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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8 , JULY 1978
(9) E. Glueckauf, Trans. Faraday SOC., 51, 34 (1955). (10) E. Giueckauf, Trans. Faraday SOC., 54, 1203 (1956). (1 1) W. A. Russell, D. A. Papanastassiou, T. A. Tombrello, and S. Epstein, Proc. Eighth Lunar Sci. Conf., 3791 (1977). (12) F. Helfferich, Ion Exchange”, McGraw-Hill, New York, N.Y., 1962. (13) F. H. Spedding, J. E. Powell, and H. J. Svec, J . Am. Chem. Soc., 77, 6125 (1955). (14) J. Aaltonen, Suom. Kemistil. B , 44, 1 (1971).
(15) A. Calusaru and F. Bunus, Radiochim. Acta, 18, 23 (1972).
RECEIVED for review February 3, 1978. Accepted April 24, 1978. Work supported by National Aeronautics and Space Administration grant NGL 05-002-188. Division of Geological and Planetary Sciences Contribution No. 2967(263).
Effects of pH, Ionic Strength, and Organic Modifier on the Chromatographic Behavior of Amino Acids and Peptides Using a Bonded Peptide Stationary Phase Godwin W-K. Fong and Eli Grushka” Department of Chemistry, State University of New York at Buffalo, Buffalo. New York 74274
A bonded tripeptide phase, L-Val-L-Ala-L-Pro, has been used as a statlonary phase in liquld chromatography for the separation of phenylthiohydantoin (PTH)-amlno acid and dipeptides. All 25 PTH-amino acids tested here have different capacity ratios when either acidic (pH N 2.5) or basic (pH 7.4) aqueous citrate mobile phases were used as eluents. The chromatographic behavior of some free and protected dipeptides were also studied systematically. The pH of the mobile phase, its ionic strength, and the amount of organic modifier affect the retention behavlor of the dipeptides. The retention characteristics of the free dipeptides were affected to a greater extent than those of the protected ones. The results lndlcate that mobile phases can be tailor-designed for optimum separations of the isomeric dipeptldes. Comparisons with previous bonded peptide columns are made in order to gain a better understanding of the mechanism of separation.
The analysis of amino acids, peptides, and their derivatives is of paramount importance in the determination of protein structures. Chromatographic techniques have played a major role in the separation and quantitation of amino acids and peptides as evidenced by the recent reviews of Deyl (1, 2 ) . These substances have been separated by GC, ion exchange chromatography, TLC, and HPLC (viz. 3-23). The analysis of amino acids and peptides can be improved with the use of selective stationary phases. This is especially true in the case of closely related peptides. It is clear from the literature that there is a need for a chromatographic procedure for the separation of such solutes. With this need in mind, Schott and his co-workers (24-26) used “template chromatography” to selectively separate tryptophan containing peptides. Grushka and his co-workers (27-29) have shown that peptides bonded to the solid support, can be used advantageously in the analysis of peptides and amino acid derivatives. Amino acids and dipeptide stationary phases have been used previously in GC and HPLC (viz. 30-35) for the separation of D,L amino acid enantiomers. However, the dipeptide stationary phases previously studied were coated in a conventional manner, and were used mainly in GC. As was pointed out by Hansen et al. (36), and recently reviewed by Morris and Morris (37),there is still a need for a quick chromatographic technique for the analysis of these compounds, especially the small peptides. Particularly important are the separations of geometrical and structural 0003-2700/78/0350-1154$01.00/0
isomers of peptides. D-Leu-L-Tyr and L-Leu-L-Tyr were separated by Hoffpauir and Guthrie (38)and by Blackburn and Tetley (39) on DEAE-cellulose and Dowex-50, respectively. The use of DEAE-cellulose, Sephadex G-50, XAD-2 resin, ion-exchange and amino acid analyzer for the separations of some free isomeric and diastereomeric peptides were also reported (40-49). Various derivatives of protected peptides, have been separated using hydrated silica gel (50) and TLC plates (51-53). Krummen and Frei (54) demonstrated the separation of nonapeptides by reversed phase LC. Little attention was directed toward studying the chromatographic characteristics of bonded peptide phases. The present paper investigates in detail the chromatographic properties of L-Val-L-Ala-L-Proas the bonded stationary phase in HPLC. More specifically the retention behaviors of some amino acids, phenylthiohydantoin (PTH) derivatives of amino acids, free and protected dipeptides are examined as a function of pH, ionic strength, and the amount of organic modifier in the aqueous mobile phase in order to ascertain the interaction between the solutes and the bonded peptide.
