485
Anal. Chem. 1981, 53, 485-489
should be considered suspect. These are a heavy catalytically cracked naphtha (-2), a heavy reformate (-2), a total reformate, and a polymer sample. There are a number of factors which could have contributed to the differences on some blending components and test fuels: (1) unusual density of one or more hydrocarbon types in the sample; (2) poor recovery of olefins from the CuSO4 column (This is particularly likely with a heavy sample where poor recovery of heavy olefins results in a low olefins value.); (3) the molar response of the detector to unusual compounds present in the sample; (4) poor chromatographic separation of the sample due to significant amounts of very light or very heavy components; (5) interferences in the FIA determination on highly colored samples and those which contain diolefins, heavily substituted aromatics, and compounds containing sulfur, nitrogen, or oxygen. Although the present data are insufficient to assign any discrepancy to a single cause, it is likely that differences in density and olefins recovery are more important factors than differences in the detector’s response or a poor chromato-
graphic separation. The importance of the FIA’s interferences as a contributor is entirely unknown.
ACKNOWLEDGMENT The author is indebted to J. W. Vezey for many important contributions to this work, to F. S. Jones for providing the FIA analyses, and to R. L. Bunvell of Northwestern University for several useful conversations.
LITERATURE CITED (1) Annu. Book ASTM Stand. 1977, 682. (2) (;reen, L. E.; Albert, D. K.; Barber, H. H. J . Gas Chromatogr. 1968. 4 , 319. (3) Swlages, N. L.; Brieva, A. M. J . Chromatogr. Sci. 1971, 9 , 492. (4) Suatoni, J. C.; Oarber, H. R.; Davis, 8. E. J . Chromatogr. Sci. 1975, 13, 367. (5) Myers, M. E., Jr.; Stollsteimer, J.; Wims, A. M. Anal. Chem. 1975. 47, 2010. (6) Scheffe “Analysis of Variance”; Wiley: New York, 1959; p 365.
RECEIVEDfor review December 17,1979. Accepted December 9, 1980.
Derivatization with 4-Chloro-7-nitrobenzofurazan for Liquid Chromatographic Determination of Hydroxyproline in Collagen Hydrolysate Martin Ahnoff , Inger Grundevik, Anders Arfwidsson, Jan Fonselius, and Bengt-Arne Persson Analytial Chemistry and Biochemistry, AB Hassle, S-43 1 83 Molndal, Sweden
The reaction of 4-chloro-7-nitrobenrofurazan (NBD-CI) with 4hydroxy-~proiineand Lproiine occurs 1 order of magnitude faster than for primary amino acids. In a methanol-buffer medlum, NBD derlvatives are formed partty by reactbn of the amino adds with 4-methoxy-7-nitrobenzofurazan (NBDOCH3) which, In turn, is formed by the reaction of NBD-Ci wtth the solvent. For 1-( 7-nitro-4-benzofuraranyi)4hydroxy-~~roiine (NBDHyp) fluorescence quantum elfidencies range from 0.80 in isobutyl methyl ketone to 0.01 in water (pH 9). Hydroiysls to 7-nttro-4-benzofuraranoi (NBDOH) occurs at a dgntficant rate In aqueous solutions at pH I1 1. Aqueous solutions are also subjected to light-Induced decomposition. A procedure Is described for the determination of 4-hydroxy-~-prolinein colagen hydrolysate. After reaction W h NBD-CI, the sample is injected onto a LiChrosorb RP18 column and eluted wtth phosphate buffer, pH 1.9, containing 20% CH3CN and 5 mM C7H,,S03Na. Preclsion is 95%.
