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Multipurpose resins for analysis of amino acids and ninhydrin-positive compounds in hydrolyzates and physiological fluids
ANALYTICAL
BIOCHEMISTRY
Multipurpose
3,
477-493
Resins
(1972)
for
Ninhydrin-Positive
Analysis
of Amino
Compounds
and
Physiological
JAMES
April
Fluids
V. BENSON,
12, 1972;
accepted
and
in Hydrolyzates
JR.
Hamilton Company, P. 0. Box 7500, 4960 Energy Received
Acids
Way, July
Reno,
Nevada
89602
17, 1972
In earlier publications we reported on a resin system and simple hydrolyzate procedure that would analyze the common 19 or 20 amino acids in 2 or 4 hr (I) on an automatic amino acid analyzer of the Spackman, Stein, and Moore (2) type. Then we reported the accelerated analysis of over 50 amino acids and related compounds by a procedure that could detect and resolve a greater number of ninhydrin-positive compounds than the simple procedure. The more complex procedure required a different resin system (3) and took 11 hr. In 1967, the separation of glutamine from asparagine was added to the procedure (without sacrifice of resolution elsewhere) using lithium citrate buffers on yet a third resin (4). We now report a new single-resin system capable of performing all of the above procedures with improved resolution, shorter analysis times, and reduced operating back-pressures. The new resin system uses HPAN 90 resin to resolve acidic and neutral amino acids by (I) a 2 hr hydrolyzate (2) a 4 hr hydrolyzate (3) a complex procedure and (4) a complex procedure separation) -lithium citrate
procedure, procedure, for physiological for physiological buffers,
fluids-sodium
citrate buffers,
fluids (glutamine-asparagine
and type HP-B 80 resin to resolve (1) basic amino acids using hydrolyzate
procedures, (2) basic amino acids using physiological procedures, (3) specific amino acids using a rapid screening procedure (4) peptides (6).
(5), and
The analysis time for the physiological procedure was reduced to about 6 hr and with improved resolution (from the previously reported 11.5 hr) . Copyright All rights
@ 1972 by Academic Press, of reproduction in any form
477 Inc. reserved.
90)
4
*Stock Solution: 50 mg in 10 ml 75% ethanol.
vol., liters
4
Final
78.4 32.3 10 0.4
78.4 49.2 10 0.4
Sodium citrate,2H,O, gm Cont. HCl, ml Thiodiglycol, ml Pentachlorophenol,” ml
0.20 0.066
4.30 f 0.02
0.20 0.066
7.28 jz 0.01
(HP-AN
Acidic-neut)rals
Sodium concn., N Citrate concn., M
Component
-
4 hr procedure
4
137.3 25.6 0.4
0.35 0.117
(HP-B 80)
4
78 4 46.7 10 0.4
0.20 0.066
4
78.4 32.3 10 0.4
0.20 0.066
4.30 f 0.02
4
137.3 25.6 0.4
0.35 0.117
5.36 + 0.02
Basics
pH at 25°C 5.36 k 0.02 3.49 + 0.01
(HP-AN
Acidic-neutrals
2 hr procedure
(HP-B 80)
--
Procedurea)
90)
Basics
TABLE 1 Sodium Citrate Buffers (Hydrolyzate
.;;
02 .d 8 z $ 3
ANALYSIS
OF
AMINO
TABLE 2 Sodium Citrate Buffers (Physiological
(HP-AN
Sodium cancn., N Citrate wncn., M Sodium citrate:2HzO, gm Cont. HCl, ml Thiodiglycol, ml Pentachlorophenol, ml Final vol., liters
Procedure)
Acidic-neutral
-
Component
479
ACIDS
Basics
90)
PH 3.20 + 0.01 4.30 f 0.02 4.26 + 0.02 0.20 0.066 78.4 51.0 10 0.4
0.20 0.066 78.4 32.3 10 0.4
4
-
(HP-B 80)
4
6.32 f 0.02
0.38 0.127 149.0 61.0 0.4
0.50 0.166 196.0 21.7 0.4
4
4
Acidic and neutral components (including glutamine and asparagine) can be separated in 205 min (formerly 270 min) using lithium citrate buffers. The operating column back-pressures have been reduced for all procedures. REAGENTS
Sodium Citrate Buffers. The buffers used for the analysis of the acidic, neutral, and basic amino acids, and related components of the simple and complex methodologies are presented in Tables l-2. Lithium Citrate Buffers. The preparation for the buffers used for analysis of the acidic and neutral amino acids and related components is presented in Table 3.
Lithium
TABLE 3 Citrate Buffers Acidic and neutral analyses (HP-AN 90 resin)
Component Lithium concn., N Citrate concn., M Lithium hydroxide, gm Citric acid, gm Thiodiglycol, ml Pentachlorophenol, ml Cont. HCI, ml Find vol., liters
__ pH 2.88 + 0.01 0.30 0.053 50.35 44.55 10 0.4 90.0 4
Sample diluter buffer
pH 4.15 + 0.01 pH 2.2 + 0.03 0.30 0.050 50.34 42.02 10 0.4 75
4
0.30 0.05 6.30 5.25 10 0.05 12 0.6
480
JAMES
V. BENSON,
JR.
Ninhydrin Reagent. The reagent was prepared according to the method of Moore (7) using dimethyl sulfoxide instead of methyl Cellosolve as the ninhydrin solvent. Resins. Hamilton Spherical Chromatographic Resins, type HP-AN 90, lot 111111, a 7.0% cross-linked resin; the spherical particles have a diameter of 7-20 p. Type HP-B 80 resin, lot 102041, is 7.75% crosslinked, the spherical particles having a diameter of 7-10,~. These resins are copolymers of styrene-divinylbenzene strong sulfonic acid cation exchanger. SAMPLE
Synthetic Mixture
PREPARATION
of Amino Acids and Related Compounds?
Type-H Amino Acid Calibration Standard was employed for the simple analytical procedure that is normally used for protein hydrolyzates. The standard of acidic and neutral amino acids and related compounds contains 2.500 f: 0.004 PmoleJml and was diluted, 1:4 with pH 2.2 (0.2 N) sample diluter buffer. Sample quantities and composition are given in Fig. 1. Type P-AN Amino Acid Calibration Standard was used for identification and peak quantitation by the more complex technical procedure of the acidic and neutral amino acids and related compounds. See Fig. 3. Type P-B Amino Acid Calibration Standard, for identification and quantitation of amino acid peaks and related compounds by the basic physiological procedure is shown in Fig. 6. Human Blood Plasma. A sample of Hyland Chemistry Control Serum 0369R007A12 was deproteinized with sulfosalicylic acid by a method previously described (4). See Figs. 4 and 7. Human Urine. Hyland Urine Chemistry control lot No. 0401 T0022A1.2 was prepared as previously described (3). See Figs. 5 and 8. PREPARATION
OF ION-EXCHANGE
COLUMNS
Sodium Citrate Buffer System. The resin was slurried with 2 vol starting buffer, without Brij-35. The resin slurry was then degassed in a vacuum filtration flask. The top of the flask was stoppered and the side arm of the flask was connected to a water aspirator. Aspiration was continued until the slurry was free of air bubbles. Before pouring the column,3 about 10 ml buffer was forced, with nitrogen pressure, through ‘Hamilton Company, P. 0. Box 7500, Reno, Nevada 89502. ‘Hyland Laboratories, P. 0. Box 2214, Costa Mesa, California 92626. *Precision bore borosilicate glass tubing, Wilmad Glass Co. Buena, New Jersey 08310.