EXPERIMENTAL Instrumentation. The liquid chromatographic system was described previously (28). Reagents. Water, used for the preparation of the mobile phase,
was obtained by passing distilled water through an ion exchanger treatment column (Barnstead Sybron Corp.). The water was degassed by boiling. pH measurements were made with a Fisher combination electrode (Fisher Scientific Co., Fairlawn, N.J.). All chemicals and solvents used either in the mobile phases or in the synthesis of the stationary phase, unless otherwise stated, were obtained from Fisher Scientific Co. Amino acids and some free dipeptides were bought from either Sigma Chemical Co. (St.Louis, Mo.) or from ICN Life Sciences Group (Cleveland, Ohio). PTH-amino acids were obtained from Pierce Chemical Co. (Rockford, Ill.). The silanizing agent, l-trimethoxysilyl-2chloromethylphenylethane (Y-5918)was received from the Union Carbide Corp. (Tarrytown, N.Y.). The coupling reagent used in the peptide synthesis, N,N-dicyclohexylcarbodiimidewas purchased from the Aldrich Chemical Co. (Milwaukee, Wis.). Partisil-10 was purchased from Whatman Inc., (Clifton, N.J.). Trifluoroacetyl (TFA)-di-and tripeptide methyl esters (OMe) were furnished by M. Schwarzberg. Buffer solutions of constant ionic strength were prepared as described by Elving et al. (55). Procedure. The tripeptide L-Val+ Ala-L-Pro was synthesized on the Partisil-10 through the reagent Y-5918. The synthesis was similar to that described previously (28). Two batches of the bonded tripeptide were prepared. For one batch, elemental analysis showed 13.6% carbon. This material was slurry packed 0 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Table I. k‘ Values of Amino Acids in Various Mobile Phasesa Aminoacids
(A)
His. HC1 Tyr Phe
0.20 0.43 0.47 1.05
DP
Mobile phasesb (B) (C) 0.68 0.96 1.06 2.06
0.89 0.96 1.02 1.94
1155
Table 11. k’ Values of PTH-Amino Acids in Various Mobile Phases (Column I ) Mobile phasesa (D) * * e
1.44 1.30 3.44
Bonded phase: Val-Ala-Pro. Column I. (A) = 1% Citric Acid/H,O; pH 2.5, I = 0.006 M. (B) = 1%Na Citrate/H,O t HCl; pH 5.5, I = 0.26 M. (C) = 1%Na Citrate/H,O t Citric Acid; pH 7.4, Z = 0.20 M. (D) = Distilled deionized water: pH 5.5. a
-
into two columns having the dimensions of 300 mm X 2.1 mm i.d. (Column I) and 250 mm X 3 mm i.d. (Column 11). The second batch had 12.3% carbon. The column packed with this batch had the same dimensions as Column 11, and it will be designated as Column 111.
RESULTS AND DISCUSSION The surface coverage of the peptide should be looked a t carefully. The first moiety bonded to the silica gel is the Y-5918 reagent. The % C for both batches a t this stage was between 9 and 10% which corresponds to a surface coverage of about 2.5 ymol/m2. The % C after the tripeptide synthesis indicates that not all the chlorine on the Y-5918 has reacted with the amino acids. It is not clear at this point whether this is due to steric effects or unfavorable reaction rates. A question might be raised concerning the surface that the solutes see. Our previous work (27,28) as well as the results described here, seems to indicate that the bonded peptide plays the major role in affecting the retention behavior of the amino acids and the dipeptides. Column Stability. The reproducibility in the retention times, the relative retentions, and the elution orders with the various phases used in this study indicates that the bonded tripeptide (L-Val-L-Ala-L-Pro)is stable. Since no mobile phase lower than pH 2.1 or higher than pH 7.4 was used, the possibility of loss of bonded amino acids or dissolution of silica gel is minimal. A typical peptide column usually had a life expectancy of two to three months of continuous use, provided that no drastic change in mobile phase polarity was made. The use of mobile phases of very high salt concentrations (or high ionic strengths) at low flow rates usually resulted in a drastic increase in column pressure. Analysis of Free Amino Acids. Monitoring a t 254 nm and 280 nm with a UV detector, only four amino acids, namely HistidineHCl (His.HCl), Tyrosine (Tyr), Phenylalanine (Phe), and Tryptophan (Trp) could be analyzed. Elution data of these four solutes on Column I are shown in Table I. The following trends are observed. (a) for a given pH of the mobile phase, the elution order is His.HCl< Tyr < Phe < Trp. The same elution order was observed previously on bonded L-Val-L-Ala-Mer, and LVal-L-Phe-L-Valtripeptide columns (28,29). A similar elution order for Tyr, Phe, and T r p was found by, among others, Moore and Stein (56) in ion exchange systems, Niederwieser (57) in adsorption chromatography, and Horvath et al. (58) in hydrophobic chromatography. The retention order of the amino acid follows their hydrophobicities. The terminal amine group of the bonded tripeptide stationary phase can be charged or neutral depending on the pH of the mobile phase. The data in Table I would seem to indicate that, at least for the few amino acids studied here, electrostatic interaction is not always the major factor affecting the retention order. Hydrophobic interactions between the hydrocarbon side chain of the bonded tripeptide phase and that of the solutes may
PTH-L-His. HC1 PTH-L-Arg PTH-D ,L -Thr PTH-D,L-Ser PTH-S-methylL-Cysteine PTH-L-Asn PTH-L-Gln PTH-Gly PTH-D,L-Ala PI”-D,L-Met. Sulfone PTH-L-OH-Pro PTH-L-G~u &YI”-D,L-Val PTH-D,L- Asp PTH-L-Pro PTH-D,L-Met PTH-L-Ile PTH-L-Tyr PTH-L-~U PTH-Nle PTH-D,L-Phe PTH-(5’- Carboxyl.
