LITERATURE CITED (1) D.C. Locke, J. Cbromatogr. Sci., 11, 120(1973). (2) A. Pryde. J. Chromatogr. Sci., 12, 486 (1974). (3) E. Grushka, Ed., "Bonded Stationary Phases in Chromatography", Ann Arbor Science, Ann Arbor, Mich. 1974. (4) E. Grushka and E. J. Kikta, Jr., Anal. Chem., 46, 1370 (1974). (5) M. Novotny, S. L. Bektosh, K. B. Denson, K. Grohmann, and W. Parr, Anal. Chem., 45, 971 (1973). (6) J. H. Knox and G. Vasvari, J. Chromafogr., 83, 181 (1973). (7) J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (1971). (8) J. N. Little, W. A. Dark, P. W. Farlinger, and K. J. Bonbaugh, J. Chromafogr. Sci., 8 , 647 (1970). (9) 6. L. Karger and G. Sibley, Anal. Chem., 45, 740 (1973). (IO) R. E. Majors and M. J. Hopper, J. Chromatogr. Sci., 12, 767 (1974). (11) D.C. Locke, J. Cbromatogr. Sci., 12, 433 (1974). (12) R. K. Gilpin, J. A. Korpi, andG. A. Janicki, Anal. Chem., 47, 1498(1975). (13) J. H. Knoxand A. Pryde, J. Chromatogr., 112, 171 (1975). (14) J. Asshauer and I. Halasz, J. Chromatogr. Sci., 12, 139 (1974).
(15) (16) (17) (18)
(19) (20) (21) (22) (23) (24)
K. Unger, Angew. Chem., lnt. Ed. Engl., 11, 267 (1972). R . K. Gilpin and M. F. Burke, Anal. Chem., 45, 1383 (1973). L. R. Snyder, J. Chromatogr. Sci., 7, 352 (1969). L. R. Snyder, "Gas Chromatography 1970", A. Stock and S. G. Perry, Ed., Elsevier. London 1971, pp 81-111. J. A. Schmitt, R. Altenny, R. C. Williams, and J. F. Diekman J. Chromatogr. Sci., 9, 645 (1971). R . E. Majors, ref 3, p 139. A. Diez-Cascon, A. Serra, J. Pascual, M. Gassiat, and J. Albaiges, J. Chromatogf. Sci., 12, 559 (1974). D. C. Locke, Queens College, New York. N.Y., private communication, 1976. R. P. W. Scott and P. Kucera, J. Chromatogr. Sci., 12, 473 (1974). M. J. Telepchak, Chromatographia, 6, 234 (1973).
RECEIVEDfor review December 1,1975. Accepted March 19, 1976.
High Pressure Liquid Chromatography of p-Methoxyanilides of Fatty Acids Norman E. Hoffman* and John C. Liao Todd Wehr Chemistry Building, Marquette University, Milwaukee, Wis. 53233
Saturated and unsaturated fatty acids were quantitatively converted to p-methoxyanilides to enhance their uv detectability. Rapid methods for the synthesis of the anilides with triarylphosphine reagents were developed. The p-methoxyanilides were separated by reverse phase chromatography using I O + partlcles packed in a 30-cm long column. Wateracetonitrile and water-methanol were used in hyperbolic gradient elution.
Recently, there has been interest in developing high pressure liquid chromatographic (HPLC) methods for the determination of long chain fatty acids. Durst et al. ( 1 ) have thoroughly reviewed these recent developments and pointed out the need to derivatize these acids in order t o enhance their detectability. Amides have not been used as derivatives. p-Methoxyanilides of fatty acids have a maximum in their uv spectrum a t 254 nm. These anilides also have high absorptivity a t this wavelength used in most uv detectors for HPLC. The purpose of this paper is to report a study of the preparation and chromatography of a large number of long chain saturated and unsaturated fatty acid p-methoxyanilides. Two rapid methods were developed to convert these acids to pmethoxyanilides, one using triphenylphosphine (2, 3 ) and carbon tetrachloride to prepare an intermediate acyl chloride and one using carbon tetrachloride and a reagent containing a diphenyl phosphino group bonded to a polymer ( 4 ) .Gradient elution HPLC of these anilides was studied with two solvent combinations, water and acetonitrile or methanol.