ANALYSIS
OF AMINO
ACIDS
481
the stainless-steel screen disc4 and bottom column fitting to remove any air. About 1 cm of buffer was left above the screen. The resin slurry was poured down the side of the’ column (1) until the column was filled according to the method previously described. The same buffer flow rate and column temperature were employed as described in the analysis being used. Lithium Citrate Buffer System. The Na+ form of the resin was first converted to the lithium form prior to packing the column. The resin was washed successively with 250 ml 2% lithium hydroxide solution, 200 ml deionized water, and finally 250 ml starting lithium citrate buffer. The resin was allowed to further equilibrate overnight in the starting buffer (3 vol buffer to ~1 vol resin slurry). The column was packed as described above, using the starting buffer and column temperature outlined in the analytical procedure (4).
CHROMATOGRAPHIC
Hydrolyzate
PROCEDURES
Procedure
Two Hour Methodology
Acidic and Neutral Amino Acids. The buffer flow rate was 70 ml/hr and the ninhydrin flow rate was 35 ml/hr on the 56.0 X 0.9 cm resin column of type HP-AN 90. Elution was started using the pH 3.49 sodium citrate buffer. A buffer change was made at 37 min to one having a pH of 4.30. A column temperature of 53.5”C was used throughout the analysis. The column operating back-pressure was 130 psig and the analysis time 130 min. A single-length reaction coil was used (11.25 ml). The cuvet path length was 6.6 mm for the 570 and 440 nm wavelengths. Basic Amino Acids. A 5 cm column of type HP-B 80 resin was used at a buffer flow rate of 70 ml/hr and a ninhydrin flow rate of 35 ml/hr. A single buffer, pH 5.36, and a single temperature, 53.5”C, were used throughout the analysis. The column pressure was 60 psig and the analysis time 45 min. By overlapping procedures (that is, to start the basic column and then the acidic-neutral column to drain position before completion of the basic analysis) it is possible to complete the analysis in just a little over 2 hr. ‘Stainless&eel screen resin column support, 0.9 cm diameter, 5p average particle retention size (part no. 777241, Hamilton. Company, P. 0. Box 7500, Reno, Nevada 89502.
482 Four Hour
JAMES
V. BENSON,
JR.
Methodology
Acidic and Neutral Amino Acids. Using the same HP-AN 90 resin as in the 2 hr procedure would give a slight improvement in resolution of the amino acid peaks. The controls are less critical and exact pH of the buffer is not as essential as with the more accelerated 2 hr methodology. A column temperature of 55.5”C is used for the analysis. A buffer flow of 70 ml/hr and a ninhydrin flow rate of 35 mlJhr are used. The same length 56 cm resin column employs the pH 3.28 sodium citrate buffer changing at 90 min to the pH 4.30 buffer, using a 35 min buffer gradient. Analysis time is 170 min and the column back-pressure 120 psig. Basic Amino Acids. The same resin column and procedures are used for the basic column as shown above under the 2 hr methodology at 55.5”C. Physiological Sodium Citrate
Procedures
Buffer System
The buffer flow rate of 100 ml/hr was used to pack the resin column and perform the analysis on the type HP-AN 90 resin. A ninhydrin flow rate of 50 mljhr was used. A column temperature of 32.3”C was used at the start of the analysis, changing to 62.0” at 60 min, using a 27 min time gradient. Elution was started using the pH 3.20 sodium citrate buffer. A buffer change was made at 90 min to pH 4.30. The operating back-pressure was 245 psig on the 56 cm column. Basic Amino Acids. The same HP-B 80 resin was used to chromatograph the basic amino acids and related compounds. Elution of the amino acids was started at a column temperature of 32.3”C, a buffer flow rate of 100 ml/hr, and a ninhydrin flow rate of 50 ml/hr. The column temperature was changed to 62.0” after 155 min (27 min gradient). The pH of the starting citrate buffer was 4.26, which was changed to pH 6.32 after 130 min. The operating column back-pressure was 190 psig. It is possible to complete a complex physiological procedure, in about 5 hr if an overlap procedure can be made. Lithium
Citrate
Buffer
System
A&c and Neutral Amino Acids. The same 56 X 0.9 cm column of HP-AN 90 resin can be used after first converting the resin to the lithium form. A buffer flow rate of 100 ml/hr and a ninhydrin flow rate of 50 ml/hr are used on a column maintained at 4O.O”C throughout the analysis. The first buffer, pH 2.88, is changed to pH 4.15 at 105 min. Chromatographic variables are detailed elsewhere (4). A total analysis time of 205 min has been achieved for resolving glutamine and asparagine
overlap)
(7.5
Peptide
(
hr)
a One column is programmed its last components. At that b Buffer flow rate.
hr)
screening
Rapid
6 hr plus glutamineasparagine
Physiological: 6 hr (5’10”
4 hr
Hydrolyzate: 2 bra
90
90
90
23 X 0.9
23 X 0.9
56 X 0.9
23 X 0.9
56 X 0.9
5 X 0.9
56 X 0.9
5 X 0.9
cm
cm
cm
cm
cm
cm
cm
cm
cm
column
56 X 0.9
Resin
Procedures
Buffer
Systems
cit,rate A: pH A: pH C: pH
Sodium Proc. Proc. Proc.
components
Pyridine acetate pH 3.5 (0.10 M) pH 3.1 (0.20M) pH 5.0 (2.0 M)
Li, Li,
0.053 izI Cit) 0.05OiU Cit,)
just
as the second
(0.35 A’) (0.35N) (0.20N)
system
6.46 3.23 3.28
citsate 3.20 (0.20N) 4.30 (0.2OiV) 4.26 (0.38N) 6.32 (0.5N) citrate 2.88 (0.31V 4.15 (O.3N
Sodium pH pH pH pH Lithium pH pH
citrate 3.49 (0.20N) 4.30 (0.20N) 5.36 (0.35N) cit,rate 3.28 (0.20N) 4.30 (0.2On;) 5.36 (0.35n;)
Buft’er
Sodium pH pH pH Sodium pH pH pH
and
for the time it will elute its first switched to coil position.