(A)
(B)
(C)
(D) (E)
0.15 0.31 3.32 3.72 3.90
5.68 5.29 5.05 5.44 4.06
7.57 5.85 3.60 3.71 3.25
5.67 0.24 6.63 7.38 5.90
1.28 0.78 0.79 0.95 1.07
4.20 4.79 4.84 6.43 6.49
5.67 6.76 6.91 8.60 8.72
4.06 4.60 4.29 6.15 7.13
8.10 8.56 8.38 12.12 12.79
1.41 1.00 0.98 0.84 1.25
7.53 10.36 12.93 14.33 15.99 16.61 19.11 20.47 20.72 25.71 33.02 41.53
10.61 19.93 18.25 11.64 24.14 25.63 28.89 33.02 29.69 39.46 49.11 27.67
8.02 13.76 b 6.45 12.98 22.71 b 4.52 14.36 13.76 18.22 30.45 21.48 38.43 25.93 60.06 23.45 42.54 27.05 53.90 37.36 78.91 10.95 116.70
0.89 b 0.79 b 0.86 0.84 0.77 1.22 0.72 0.72 0.93 0.89
methyl)-^-
Cysteine P T H - D , L - ~ ~ 78.82 146.13 84.98 224.75 1.07 PTH(ePheny1113.85 b 169.11 b 1.46 thiocarbamyl) L Lysine YI’H-L-Cysteic b 13.25 7.26 b b Acid (K-salt) a (A)-(D) = same as in Table I. ( E ) = Methanol. Peaks did not elute in reasonable times. determine their elution sequence. (b) Table I shows the pH dependence of the capacity ratio, k’. Phe and T r p behave similarly, namely their k’ values increased at first as the pH was raised, and then decreased slightly as the pH was further increased. The capacity ratio of Tyr seems to level off at basic pH’s, whereas the h’value for His.HC1 seems to increase continuously with the pH. The pH dependence observed here seems to agree with the theoretical arguments of Horvath et al. (58). (c) The differences in the capacity ratios of the same amino acids in columns B and D of Table I are noteworthy. The pH of both mobile phases was about 5.5. The one containing the citrate (and chloride) anions, having the ionic strength of I = 0.26 M, in general, gave smaller k’values than those observed with the deionized water mobile phase, where I is assumed to be about 0. The bonded peptide at pH 5.5, most likely is partially charged, and the retention behavior (but not elution sequences) of the amino acids is a function of not only hydrophobic, but also electrostatic interactions. As discussed by, among others, Pahlman et ai. (59) a t very low ionic strength, electrostatic forces predominate and the larger k ’ values may be a reflection of these forces. Alternatively the arguments used by Horvath et al. (58) can also be used to explain the decrease in k’ as I increases. The data shown in Table I are not sufficient to draw any conclusions concerning the retention mechanism of free amino acids and a more detailed study of the effect of pH, ionic strength, and nature of the anions in solution is needed. Analysis of PTH Derivatives of Amino Acids. The capacity factors of 25 PTH-amino acids, obtained in Column I, are shown in Table 11. Similar to the behavior on the L-Val-L-Phe-L-Valcolumn (29),all but the acidic PTH-amino
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
acids were eluted very fast (k’values less than 1.5) when using neat methanol as the mobile phase. Unlike the L-Val-LPhe-L-Val column, almost all the PTH-amino acids were eluted with deionized distilled water (pH -5.5). The four exceptions were the acidic amino acids and PTH-(ephenyl- thiocarbamy1)-L-lysine. With a mobile phase having the same pH as distilled deionized water, but a t an ionic strength I = 0.26 M, all the PTH-amino acids, except PTH-L-Arg, PTH-L-Pro and PTH-L-His-HC1have a smaller k’ value than with the water mobile phase. Of the above mentioned three amino acid derivatives, the capacity ratio of the PTH-Arg changed the most, perhaps since argenine is very soluble in water. The citrate buffer in general seems to salt out these three solutes. Under acidic conditions, all the P T H derivatives have different k’values, although some values are very close; e.g., PTH-L-Gln and PTH-Gly. The basic amino acids (His-HC1 and Arg) were eluted very close to the void volume, as was observed before (29). The elution order is of particular interest since it was found to be similar to that on the L-Val-LPhe-L-Val column (29), with several exceptions. Unlike the previous study, PTH-S-methyl-L-cysteine eluted before PTH-L-Asn; PTH-D,L-Met.