Chemical reactions are extensively used in conjunction with chromatography to improve chromatographic properties as in gas chromatography (GC) or to facilitate detection as in thin-layer chromatography. In liquid chromatography (LC) pre- or postcolumn reaction is increasingly used to enable quantitation by W or fluorescence detection of nonresponding compounds such as amino acids. Information on derivatization techniques for amino compounds has been compiled by Blau and King (1) and recently by Knapp (2). Among reagents 0003-2700/6110353-0485801.0010
used, 4-chloro-7-nitrobenzofurazan (NBD-C1, Figure 1) is suggested for both primary and secondary amines. This particular reagent has been the subject of studies both in organic synthesis (3-7) and in papers concerning analytical methods (8-12). However, the reactivity for secondary amino acids as contrasted with primary amino acids has not been studied thoroughly and neither have reaction yields and spectroscopic properties of the formed derivatives. In the present investigation 4-hydroxy-~-proline(Hyp) was assayed in collagen hydrolysate. Recent methods for hydroxyproline determination are based on amino acid analyzers (11,13),on GC (14,15) or on the classical colorimetric method with Chloramine T and Ehrlich’s reagent (16). NBD-Cl was chosen as a reagent for derivatization of hydroxyproline for subsequent LC separation since such a method seemed to combine simple pretreatment of the samples with sensitive detection and selectivity against, for example, primary amino acids. The reaction mechanism and conditions for analytical application were examined. The properties of the derivative formed were studied regarding stability, fluorescence detection, liquid-liquid partition, and chromatographic separation. The analytical method developed has been used routinely for the analysis of hydroxyproline in collagen hydrolysate.
EXPERIMENTAL SECTION Reaction Studies. Reactions of NBD-C1 with amino acids were carried out as follows: Samples, buffer (K2B4O7-4H20, 0.4 mol/L), and NBD-Cl (E. Merck) dissolved in an organic solvent, usually methanol, were mixed in screw-capped teat tubes and kept in a thermostated water bath for definite periods of time. To stop the reaction, we cooled the mixture in ice-water and acidified with 0 1981 American Chemical Society
488
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
Table I. Partition Constants of the NBD Derivative of Hydroxyproline between Water (pH 1) and Some Organic Solvents
[
CI
NBD-CI
)-COOH
HO
NBD-Hyp
organic solvent isobutyl methyl ketone ethyl acetate diethyl ether dichloromethane
log KD 1.22 1.10 -0.03
-0.96
maxima and molar absorptivities were determined with a Perkin-Elmer 555 spectrophotometer. A corrected fluorescence emission spectrum of the derivative in isobutyl methyl ketone was recorded on an Aminco Bowman SPF 550 spectrofluorometer. &o . .N/ The quantum efficiency was determined relative to fluorescein, which has quantum efficiency 4 = 0.93 in 0.01 mol/L NaOH (17). Correction was made for refractive index differences between the OCH, OH solvent and water. Time-resolved measurements with single photon counting technique showed only one fluorescent species, NBD-OCH3 "I-OH having a lifetime in isobutyl methyl ketone of 7 = 9.4 f 0.1 ns. Quantum efficiencies of the derivative in other solvents were Flgure 1. Structural formulas. determined on a simple instrument (Aminco Bowman SPF ratio HC1 to pH 2. The pH of the reaction mixture was varied between spectrofluorometer)with the derivative in isobutyl methyl ketone 8.5 and 10.5. Since optimum reaction rate was found near pH as a reference. Synthesis of 4-Methoxy-7-nitrobenzofurazan (NBD-OC9.5, this pH was used throughout the study. Preliminary experiments had shown similar temperature deHa). NBD-OCH3 (Figure 1)was synthesized from NBD-Cl and pendence for the reactions with different amino acids as well as CH30Na in CH30H according to the literature (4). The yield was with soIvent components. Ten degrees increase in temperature 450 mg (46%): mp 114-115 OC (Buchi 510, 1°/min); lH NMR (CDC13) 6 8.7 (d, J = 9 Hz, 1 H), 6.8 (d, J = 9 Hz, 1 H), 4.3 (8, raised the reaction rate of hydroxyproline by a factor of 1.7. In 3 H). the present work 60 "C was used in all