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. In “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.
RECEIVEDfor 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 5 2 2 4 2
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
490
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
Chemical Co., Chemalog, and Aldrich Chemical Co. Some diastereomeric peptides were synthesized by reaction with (tertbutoxycarbonyl-L-amino acid)-N-hydroxysuccinimideesters (8). Acetonitrile was obtained as the chromatographic grade and the water was purified by passing distilled water through a mixed bed ion exchanger, an activated charcoal column, and finally through a 2-pm stainless steel fiiter. Inorganic acids, bases, and salts were analytical reagent grade. Instrumentation. A Waters Model 202 LC equipped with a Model 6000 pump, a U6K injector, and either a Waters fixed wavelength UV detector or a Tracor Model 970 variable-wavelength detector was used. The gradient LC used was an Altex Model 332. The stationary phase was a 10-pm spherical macroporous polystyrene-divinylbenzene copolymer, 4.1 mm X 150 mm column (PRP-1) obtained from Hamilton Co. Procedures. Sample solutions (about 1mg/mL) were prepared by dissolving milligram quantities of the analyte in several milliliters of HzO, ethanol, or their mixture. These were kept in 6-mL Hypovials fitted with Hycar Septa and sealed with aluminum caps (Pierce Chemical) and stored in a refrigerator when not in use. Pressure Lok Series B-110 10-pL or 25-pL syringes (Precision Sampling Corp.) were used to inject 1-to 4-pL amounts of the samples. Flow rates were generally 1mL/min and inlet pressure ranged from 500 to lo00 psi depending on eluting conditions. Either 254 or 208 nm was used for detection. Mixed solvents are expressed as percent by volume. Buffered solutions were phosphate buffers when possible or were HC1 or NaOH solutions. Ionic strength was maintained at 0.10 M by adding appropriate amounts of NaCl to the eluting mixture and the column temperature was controlled at 25 "C. Capacity factors were calculated as described elsewhere (1). Vowas determined by observing the retention volume for samples that were not retained by the column at the given eluting condition.
RESULTS AND DISCUSSION Amino acids (AA), peptides, and certain amino acid derivatives are ampholytes that change from a cationic species in acidic solution to a neutral species a t an intermediate pH and f m d y to an anionic species in basic solution. Furthermore the intermediate species is a polar zwitterion. These transformations are represented by the following equilibria: H,&CHCOzH
-t H20
& H30+
H36CHC02-
-I- H 2 0
% + 7 H30
I R I
R
4-
H3hCHC02-
(1)
HzNCHC02-
(2)
I R
h
If additional ionization sites are present, for example on the AA side chain, ionization at these sites will also contribute to the overall charge on the species as a function of pH. The presence of the charged sites has a significant effect on the level of retention of AA and derivatives in reversedphase LC (1, 9, 10). Thus, for the amphoteric AA and derivatives, retention passes through a minimum at the intermediate pH if the species has zwitterion properties; if it does not have this property, retention is a t a maximum at these conditions. Retention of monoprotic and polyprotic acids and bases in reversed-phase LC is also significantly influenced by pH; in these cases retention is low when the sample is in the charged form and large when in the uncharged form. In general, for polyprotic acids and bases, the first ionization has the greatest effect on the retention. Equations that relate the change in capacity factor, k', with pH and account for all the equilibria have been derived and verified for the retention of organic acids, bases, and ampholytes on reversed stationary phases (9, 10). Both alkylmodified silica and the XAD copolymers have been considered. The studies described here and others that focus on weak acids and bases demonstrate that the equations previously
IMIC Strength, M 0004 004
4
/ / !
a L-Phe-Gly b L-Phe-L-Ser c Gly-L-Phe d L-Ser-L-Phe
8
Oi4
k' 4.
2CHSCN 0
5
EtOH
10 15 20 Percent Organic Solvent
25
30
Figure 1. Retention of several dipeptides on PRP-1 as a function of solvent composition and ionic strength. For the variable solvent study pH 11.00, buffer was 0.01 M, and ionic strength was 0.10 M; for the knic strength study pH 11.00, buffer was 1.0 X lo4 M, and the solvent
was
5 % CH,CN-95%
H,O.