-Sulfone before PTH-OH-Pro; P T H - D , L - A s ~before PTH-L-Pro; and PTH-L-Ile before PTH-L-Tyr. All k’values on the present column are smaller than the corresponding ones on the L-Val-L-Phe-L-Valcolumn (29). One reason for the lower k’values may be due to the relative small amount of the bonded tripeptide in the present L-Val-L-Ala-L-Procolumn. In addition, each of the two bonded phases can have their own specific interactions with the solutes. The early elution of the basic amino acids, when an acidic mobile phase is used, is explained by the fact that the bonded peptide can act as a weak anion exchanger. With the more conventional stationary phases, Le., cation exchangers or reversed phases, the P T H derivatives of the basic amino acids elute after the acidic ones. In particular, PTH-arginine elutes last under acidic conditions. The P T H amino acids with uncharged polar groups elute after the basic derivatives and before most of the hydrophobic ones. The tyrosine derivative elutes much later than the rest of the polar amino acids since it is the least soluble of that group. The two acidic amino acids elute after the uncharged polar amino acids (excluding tyrosine). The retention order of the hydrophobic P T H derivatives follows their hydrophobic properties. The retention order observed here as well as that with other bonded peptides (29) does not resemble the order obtained in reversed phase chromatography (22), adsorption chromatography (18,231 or cation exchange chromatography (2). Figure 1 shows a chromatogram of 13 PTH-amino acids eluted with a 1%citric acid buffer (pH -2.5) within 66 min. No attempts were made to optimize the resolution and the number of PTH-amino acids separated. However, it is clear from Table I1 that a methanol gradient can be used for the rapid separation of a large number of the 25 PTH-derivatives studied here. The poor efficiency of the more retained peaks in the chromatogram shown in Figure 1 should be noted. The poor efficiency can be a result of slow desorption kinetics from the bonded peptides, of residual silanol groups, or of inefficient packing procedure. The presence of silanol should also affect the retention behavior. However, our previous studies (27, 28) indicate that the peptide controls the retention orders and selectivities. The column efficiencies for the early peak, e.g. PTH-Arg, can be as low as 0.1 mm, depending on the mobile phase used. It would seem then, that slow desorption kinetics are controlling the efficiencies. Further studies are underway
MINUTES
Figure 1. Separatlon of PTH-amlno acids, column I. Moblle phase: 1% citric acid/water (pH -2.5). 1 mL/mln. Chart speed, 4 mm/mln. Changed to 2 mmlmin after 18 min
to improve the chromatographic efficiencies of the peptide columns. With a basic buffer of pH -7.4, several changes in the retention orders of the PTH-amino acids occur. The terminal amine on the bonded peptide is partially protonated at that pH. The imidazolium group in His is probably less than 10% protonated whereas the guanidinium group of Arg is still positively charged. As a result, the retention order of His and Arg is the reverse of that which occurred at pH -2.5. Also, they are no longer the first solutes to elute from the column. The capacity ratios of most of the hydrophobic P T H derivatives are larger with the basic mobile phase (pH 7.4) than with the acidic one (pH 2.5). However, the retention order is the same in both mobile phases. The change in the k’values of the uncharged polar amino acids between these two mobile phases is not systematic; however, the retention order is the same with both mobile phases. At the p H of 7.4, PTH-aspartic acid is eluted before PTH-glutamic acid. The capacity ratios of the acidic P T H derivatives a t pH 7.4, are similar in value to the other polar derivatives. The drastic decrease in the k’ values of the cysteine derivative and of the PTH-cysteic acid salt is to be noticed. Separation of PTH-amino acid a t a pH of 7.4 can be