experiments, unless othSynthesis of 7-Nitro-4-benzofurazanol(NBD-OH). erwise stated. The chosen concentrations of NBD-Cl and methanol in the NBD-OH (Figure 1)was prepared by demethylating NBD-OCHB in aqueous NaOH according to the literature (4): yield 400 mg mixture were 8.3 mmol/L and 33%, respectively. Neither the (50%); mp 200 OC (Kofler Heizbank); 'H NMR (CDBOD)6 8.7 concentration of NBD-C1nor the water content could be increased (d, J = 9 H, 1 H), 6.8 (d, J = 9 Hz, 1 H). appreciably due to the limited solubility of NBD-Cl. Since only Stability of NBD-Hyp in Solution. Decomposition of slightly higher reaction rates were observed for higher methanol NBD-Hyp in solution was studied by spectrophotometry. The concentrations,a low methanol concentration was chosen for better absorption spectrum of NBD-Hyp differed sufficiently from compatability with the liquid chromatographic system. spectra of formed products to permit measurement of remaining Results from the following reaction experiments will be disNBD-Hyp as low as 5% of the initial concentration. cussed in the next section: (a) reaction of NBD-Cl in methamol/L) in methanol-water Spectra of NBD-Hyp (0.25 x nol-buffer at 60 "C with hydroxyproline, proline, and alanine; (30/70) were recorded at regular intervals to follow the decom(b) determination of absolute yields of the NBD derivative of position under alkaline, essentially dark, conditions. Decompohydroxyproline (NBD-Hyp, Figure 1)in reactions with methanol, sition was also studied in solutions of NBD-Hyp (0.25 X lo-' acetonitrile, or acetone as organic solvent; (c) reactions of NBD-Cl mol/L) in water, water-methanol, or methanol which were e@ with the methanol-water solvent. In all experiments, NBD-C1 to light. Solutions were kept in Pyrex glass test tubes attached and reaction products were quantitated with liquid chromatogto the inside of a window and were thus exposed to daylight raphy with UV or fluorescencedetection. W absorption was used filtered through glass. Spectra were recorded after 0, ca. 30, and to detect all types of NBD compounds. Synthesis of 1-(7-Nitro-4-benzofurazanyl)-4-hydroxy-~- ca. 60 min exposure. Analysis of Hydroxyproline in Collagen Tissue. The liquid proline (NBD-Hyp). A 2.5-g sample (30 mmol) of NaHC03 and chromatograph consisted of an LDC Model 711-47 pump, a 1.3 g (10 mmol) of 4-hydroxy-~-proline(Sigma) were dissolved Rheodyne microsampling valve provided with a 20-pL loop, a in H20 (20 mL). A 2.0-g sample (10 mmol) of NBD-Cl (Merck) separation column of stainless steel (150 mm in length, 4.5 mm dholved in 80 mL of methanol was added. The reaction mixture i.d.), a Schoeffel FS 970 fluorescence detector, equipped with a was warmed to 55 "C, stirred at room temperature for 30 min, deuterium lamp, a Corning 7-59 primary filter, and a 47C-nm cutoff and then acidified to pH 1.5 with 2 mol/L HCl (14 mL). Most secondary filter. The excitation monochromator was set at 345 of the solvent was evaporated and water was added. The hynm. droxyproline derivative precipitated as an oil and crystallized on The separation column was packed with LiChrosorb RP 18, scratching to yield 2.0 g (68%): mp 95-120 OC (dec, Buchi 510, 5 pm (E, Merck). The mobile phase was composed of 20% 2"/min); 'H NMR (Varian EM-390,90 MHz, CD,OD, 60 "C) 6 acetonitrile in phosphate buffer solution (pH 1.9, I = 0.05) con8.6 (d, J = 9 Hz, 1 H), 6.3 (d, J = 9 Hz, 1 H), 5.6-5.2 (m, 1 H), mol/L (Eastman, Kodak). taining 1-heptane sulfonate 5 X 4.7 (m, 1H), 4.3-3.8 (m, 2 H), 2.8-2.3 (m, 2 H); 13CNMR (Varian The mobile phase was degassed in an ultrasonic bath for 20 min CFT-20,20 MHz, CD30D,60 "C) 6 173.2,145.3,144.7, 144.6,136.1, before use. The flow rate was 1mL/min. The blood vessels were 122.5, 102.7,68.7,63.2, 59.3,39.0; mass spectrum (Varian MAT homogenized, and the collagen was extracted by the method of 448, electron impact 70 eV) m / e (relative intensity) 294 (M, 2), Fitch et al. (18). The collagen was hydrolyzed in 0.5 mL of 230 (86), 81 (96), 51 (100). hydrochloric acid (6 mol/L) at 253 Pa for 6 h and then neutralized. Purity was estimated at 95 & 2% by nitrogen determination The hydrolysates contained about 250 pg of protein/mL, about and by titration of the carboxylic acid function. 