Table I. Retention of Amino Acids on PRP-la capacity factor, k' amino acid pH 1.60 pH 5.00 pH 11.00 Nonpolar 0.63 0.52 0.43 GlY 0.65 0.52 0.47 D,L-Na 0.65 0.54 2.92 D,L-Leu D ,L -Pro 0.63 0.54 0.82 3.20 1.22 1.95 D,L-Met 21.5 6.69 16.8 D,L-Phe 21.7 38.7 D , L - T ~ ~ > 85 Polar (Uncharged) D,L-Thr 0.46 0.63 0.54 0.48 0.56 0.52 D,L-Gln Basic 0.56 0.82 0.62 D ,L-Lys 0.49 0.60 0.47 D,L-His Acidic 0.41 0.58 0.55 D,L-GIu 5.50 1.56 0.52 D,L-nr Eluting condition was 100% H,O, 0.01 M phosphate buffer or HCl (pH 1.60) with NaCl added to give ionic strength of 0.10 M.at a flow rate of 1.00 mL/min and a V,,= 1.30 mL. derived which indicate how k'changes with pH for XAD and alkyl modified silicas (9,10) as the stationary phase apply also to the PRP-1. Since the efficiencies on the PRP-1 are similar to those obtained on alkyl-modified silica,the emphasis in this study centered on the eluting conditions where only the PRP-1 can be used, namely, strongly acidic and basic conditions. This is particularly significant since generally it is under these conditions where the AA is in the cationic and anionic form, respectively, that the best resolution is obtained for their separation. Solvent and Ionic Strength Effects. Figure 1shows that as the percent organic solvent in an aqueous mixture is increased, retention on the PRP-1 decreases sharply. Furthermore, the eluting power changes in order CHSCN > EtOH > MeOH. If the ionic strength of the eluting mixture is increased, retention increases. This is also shown in Figure 1. Peak shapes, in general, are better defined a t high ionic strength and differences in retention are the largest at this condition. These trends are similar to those observed when using the XADs as the stationary phase (I). Amino Acids. Table I lists the k'values for several AA on PRP-1 as a function of pH. The AA are grouped according
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
a. DL-Ala b. L-Pro c. DL-Met d. DL-Leu e. DL-Pho
Flgure 2. Separation of several amino acMs on PRP-1 using (A) pH 1.6 and (B) pH 11.00 mobile phase. Ionic strength was 0.10 M, buffers were 0,010 M, Row rates were 1.00 mL/min, and the solvent used was 100% H 2 0 for 4 min followed by a linear gradient to 15% CH,CN85% H 2 0 after an additional 15 min.
to the properties of the side chain. If the ionic strength is descreased or if the percent organic modifier is increased, retention decreases. Retention on the PRP-1, like that on the XAD (1,9), passes through a minimum at an intermediate pH depending on the structure of the side chain. For nonpolar side chains the minimum is well-defined particularly for the AA that have high retention. The presence of polar side chain groups reduces retention in basic solution, where the AA is in an anionic form so that the minimum at the intermediate pH is no longer observed. If the side chain is acidic or basic, ionization at this site also reduces retention. Thus, for a basic side chain, the effect is the largest in acidic solution where the AA can exist as the multivalent cation depending on the K, values, while if the side chain is acidic the effect is the greatest in basic solution where the multivalent anion is present. Figure 2 illustrates a separation of a six-component AA mixture using an acidic (A) and a basic (B)mobile phase. Resolution is significantly improved in the basic mobile phase, a condition not readily obtained with the alkyl-moditid silica, over an acidic one. The background drift is due to the gradient. Increasing the column length and/or changes in gradient should permit the separation of mixtures of the more closely related AA. Peptides. Peptides, like AA, have terminal acidic -C02H and basic -NH2 groups and exist as zwitterions. Thus, their retention should be similar to AA in that k’is at a minimum at their isoelectric point pH. Similarly, the side chains of the AA subunits in the peptide chain should influence retention since additional protonic equilibria can occur at acidic and/or basic groups on the side chains or via the polar or nonpolar properties of the side chains. Table I1 lists k’ data as a function of pH for several dipeptides of general structures I and 11. Since one side chain, H 2 N C H CON H C H CO2 H