obtained. However, the efficiency of the column a t that pH is worse than that a t an acidic pH. When comparing the k ’ values obtained with the three citrate buffers (pH -2.5, 5.5, and 7.4) the following points of interest are observed. (a) The k’ values of the basic amino acids first increase sharply when changing from a pH of 2.5 to 5.5, and then they level off. (b) The capacity ratios of the acidic amino acids, except Glu, decrease as the pH is increased. (c) All other PTH-amino acids have higher k’values when using the pH 5.5 buffer than with mobile phases having pH of 2.5 or 7.4. (d) The capacity ratios of all P T H derivatives with the exception of Arg, Pro, and His-HC1, were the largest when deionized distilled water, pH 5.5, was used as the mobile phase. Electrostatic interactions may be the reason for these observations.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Table 111. k’ Values of Dipeptides in Various Mobile Phases (Column I)
7
li
Mobile phasesa Gly-L-Trp ~-?kp-Gly Gly-L-Tyr L-Tyr-Gly Gly-L-Phe L-Phe-Gly L-Val-L-Phe L-Phe-L-Val L-Trp-L-Tyr L-Trp-L-Trp L-Tyr-L-Phe
(A) 0.47 0.24 0.17 0.15 0.22 0.11 0.26 0.15 0.49 1.81 0.36
(B)
(0
2.62 2.18 1.10 0.98 1.28 1.17 1.58 1.42 5.73 22.70 2.64
3.44 3.83 1.16 1.38 1.35 1.74 2.32 2.74 21.18 103.60 8.03
1157
(D) (E) 9.18 17.62 1.71 3.78 1.83 4.67 4.96 9.06 b b b
b b b b b b b b b b b
INJ. I
For mobile phases (A)-(E), see Tables I and 11. Peaks did not elute in reasonable times. a
Analysis of Dipeptides. Table I11 shows the capacity ratios of 11dipeptides eluted with various mobile phases. The first eight dipeptides represent four isomeric pairs. The capacity ratio for each dipeptide, without exception, was the largest with the distilled deionized water mobile phase. In fact, the k‘values of the last three dipeptides in Table 111were very large and they did not elute in reasonable times. When a citrate buffer, having the same pH as the water (pH 5.5) was used as the mobile phase, the value of the capacity ratios decreased. That decrease is drastic for the last three dipeptides in Table 111. Since the only difference between these two mobile phases is the presence of citrate and chloride ions in the buffer solution, the explanation for the retention behavior must be related to the anions. The ionic strength ( I ) of the citrate buffer is 0.26 M whereas I for the water mobile phase is close to zero. Since the bonded phase is most likely charged at pH of 5.5, an increase in the ionic strength can cause desorption of peptides as discussed by Hjerten (60). Similarly, Horvath et al. (58) have shown that the capacity ratio of an ionizable solute can decrease when the ionic strength is increased from 0 to about 0.5 M. A comparison of the k’ values obtained with the three buffered mobile phases (columns A, B, and C in Table 111) shows a reversal of retention orders between members of each isomeric pair as the pH is increased. This is in agreement with the results obtained with Val-Phe-Val bonded peptide stationary phase (29). The pH dependence of the capacity ratio will be discussed shortly. Independent of the pH of the mobile phases used in this study, it is interesting to note the retention order of dipeptides having a common amino acid Gly-Tyr C Gly-Phe C Gly-Trp, and Tyr-Gly C Phe-Gly C Trp-Gly. The exception to the above is that Phe-Gly C Tyr-Gly in the acidic buffer. As discussed previously (29),this retention order is similar to that of the PTH derivatives of the “different” amino acids in the dipeptides. The same retention behavior can be seen with other hydrophobic dipeptides, e.g. Gly-Phe < Val-Phe < Tyr-Phe, and Trp-Gly C Trp-Tyr C Trp-Trp, which agrees with the retention order of Gly, Val, Tyr, and Trp PTH derivatives. The hydrophobic dipeptides should have properties similar to the hydrophobic amino acids. It is reasonable, therefore, that the retention order is determined by the amino acids which are not common in the series of the dipeptides studied here. Several additional trends should be noticed: (a) Unlike the case with the P T H derivatives, the capacity ratios of the dipeptides are not the largest when the mobile phase is a buffer at pH 5.5; rather k’increases with the pH. (b) Because of the low k ’values with the very acidic buffer, separation of the dipeptides is difficult with the mobile phase whose pH