80
HP-B
to elute to drain position time it can be automatically
80
HP-AN
Acidic-neutral
80
80
80
90
type
HP-B
HP-B
HP-B
Basic
Basic
HP-AN
Acidic-neutral
HP-AN
HP-B
Basic
Acidic-neut,ral
HP-AN
Resin
4. L’hromatographic
Acidic-neutral
Analysis
‘IAM&
ml/hr)
ml/hr)
column
is eluting
450 psi (90 ml/hr)
165 (90 ml/hr)
255 (100
190 (100
245 (100 ml/hr)
60 (70 ml/hr)
120 (70 ml/hr)
60 (70 ml/hr)
130 (70 ml/hr)*
Column backpressure
484
JAMES
V. BENSON,
JR.
without loss of resolution of the other amino acids and related compounds. Peptides. Tryptic hydrolyzates of hemoglobin, lysozyme, and ribonuclease can be analyzed on a 23 X 0.9 cm column of type HP-B 80 resin. An analysis time of 7.5 hr for each of these peptide mixtures is possible using pyridine-acetate developers as previously described (6). Rapid Screening
Procedure
Amino acids related to the specific diseases-leucinosis, homocystinuria, and hyperprolinemia-may be separated in less than 60 min. The amino acids proline, valine, methionine, alloisoleucine, isoleucine, and leucine may be resolved on a 23 cm column of HP-B 80 resin. The pH 3.23 (0.35 N) sodium citrate buffer is used at a column temperature of 55°C. For the amino acids associated with phenylketonuria, tyrosinosis, and histidinemia-tyrosine, phenylalanine, and histidine are resolved in $8 min. The third buffer, pH 3.28 (0.2 N), on the same column will resolve aspartic acid, glutamic acid, proline, glycine, alanine, and cystine in .@ min. The chromatographic procedures and column preparation are described in detail elsewhere (5). All procedures with their respective buffer systems are summarized in Table 4. RESULTS (A)
Two
Hour
Hydrolyzate
System
(1) Acidic and neutral amino acids (type HP-AN 90 resin). The threonine-serine peak height-to-valley absorbance ratio was 0.09 and for serine-glutamic acid the ratio was 0.21 as shown in Fig. 1. The pH of the first buffer was adjusted to center cystine between proline and glycine and separation was almost complete. The separation of the leueines was almost baseline and the tyrosine-phenylalanine peak heightto-valley ratio was 0.05. (2) Basic amino acids (type HP-B 80 resin). ,Complete separation of tryptophan, which was eluted ahead of lysine, was achieved on the short 5 cm column. The internal standard, #a-amino-/3-guanidinopropionic acid, was well separated from ammonia and arginine, and is also shown in Fig. 1. (B) Four
Hour
Hydrolyzate
System
(1) Acidic and neutral amino acids (type HP-AN 90 resin). The separation of methionine sulfone in this system was complete. Peak height-to-valley ratio of threonine-serine was 0.095. Homocitrulline was
ANALYSIS
OF
AMINO
ACIDS
485
Fm. 1. Analysis of amino acid calibration mixture (type H) containing 0.10 pmole of each amino acid by 2 hr procedure. Basic amino acids and ammonia (short curve on left) are on 5 cm column. Acidic and neutral amino acids are on 56 cm column.
added to the sample and eluted just before valine. Alloisoleucine was we11 separated from methionine and isoleucine. Norleucine was added as an internal standard and eluted just after leucine. Tyrosine and phenylalanine peaks were well resolved. See Fig. 2. (8) Basic amino acids. The same component,s were added to sample as shown in the chromatograms of Fig. 2. A slightly longer column (7 cm) then was used for the basic column in Fig. 1, resulting in a slightly longer analysis time with little improvement in peak resolution. (C) Physiological
Sgs tern (Sodium Citrate Buffers)
(1) Acidic and neutd amino acids (t,ype HP-AN 90). The acidic and neutral amino acids of a synthetic calibration mixture are shown in Fig. 3 at a flow rate of 100 ml/hr. The aspartic acid-threonine peak height-to-valley absorbance ratio was 0.25; the same ratio as reported earlier (3), using only a 70 mlJhr buffer flow rate on a resin column capable of performing only a physiological procedure and requiring 2.5 hr longer. Separation of proline-glutamic acid-citrulline was almost complete and the peak height-to-valley ratio of tyrosine-phenylalanine was 0.15.
486
FIQ.
amino
JAMES
2. Analysis of amino acid by 4 hr procedure.
acid
V. BENSON,
calibration
mixture
JR.
containing
0.10 &mole
of each
FIG. 2. Analysis of synthetic mixture (type P-AN) of acidic and neutral amino acids and related compounds found in physiological fluids on 56 cm resin column (HP-AN 90 resin). The following amino acids and their respective amounts (pmole) are : phosphoserine, phosphoethanolamine, taurine, asparagine, sarcosine, cystathionine, and p-alanine (0.050) ; glycerophosphoethanolamine and norleucine (0.060) ; urea (1.500) ; citrulline, a-aminoadipic acid, and a-amino-n-butyric acid (0.025). AU others were present in 0.100 pmole quantities.
ANALYSIS
OF
AMINO
487
ACIDS
FIG. 4. Amino acid composition of protein-free human blood plasma on spherica resin. A sample corresponding to 0.75 ml was used for determination of acidic and neutral components.
FIG. 5. Determination Sample
load
was 0.50 ml
of acidic urine.
and
neutral
amino
acids
of normal
human
urine.
488
JAMES
V. BENSON,
JR.
The analysis of human plasma is shown in the chromatogram of Fig. 4. A sample corresponding to 0.75 ml was analyzed. The chromatographic analysis of human urine is presented in Fig. 5. A 0.50 ml sample was chromatographed for the acidic and neutral components.. The taurineurea peaks, a difficult to separate doublet, are adequately resolved in urine. (2) Basic amino acids (type HP-B 80). Analysis of a synthetic mixture of amino acids and related compounds is shown in Fig. 6. There are several peaks which are difficult to resolve and which appear in many physiological samples. These amino acid peaks are ethanolamine and ammonia, which are present in urine and other physiological samples. It is essential that separation of these peaks be a resin requirement for the complex methodologies. Lysine and 1-methylhistidine are another difficult to separate area of the analysis and, as shown on the chromatogram, are well separated. The chromatographic analysis of human plasma is presented in Fig. 7. A 0.75 ml sample was chromatographed for the basic components. The analysis of human urine is shown in Fig. 8. A sample size corresponding to 0.60 ml was analyzed.
_. 6. Analysis of synthetic mixture (type P-B) of basic ammo acids and related compounds on 23 cm resin column (HP-B 30 resin). The following ammo acids and their amounts (#mole) are: anserine (0.050) and creatinine (0.660). All others were present in 0.100 pmole quantities. FIG.
ANALYSIS
OF
AMINO
489
ACIDS
FIQ. 7. Analysis of basic amino acids of human blood plasma. A sample corresponding to 0.75 ml plasma was added to the column.
L
do
do
40
$0.
$0
FIG. 8. Determination of basic amino acids and related compounds of normal deammoniated urine on spherical resin. A sample corresponding to 0.60 ml was analyzed.
490
JAMES
V.
BENSON,
JR.
