nomenon, some peaks are poorly resolved or not resolved at all. Figure 2 shows the separation of p-methoxyanilides prepared by the polystyryl-diphenylphosphine procedure also. The solvent system was water-methanol. As with wateracetonitrile, retention time increases with molecular weight for a given degree of unsaturation, and increasing unsaturation leads to decreasing retention time. In water-methanol, unlike water-acetonitrile, the gap in retention time between two acids differing by a given number of carbons decreases with increasing retention time. To test the quantitative applicability of the chromatography, a study was made of the yields of the anilides and the relationship of peak height ratio to mole ratio of acids. The yield of anilides averaged 1ooOh.A plot of the peak height ratio of Clo anilide to C12 anilide vs. the mole ratio of the two over a range for 0.1 to 10 gave a straight line that intercepted the abscissa at zero. Thus, by using ap-methoxyanilide as an in-
ternal standard, this HPLC method can be used for quantitative determination of fatty acids. ACKNOWLEDGMENT
The authors thank S. L. Regen and D. P. Lee for helpful suggestions in the preparation and use of polystyryl-diphenylphosphine reagent. LITERATURE CITED (1)
H. D. Durst, M. Milano, E. J. Kikta, Jr., S.A. Connelly,and Eli Grushka, Anal.
(2) (3) (4) (5) (6) (7)
Chem., 47, 1797 (1975). L. E. Barstow and V. J. Hruby, J. Org. Chem., 38, 1305 (1971). J. B. Lee, J. Am. Chem. SOC.,88, 3440 (1966). P. Hodge and G. Richardson, Chem. Commun., 622 (1975). H. M. Relles and R. W. Schluenz, J. Am. Chem. SOC.,96,6469 (1974). D. C. Sheehan and G. P. Hess, J. Am. Chem. SOC.,77, 1067 (1955). R. F. Borch, Anal. Chem., 47,2437 (1975).
RECEIVEDfor review February 12,1976. Accepted April 15, 1976.
Separation of Amino Acids by High Performance Liquid Chromatography Ernst Bayer," Edgar Grom, Berthold Kaltenegger, and Rainer Uhmann lnstitut fur Organische Chemie der Universitat Tubingen, Germany
Amino acids are separated as dansyl derivatives on slllca gel with high performance liquid chromatography. The common protein amino acids are separated in 30 mln. Using a fluorometer, amino acid concentrations below pmol/ml can be detected in the eluent. The effects of pH, time, and dansyl chloride concentration on the dansylation of amino acids were Investigated and the response factors for quantitativeanalysis of the common amino acids measured.
The standard procedure for separation and estimation of amino acids is ion-exchange chromatography as has been developed by Stein and Moore ( I ) , using the ninhydrin reaction. The use of fluorometric detection instead of the ninhydrin reaction has been recently proposed, the amino acids being converted to the fluram derivatives (2). However the advantages of high pressure liquid chromatography, especially the high flow rate of the mobile phase cannot be fully realized with ion-exchange chromatography. The plastic ion-exchange beads are more compressible than solid inorganic materials, and therefore the flow rate of the mobile phase does not vary directly with the applied pressure. Additionally, the eluents used in ion-exchange chromatography are not suitable for extremely sensitive fluorometric estimation. T o use the advantages of modern high performance liquid chromatography, a separation on silica gel seemed to be more appropriate. Because free amino acids require very polar mobile phases for elution from silica1gel, more lipophilic derivatives should be prepared. These derivatives should be detected sensitively. The preparation of derivatives prior to separation is no disadvantage in comparison to the ion-exchange chromatography of amino acids, since a reaction is also performed, the ninhydrin reaction, after separation in the flow system of an amino acid analyzer. If the derivatives are prepared prior to separation, no elaborate flow system is necessary, simplifying the instrumentation. The most sensitive 1106
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