0
13
6
9
21
33
45
MINUTES
Figure 2. Separation of dipeptides, column I. Mobile phase: 1% sodium citratelwater (pH -7.4) 0.35 mL/min. Chart speed, 8 mm/mln. Changed to 2 mm/min after 9 min
is 2.5. At the pH of 7.4 the separation is quite easy and Figure 2 shows a chromatogram of seven dipeptides including Gly-Phe/Phe-Gly and Gly-Trp/Trp-Gly isomeric pairs. Again no attempts were made to optimize the resolution. Finally, it should be pointed out that the capacity factors of the dipeptides, in general, are larger than those of the amino acids, except with the very acidic buffer where the reverse is observed. Schott et al. (24-26) have shown that interactions between peptides and bonded oligonucleotides could form the basis of specific recognition of peptide sequences, and thus the selective separation of peptides. In the system reported by them, it was maintained that selectivity toward Trp containing peptides was achieved. Examination of the data shown in Table I11 indicates that in the present system tryptophancontaining peptides are also retained longer than the rest of the peptides studied. Hence, the results of Schott et al. can be explained in terms of hydrophobic considerations as well as in terms of specific interaction with the bonded nucleotides. The results described above indicate that a more systematic study of the effect of pH and ionic strength on the retention behavior is needed. The following section looks at the behavior of free and protected dipeptides under different conditions. Effects of pH and Ionic Strength of Mobile Phases on t h e k’of t h e Peptides. Free Dipeptides. Based on a study of the chromatographic properties of dipeptides on ion exchangers, Haworth (8)proposed an empirical relationship for calculating the relative elution volumes of dipeptides from those of the constituent amino acids: Rpep = 1 . 5 0 [ R ~+ R c ] - [0.0101 RNRc]- 28.6
where Rpepis the relative retention volume of the dipeptide, RN and Rc are the relative retention volumes of the constituent N-terminal and C-terminal amino acids, respectively. Haworth reported that discrepancies occurred in a few cases, e.g., structural isomers of dipeptides. The above relationship indicates that isomeric dipeptides should have identical retention volumes. However, it is known that isomeric dipeptides can be separated (viz. 29, 61, 62). To understand the separation mechanism, a careful evaluation of the pH and the ionic strength effects should be made. Table IV shows the k’dependence of free dipeptides on the pH of the mobile phase. Figure 3 presents in a graphical form
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Table IV. 12’ Values of Free Dipeptides in Mobile Phases Having Various pH’s and Ionic Strengths (Column 111) H2O (pH 5.5)
Dipeptides Gl y -L -Trp
~-Trp-Gly G l y --Phe ~ Gly-L-Phe L-Phe-Gly Gly-L-Tyr L-Tyr-Gly L-Phe-L-Val L -Val- L-Phe D -Leu-L-Tyr L-Leu-L-Tyr L-Tyr-L-Leu L-Ile-L-Tyr L -Tyr-L-Ile L-Phe-L-Tyr L-Tyr-L-Phe L-Trp-L-Tyr ~-Trp-~-Trp a
6.47 11.50 1.92 1.92 5.10 1.87 4.67 9.42 5.83 2.50 6.90 13.5 8.33 12.9
... ...
. I .
...
I = 0.5 M
I = 1.OM pH 7.15
pH 5.0
pH 3.8
pH 2.1
pH 7.4
pH 5.2
1.91 2.20 0.58 0.61
1.55 1.31 0.38 0.38 0.41 0.32 0.27 0.73 0.77 0.82 0.82 0.80 0.86 0.73 1.75 1.89 5.50 25.4
1.65 1.30 0.46 0.46 0.43 0.36 0.28 0.72 0.86 0.87 0.86 0.90 0.88 0.80 1.82 1.92 5.36 23.9
1.60 0.84 0.34 0.33 0.21 0.26 0.12 0.48 0.65 0.72 0.65 0.67 0.69 0.60 1.35 1.56 4.26
2.67 3.45 0.65 0.62 1.09 0.55 0.88 2.38 1.77 1.11 1.88 2.56 2.09 2.59 9.73 10.1
1.57 1.20 0.43 0.43 0.42 0.30 0.28 0.60 0.69 0.72 0.76 0.70 0.79 0.70 1.72 1.83 5.26
0.88
0.46 0.58 1.43 1.15 1.09 1.32 1.61 1.29 1.45 4.65 4.66 14.2 a
...
... ...