EXPERIMENTAL Apparatus. A model ALC 202 liquid chromatograph equipped with a model 660 solvent programmer, model 6000 pumps, and a model 400 loop injector (Waters Associates, Milford, Mass.) was used. This chromatograph has a uv detector that was operated at 254 nm. Throughout all the work reported here, the chromatographic solvent flow rate was 1ml/min. A %-in.0.d. by 30 cm K-Bondapak CIS column (Waters Associates) was used. The column's plate number was 3000 measured with p-methoxylauranilide and a solvent of 83% acetonitrile and 17% water. 1104
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Gradient elution was started at 100%water and continued to 100% organic solvent (acetonitrile or methanol) in a hyperbolic manner. "Curve 2" of the Waters programmer was used. The time setting for the program was 40 min. Reagents. All reagents were used as purchased without further purification. Saturated straight chain fatty acids c14, CIS, c16, CIS, C20, C22, C24, and unsaturated fatty acids palmitoleic (Clc:~),oleic ( C l ~ l )linoleic , CIS:^), linolenic (C18:3),arachidonic (CZO:~), erucic (CZZ:~), 4,7,10,13,16,19-docoshexaenoic (C22:6),nervonic (c24:1)acids were purchased from Applied Science Laboratories, State College, Pa. Straight chain saturated fatty acids c6, cs, (210, Cl2, carbon tetrachloride, chloroform, ethyl acetate, triphenylphosphine, chlorodiphenylphosphine, p-methoxyaniline, N,N'-dicyclohexylcarbodiimide, spectrophotometric grade methanol and acetonitrile were obtained from Aldrich Chemical Co., Milwaukee, Wis. Polystyryldiphenylphosphine reagent was synthesized ( 5 ) from cross-linked polystyrene (2% divinylbenzene, 200-400 mesh) obtained from Bio-Rad Laboratories, Richmond, Calif. Phosphorus analysis showed 79% of the phenyl rings of the polymer were substituted with diphenylphosphino groups. Carbon tetrachloride and ethyl acetate were dried over Linde Type 4A molecular sieves (ALFA Products, Beverly, Mass.). Derivative Formation. a.Triphenylphosphine Method. A mixture of fatty acid, 5-10 mg of each (about 0.7 mmol total acids), 0.5 g (1.9 mmol) triphenylphosphine and 2 ml dry carbon tetrachloride were placed in a 15-ml vial. The vial was sealed with a Teflon-lined screwcapand placed in an 80 "C oil bath for 5 min. Cloudinessappears when the reaction is complete. After withdrawing and cooling to room temperature, the vial contents were mixed with 0.5 g (4.1 mmol) of p-methoxyaniline dissolved in 8 ml of dry ethyl acetate. The vial was returned to the oil bath (80 "C) for 1h. An insoluble oil appears a t this point but, because it is insoluble, it has no effect on chromatography. If the temperature of the bath is raised to 140 "C, the first step can be completed in 2 min and the second step in 20 min. b. Polystyryl-Diphenylphosphine Method. A mixture of fatty acids as in a.,1.4 g (4.4 mmol phosphorus) of polystyryl-diphenylphosphine reagent, and 2 ml of carbon tetrachloride was placed in a 15-ml vial sealed with a Teflon-lined screwcap. The vial was heated for 5 min at 80 OC in an oil bath with gentle shaking. The vial was withdrawn from the oil bath, cooled to room temperature, and a solution of 0.5 g (4.1mmol) of p-methoxyaniline in 8 ml of ethyl acetate was then added. The vial was returned to the oil bath (80 "C) for an additional 10 min of heating. If the temperature of the bath is raised to 140 OC, the first step can be completed in 2 min and the second step in 3 min. c. N,N'-Dicyclohexylcarbodiimide(DCC)Method. This method was used to prepare the anilides for identification purposes. Because it is an established method, the products were used to compare
I
I
I
I
I
'ha
I
I
I
I
I
I
I
I
I 1
Minuti: Figure 1. Water-acetonitrile gradient elution HPLC of fatty acid p
methoxyanilides Peak: (a)prnethoxyaniline, (b) unknown, ( C ) c6, (d) Cs, (e)CIO, (f) CIZ~ (9) c18:3~ (h) c14 + (i) C20:4 + CIS:I; (j) CI~:Z, (k) Cl5, (I)cl6, (rn) C~S:I, (n) C17. (0)
+ C221, (4)
CIS, (p) CZO
C241,
(r) C22, (s) c24
chromatographically to the products of methods a. and b. and, thereby, prove their anilide structure. The structure of the DCC method products was further characterized by NMR, ir, and uv. A second purpose for using this method was to prepare anilides on a 1-g scale so that they could be purified and used in studying the yields of anilides obtained by methods a. and b. A typical derivative preparation follows ( 6 ) .Lauric acid, 2.0 g, and 2.5 g DCC were dissolved in 25 ml of chloroform. This solution was added to a solution of 3.5 g p-methoxyaniline in 25 ml chloroform. The resulting solution was allowed to stand at room temperature for 12 h or longer. Glacial acetic acid, 1ml, was added to destroy excess DCC. Dicyclohexyl urea was filtered off, and the filtrate was washed with 5% hydrochloric acid followed by 5% sodium bicarbonate. On addition of petroleum ether, the desired p-methoxylauranilide