40 pg (0.3 pmol) of this being hydroxyproline. After this prepK, in water of the carboxylic acid group was 2.5 (determined treatment the collagen hydrolysates were either analyzed imby potentiometric titration as well as by examination of the pH mediately or frozen. dependence of the partition to aprotic solvents). A 100-pL sample of the collagen hydrolysate was mixed with Partition constants (KD= CC /),, of the derivative between 100pL of potassium tetraborate buffer (0.4 mol/L) in a g h tube water (pH 1)and ethyl acetate, isobutyl methyl ketone, diethyl with a screw cap. After addition of 100 pL of NBD-C1 reagent ether, or dichloromethane were determined (Table I). (25 mmol/L of methanol) the tube was incubated at 60 "C in a Light absorption and fluorescence characteristics of the dewater bath for exactly 3 min and protected from light. The rivative dissolved in various solvents were measured. Absorption
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 h
0
0
HVD P c
e
-e
"1
A NED-OH V NBD-OCH3 0
derivatization reaction was quenched by the addition of 50 pL of hydrochloric acid 1 mol/L and by immersion of the tube in an ice bath. Twenty microliters of the reaction mixture was injected onto the chromatographic column. Standard samples consisting of 100 pL of hydroxyproline (500 pmol/L in phosphate buffer pH 7) were analyzed according to the method. The amounts of hydroxyproline were calculated by comparison of the peak heights of the unknown samples to those of the standard samples.
RESULTS AND DISCUSSION Reaction Rates of Secondary a n d P r i m a r y Amino Acids. The reaction rates of hydroxyproline (Hyp) and proline (Pro) were 1order of magnitude higher than those of primary amino acids. The reaction courses of hydroxyproline and alanine (Ala) at 60 OC are shown in Figure 2. Reaction conditions at which hydroxyproline reacts with quantitative yield, e.g., 8.3 mmol/L NBD-C1 at 25 "C, 20 min or 60 "C, 3 min, are milder than those described by others for completed reactions of amines (I),indicating that hydroxyproline and proline are fast reacting amines. A short reaction time will favor the selective derivatization of the secondary amino acids (Figure 2). Reaction Yields in Different Solvents. A reaction yield of ca. 95% was obtained for hydroxyproline reacted in methanolic solution. When acetonitrile was used instead of methanol, a significantly lower yield, 65%,was obtained. An even lower yield, 15%, was obtained with acetone. This is noteworthy since the use of acetonitrile and a ketone as reaction solvents are described in the literature (8, 12). The same observations were made for proline. Reaction Mechanism. Dal Monte et al. (6) have shown that the reaction of NBD-Cl with methoxide ion in methanol is second order (first order in each reactant). The reaction of NBD-CI with a neutral electrophile such as an amine involves the elimination of a proton and therefore is somewhat different. Nevertheless, SN reactions of neutral nucleophiles with aromatic halides are generally second order according to Miller (19).Experiments were carried out to see whether the reaction of NBD-C1 with amino acids actually is second order. However, interpretation was made difficult by competing solvolytic reactions. In a buffered water-methanol medium solvolytic reactions are rapid enough to considerably decrease the concentration of NBD-C1 during its reaction with amino acids. Consequently, pseudo-first-order conditions could not be maintained in the experiments. Solvolytic Reactions. Liquid chromatographic analysis of an NBD-C1-methanol-buffer mixture revealed the formation of two substances. They were tentatively identified as 7-nitro-4-benzofurazanol(NBD-OH, Figure 1) and 4methoxy-7-nitrobenzofurazan(NBD-OCH3,Figure 1). This was confirmed by comparison to synthesized NBD-OH and NBD-OCH3 Figure 3 shows the formation of NBD-OH and NBD-OCH3. It appears that NBD-OCH3 is an intermediate, which reacts further to give NBD-OH. Reaction of Amino Acids w i t h 4-Methoxy-7-nitrobenzofurazan (NBD-OCH3). A close examination of the formation of the hydroxyproline and alanine derivatives
NED-CI
/
10 20 reaction tlme (min)
F b r e 2. Formation of NBD derivatives of hydroxyprolhreand alanine. Conditions: 8.3 rnrnoi of NBD-Ci/L in rnethanol/borate buffer pH 9.5 (33/67),60 OC.