...
pH3.8 1.42 1.02 0.36 0.35 0.27 0.26 0.17 0.50 0.59 0.63 0.65 0.57 0.64 0.54 1.40 1.31 4.03 17.2
pH 2.2 0.88 0.43 0.13 0.13 0.00 0.13 0.01 0.12 0.25 0.34 0.28 0.26 0.26 0.24 0.67 0.76 2.13 9.96
Solute did not elute in reasonable time.
ti I /
’
L-TYR-GLY
00
0
2
4
6
8
PH Figure 3. Plot of capacity factor ( k ’ ) vs. pH for free dipeptides
some of the data in the table at ionic strength of 1.0 M. As expected the h’ values increase as the p H of the eluent is increased, and the elution order of isomeric dipeptides is a function of the pH. The pH a t which the elution order reverses, as seen from Figure 3, is dependent on the nature of the dipeptides. The similarity of the k’vs. pH plots to titration curves of peptides should be noticed. The titration curves of the two peptides in each isomeric pair should be different since the terminal groups are different. Noda et al. (43) in their study of some diastereomers of leucyl dipeptides by ion-exchange chromatography have also found an inversion of the elution order as a function of the pH. They have shown that the titration curves of the diastereomers crossed around pH of 5 , the p H where the inversions in the elution order occurred. Table IV indicates that in the present study the retention order of the diastereomers, D,L-Leu-L-Tyris also pH dependent. Electrostatic arguments have been used previously to explain the retention reversal (29). Perhaps a better explanation might lie in the shielding effects of the water
molecules (63). At the low pH’s the solutes have a net positive charge due to the large extent of the protonation of the terminal amine group. Water molecules, forming hydrogen bonds with the protonated amine, can shield part of the peptide, weakening the interaction with the bonded phase. The retention order is thus affected by the portion of the peptide which is shielded. For example, under acidic conditions the T r p group in the dipeptide Trp-Gly is shielded, while in Gly-Trp it is the Gly moiety which is masked by the water. Since Trp is more hydrophobic than Gly, it is expected that Gly-Trp will elute after Trp-Gly. The inverse would be expected with a basic mobile phase. Table IV shows that such is the case. Table IV indicates that with mobile phases having pH value less than 4, the k’ values increase as the ionic strength is increased. Depending on the solute, the capacity ratio increases, decreases or remained relatively unchanged as I increases when the mobile phase is a buffer a t pH of about 5. The capacity ratios decrease as I is increased when a basic mobile phase is used. The largest capacity ratios were obtained when distilled deionized water (pH -5.5) was used as the mobile phase. The increase in h’with I a t the lower pH’s can be explained by the results of Horvath et al. (58). They reasoned that the increase in k’ as a function of the ionic strength is due to an increase in the surface tension of the aqueous phase when the salt concentration increases. Hjerten (60) has indicated that with charged hydrophobic beds the effects of a salt on the retention can be magnified due to electrostatic interactions, especially at low salt concentrations. As the pH of the mobile phase is increased, the bonded peptide loses its charge and a t a pH of 7.4 it is only partially protonated. However, the solute has a net negative charge. Therefore, an explanation of the behavior of k’as a function of I at pH of 2.2 and 7.4 might lie in electrostatic interactions. Okai et al. (44) found that a t pH 7 and above, the diastereomers of L-Leu-D,L-Tyr co-eluted from the ion-exchange column of Dowex 50W-X8 in the amine form. In the present tripeptide column, the terminal group is also an amine. However, it was found here that good resolution of the Leu-Tyr diastereomers could be obtained with the basic mobile phases. Poorer resolutions were observed with acidic mobile phases. Figure 4 shows the separation of the diastereomers and other Tyr based dipeptides with a pH 7.4 buffered mobile phase. Protected Peptides. The behavior of some TFA-di- and tripeptide methyl esters as a function of p H at constant ionic
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978 LL
4
(8)
1159
TFA-GLY-L-TYR-OM$
i; 2
0
8 -
6 ,
I
4
2
0
’
MINUTES
Flgure 4. Separation of tyrosin based dipeptides, Column 11. Mobile phase: 1% sodium citrate solution (pH 7.4), 0.96 mL/min. X = 254 nm I
I 0 :TFA- L-PHE-GLYWAL- OMB 0 =TF+C-VAL-GLY-L-FHE-OMe
/ = / OM
Figure 6. Plot of capacity factor ( k ’ ) vs. volume % methanol in water for (A) free peptides, (B) protected peptides
0 ETFA-L-PRO-L-PHE-We 0 :TFA-L-PRO-DPHE-CMa A ;TFA-LALA-L-FHE-CMe .=1M-L?dE-L-ALA-OMe
rn :TFA-L-TYR-GLY-OMe
200-
:TFA-GLY-kTM).CMe L.TYR70% MeOH/H20 was used. On the other hand, more than unit resolution was obtained with 25% MeOH/H20 mobile phase. Solubility is certainly a major factor responsible for the variation of h'values. The protected dipeptides are rather soluble in MeOH. The resolution of TFA-G~Y-LTyr-OMe from TFA-Gly-L-Tyr-(OMe)2demonstrates the selective interactions between the solutes, the stationary phase and the aqueous mobile phase a t high water content.
