, the results shown here are typical;
*
identical results have been consistently obtained from batch to batch. Another resin system has been developed which will allow two complete analyses of physiological fluids in 24 hours; the details of this analysis are to be reported, later. ACKNOWLEDGMENT
The authors gratefully acknowledge the editorial assistance of P. B. Hamilton and the technical assistance of Jean Cormick. LITERATURE CITED
Benson, J. V., Jr., Patterson, J. A., Federation Proc. 23:2. 371 11964). (2) “Beckman Technical Bulletin;” T B (1)
6028A, March 1962. (3) Dallavalle, J. M., “Micromeritics,” 2nd ed., pp. 134-43, Pitman Publishing CorD.. New York. 1948. (4) kamiiton P. B., Ann. N . Y . Acad.
Sci. 102, 5: (1962). (5) Hamilton, P.‘ B., ANAL. CHEM. 35, 2055 (1963) :hemica1 Engineers’ (6) ~, Perrv. J.’ H.. HandKook,” 3rd ed., pp. 327-340, McGraw-Hi 11, New York, 1950. (7) Spackman, D. H., Stein, W. H., Moore, S., ANAL.CHEM.30,1190 (19,58). 18) SDackman. D. H.. Fedei.ation Proc. 22:&2, 244 (1963). ~I
RECEIVED for review November 2, 1964. Accepted May 24, 1965.
ion Exchange Separation of Americium and Cerium HAZEL
D. PERDUE
and HARRY G. HICKS
Lawrence Radiation Laboratory, University o f California, Livermore, Calif.
b Tracer quantities of americium-24 1 ( 1 06 disintegrations per minute) and 30 mg. of cerium have been separated quantitatively using Dowex 50W X12, 100- to 200-mesh, ion exchange resin and ammonium lactate as the eluant. In the present procedure small cdumns are used, and the use of a fraction collector is eliminated.
C
can be separated from the rare earths by extraction (3) of Ce(1V) by diethylhexyl phosphoric acid; radiochemical analyses for fission product cerium have been developed using this property. Usually the cerium is back-extracted, precipitated as the oxalate, ignited, and weighed. In this case, there is a small amount of phosphate (either organic or inorganic) carried along with the cerium, which renders the composition of the ignited oxalate indefinite. To eliminate the contamination, the cerium is absorbed ERIUM
1 1 10
e
ANALYTICAL CHEMISTRY
on cation exchange resin, washed well with dilute nitric acid, and eluted rapidly with ammonium lactate before the oxalate precipitation. When the analysis is made from gram amounts of plutonium, there is, in addition, a large amount of Am*41(IO9 disintegrations per minute) present from the decay of Puzrl. The oxidation of Ce(II1) to Ce(1V) with in strong H N 0 3 solutions will oxidize a small fraction (-0.1%) of the Am(II1) to Am(V) which extracts into diethylhexyl phosphoric acid ( I ) and is not subsequently separated from the cerium. The radioactivity of the remaining Amzr1 is enough to interfere seriously with the measurement of CeI4l and Ce144 radioactivities. The object of this study is to find a rapid, simple separation of americium from 30 mg. of cerium that is compatible with the existing radiochemical procedures. The relatively large difference in cation exchange behavior between Ce(II1) and Am(II1)
(4) suggested that the separation could be carried out using a variation of the ion exchange procedure already in use. EXPERIMENTAL
Apparatus. The ion exchange column was 9 cm. long, 6 mm. in diameter, and had a reservoir 16 mm. in diameter. Solutions were added to the reservoir a t the top of the resin column and allowed to flow by gravity into collection tubes. Gamma radiations from Am241and Ce144-Pr*44 in the collection tubes were counted directly by a well-type NaI(T1) scintillation crvstal. a photomultiplier tube, an aiplifier, and a scaling unit. Reaeents. The Dowex 50W X12 100- co 200-mesh resin used was purchased from the BioRad Co., Richmond, Calif., and was used without further treatment. Lactic acid was purchased from J. T. Baker and was used without further purification. Ammonium lactate solutions were prepared by diluting t h e