FIG. 9. Analysis of synthetic mixture of amino acids and related compounds using lithium citrate buffers on 56 cm column of HP-AN 90 resin for acidic and neutral compounds.
(D) Physiological
System
(Glutamine-Asparagine
Separation)
(1) Acidic and neutral amino acids (type HP-AN 90). Development of the lithium citrate buffer system allows the separation of glutamine and asparagine, without interference from other amino acids and related compounds. At a buffer flow rate of 100 ml/hr there was little if any loss of peak resolution over that previously reported. A faster analysis time than previously reported was achieved, 205 min. See Fig. 9. DISCUSSION
Earlier, the performance of two spherical resins, type AA-15 and type AA-27 for the chromatography of amino acids was reported (1). With these resins, analysis time was reduced significantly compared with the systems using the classical pulverized resins for the analyses of hydrolysates. The introduction of spherical resins made it possible to accelerate the analysis of amino acids and to use shorter columns. Higher buffer flow rates were possible. The new resins resulted in higher and more discrete chromatographic peaks, as compared with those obtained using pulverized resins, because the amino acids were being eluted from the column in narrower bands. Much of the improvement in peak resolution was also due to advancements in design and reliability of the amino acid analyzers using these improved resins.
ANALYSIS
OF
AMINO
ACIDS
491
The goal of this work has been to further optimize the resin and chromatographic procedures for the separation of amino acids employing the complex physiological procedures and also the simple procedure for hydrolyzates, That these aims have been met adequately is demonstrated by the chromatographic results obtained. In 1969, Long and Geiger (8) made two significant observations: (a) any resin available which is capable of resolving the components of a hydrolyzate would probably be unable to separate a physiological fluid, and (b) though one may try to control variables from batch to batch, gross variations in capability appear in resins. The latter point has been thoroughly covered by Hamilton (9), who lists five departures from ideality which account for variations in repetitive batches: (1) irregular shape, (2) wide range of diameter, (3) variations in cross-linking from bead to bead, (4) nonhomogeneity, and (5) incomplete sulfonation. To find the optimal resin for separating acidic and neutral components in fluids, a wide range of cross-linkings (&lO%) in 1/h% increments was examined. Four procedures were established (2 and 4 hr hydrolyzate, sodium citrate and lithium citrate buffers for physiological) and performed identically on each individual resin. Results were studied and the single resin which produced the best results (7.0% cross-linked) was selected as the cross-linking most likely to succeed at performing all functions. Next, each of the four methodologies was studied individually to optimize resolution via small changes (buffer pH, column temperature, flow rate, etc.). Thus, each method alone was perfected; they equaled or bettered the performance of previous resins. A similar study of resins for resolution of basic components revealed that ethanolamine/ammonia and lysine 1-methylhistidine were most affected by variations in cross-linking. A 7.75% cross-linked polymer was selected as the optimal resin for basic amino acids. The reproducible nature of these resins can be attributed to greater attention to process variables. All resins in this study were made in smallbatch processes. Certain variations are inherent in large-batch production: e.g., use of practical grade monomers, wide particle size distribution, and consequent wide variation in cross-linking. By employing purified monomers and small-scale operation we were able to prepare a polymer relatively free of linear material and to narrow the size range of the batch (10). Atkin and Ferdinand noted the effect of proportions of m-DVB and p-DVB in the styrene (11). m-DYE3 gives uniform cross-linking spaced evenly throughout the bead and sulfonates faster than p-DVB. p-DE%, on the other hand, gives a tightly cross-linked nucleus. TQ eliminate
492
JAMES
V. BENSON,
JR.
this variability, the same lot of divinylbenxene was used in all polymerizations. The ultimate criterion of batch reproduction is column performancedoes the resin separate the components according to the methodology? Resins were made specifically in an effort to reproduce HP-AN 90 once it was apparent that this was the optimal resin. The result was several batches, all giving the same resolving capabilities as the model resin. Mixing of batches produced no loss in resolution. SUMMARY
The newly available spherical cation-exchange resins HP-AN 90 and HP-B 80 are capable of performing various analyses of amino acids and peptides at low backpressures. These multipurpose resins can perform: a 2 or 4 hr simple (approximately 20 amino acids) procedure used with protein hydrolyzates, or a complex (50 or more amino acids) technical procedure (for physiological fluids), or a rapid screening procedure dealing with specific amino acids as they relate to clinical disease or biomedical studies, or the analysis of peptide samples. Analysis time for acidic and neutral amino acids in a physiological fluid, using sodium citrate buffers, has been reduced from the previously reported 11.5 hr to about 6 hr. With lithium citrate buffers, the chromatographic separation of glutamine and asparagine without sacrifice of resolution of other amino acids can now be completed in 205 min using the same HP-AN 90 resin. It is now possible to analyze four complex samples (acidic, neutral, and basic components) per day. REFERENCES J. V., JR., AND PATTERBON, J. A., Accelerated automatic chromatographic analysis of amino acids on a spherical resin, And. Chem. 37, 1108 (1965). SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Automatic recording apparatus for use in the chromatography of amino acids, Anal. Chem. 30, 1190 (1958). BENEW)N, J. V., JR., AND PATTERSON, J. A., Accelerated chromatographic analysis of amino acids commonly found in physiological fluids on a spherical resin of specific design, Anal. Biochem. 13, 265 (1965). BENSON, J. V., JR., GORGON, M. J., AND PATTERSON, J. A., Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamine and asparagine, Anal. Biochem. 18, 228 (1967). BENMN, J. V., JR., C~RMICK, J., AND PATTERSON, J. A., Accelerated chromatography of amino acids associated with phenylketonuria, leucinosis (maple syrup urine disease), and other inborn errors of metabolism, Anal. Biochem. 18, 481 (1967). BENMN, J. V., JR., JONES, R. T., CORMICK, J., AND PAWERSON, J. A., Accelerated automatic chromatographic analysis of peptides on a spherical resin, Anal. Biochem. 16, 91 (1966).
1. BENSON, 2. 3.
4.
5.
6.
ANALYSIS
OF
AMINO
ACIDS
493
7. MOORE, S., Amino acid analysis: aqueous dimethylsulfoxide as solvent for the ninhydrin reaction, J. Biol. Chem. 243, 6281 (1968). 8. Lola, C. L., AND GEIGER, J. W., Automatic analysis of amino acids: effect of resin cross-linking and operational variables on resolution, Anal. Biochem. 29, 265 (1969). 9. HAMILTON, P. B., in “Advances in Chromatography,” Vol. 2 (J. C. Giddings and R. A. Keller, eds.), Chapter 1. Dekker, New York, 1966. 10. RAHM, J., WEINOVA, H., AND PROCHAZKA, Z., The effect of the conditions of preparation of cation-exchange resins on their resolution efficiecny in ion-exchange chromatography of amino acids, J. Chromatogr. 60, 256 (1971). 11. ATKIN, G. E., AND FERDINAND, W., Accelerated amino acid analysis with lithium citrate buffers, J. Chromalo~r. 62, 373 (1971).