I
CH2Ph
I
I R
H 2 N CH CONH CH C 0 2 H
I
R
I
CH2Ph
I1 the -CH2Ph group from the Phe subunit, is fmed, the influence on retention of other side chains with polar, nonpolar, or ionizable properties can be established. Furthermore, by changing pH the effect of the location of the side chain relative to the charged site will be observed. A higher percent CH&N was used in studying several dipeptides because of their high retention on the PRP-1. A minimum is observed in all cases except for the Tyr and Asp dipeptides which have acidic side chains. The additional
401
Table 11. Retention of Dipeptides on PRP-lU capacity factor, k’ dipeptide pH 1.6 pH 5.00 pH 11.00 Nonpolar 3.50 1.59 3.79 L-Phe-Gly 5.07 L -Phe-L-Ala 3.34 0.98 2.79 1.85 0.46 L-Phe-L-Val 7.17 L-Phe-L-Leub 5.79 1.65 L-Phe-L-Trp 24.5 7.38 31.8 Gly-L-Phe 7.25 1.37 2.20 L -Ala-L-Phe 7.69 1.38 3.63 2.77 L-Val-L-Pheb 2.87 0.69 L-Leu-L-Pheb 7.19 6.66 2.13 L-Trp-L-Pheb 31.0 9.05 24.8 Polar (Uncharged) 2.53 L-Phe-L-Ser 1.78 0.69 L-Phe-L-Thr 2.14 1.21 5.38 L-Ser-L-Phe 6.37 1.15 1.55 L-Thr-L-Phe 8.14 1.45 2.18 Basic 7.71 L -Phe-L-Arg 1.48 0.82 3.49 ~-Arg-~-Phe 4.48 1.21 Acidic L -Phe-L-Asp 0.65 0.41 0.64 L-Phe-L-Tyr 3.43 0.87 0.71 L-Asp-L-Phe 0.28 0.76 0.36 L-Tyr-L-Pheb 5.51 1.31 0.59 Eluting condition was 5%CH,CN-95% H,O, 0.010 M phosphate buffer or HCI (pH 1.60) with NaCl added to give an ionic strength of 0.10 M at a flow rate of 1.00 mL/ Same as ( a ) except that the min and V , = 1.27 mL. CH,CN-H,O ratio was 13:87.
side chain ionization site, which can lead to a double negatively charged species in basic solution, and its location in the dipeptide will influence the retention. For example, compare the retention of Phe-Asp and Phe-Tyr to Asp-Phe and TyrPhe in basic solution. When the hydrophobic -CH2Ph side chain is at subunit one and the two negative charge sites are in subunit two, retention is higher than that when one negative site is on the side chain in subunit one and the -CH2Ph is adjacent to the terminal negative site in subunit two. Retention of the Arg dipeptides, which have a basic side chain group, passes through a minimum like the other dipeptides. Apparently, the influence of the -CH2Phgroup and its location in the dipeptide has a greater influence on retention than that due to the additional basic site. For the other dipeptides retention increases as the hydrophobic property of the side chain increases. Thus, retention of the dipeptide is in the order Leu > Val > Ala > Gly regardless of whether these are at subunit one or two in the dipeptide or whether the solution is acidic or basic. However, the position of the -CH2Ph group in the side chain has a strong influence on retention. When this group is one subunit away from the charge site in the dipeptide as in Phe-AA in basic solution or AA-Phe in acidic solution (see structures I and 11), retention is higher than for the reverse or AA-Phe and PheAA, respectively. If a polar group is present as a side chain, retention decreases. For example, Ser and Thr dipeptides contain a hydroxyl side chain group and their retention at all pH values is much less than the corresponding dipeptides without these groups such as the Ala and Val containing dipeptide. Even with polar groups, small differences influence the retention. Thus, the Thr dipeptide is more retained than the Ser dipeptide because the former has an additional methyl group on the side chain.