487
z__p 10
30
L5
60
reaction time (min)
Fbure 3. Formation of NBD-OCH:, and NBD-OH from NBD-Ci during reaction of the latter with the methand-aqueous buffer solvent. Same conditions as in Figure 2.
Table 11. Spectroscopic Properties of the NBD Derivative of Hydroxyproline in Some Solvents absorption fluorescence emission E x 10-4, h, Lmol-I h, solvent nm cm-l nm ~
water, pH 9 water, pH 1.5 methanol + mol/L NH, methanol + mol/L HC1 isobutyl methyl ketone &chloromethane
500 355 485 348 486 345 410 336
3.1 0.73 2.8 0.69 .... 2.6 0.81 2.2 0.77
-461 462 333
536
o'olo
532
o*020
532
0.056
-2.0
526 519
o.80
2.0 0.71
513
O."
9 = quantum efficiency.
(Figure 2) reveals that the rate of reaction is not related to the concentration of NBD-C1 as would be expected. The rate of formation of the alanine derivative actually increases during the first minutes and is highest when less than 20% of the NBD-C1 is left. Similarly, the reaction rate of hydroxyproline seems not to follow the decrease in NBD-Cl concentration. This suggests that NBD-OCH3 is a reactive intermediate, responsible for formation of the main part of NBD-Ala and a considerable part of NBD-Hyp. Reaction experiments with hydroxyproline and alanine, where NBD-C1 was replaced by NBD-OCH,, confirmed that NBD-OCH3 reacts faster than NBD-Cl, giving the normal NBD derivatives. This is in accordance with kinetic studies by Di Nunno et al. (20) on the reaction of methoxide ion with substituted 7-nitrobenzofurazans. They reported a two times higher rate constant for NBD-OCH3 than for NBD-Cl. NBD ethers should be of interest as reagents and are presently studied. Spectroscopic Properties of NBD-Hyp. The high quantum efficiency in medium polar solvents and the high absorptivity (Table 11) make fluorescence detection in those solvents favorable. The quantum efficiencies are similar to those reported for dansyl and bansyl derivatives of amino acids (21).However, the 40-80-fold decrease in quantum efficiency when changing from isobutyl methyl ketone to water as solvent is more drastic than the 10-fold decrease observed for DNStryptophan when changing from dioxane to water (21).Thus, for NBD derivatives in aqueous solutions, fluorescence detectors may not be superior to UV detectors in terms of sensitivity, although their selectivity against, e.g., NBD-CI and NBD-OCH3 is higher. Stability of NBD-Hyp in Solution. Under conditions relevant for analytical work, base-catalyzed hydrolysis and light-induced decomposition are the two most important processes leading to destruction of NBD-Hyp in solution.