detectors in liquid chromatography are fluorometers. Therefore, derivatives with strong fluorescence were chosen. The fluram derivatives were not applied since secondary amines (e.g., proline) are not easily derivatized and this method still maintains the separation on ion exchangers. Also the pyridoxyl derivatives of amino acids ( 3 )were not considered since they, too, are separated on ion exchangers. The l-N,N'-dimethylaminonaphthalene-5-sulfonyl derivatives of amino groups (dansyl derivatives) show very strong fluorescence and already have been used in protein chemistry (4, 5 ) and in thin-layer chromatography (6-8). The preparation of these derivatives is fast and quantitative (9),and their strong fluores6ence enables detection a t concentrations at least four to five orders of magnitude lower than is possible with the ninhydrin reaction. This would then be an even more sensitive method for the determination of amino acid derivatives than gas chromatography. A low detection limit for amino acids is extremely important in biochemistry. The same principles of separation and detection can also be applied to peptides and amines. EXPERIMENTAL High Pressure Liquid Chromatography Unit and Detector. A self-constructed chromatography unit was used. A scheme of the instrument is shown in Figure 1. The fluorescence detector (double monochromator, Winopal, Isernhagen) enables the selection of the wavelength for excitation and emission in between the 10-nm band width. The cell volume is 8 p1. For the dansyl derivatives, 340 nm was selected as the primary and 510 nm as the secondary wavelength. Columns and Mobile Phases. In general, columns of 30-50 cm length and 3-mm internal diameter were used. The column was filled with Li Chrosorb SI 60, particle diameter 5 pm (Merck, A.G., Darmstadt) and in the case of reversed phase chromatography with Li Chrosorb RP 8, 10 pm, according to the procedure described by Strubert (IO).The column temperature was 65 O C and the pressure, 230 atm. According to Figure 1, a dual column system with a three-way valve was constructed. Gradient elution was used in column 1. Eluent A was benzene-pyridine-acetic acid (5050.5v/v). To 50 ml of eluent A was added the amount of eluent B pyridine-acetic acid (303; v/v)
i Figure 1. Scheme of the high pressure liquid chromatograph (1) Reservoir for eluent, (2) pump, (3) pulsation damper, (4)three-way valve, (5) sample injection, (6) column 1, (7) temperature bath, (8) fluorometer, (9) recorder, (10) column 2
Table 1. Relative Calibration Factors for Chromatographic Estimation of Amino Acids.a Dansyl amino acids
LYS Met Phe Pro TYr
Val
Leu Ile Glu
Dansyl amino Calibration factor acid DNS-X = DNS-Gly 3.17 2.01 1.91 1.45 1.26 1.22 1.14 1.08 1.02
a
10
30
Minutes Calibration factor DNS-X = DNS-Gly 1.00 0.91 0.86 0.84 0.45 0.43 0.36 0.35 0.31
Figure 2. Gradient change for amino acid analysis (Figure 3) To a mixture of benzene-pyridine-acetic acid (50:5:0.5v/v), the indicated amount (ml) of pyridine-acetic acid (30:3; v/v) Is added continuously during elution
a
21
a Dansyl-glycine = 1.00. Dansylation and separation was performed as described in the Experimental section.
K
‘
in the time scale given in Figure 2. Reequilibration after gradient elution is achieved by passing eluent A through column 1for 5 min. The dansyl derivatives of lysine, histidine, and arginine can only be separated in a reasonable time with an eluent containing lower alcohols. In a single column system, the solvent has to be changed to eluent C: benzene-pyridine-acetic acid-methanol (100:505:50). Since reequilibration to eluent A takes too much time, it is advisable to use the dual-column system. After 25 minutes, the mixture of dansyl amino acids is injected into column 2 and eluted with eluent C by switching the three-way valve. The flow rate was always 1 ml/min. Between to mol dansyl derivatives in 0.2-0.5 pl of the mixture obtained after dansylation were injected in all cases. The separation is also possible by reversed phase chromatography. Dansylation. Two to 10 X lov8mol amino acids are dissolved in 20-100 p1 distilled water; 20-100 p1 Titrisol-buffer pH 9.0 (Merck A.G., Darmstadt), and 20-100 p1 of a solution of 27 mg dansyl chloride (Serva Feinbiochemica, Heidelberg) in 10 ml of water-free acetone are added. The dansyl chloride should be in 10-to 20-fold molar excess to the total amino acid content. After 1 h at room temperature and in the dark, the solvents are evaporated under vacuum. The dry residue is dissolved in 100 pl acetone/l N HC1(19:1,v/v) and 0.1-1.0 p1 of this solution injected into the chromatograph by the “stop-flow” method. For quantitative estimation the peak areas are measured and the response factors of Table I used for the calculation of the amount of amino acid. Under the conditions described only lysine forms a didansyl derivative. It is recommended that the relative response factors of the amino acids be determined, since the fluorescence is very sensitive to impurities of the solvents. The solvents have to be dried. If less material is available,the dansylation can also be performed with to mol amino acids in the same volumes of buffer and solvents described above. The molar ratio of dansyl chloride to total amino acids is always kept at approximately 10-2O:l.