492
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
Table 111. Retention of (Ala), and Other Tripeptides on PRP-1 peptide D,L-&a (L-Ala), ( L -Ala), (L-44,
(L-Ala), peptide
capacity factor, k‘ pH 1.75 pH 5.20 pH 11.00 0.52 0.68 1.15
0.51 0.47 0.57
3.81 7.86
1.02 1.98
0.42 0.70 0.95 1.54 2.78 6.32
A
capacity factor, k‘ pH 1.56 pH 5.00 pH 11.00
Gly -Gly-Gly 0.54 0.42 0.51 Gly-Gly-L-Ala 0.83 0.44 0.58 Gly-Gly-~-Vd 5.05 0.90 1.50 Gly-Gly-L-Leu 24.4 3.20 6.00 Gly-Gly-L-Phe > 20 10.6 20.2 L- Ala-Gly-Gly 0.62 0.46 0.68 L-Val-Gly-Gly 1.53 0.91 3.43 ~-Le~-Gly-Gly 5.22 2.82 16.3 L-Phe-Gly-Gly 22.6 12.4 >>20 Eluting condition was 100% H,O, 0.01 M phosphate buffer or HCl (pH 1.60) with NaCl added to give ionic strength of 0.10 M at a flow rate of 1.00 mL/min and a V, = 1.30 mL. The effects of peptide structure on retention on PRP-1 are similar to that observed on XAD-2 and -4where the influences of these factors have been discussed in greater detail (I, 9). Since efficiency is improved significantly on the microparticle PRP-1 over that observed on the large XAD particles, many additional separations particularly of closely related dipeptides are possible. The data in Table I1 were used to predict the separation of several different dipeptide mixtures; these were carried out with both an acidic and basic mobile phase. For complex mixtures a CH3CN-H20 gradient was also used. Depending on the mixture, resolution tends to be more favorable with the basic solution; however, some mixtures that are easily separated with an acidic mobile phase are not separated in a basic one (at the same solvent mixture) and vice versa. For example, L-Phe-Gly and L-Ala-L-Phe are easily separated in an acidic mobile phase but not a basic one while the reverse was found for a Gly-L-Phe and L-Ala-L-Phe mixture. Also, elution orders for the two mobile phases can be reversed. For example, for a L-Phe-L-Ala and L-Ala-L-Phe mixture the former peptide appears first in an acidic mobile phase and second in a basic one. Many other examples illustrating these observations are apparent from the data in Table 11. Peptide Chain Length. Increasing the peptide chain length via a repeating AA subunit increases the retention. This is illustrated in Table I11 where k’ data for a series of Ala peptides are listed as a function of pH. Like the AA and dipeptides, a minimum even for the longer chain peptides is observed at the zwitterion pH. As the chain length increases retention in acidic solution increases more rapidly than that in basic solution. Plotting the k’values in Table 111vs. chain length indicates that an increase in retention is gradual up to 4 units, after which it increases sharply. The increase is also greater in acidic and basic solution over that observed at the zwitterion pH. Separation of the Ala peptides through 6 units is shown in Figure 3 with both an acidic (A) and basic (B) eluting condition. Introducing CH3CN as a gradient will reduce the separation time for the longer peptides. It also appears that a zwitterion pH would be a suitable eluting condition for the longer chain peptides since retention is lower a t this pH and less organic modifier would be required for elution.
0
5 m,
IO
15
Figure 3. Separation of (Ala),, peptides on PRP-1 using (A) pH 1.60 and (B) pH 11.00 mobile phase. Ionic strength was 0.10 M, buffers were 0.010 M, fbw rate was 1.00 W m i n , and the sokent was 100% HZO.