488
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 I 5106\
I
0
1
2 time ( h l
3
L
Flgwe 4. Base-catalyzed hydrolysis of 0.25 X lo-' MI of NBD-Hyp/L of methanol-water (30170): (A) initially added lo3 mol of NaOH/L; (B) Initially added lo-' mol of NaOH/L.
Decomposition by hydrolysis in alkaline media (Figure 4) closely followed an exponential course. The decomposition rate was directly related to the concentration of hydroxyl ions. Base-catalyzed hydrolysis of NBD derivatives of secondary amines has been mentioned by Aboderin et al. (ZZ),who reported a half-life of 20.5 min at pH 10.3 for morpholine. Although the hydroxyproline residue is a weak leaving group in nucleophilic substitution reactions, substitution by a hydroxyl ion occurs since the formed phenol is acidic (pK, < 2) and is stabilized as phenolate ion. Contrary to the secondary amines, primary amines form NBD derivatives which, according to Aboderin et al. (22),are weak acids and are stable anions in alkaline media. Solutions of NBD-Hyp in water and in water-methanol were sensitive to light at all pH levels tested (pH 1.5-10). When dissolved in methanol-water (30/70) and exposed to daylight (see Experimental Section) 50% decomposed within 25 min. Acidification to pH 1.5 increased the half-life to ca. 40 min. When dissolved in methanol alone, NBD-Hyp was resistant to light provided that the solution was acidified with a small amount of hydrochloric acid. More than 98% then remained after 1 h of exposure to light. A light-exposed methanolic solution of NBD-Hyp was analyzed by reversephase LC, using a neutral mobile phase. A large number of fluorescent components were found which eluted later than the derivative itself. They were not retained by adding quaternary amines as ion-pairing cations to the mobile phase. We assume that the carboxyl group was removed by the lighbinduced decomposition. Long exposure of acidic aqueous solutions to daylight resulted in almost colorless solutions. Determination of Hydroxyproline in Collagen Hydrolysate. The reaction conditions used in the analytical procedure have been adapted to give a quantitative recovery of the NBD derivative of hydroxyproline. Standard curves were linear up to more than 1250 pmol/L hydrolysate. Both excessive reaction time or temperature and higher concentration of NBD-C1 would mainly promote the formation of derivatives of the primary amino acids present in the collagen hydrolysate. Since neither NBD-C1 itself nor the corresponding phenol or the methoxy compound interfered in the chromatographic separation with fluorometric detection, the derivatization mixture was injected directly after the reaction was quenched by addition of acid and cooling. This was possible due to the solvent composition being similar to that of the mobile phase. NBD-OH eluted after NBD-Hyp and was well separated, while NBD-OCH:, and NBD-C1 had 3 times the retention time of NBD-Hyp. This did not affect the intervals of injection of samples owing to the low fluorescence response of these components. The chromatographic separation of NBD-Hyp from other sample components was performed with a mixture of phosphate buffer (pH 1.9) and acetonitrile as the eluent. Under these conditions the NBD derivatives of the amino acids are present mainly in nondissociated forms in the mobile phase
2
0
6
4
10
retentionvolume (ml)
Flgue 5. Retenth of NBD derivattves of lysine, Serine, hydroxyproline, and water (w) In some chromatographic systems: column, LChrosorb RP 18,5 pm, 150 mm long by 4.5 mm 1.d.; mobile phase, phosphate buffer (pH 1.9)plus organic modifier, I = 0.05.
NBrHypl I OOZPA
Ii
!I
h
I! 1;
retention volume Iml)
Figure 6. Chromatogram of a collagen hydrolysate containing 400 pmol of hydroxyprollne/L: column, LiChrosorb RP18, 5 pm, 15 mm long by 4.5 mm i.d.; mobile phase, 20% CH,CN In phosphate buffer (pH 1.9, I = 0.05)with 5 mM C,H,,S03Na. Number of theoretical plates ( N ) for Hyp Is 3000.