RESULTS A N D DISCUSSION Chromatographic Separation. Figure 3 shows a chromatogram of the common amino acids. Remarkable is the s h o r t separation t i m e of only 30 m i n for t h e protein a m i n o acids. T h e chromatogram was performed with the dual-colu m n system described i n t h e Experimental section. T h e r e is
JO
25
20
rs
IO
Flgure 3. Separation of dansyl amino acids according to conditions described in the Experimental section Column 50 cm (0.3 cm I$), filled with Li Chrosorb SI 60, 5 pm. Pressure 230 atm. Temperature 65 ‘C. Gradient benzene-pyridine-acetic acid (Figure 2) and dual-column system, Flow rate 1 ml/min. Column 2 isocratic with benzenev/v). The peaks correspond to pyridine-acetic acid-methanol (100:50:0.5:50: (1) Dansyl chloride, (2) dansyl amide, dansyl derivatives of (3) isoleucine, (4) leucine, (5) valine, (6) proline, (7) phenylalanine,(8) methionine, (9) alanine, (IO) lysine (didansyl derivative) (11) tyrosine, (12) glycine, (13) tryptophan, (14) glutamic acid, (15) threonine, (16) serine, (17) aspartic acid, (19) cystine, (20) histidine, (21) arginine. (18) corresponds to compounds 1-1 7
a variety of solvent combinations which a r e capable of separating the amino acids, also under isocratic conditions. However, the t i m e for separation is longer. Benzene has been chosen because t h e fluorescence of t h e dansyl derivatives is higher in this solvent. Small variations in t h e amounts of acetic acid and pyridine influence very strongly t h e relative retentions of the amino acids. Figures 4 and 5 demonstrate this effect. The chromatogram in Figure 3 using the dual-column system and a slight gradient is optimized a n d , as far as we know, the shortest separation t i m e ever achieved for a total protein amino acid analysis. The column used had approximately 7.000 theoretical plates per m or 10 plates per second. Because of t h e optimal separation conditions, one peak follows immediately after the o t h e r with no t i m e between peaks. It is probable that the separation can be achieved in even shorter t i m e by using columns with higher plate n u m b e r s ( 1 1 ) . ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1107
'
50
p.,E
8
Benzene/ Acetic A c i d / ISopropyl A(cohol
s 30
20
10
Figure 4. Change of the retention of amino acids with the concentration of pyridine
Brnzme / Pyridine / Isoprrrprl Alrohol
I
5
1
minuter
I
I
1
20
m
0
Flgure 6. Reversed phase chromatography of dansyl amino acids
on
Li Chrosorb RP 8 (Merck A.G.) 10 wm
1
20
J
3
Column 50 cm (0.3 cm 4). Temperature 45 OC. Pressure 140 atm. Flow rate 1.5 ml/min. Eluent aqueous 0.01 M Na2HP04 buffer-methanol (5020) to which 1.5 ml methanollmin is added. The peaks correspond to (1) Dimethylaminonaphthalene-5-sulfonic acid, (2) dansyl derivatives of (2) histidine, (3) serine, (4) glycine, (5)threonine, (6)alanine, (7) arginine, (8) proline, (9) valine, (IO) methionine, (11) phenylalanine, (12) isoleucine,(13) leucine, (14) cystine, (15) aspartic acid, (16) tryptophan, (17) lysine (didansyl derivative), (18) tyrosine (didansyl derivative), (19) dansyl amide, (20) dansyl chloride
LHI
*cu
Pho
I
l
i
2
I
I
I
I
I
3 4 s c 7 relative referhon
C
I
l
i o 1 0
Figure 5. Change of the retention of amino acids with the concentration of acetic acid
The separation is more efficient at elevated temperatures, an observation not generally made in liquid chromatography. This is explained by a reduction of hydrogen bonding of polar compounds at 65 OC, and consequent reduction of tailing which is observed at room temperature. The fluorescence detector does not respond to other impurities if protein hydrolysates of natural samples are analyzed. Other compounds are not detected, the baseline shows less noise, and quantitative analysis is more reliable. Choosing the excitation and emission wavelength of dansyl amino acids also allows a discrimination from other fluorescent compounds-e.g., the N,N'-dimethylaminonaphthalenesulfonicacid, which is always formed from dansyl chloride by partial hydrolysis shows other excitation and emission wavelengths than the dansyl derivatives of amino acids. Accompanying compounds are the dansyl chloride and amide, showing emission at approximately the same wavelengths. They appear first in the chromatogram and do not interfere. The aliphatic neutral amino acids are first eluted. The higher the molecular weight of the amino acid, the lower is the 1108
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
10
io
3b
v
40
50
I
-
60
Min.