Table IV. Retention of Diastereomers on PRP-1 a capacity factor, k’ PH PH PH peptide 1.6 5.25 11.00 L-Ala-L-Ser, D-Ala-D-Ser 0.34 0.31 0.51 L-Ala-D-Ser, D-Aa-L-Ser 0.34 0.31 0.51 1.67 0.56 1.26 L-Ala-L-Met, D-Ala-D-Met L-Ala-D-Met, D-Ala-L-Met 2.80 0.89 1.40 L-Ala-L-Leu, D-Aa-D-hu 2.88 0.70 1.73 L-Ala-D-Leu, D-Ala-L-Leu 5.87 1.35 2.00 L-Ala-L-Phe, D-Ma-D-Phe 7.75 1.67 4.70 L-Ala-D-Phe, D-Ala-L-Phe 14.5 3.46 5.30 L-Leu-L-Na, D-Leu-D-Alab 0.62 0.60 0.69 L-Leu-D-Ma, D-Leu-L-Alab 0.97 0.60 0.70 0.67 1.19 L-Leu-L-Val, D - L ~ u - D - V ~1.11 ~ 3.14 1.42 1.29 L-Leu-D-Val, D-Leu-L-Vdb L-Leu-L-Leu, D-LeU-D-LeUb 2.51 1.11 2.21 L-LeU-D-kU, D-Leu-L-Leub 7.18 3.68 2.68 L-Leu-L-Phe,D-Leu-D-Pheb 5.29 2.25 5.01 L-Leu-D-Phe, D-Leu-L-Pheb 13.8 5.56 5.74 L-Ala-L-Ala-L-Ala 1.15 0.57 0.95 L-Aa-L-Ala-D-Ala 1.94 0.69 1.39 3.64 1.18 1.74 L-Ala-D-Ala-L-AlaC Eluting condition was 5%CH,CN-95% H,O,0.010 M phosphate buffer or HCl (pH 1.60) with NaCl added to give an ionic strength of 0.10 M at a flow rate of 0.98 mL/ min and V , = 1.27 mL. Same as ( a ) except CH,CN:H,O ratio was 15:85. Same as ( a ) except solvent was 100% H,O. Table I11 also lists k’data for a series of tripeptides where one AA subunit with a nonpolar side chain is changed at either subunit one or three. Since the other two AA subunits are the same, the change in retention with pH is a function of the nonpolar side chain and its location in the tripeptide relative to the changed sites. As with the AA and dipeptides, retention is at a minimum at the zwitterion pH and retention changes in the order Phe > Leu > Val > Ala > Gly. This order is observed throughout the pH range studied. In acidic solution the increase in retention is the largest when the variable A4 side chain is a t subunit three since at this condition the side chain is located in the tripeptide a t the end opposite to the charged site. In basic solution the reverse is observed; that is, retention is larger for the tripeptide series where the available AA side chain is at subunit one and is at the end opposite to the charge site. Examination of the k’data for the Gly tripeptides in Table I11 reveals that many different separations are possible. Although not shown here, six- and seven-component mixtures were successfully separated by using acidic and basic solution
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
and a CH3CN-H20 gradient. Because of the structural effect on retention, resolution is more favorable in acidic solution when the AA subunit changes at position 3 and better in basic solution when the change is at position 1. If the mixture contains tripeptide members of both series, elution orders can be reversed when comparing the two eluting conditions. For example, Gly-Gly-~-Valwould elute before ~-Val-Gly-Glyin basic solution; in acidic solution the reverse order is obtained. Peptide Diastereomers. Resolution of a DL-AA mixture was not obtained on the PRP-1 at any pH or CH3CN-H20 condition. However, if two or more chiral centers are present, as in the case of a peptide, the stereochemistry of the individual AA subunits has an effect on the retention. Retention data for several di- and tripeptide diastereomers on PRP-1 as a function of pH are shown in Table IV. Resolution of the LL and DD mixture or of the LD and DL mixture was not found. The LL and DD dipeptide retention is always less than that for the LD and DL form and is consistent with the stereochemistry of the dipeptide. Similar results were found with an alkyl-modified silica stationary phase (11). The conformations for a typical dipeptide, such as DLAla-DL-Ala, are H
H
i!