BIOCHEMISTRY
Multipurpose
3,
477-493
Resins
(1972)
for
Ninhydrin-Positive
Analysis
of Amino
Compounds
and
Physiological
JAMES
April
Fluids
V. BENSON,
12, 1972;
accepted
and
in Hydrolyzates
JR.
Hamilton Company, P. 0. Box 7500, 4960 Energy Received
Acids
Way, July
Reno,
Nevada
89602
17, 1972
In earlier publications we reported on a resin system and simple hydrolyzate procedure that would analyze the common 19 or 20 amino acids in 2 or 4 hr (I) on an automatic amino acid analyzer of the Spackman, Stein, and Moore (2) type. Then we reported the accelerated analysis of over 50 amino acids and related compounds by a procedure that could detect and resolve a greater number of ninhydrin-positive compounds than the simple procedure. The more complex procedure required a different resin system (3) and took 11 hr. In 1967, the separation of glutamine from asparagine was added to the procedure (without sacrifice of resolution elsewhere) using lithium citrate buffers on yet a third resin (4). We now report a new single-resin system capable of performing all of the above procedures with improved resolution, shorter analysis times, and reduced operating back-pressures. The new resin system uses HPAN 90 resin to resolve acidic and neutral amino acids by (I) a 2 hr hydrolyzate (2) a 4 hr hydrolyzate (3) a complex procedure and (4) a complex procedure separation) -lithium citrate
procedure, procedure, for physiological for physiological buffers,
fluids-sodium
citrate buffers,
fluids (glutamine-asparagine
and type HP-B 80 resin to resolve (1) basic amino acids using hydrolyzate
procedures, (2) basic amino acids using physiological procedures, (3) specific amino acids using a rapid screening procedure (4) peptides (6).
(5), and
The analysis time for the physiological procedure was reduced to about 6 hr and with improved resolution (from the previously reported 11.5 hr) . Copyright All rights
@ 1972 by Academic Press, of reproduction in any form
477 Inc. reserved.
90)
4
*Stock Solution: 50 mg in 10 ml 75% ethanol.
vol., liters
4
Final
78.4 32.3 10 0.4
78.4 49.2 10 0.4
Sodium citrate,2H,O, gm Cont. HCl, ml Thiodiglycol, ml Pentachlorophenol,” ml
0.20 0.066
4.30 f 0.02
0.20 0.066
7.28 jz 0.01
(HP-AN
Acidic-neut)rals
Sodium concn., N Citrate concn., M
Component
-
4 hr procedure
4
137.3 25.6 0.4
0.35 0.117
(HP-B 80)
4
78 4 46.7 10 0.4
0.20 0.066
4
78.4 32.3 10 0.4
0.20 0.066
4.30 f 0.02
4
137.3 25.6 0.4
0.35 0.117
5.36 + 0.02
Basics
pH at 25°C 5.36 k 0.02 3.49 + 0.01
(HP-AN
Acidic-neutrals
2 hr procedure
(HP-B 80)
--
Procedurea)
90)
Basics
TABLE 1 Sodium Citrate Buffers (Hydrolyzate
.;;
02 .d 8 z $ 3
ANALYSIS
OF
AMINO
TABLE 2 Sodium Citrate Buffers (Physiological
(HP-AN
Sodium cancn., N Citrate wncn., M Sodium citrate:2HzO, gm Cont. HCl, ml Thiodiglycol, ml Pentachlorophenol, ml Final vol., liters
Procedure)
Acidic-neutral
-
Component
479
ACIDS
Basics
90)
PH 3.20 + 0.01 4.30 f 0.02 4.26 + 0.02 0.20 0.066 78.4 51.0 10 0.4
0.20 0.066 78.4 32.3 10 0.4
4
-
(HP-B 80)
4
6.32 f 0.02
0.38 0.127 149.0 61.0 0.4
0.50 0.166 196.0 21.7 0.4
4
4
Acidic and neutral components (including glutamine and asparagine) can be separated in 205 min (formerly 270 min) using lithium citrate buffers. The operating column back-pressures have been reduced for all procedures. REAGENTS
Sodium Citrate Buffers. The buffers used for the analysis of the acidic, neutral, and basic amino acids, and related components of the simple and complex methodologies are presented in Tables l-2. Lithium Citrate Buffers. The preparation for the buffers used for analysis of the acidic and neutral amino acids and related components is presented in Table 3.
Lithium
TABLE 3 Citrate Buffers Acidic and neutral analyses (HP-AN 90 resin)
Component Lithium concn., N Citrate concn., M Lithium hydroxide, gm Citric acid, gm Thiodiglycol, ml Pentachlorophenol, ml Cont. HCI, ml Find vol., liters
__ pH 2.88 + 0.01 0.30 0.053 50.35 44.55 10 0.4 90.0 4
Sample diluter buffer
pH 4.15 + 0.01 pH 2.2 + 0.03 0.30 0.050 50.34 42.02 10 0.4 75
4
0.30 0.05 6.30 5.25 10 0.05 12 0.6
480
JAMES
V. BENSON,
JR.
Ninhydrin Reagent. The reagent was prepared according to the method of Moore (7) using dimethyl sulfoxide instead of methyl Cellosolve as the ninhydrin solvent. Resins. Hamilton Spherical Chromatographic Resins, type HP-AN 90, lot 111111, a 7.0% cross-linked resin; the spherical particles have a diameter of 7-20 p. Type HP-B 80 resin, lot 102041, is 7.75% crosslinked, the spherical particles having a diameter of 7-10,~. These resins are copolymers of styrene-divinylbenzene strong sulfonic acid cation exchanger. SAMPLE
Synthetic Mixture
PREPARATION
of Amino Acids and Related Compounds?
Type-H Amino Acid Calibration Standard was employed for the simple analytical procedure that is normally used for protein hydrolyzates. The standard of acidic and neutral amino acids and related compounds contains 2.500 f: 0.004 PmoleJml and was diluted, 1:4 with pH 2.2 (0.2 N) sample diluter buffer. Sample quantities and composition are given in Fig. 1. Type P-AN Amino Acid Calibration Standard was used for identification and peak quantitation by the more complex technical procedure of the acidic and neutral amino acids and related compounds. See Fig. 3. Type P-B Amino Acid Calibration Standard, for identification and quantitation of amino acid peaks and related compounds by the basic physiological procedure is shown in Fig. 6. Human Blood Plasma. A sample of Hyland Chemistry Control Serum 0369R007A12 was deproteinized with sulfosalicylic acid by a method previously described (4). See Figs. 4 and 7. Human Urine. Hyland Urine Chemistry control lot No. 0401 T0022A1.2 was prepared as previously described (3). See Figs. 5 and 8. PREPARATION
OF ION-EXCHANGE
COLUMNS
Sodium Citrate Buffer System. The resin was slurried with 2 vol starting buffer, without Brij-35. The resin slurry was then degassed in a vacuum filtration flask. The top of the flask was stoppered and the side arm of the flask was connected to a water aspirator. Aspiration was continued until the slurry was free of air bubbles. Before pouring the column,3 about 10 ml buffer was forced, with nitrogen pressure, through ‘Hamilton Company, P. 0. Box 7500, Reno, Nevada 89502. ‘Hyland Laboratories, P. 0. Box 2214, Costa Mesa, California 92626. *Precision bore borosilicate glass tubing, Wilmad Glass Co. Buena, New Jersey 08310.