and can be retained as carboxylic acids on the stationary phase. A higher pH of the mobile phase would decrease the retention, which could be compensated for by lowering the content of the organic modifier. This is, however, disadvantageous since the fluorescence detector response is lowered and samples can no longer be injected undiluted. Mobile phases with neutral pH and with quaternary ammonium ions were also tested but gave poor resolution of the amino acid derivatives. Some chromatographic systems with different composition of the mobile phase were tested and examples are given schematically in Figure 5. Acetonitrile gave better resolution than methanol. With a mobile phase containing only acetonitrile and buffer solution (pH 1.9) the NBD derivatives of lysine (Lys) and serine (Ser) eluted too close to hydroxyproline. Two different derivatives are formed by lysine because of the two amino groups available for reaction. Both of them seem to be monoderivatives since they can be retained by the addition of ion-pairing heptanesulfonate, while the retention of hydroxyproline and serine are not affected (Figure 5). The relative recovery for hydroxyproline in collagen hydrolysate compared to a pure aqueous test solution waa greater than 95%. The repeatability on the analysis of identical portions of a collagen hydrolysate containing 400 pmol of hydroxyproline/L was tested and gave a relative standard deviation of less than 5%. A chromatogram from an authentic sample is shown in Figure 6.
489
Anal. Chem. 1981, 5 3 , 489-495
ACKNOWLEDGMENT Kerstin Martinson, Department of Organic Chemistry, AI3 Hassle, gave technical assistance in the preparation of the NBD compounds. Jan-Erik Lofroth, Department of Physical Chemistry, University of Gothenburg, performed the timefluorescence measurements and gave advice and assistance in the quantum efficiency measurements.
LITERATURE CITED Blau, K.; King, G. “Handbook of Derhratkes for Chromatography”; Heyden: London, 1977. Knapp, D. R. “Handbook of Analytical Derivatiratbn Reactions”; Wiley: New Y a k , 1979. Bouiton, A. J.; Ghosh, P. B.; KatrMy, A. R. J. Chem. Soc. B 1966, 1004- 10 11. Dai Monte, D.; Sandri, E.; Mazzaracchb, P. Boll. Scl. Fac. Chhn.I d . Bologna I968 26, 185-180. Chem. Abstr. 1989, 70, 115074q. Ghosh, P. B. J. Chem. Soc. B1968, 334-338. Dal Monte, D.; Sandri, E.; Di Nunno, L.; Fiorb, S.; Todesco, P. Chlm. Ind. (Milan) 1971, 53, 940-942. Ah-Kow, G.; Terrier, F.; Lessard, F. J . Org. Chem. 1978, 43, 3578-3584. Lawrence, J. F.; Frei, R. W. Anal. Chem. 1972. 44, 2048-2049. Kiimisch, H.J.; Stadler, L. J . Chromatcgr. 1974, 90, 141-148.
( I O ) Wolfram, J. H.; Feinberg, J. I.; Doerr, R. C.; Fkkller, W. J . ChromatOgr. 1977, 132, 37-43. (11) Roth, M. Clln. Chlm. Acta 1978, 83, 273-277. (12) Krd, 0. J.; BanOvsky,J. M.; Mannan, C. A.; Pickering, R. E.; Kho, B. T. J . Chfomatogr. 1970, 163, 383-389. (13) BMien, P.; Meliet. M. Anal. Biochem. 1979, 94, 313-321. (14) Maklta. M.; Yamamto. S.; Tsudaka, Y. Clln. Chlm. Acta 1978, 88, 305-310. (15) Woiwode, W.; List, D.; Weichardt, H. J . Clln. Cham. C//n. B&che171. 1979. 17, 251-258. (18) Verch, R. L.; Wallach, S.; Peabody, R. A. Clln. Chim. Acta 1979, 96, 125-130. (17) Martin, M. M.; Undqvist. L. J . Lumln. 1975, 10, 381-390. (18) Fit&, S. M.; Harkness, M. L. R.; Harkness, R. D. Nature (London) 1955, 176, 183. (19) Mlller, 1988; p J. 204. “Aromatic Nucleophilic Substitution”; Elsevier: Amsterdam, (20) DI Nunno, L.; Florio, S.;Todesco, P. E. J. Chem. Soc.,Perkln Trans. 2 1975. 14, 1489-1472. (21) Seller, N.; Demisch, L. I n “Handbook of Derivatives for Chromatogaphy”; Blau, K., King, G., Eds.; Heyden: London, 1977; pp 355-356. (22) Aboderin, A. A.; Semakula, R. E. K.; Boedefeld, E.; Kenner, R. A. F€BS Lett. 1973, 34, 90-94.