L 2
DA
For the LD and DL forms, both AA side chain groups are on the same side. This provides a concentrated hydrophobic center for interaction with the PRP-1 surface and hence a high retention. In contrast, the LL and DD forms have side chain groups on opposite sides, and this reduction in the hydrophobic center decreases the retention. The observed increase in retention due to individual hydrophobic side chains or decrease due to polar ones is consistent with the previous discussion considering side chain effects within individual AA. If the k’data in Table IV are plotted vs. pH, retention for the LL and DD forms undergoes a greater decrease at the zwitterion pH than that for the LD and DL forms. This is consistent with the dipeptide conformations. In the LL and DD forms the terminal charged sites are on opposite sides and have a greater effect on the hydrophobic interaction than for the LD and DL forms where the two charged sites are on the same side. The k’data in Table IV also indicate that a single charged site has a greater effect on retention in acidic solution where the -NH3+ group is present over basic solution where the -COP- group is present. Furthermore, the retention difference between the LL-DD and LD-DL dipeptides is greater in acidic solution. This suggests either that the -NH3+ group is better able to participate in an interaction along with the hydrophobic center with the stationary phase or that the -COP- charged site is better able to disrupt the interaction. These unique structural properties indicate that the optimum mobile phase for the separation of complex mixtures of dipeptide diastereomers is in the order zwitterion pH > acidic pH > basic pH. Figure 4 illustrates the separation of a complex mixture of dipeptide diastereomers at the zwitterion pH. Similar complex mixtures were separated with acidic and basic solutions, and the resolution found was as expected when comparing the three conditions. One major advantage of the acidic or basic mobile phase is that elution orders can often
403
d. (L-Dl (D-LNLeu-Alo) e. (L-L) (D-D) (Leu-VoI) f. (L-L) (D-D) (Ala-Phe) g. (L-D) (D-L) (Ala -Phe) h. (L-L)(D-D) (Leu-Leu) i . (L-Dl(D-L) (Leu-Val) j . (L-Ll(D-D) (Leu-Phel k. (L-D) (D-L) (Leu-Leu) I. (L-D)(D-L) (Leu-Phe)
A
0
IO
ml
20
30
Figwe 4. Separation of peptide diastereomers on FJRP-1 at pH 5.25. Ionic strength was 0.10 M, buffers were 0.010 M, Row rate was 1.00 mL/min, and the solvent used was 3.3% CH3CN-96.7% H,O for 20 min followed by a linear gradient to 15% CH3CN after 3 mln and
isocratic elution at 15% CH3CN.
be altered between the two conditions. Similar diastereomer separations were not possible on 45-65-pm XAD-2 or -4 copolymers as the stationary phase, except for those cases where retention differences were very large (1,9). There is considerable difference in the retention of the (Ala), diastereomers as shown in Table IV for the different pH conditions. The elution order found is consistent with the orientation of the side chains and terminal charged sites within the different (Ala), conformers (11). For the three (Ala)3 diastereomers studied the acidic eluting agent provides the optimum condition for separation; the basic and zwitterion pH condition are nearly equivalent in their ability to resolve the mixture. Amino Acid Derivatives. Complex mixtures of AA are often separated after their conversion to one of several derivatives. Usually the derivatives are more easily separated and/or detected at a lower concentration level because of the structure of the derivative. The three major derivatives of AA of general structure H2NCHRC02Hare the DNP (1111, Dansyl (IV), and PTH (V) derivatives.
& j ’ “02
I
HNFHC02H
R DNP Ill
F;J(CH3)2
I
S02NHyHC02H
R Dansyl I V
In considering each type of AA derivative the only structural change within the family is the R group introduced as the side chain on the AA. Thus, the polar, nonpolar, acidic, or basic
494
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 DNP Derivatives
a
0
20
40
ml
60
80
Flgure 5. Separation of amino acid DNP derivatives on PRP-1 using (A) pH 1.60 and (B) pH 11.00 mobile phase. Ionic strength was 0.10 M, bufferswere 0.010 M, Row rates were 1.00 mUmln, and the solvent was In (A) 3 5 % CH3CN-65% H,O for 10 min followed by a linear gradient to 4 5 % CH3CN after an additional 10 min and in (B) 1 2 % CH3CN for 20 mln followed by a linear gradient to 2 5 % CH3CN after
an additional 40 min.