Flgure 7. Dependence of the dansylation of amino acids upon time. Dansylation according to description in the Experimental section
retention time. Lysine forms the didansyl derivative and appears before glycine in the chromatogram. If a hydrogen of neutral amino acid is substituted by a hydroxylic or a carboxylic group, the retention increases approximately twofold. Histidine, cystine, and arginine are very strongly retarded. Aromatic amino acids appear later than aliphatic amino acids with comparable carbon number. For separation also, reversed phase chromatography can be utilized, as is shown in Figure 6. For the separation of dansyl peptides, reversed phase chromatography is an extremely valuable additional method. Dansylation. The reaction of dansyl chloride with amino acid goes to completion in alkaline pH. However at pH values
to
5
0
mihufes
Flgure 8. Chromatogram of 68 fmol dansyl leucine. Column as described in Figure 3. lsocratic elution with eluent A
1
1
@
above 9.5, a base-catalyzed hydrolysis of the dansyl chloride occurs (8,9,12).Therefore pH 9 has been selected. Among the investigated buffers, the borate buffer gave the most consistent results. Under these conditions, all primary and secondary amino groups are dansylated, with the exception of the NH groups of heteroaromatic amino acids like histidine and tryptophane. Lysine forms exclusively the didansylated product and OH groups are not dansylated under the conditions described in the Experimental section. The complete dansylation takes approximately 20-40 min, depending upon the structure of the amino acid (see Figure 7 ) .Therefore we recommend generally 1h a t room temperature. An approximately 10-foldmolar excess of dansyl chloride to the total amino acids is used. A larger excess is not advisable since conversion to dansyl amide and hydrolysis to the sulfonic acid occur. Sensitivity and Quantitative Estimation. In the chromatographic system using the eluents described, 0.1 pmol of dansyl amino acid can be detected. Figure 8 shows the chromol) of dansyl lysine corrematogram of 68 fmol(68 X sponding to approximately 10 pg of lysine. The fluorescence of each derivatized amino acid is directly proportional to the concentration of the amino acid over a range of a t least 5 orders of magnitude. The concentration is measured by integrating the area of the chromatographic peak. Figure 9 shows the response of the fluorescence detector in dependence from the amino acid concentration. The relative response factors for the different amino acids are summarized in Table I. These response factors are obtained by dansylating the amino acids and chromatographing them according to the conditions described in Figure 3 and in the Experimental section. If other chromatographic eluents are used, the response factors can vary and an estimation is advisable. However, the linearity of the response is preserved under all conditions.
lo-*
I
I
w*n
fU*
Flgure 9. Linearity of calibration factor
I
?O-a
1
W-*
i
Md
DNS-QS
for didansyl lysine
The dansyl derivatives should be kept under exclusion of light and air and the analysis carried out as soon as possible after their preparation. This new quantitative amino acid analysis is several orders of magnitude more sensitive than the ninhydrin reaction, and the time of chromatography is approximately a tenth of that of the Stein and Moore procedure. Even if the dansylation time is included, the total procedure still is much faster. However, during dansylation, no valuable instrument is occupied. Since peptides can also be separated as rapidly and detected as sensitively as amino acids ( 1 3 ) ,this method opens new frontiers for sequence analysis of proteins where sensitivity and time is an important factor. LITERATURE CITED (1) D. H. Spackman, W. H. Stein, and S. Moore, Anal. Cbem. 30,1190 (1958). (2)S.Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele, Science, 178, 871 (1972). (3)H.-W. Lange, N. Lustenberger. and K. Hempel, Z. Anal. Chem. 261, 337
(1972). (4)G. Weber, Biocbem. J., 51, 155 (1952). (5)B. S.Hartley and V. Massey, Biocbim. Biophys. Acta, 21, 58 (1956). (6) 2. Deyl and J. Rosmus. J. Cbromatogr.,20, 514 (1965). (7)N. Seiler and M. Wiechmann, Z.Anal. Cbem., 220, 109 (1966). (8)V. A. Spivak, V. V. Shcherbukhin, V. M. Orlow, and J. M. Varshavsky. Anal. Biocbem., 39, 271 (1971). (9)C.Gros and B. Labouesse, Eur. J. Biocbem., 7, 463 (1969). (IO) W. Strubert, Cbromatograpbia, 6, 50 (1973). (11) R. Endele, I. Halasz, and K. Unger, J. Cbromatogr., 99, 377 (1974). (12) W. R. Gray, Methods Enzymol., 11, 139 (1967). (13)E. Bayer, E. Grom, and R. Uhmann in "Peptides 1974",Academic Press, New York, 1975,p 247.
RECEIVEDfor review December 1,1975. Accepted March 22, 1976. We gratefully acknowledge the support of this work by the Deutsche Forschungsgemeinschaft.
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