RECEIVED for review August 25,1980. Accepted November 25, 1980.
Liquid Chromatographic Separation of Amino Acids, Peptides, and Derivatives on a Porous Polystyrene-Divinylbenzene Copolymer Liad Iskandaranl and Donald J. Pietrzyk’ Chemistry Department, University of
Iowa, Iowa City, Iowa 52242
PRP-1, a porous efficient 10-pm spherlcal polystyrene-diVinylbenzene copolymer, was evaluated as a stationary phase for the chromatographic retention of amino acids, peptides, and amino acid derlvatlves. PRP-1, which Is a reversedphase adsorbent, is stable In acklk and basic sdutlons. Ionk strength, solvent composition, and pH were major eluting varlables. Effects d slde chains In amho ackls, peptides, and DNP, Dansyl, and PTH derivatives were evaluated. The positlon of the side chaln relative to the charge sites Influences the retention. The effects of chlrai centers and chaln length were evaluated. Several complex mlxtures were separated. Efficiencles were comparable to those obtained wlth alkyl-modified silica as the statlonary phase.
Reversed-phase liquid chromatography (LC) employing alkyl modified silica as the stationary phase is currently the major LC technique employed for the separation of complex mixtures. However, hydrophobic type polymers have also been successfully used in reversed-phase LC. The major types studied have been polystyrene-divinylbenzene copolymers (1-6). Typical of this group, which are highly and permanently porous with large surface areas, are the Amberlite XAD-1, -2, and -4 copolymers. XAD copolymers, which are reversed-phase adsorbents and are similar to the alkyl modified silicas in their LC performance, exhibit several advantages not found with the modified silicas. For example, the XADs are chemically and physically stable throughout the pH range 1-14, they generally exhibit higher retention under similar conditions, they have larger
loading capacities, and they respond to a variety of eluting variables including pH, type of organic modifer, waterorganic modifier ratio, and presence of counterions. Perhaps their main disadvantages compared to the alkyl modified silicas have been that the XADs or similar copolymers are not commercially available in uniform, spherical microparticles in either bulk form or prepacked columns. Thus, to successfully use an XAD column, the operator must crush large XAD particles, sieve them, and subsequently pack the column (1). Most applications of XAD columns were with irregular particles with a size range falling somewhere within the range of 25-75 pm. If care is exercised to isolate microirregular XAD particles, for example 6-pm particles, efficiencies similar to those found for the alkyl modified silica can be obtained (2). Recently, Hamilton Co. made available a polystyrene-divinylbenzene copolymer (PRP-1) that is a spherical, uniform 10-pm particle with a high pore volume (0.76 mL/g) and a large surface area (415 m2/g). These particles can be readily packed into highly efficient columns and have been used to separate nucleosides and related bases (7). In this report the retention of amino acids, amino acid derivatives, and small chain peptides on PRP-1is described. Since the PRP-1 is stable in acidic and basic solutions, unlike the alkyl modified silicas,this study focuses on the applications of these kinds of eluting conditions. With these data it is possible to predict separations, to discuss the influence of amino acid structure on retention, and to establish quantitatively the equilibria that influence the retention.
EXPERIMENTAL SECTION Reagents. Amino acids, amino acid derivatives,and peptides were obtained from Eastman Kodak Chemical Co., Sigma
0003-2700/81/0353-0489$01.00/00 1981 American Chemical Society