properties of the side chain as a major influence on retention are still retained. However, the influence of ionization at the terminal -NH2 and -C02H group within the AA is modified since derivative formation leads to blockage of either or both of these sites. For example, the terminal -NH2 group in the DNP is blocked and in aqueous solution the AA as the DNP derivative is no longer a diprotic ampholyte but is a monoprotic weak acid. Hence, its retention on PRP-1 as a function of pH should be high in acidic solution where it is un-ionized and low in basic solution where it is ionized. Even though the terminal AA -NH2 group is blocked in the Dansyl, this derivative is a triprotic ampholyte due to ionization a t the -N(CH&, -NH-, and -C02H group, and its retention should pass through a minimum or maximum as a function of pH depending on whether it exists as a zwitterion or not. Formation of the P T H derivative blocks both the terminal -NH2 and -C02H groups. However, the newly formed ring should exhibit weakly acidic and basic properties and its retention should pass through a maximum typical of a nonzwitterion type ampholyte. The LC data for these derivatives on XAD-2 and -4, in general, are consistent with these anticipated results (I). Futhermore, since equations can be derived which relate how k'changes with H+ by accounting for all protonic equilibria, it should be possible to determine the K , values for the DNP and Dansyl derivatives from the LC data (1,9, 12). Table V lists k'data for a series of DNP, Dansyl, and PTH derivatives on PRP-1 as a function of pH. Examples were chosen to emphasize the major retention effects attributable to the AA side chain. Since the retention due to the side chain and/or pH can lead to a very high retention, the percent CH3CN was increased to keep retention times small. For the DNP derivatives without ionization sites on the side chain, retention is high in acidic solution and low in basic solution which is typical of a monoprotic acid. (It should be noted that the k'data for the DNP derivatives at pH 1.50 are all a t 20% more CH,CN than listed because retention times were very large a t this pH.) If a basic group is on the side chain as in the Lys- and Arg-DNP, retention is low in acidic solution since this group is ionized. If the side chain group is acidic, as in the Asp and Glu-DNP, retention is much lower in basic solution because these derivatives (diprotic acids) are doubly ionized. Figure 5 shows the separation of a complex AA-DNP mixture using an acidic (A) and basic (B) eluting mixture with
U-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 Dansvl
.
Derivatives
.
495
-PTH Derivatives
P D L-Tr p
DL-Phe
DL-Lnu
mlI0
0
IOm1 20
Flgwe 6. Separation of amino acid Dansyl derivatives on PRP-1 using (A) pH 1.75, (B) pH 3.60, and (C) pH 11.00 mobile phase. Ionic
strength was 0.10 M, buffers were 0.010 M, flow rates were 1.00 mL/min, and the solvent in (A) and (C) was 2 5 % CH3CN-75% H,O; the solvent in (B) was 2 0 % CH,CN for 12 mln followed by a linear gradient to 50% CH3CH after an additional 20 min.