ANALYSIS
OF AMINO
ACIDS
481
the stainless-steel screen disc4 and bottom column fitting to remove any air. About 1 cm of buffer was left above the screen. The resin slurry was poured down the side of the’ column (1) until the column was filled according to the method previously described. The same buffer flow rate and column temperature were employed as described in the analysis being used. Lithium Citrate Buffer System. The Na+ form of the resin was first converted to the lithium form prior to packing the column. The resin was washed successively with 250 ml 2% lithium hydroxide solution, 200 ml deionized water, and finally 250 ml starting lithium citrate buffer. The resin was allowed to further equilibrate overnight in the starting buffer (3 vol buffer to ~1 vol resin slurry). The column was packed as described above, using the starting buffer and column temperature outlined in the analytical procedure (4).
CHROMATOGRAPHIC
Hydrolyzate
PROCEDURES
Procedure
Two Hour Methodology
Acidic and Neutral Amino Acids. The buffer flow rate was 70 ml/hr and the ninhydrin flow rate was 35 ml/hr on the 56.0 X 0.9 cm resin column of type HP-AN 90. Elution was started using the pH 3.49 sodium citrate buffer. A buffer change was made at 37 min to one having a pH of 4.30. A column temperature of 53.5”C was used throughout the analysis. The column operating back-pressure was 130 psig and the analysis time 130 min. A single-length reaction coil was used (11.25 ml). The cuvet path length was 6.6 mm for the 570 and 440 nm wavelengths. Basic Amino Acids. A 5 cm column of type HP-B 80 resin was used at a buffer flow rate of 70 ml/hr and a ninhydrin flow rate of 35 ml/hr. A single buffer, pH 5.36, and a single temperature, 53.5”C, were used throughout the analysis. The column pressure was 60 psig and the analysis time 45 min. By overlapping procedures (that is, to start the basic column and then the acidic-neutral column to drain position before completion of the basic analysis) it is possible to complete the analysis in just a little over 2 hr. ‘Stainless&eel screen resin column support, 0.9 cm diameter, 5p average particle retention size (part no. 777241, Hamilton. Company, P. 0. Box 7500, Reno, Nevada 89502.
482 Four Hour
JAMES
V. BENSON,
JR.
Methodology
Acidic and Neutral Amino Acids. Using the same HP-AN 90 resin as in the 2 hr procedure would give a slight improvement in resolution of the amino acid peaks. The controls are less critical and exact pH of the buffer is not as essential as with the more accelerated 2 hr methodology. A column temperature of 55.5”C is used for the analysis. A buffer flow of 70 ml/hr and a ninhydrin flow rate of 35 mlJhr are used. The same length 56 cm resin column employs the pH 3.28 sodium citrate buffer changing at 90 min to the pH 4.30 buffer, using a 35 min buffer gradient. Analysis time is 170 min and the column back-pressure 120 psig. Basic Amino Acids. The same resin column and procedures are used for the basic column as shown above under the 2 hr methodology at 55.5”C. Physiological Sodium Citrate
Procedures
Buffer System
The buffer flow rate of 100 ml/hr was used to pack the resin column and perform the analysis on the type HP-AN 90 resin. A ninhydrin flow rate of 50 mljhr was used. A column temperature of 32.3”C was used at the start of the analysis, changing to 62.0” at 60 min, using a 27 min time gradient. Elution was started using the pH 3.20 sodium citrate buffer. A buffer change was made at 90 min to pH 4.30. The operating back-pressure was 245 psig on the 56 cm column. Basic Amino Acids. The same HP-B 80 resin was used to chromatograph the basic amino acids and related compounds. Elution of the amino acids was started at a column temperature of 32.3”C, a buffer flow rate of 100 ml/hr, and a ninhydrin flow rate of 50 ml/hr. The column temperature was changed to 62.0” after 155 min (27 min gradient). The pH of the starting citrate buffer was 4.26, which was changed to pH 6.32 after 130 min. The operating column back-pressure was 190 psig. It is possible to complete a complex physiological procedure, in about 5 hr if an overlap procedure can be made. Lithium
Citrate
Buffer
System
A&c and Neutral Amino Acids. The same 56 X 0.9 cm column of HP-AN 90 resin can be used after first converting the resin to the lithium form. A buffer flow rate of 100 ml/hr and a ninhydrin flow rate of 50 ml/hr are used on a column maintained at 4O.O”C throughout the analysis. The first buffer, pH 2.88, is changed to pH 4.15 at 105 min. Chromatographic variables are detailed elsewhere (4). A total analysis time of 205 min has been achieved for resolving glutamine and asparagine
overlap)
(7.5
Peptide
(
hr)
a One column is programmed its last components. At that b Buffer flow rate.
hr)
screening
Rapid
6 hr plus glutamineasparagine
Physiological: 6 hr (5’10”
4 hr
Hydrolyzate: 2 bra
90
90
90
23 X 0.9
23 X 0.9
56 X 0.9
23 X 0.9
56 X 0.9
5 X 0.9
56 X 0.9
5 X 0.9
cm
cm
cm
cm
cm
cm
cm
cm
cm
column
56 X 0.9
Resin
Procedures
Buffer
Systems
cit,rate A: pH A: pH C: pH
Sodium Proc. Proc. Proc.
components
Pyridine acetate pH 3.5 (0.10 M) pH 3.1 (0.20M) pH 5.0 (2.0 M)
Li, Li,
0.053 izI Cit) 0.05OiU Cit,)
just
as the second
(0.35 A’) (0.35N) (0.20N)
system
6.46 3.23 3.28
citsate 3.20 (0.20N) 4.30 (0.2OiV) 4.26 (0.38N) 6.32 (0.5N) citrate 2.88 (0.31V 4.15 (O.3N
Sodium pH pH pH pH Lithium pH pH
citrate 3.49 (0.20N) 4.30 (0.20N) 5.36 (0.35N) cit,rate 3.28 (0.20N) 4.30 (0.2On;) 5.36 (0.35n;)
Buft’er
Sodium pH pH pH Sodium pH pH pH
and
for the time it will elute its first switched to coil position.