precipitated. It was filtered and recrystalized from methanol-water. The yield of anilide was 1.0 g, mp 102-103 OC. Chromatographic Procedure. In all runs, 2 ~1 of the solution containing the anilides was injected with a lO-,ul syringe. When derivative formation procedure a.or b. was used the anilides were in the ethyl acetate-carbon tetrachloride solution described above. The gradient elution program was begun immediately upon sample injection. However, the model 400 injector has a 2-ml solvent loop between the pumped solvent and the point of injection. Therefore, 2 ml of programmed solvent must be pumped before it reaches the point of injection (approximatelythe head of the column).Gradient curves, Figures 1 and 2, plot the solvent composition at the head of the column and show this 2-ml lag. Quantitative Studies. To determine yields of the anilides, a given acid was removed from the fatty acid mixture and derivative formation procedure a. or b. was performed. The anilide of the missing acid, prepared by procedure c., was then added in known amounts to the a . or b. derivative solution. The solution was chromatographed and areas were compared to determine derivative yields by procedure a. or 6 . All derivatives had the same molar absorptivity as determined by a Cary 14 uv spectrophotometric measurement. Capric acid in varying amounts and lauric acid in fixed amounts were converted to their anilides by procedure a. Solutions covering the mole ratio of Clo to Clz of 0.1 to 10 were chromatographedand the peak height ratios of Clo to Clz were determined.
RESULTS AND DISCUSSION After several different methods of amide synthesis were tried, a preparation t h a t was simple, rapid, and quantitative was chosen. T h e fatty acid was converted to an acyl chloride and, without isolating it, the chloride solution was heated with p-methoxyaniline
R + 2 p-CH,0C6H4NH2
RCCl
3
P - C HaOCeH4N H C O R
+
P - C H ~ O C ~ H ~ N HI S C
T h e p-methoxyanilide formed needed no purification or concentration but was ready for injection in the solution in which it was prepared. Two procedures were used t o form the
acyl chloride. Procedure a. used triphenylphosphine ( 2 , 3 )in the chloride preparation
f.l
RCOH
+
+
(C~HS)~P
CC14 RCOCl
4-
(C6H&PO
+
CHCI,
Procedure b. used a triarylphosphine also, but one of the aryl groups was a phenyl group of a polystyrene polymer ( 4 )
8
RCOH
+
pOlYmer-C6H4P(C&)2
RCOCI
+
+
CC14
poIymer-C6H4PO(C6H&
+
CHCI,
At present, the polystyryl-diphenylphosphine reagent is not available commercially. Therefore, procedure a. has the advantage of using commercially available reagent, triphenylphosphine. Procedure b. has the advantage t h a t no organophosphine or phosphine oxide is in the solution after anilide formation and, therefore, these phosphorus compounds cannot interfer with chromatographic separation. With water-methanol, hexanoic acid anilide cannot be resolved from triphenylphosphine or its oxide, and, therefore, procedure a. could not be used with this acid and this solvent. Another advantage of procedure b. is t h a t i t is faster. The p-methoxyanilides have good absorption at 254 nm as indicated by a molar absorptivity of 2.43 X lo41. mole-l cm-l in methanol. The absorption makes for good detectability. Thus, 5 ng of lauric acid gave a peak of 5%of full scale above a baseline with no visible noise. Figure 1shows the separation of fatty acid p-methoxyanilides obtained by using polystyryl-diphenylphosphine reagent. The solvent was water-acetonitrile. For a given degree of saturation, retention time increases with molecular weight. T h e gap in retention time between two acids differing by a given number of carbons, e.g., Ce and CS, and CIS and Cm, increases with increasing retention time. This increasing separation is probably the result of the program in addition to the kind of organic solvent constituent. Borch (7) observed the opposite separation change with phenacyl esters of fatty acids. His program had a more rapid change in acetonitrile concentration in the second half of the program whereas, with p-methoxyanilides, the rapid change in acetonitrile concentration occurred a t the beginning of the program. Figure 1 shows increasing unsaturation decreases retention time significantly. Thus, C l ~ : elutes 1 close t o C15, C18:~before C14 and with (214. As a consequence of this unsaturation pheANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1105
nomenon, some peaks are poorly resolved or not resolved a t 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. T o 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 t h a t intercepted the abscissa a t 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 silica1 gel, 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 a t 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 t o 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)