a CH3CN-H20gradient. The elution order is the same in both conditions except for the DNP derivatives that have ionizable sites on the side chain for reasons outlined previously. For complex mixtures the basic eluting condition, which cannot be used with the alkyl modified silica stationary phase, is preferred because of greater differences in selectively and subsequently better resolution. An intermediate pH can also be used, but this condition does not provide the best selectivity and resolution for complex mixtures. The retention of the Dansyl derivatives in Table V is typical of ampholytm without zwitterion properties (9);that is, if the k'data are plotted vs. pH a maximum occurs at about pH 3.5. If the AA side chains have acidic or basic properties, retention is reduced in basic and acidic media, respectively, because of dissociation at these sites, while retention of the other Dansyl derivatives corresponds to the polar or nonpolar properties of the side chain. Figure 6 illustrates the separation of several Dansyl derivatives using an acidic (A), basic (C), and an intermediate pH (B) corresponding to the maxima of a It '-pH plot. Elution orders are essentially the same in the three eluting conditions except where ionization of the side chain occurs. Slight differences in the acidic condition are probably due to the fact that pH 1.,75is not sufficiently acidic enough to convert all of the Dansyl derivatives to the fully protonated form. For complex mixtures the optimum eluting condition in terms of selectively and resolution is at the intermediate pH, particularly if a CH3CN-H20 gradient is also employed. The retention data for the PTH derivatives in Table V are typical of an ampholyte with weak acid and very weak basic properties, since retention is very high in acidic solution and does not decrease until a very basic solution is obtained. The weak amphoteric property is indicated by a maximum at about pH 4-6 in the k'-pH plot. The influence of the side chain is consistent with the influence of the side chains with previous observations. For example, the ones with the nonpolar side chains are more retained than those with polar ones. An acidic side chain reduces retention not only earlier but to a greater extent due to the second stronger ionization step while those with basic side chains have reduced retention in acidic solution. Figure 7 illustrates a separation of a PTH mixture by using an acidic (A) and an intermediate pH (B) as the eluting
ml
ml
Figure 7. Separation of amino acid PTH derivatives on PRP-1 using (A) pH 1.65 and (B) pH 5.50 mobile phase. Ionic strength was 0.10 M, buffers were 0.010 M, flow rates wwe 1.00 Wmin,and the sotvent
in (A) was 2 5 % CH3CN-75% H,O up to 10 min followed by a linear gradient to 60% CH3N after an additional 20 min and in (B) was 2 0 % CH3CN-80% H,O for 8 mln followed by two linear gradient steps to 4 2 % CH3CN at an addiiional 5 mln and 55% CH,CN at an additional 25 min. condition. Elution orders in the two differ because of the maxima for the derivatives and because of acidic and/or basic side chain groups. Separations. The examples were chosen to illustrate that not only resolution but also elution order is significantly different when comparing acidic, basic, and intermediate pH eluting conditions. Gradients were used in most examples, however, this is not always necessary for simpler mixtures. Examination of the k 'data reveals that many other mixtures can be separated and provides a basis for predicting the elution order when extending the application of the PRP-1 column to AA, peptides, and AA derivatives that were not studied here. In general, plate numbers, calculated by N = 16(t,/w)*, range from 3000 to 7000 plates/m and depend on the type of mobile phase used. Efficiencies over 20000 plates/m have been reported for the PRP-1 column (5).
LITERATURE CITED (1) Kroeff, E. P.; Pletrzyk, D. J. Anal. Chem. 1978, 50. 502-511. (2) Baum. R. G.; Saetre, R.; Cantwell, F. F. Anal. Chem. 1980, 52, 15-19. (3) Mohammed, H. Y.; Cantwell, F. F. Anal. Chem. 1978, 50, 491-496. (4) Mats&, R.; Yamamlya, T.; Tatsuawa, M.; Ejima, A.; Takai, N. J . Chromatogr. 1979, 173, 75-87. (5) Walton, H. F. Anal. Chern. 1980, 52, 15R-27R. (6) Cantwell, F. F.; Puon, S. Anal. Chem. 1979, 51, 623-632. (7) Lee, D. P.; Klndsvatw, J. H. Anal. Chem. 1980, 52, 2425-2428. (8) Bodanszky, M.; Klausner, Y. S.; Ondettl, M. A. "Peptide Synthesis", 2nd ed.; Wlley: New York, 1976. (9) Kroeff, E. P.; Rotsch. T. D.; Pietrtyk, D. J. Anal. Chem. 1978, 50, 497-502. (10) Horvath, C.; Melandar, W.; Molnar, I. Anal. Chem. 1977, 49, 142-154. (11) Kroeff, E. P.; Plelrzyk, D. J. Anal. Chem. 1978, 50, 1353-1358. (12) Palallkit. D.; Block, J. H. Anal. Chem. 1980, 52, 624-630.
RECEIVED for review August 25,1980. Accepted November 17,1980. Part of this work was presented at the 16th Midwest Regional Meeting of the American Chemical Society, Lincoln, NE, 1980, and at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, 1981. This investigation was partly supported by Grant CA 1855505 awarded by The National Cancer Institute, DHEW, and by Grant CHE 7913203 awarded by The National Science Foundation.