80
HP-B
to elute to drain position time it can be automatically
80
HP-AN
Acidic-neutral
80
80
80
90
type
HP-B
HP-B
HP-B
Basic
Basic
HP-AN
Acidic-neutral
HP-AN
HP-B
Basic
Acidic-neut,ral
HP-AN
Resin
4. L’hromatographic
Acidic-neutral
Analysis
‘IAM&
ml/hr)
ml/hr)
column
is eluting
450 psi (90 ml/hr)
165 (90 ml/hr)
255 (100
190 (100
245 (100 ml/hr)
60 (70 ml/hr)
120 (70 ml/hr)
60 (70 ml/hr)
130 (70 ml/hr)*
Column backpressure
484
JAMES
V. BENSON,
JR.
without loss of resolution of the other amino acids and related compounds. Peptides. Tryptic hydrolyzates of hemoglobin, lysozyme, and ribonuclease can be analyzed on a 23 X 0.9 cm column of type HP-B 80 resin. An analysis time of 7.5 hr for each of these peptide mixtures is possible using pyridine-acetate developers as previously described (6). Rapid Screening
Procedure
Amino acids related to the specific diseases-leucinosis, homocystinuria, and hyperprolinemia-may be separated in less than 60 min. The amino acids proline, valine, methionine, alloisoleucine, isoleucine, and leucine may be resolved on a 23 cm column of HP-B 80 resin. The pH 3.23 (0.35 N) sodium citrate buffer is used at a column temperature of 55°C. For the amino acids associated with phenylketonuria, tyrosinosis, and histidinemia-tyrosine, phenylalanine, and histidine are resolved in $8 min. The third buffer, pH 3.28 (0.2 N), on the same column will resolve aspartic acid, glutamic acid, proline, glycine, alanine, and cystine in .@ min. The chromatographic procedures and column preparation are described in detail elsewhere (5). All procedures with their respective buffer systems are summarized in Table 4. RESULTS (A)
Two
Hour
Hydrolyzate
System
(1) Acidic and neutral amino acids (type HP-AN 90 resin). The threonine-serine peak height-to-valley absorbance ratio was 0.09 and for serine-glutamic acid the ratio was 0.21 as shown in Fig. 1. The pH of the first buffer was adjusted to center cystine between proline and glycine and separation was almost complete. The separation of the leueines was almost baseline and the tyrosine-phenylalanine peak heightto-valley ratio was 0.05. (2) Basic amino acids (type HP-B 80 resin). ,Complete separation of tryptophan, which was eluted ahead of lysine, was achieved on the short 5 cm column. The internal standard, #a-amino-/3-guanidinopropionic acid, was well separated from ammonia and arginine, and is also shown in Fig. 1. (B) Four
Hour
Hydrolyzate
System
(1) Acidic and neutral amino acids (type HP-AN 90 resin). The separation of methionine sulfone in this system was complete. Peak height-to-valley ratio of threonine-serine was 0.095. Homocitrulline was
ANALYSIS
OF
AMINO
ACIDS
485
Fm. 1. Analysis of amino acid calibration mixture (type H) containing 0.10 pmole of each amino acid by 2 hr procedure. Basic amino acids and ammonia (short curve on left) are on 5 cm column. Acidic and neutral amino acids are on 56 cm column.
added to the sample and eluted just before valine. Alloisoleucine was we11 separated from methionine and isoleucine. Norleucine was added as an internal standard and eluted just after leucine. Tyrosine and phenylalanine peaks were well resolved. See Fig. 2. (8) Basic amino acids. The same component,s were added to sample as shown in the chromatograms of Fig. 2. A slightly longer column (7 cm) then was used for the basic column in Fig. 1, resulting in a slightly longer analysis time with little improvement in peak resolution. (C) Physiological
Sgs tern (Sodium Citrate Buffers)
(1) Acidic and neutd amino acids (t,ype HP-AN 90). The acidic and neutral amino acids of a synthetic calibration mixture are shown in Fig. 3 at a flow rate of 100 ml/hr. The aspartic acid-threonine peak height-to-valley absorbance ratio was 0.25; the same ratio as reported earlier (3), using only a 70 mlJhr buffer flow rate on a resin column capable of performing only a physiological procedure and requiring 2.5 hr longer. Separation of proline-glutamic acid-citrulline was almost complete and the peak height-to-valley ratio of tyrosine-phenylalanine was 0.15.
486
FIQ.
amino
JAMES
2. Analysis of amino acid by 4 hr procedure.
acid
V. BENSON,
calibration
mixture
JR.
containing
0.10 &mole
of each
FIG. 2. Analysis of synthetic mixture (type P-AN) of acidic and neutral amino acids and related compounds found in physiological fluids on 56 cm resin column (HP-AN 90 resin). The following amino acids and their respective amounts (pmole) are : phosphoserine, phosphoethanolamine, taurine, asparagine, sarcosine, cystathionine, and p-alanine (0.050) ; glycerophosphoethanolamine and norleucine (0.060) ; urea (1.500) ; citrulline, a-aminoadipic acid, and a-amino-n-butyric acid (0.025). AU others were present in 0.100 pmole quantities.
ANALYSIS
OF
AMINO
487
ACIDS
FIG. 4. Amino acid composition of protein-free human blood plasma on spherica resin. A sample corresponding to 0.75 ml was used for determination of acidic and neutral components.
FIG. 5. Determination Sample
load
was 0.50 ml
of acidic urine.
and
neutral
amino
acids
of normal
human
urine.
488
JAMES
V. BENSON,
JR.
The analysis of human plasma is shown in the chromatogram of Fig. 4. A sample corresponding to 0.75 ml was analyzed. The chromatographic analysis of human urine is presented in Fig. 5. A 0.50 ml sample was chromatographed for the acidic and neutral components.. The taurineurea peaks, a difficult to separate doublet, are adequately resolved in urine. (2) Basic amino acids (type HP-B 80). Analysis of a synthetic mixture of amino acids and related compounds is shown in Fig. 6. There are several peaks which are difficult to resolve and which appear in many physiological samples. These amino acid peaks are ethanolamine and ammonia, which are present in urine and other physiological samples. It is essential that separation of these peaks be a resin requirement for the complex methodologies. Lysine and 1-methylhistidine are another difficult to separate area of the analysis and, as shown on the chromatogram, are well separated. The chromatographic analysis of human plasma is presented in Fig. 7. A 0.75 ml sample was chromatographed for the basic components. The analysis of human urine is shown in Fig. 8. A sample size corresponding to 0.60 ml was analyzed.
_. 6. Analysis of synthetic mixture (type P-B) of basic ammo acids and related compounds on 23 cm resin column (HP-B 30 resin). The following ammo acids and their amounts (#mole) are: anserine (0.050) and creatinine (0.660). All others were present in 0.100 pmole quantities. FIG.
ANALYSIS
OF
AMINO
489
ACIDS
FIQ. 7. Analysis of basic amino acids of human blood plasma. A sample corresponding to 0.75 ml plasma was added to the column.
L
do
do
40
$0.
$0
FIG. 8. Determination of basic amino acids and related compounds of normal deammoniated urine on spherical resin. A sample corresponding to 0.60 ml was analyzed.
490
JAMES
V.
BENSON,
JR.
FIG. 9. Analysis of synthetic mixture of amino acids and related compounds using lithium citrate buffers on 56 cm column of HP-AN 90 resin for acidic and neutral compounds.
(D) Physiological
System
(Glutamine-Asparagine
Separation)
(1) Acidic and neutral amino acids (type HP-AN 90). Development of the lithium citrate buffer system allows the separation of glutamine and asparagine, without interference from other amino acids and related compounds. At a buffer flow rate of 100 ml/hr there was little if any loss of peak resolution over that previously reported. A faster analysis time than previously reported was achieved, 205 min. See Fig. 9. DISCUSSION
Earlier, the performance of two spherical resins, type AA-15 and type AA-27 for the chromatography of amino acids was reported (1). With these resins, analysis time was reduced significantly compared with the systems using the classical pulverized resins for the analyses of hydrolysates. The introduction of spherical resins made it possible to accelerate the analysis of amino acids and to use shorter columns. Higher buffer flow rates were possible. The new resins resulted in higher and more discrete chromatographic peaks, as compared with those obtained using pulverized resins, because the amino acids were being eluted from the column in narrower bands. Much of the improvement in peak resolution was also due to advancements in design and reliability of the amino acid analyzers using these improved resins.
ANALYSIS
OF
AMINO
ACIDS
491
The goal of this work has been to further optimize the resin and chromatographic procedures for the separation of amino acids employing the complex physiological procedures and also the simple procedure for hydrolyzates, That these aims have been met adequately is demonstrated by the chromatographic results obtained. In 1969, Long and Geiger (8) made two significant observations: (a) any resin available which is capable of resolving the components of a hydrolyzate would probably be unable to separate a physiological fluid, and (b) though one may try to control variables from batch to batch, gross variations in capability appear in resins. The latter point has been thoroughly covered by Hamilton (9), who lists five departures from ideality which account for variations in repetitive batches: (1) irregular shape, (2) wide range of diameter, (3) variations in cross-linking from bead to bead, (4) nonhomogeneity, and (5) incomplete sulfonation. To find the optimal resin for separating acidic and neutral components in fluids, a wide range of cross-linkings (&lO%) in 1/h% increments was examined. Four procedures were established (2 and 4 hr hydrolyzate, sodium citrate and lithium citrate buffers for physiological) and performed identically on each individual resin. Results were studied and the single resin which produced the best results (7.0% cross-linked) was selected as the cross-linking most likely to succeed at performing all functions. Next, each of the four methodologies was studied individually to optimize resolution via small changes (buffer pH, column temperature, flow rate, etc.). Thus, each method alone was perfected; they equaled or bettered the performance of previous resins. A similar study of resins for resolution of basic components revealed that ethanolamine/ammonia and lysine 1-methylhistidine were most affected by variations in cross-linking. A 7.75% cross-linked polymer was selected as the optimal resin for basic amino acids. The reproducible nature of these resins can be attributed to greater attention to process variables. All resins in this study were made in smallbatch processes. Certain variations are inherent in large-batch production: e.g., use of practical grade monomers, wide particle size distribution, and consequent wide variation in cross-linking. By employing purified monomers and small-scale operation we were able to prepare a polymer relatively free of linear material and to narrow the size range of the batch (10). Atkin and Ferdinand noted the effect of proportions of m-DVB and p-DVB in the styrene (11). m-DYE3 gives uniform cross-linking spaced evenly throughout the bead and sulfonates faster than p-DVB. p-DE%, on the other hand, gives a tightly cross-linked nucleus. TQ eliminate
492
JAMES
V. BENSON,
JR.
this variability, the same lot of divinylbenxene was used in all polymerizations. The ultimate criterion of batch reproduction is column performancedoes the resin separate the components according to the methodology? Resins were made specifically in an effort to reproduce HP-AN 90 once it was apparent that this was the optimal resin. The result was several batches, all giving the same resolving capabilities as the model resin. Mixing of batches produced no loss in resolution. SUMMARY
The newly available spherical cation-exchange resins HP-AN 90 and HP-B 80 are capable of performing various analyses of amino acids and peptides at low backpressures. These multipurpose resins can perform: a 2 or 4 hr simple (approximately 20 amino acids) procedure used with protein hydrolyzates, or a complex (50 or more amino acids) technical procedure (for physiological fluids), or a rapid screening procedure dealing with specific amino acids as they relate to clinical disease or biomedical studies, or the analysis of peptide samples. Analysis time for acidic and neutral amino acids in a physiological fluid, using sodium citrate buffers, has been reduced from the previously reported 11.5 hr to about 6 hr. With lithium citrate buffers, the chromatographic separation of glutamine and asparagine without sacrifice of resolution of other amino acids can now be completed in 205 min using the same HP-AN 90 resin. It is now possible to analyze four complex samples (acidic, neutral, and basic components) per day. REFERENCES J. V., JR., AND PATTERBON, J. A., Accelerated automatic chromatographic analysis of amino acids on a spherical resin, And. Chem. 37, 1108 (1965). SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Automatic recording apparatus for use in the chromatography of amino acids, Anal. Chem. 30, 1190 (1958). BENEW)N, J. V., JR., AND PATTERSON, J. A., Accelerated chromatographic analysis of amino acids commonly found in physiological fluids on a spherical resin of specific design, Anal. Biochem. 13, 265 (1965). BENSON, J. V., JR., GORGON, M. J., AND PATTERSON, J. A., Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamine and asparagine, Anal. Biochem. 18, 228 (1967). BENMN, J. V., JR., C~RMICK, J., AND PATTERSON, J. A., Accelerated chromatography of amino acids associated with phenylketonuria, leucinosis (maple syrup urine disease), and other inborn errors of metabolism, Anal. Biochem. 18, 481 (1967). BENMN, J. V., JR., JONES, R. T., CORMICK, J., AND PAWERSON, J. A., Accelerated automatic chromatographic analysis of peptides on a spherical resin, Anal. Biochem. 16, 91 (1966).
1. BENSON, 2. 3.
4.
5.
6.
ANALYSIS
OF
AMINO
ACIDS
493
7. MOORE, S., Amino acid analysis: aqueous dimethylsulfoxide as solvent for the ninhydrin reaction, J. Biol. Chem. 243, 6281 (1968). 8. Lola, C. L., AND GEIGER, J. W., Automatic analysis of amino acids: effect of resin cross-linking and operational variables on resolution, Anal. Biochem. 29, 265 (1969). 9. HAMILTON, P. B., in “Advances in Chromatography,” Vol. 2 (J. C. Giddings and R. A. Keller, eds.), Chapter 1. Dekker, New York, 1966. 10. RAHM, J., WEINOVA, H., AND PROCHAZKA, Z., The effect of the conditions of preparation of cation-exchange resins on their resolution efficiecny in ion-exchange chromatography of amino acids, J. Chromatogr. 60, 256 (1971). 11. ATKIN, G. E., AND FERDINAND, W., Accelerated amino acid analysis with lithium citrate buffers, J. Chromalo~r. 62, 373 (1971).