CONCLUSIONS T h e results presented above demonstrate further the usefulness of bonded peptide phases for the separation of amino acids and their derivatives, of isomeric dipeptides as well as of diastereomers. The retention orders seem to indicate that the retention mechanism is a combination of anion exchange and hydrophobic interactions. It is shown that, for a given bonded peptide phase, the retention order of the peptides, especially the hydrophobic ones, follows the degree of hydrophobicity of the hydrocarbon side chains of the solutes. Elution orders of the dipeptides have been shown to resemble the elution orders of their amino acid constituents. The pH dependence of the capacity ratios of the isomeric dipeptides seems to resemble the titration curves of the solutes. The understanding of the effects of ionic strength, pH, and alcohol content in the aqueous mobile phase allows one to design eluents which optimize the separation of a given series
11.8
11.1
9.69
8.18
8.60 4.45 4.45 5.15 4.91 3.18 3.62 7.71 9.35 8.29
of peptides and/or PTH-amino acids. For example, the use of 80% MeOH/H20 as mobile phase can yield the baseline resolution of any of the free isomeric dipeptides studied here.
ACKNOWLEDGMENT We thank M. Schwarzberg of Syva Research Institute for the gift of the protected peptide.
LITERATURE CITED J. Rosmus and 2. Deyi, J . Chromatogr., 70, 221 (1970). Z. Deyi, J . Chromatogr., 127,91 (1976). H. Lindiey and P. C. Davis, J . Chromatogr., 100, 117 (1974). C. W. Gehrke and H. Takeda, J . Chromatogr., 76,63 (1973). (5) 0. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem., 30, 1190 (1958). (6) J. G. Heathcote, R. J. Washington, B. J. Keogh, and R. W. Glanviiie. J . Chromatogr., 65,397 (1972). (7) F. Giiiberti and A. Niederwieser, J . Chromatogr., 66,261 (1972). (8) C. Haworth, J . Chromatogr., 67,315 (1972). (9) C. Martei and D. J. Pheips, J . Chromatogr., 115, 633 (1975). (IO) P. B. Hamilton nd M. F. Low, Biochem. Med., 6, 193 (1972). (11) M. F. Low and P. B. Hamilton, Biochem. Med., 8, 485 (1973). (12) R. S. Ward and A. Peiter, J . Chromatogr. Sci., 12, 570 (1974). (13) A. Arendt, A. Kotodziejczyk, and T. Sokotowska, Chromatographia, 9, 123 (1976). (14) J. N. Manning and S. Moore, J . Biol. Chem., 243, 5591 (1966). (15) A. Haag and K. Langer, Chromatographia, 7, 659 (1974). (16) E. W. Matthews, P. G. H. Byfieid, and I.MacIntyre, J . Chromatogr., 110, 369 (1975). (17) A. P. Graffeo, A. Haag, and B. L. Karger, Anal. Lett., 6, 505 (1973). (18) G. Frank and W. Strubert, Chromatographia, 6,522 (1973). (19) J. X. deVries, R. Frank, and C. Birr, F€BS Left., 55, 65 (1975). (20) M. R. Downing and K. G. Mann, Anal. Biochem., 74, 298 (1976). (21) C. Boiiet and M. Caude, J . Chromatogr., 121, 323 (1976). (22) C. J. Zimmerman, E. Appelia, and J. J. Pisano, Anal. Biochem., 77,569 (1977). (23) H. Bridgen, A. P. Graffeo, 8. L. Karger and M. D. Waterfield, in "Instrumentation in Amino Acid Sequence Analysis", R. N. Perham, Ed., Academic Press, London, 1975, p 111. (24) H. Schott, H. Eckstein, and E. Bayer, J . Chromatogr., 99, 31 (1974). (25) H. Schott, H. Eckstein, I. Gatfieid, and E. Bayer, Biochemistry. 14,5541 (1975). (26) H. Schott, H. Eckstein, and E. Bayer, Biochem. Biphys. Acta, 432, 1 (1976). (27) E. Grushka and R. P. W. Scott, Anal. Chem., 45, 1626 (1973). (28) E. J. Kikta, Jr., and E. Grushka, J . Chromatogr., 135, 367 (1977). (29) G. W-K. Fong and E. Grushka, J . Chromatogr., 142, 299 (1977). (30) E. Gii-Av and D. Nurok, Adv. Chromatogr., 10,99 (1974). (31) J. A. Corbin, E. J. Rhoad, and L. B. Rogers, Anal. Chem., 43,327 (1971). (32) W. A. Koening, K. Stoeiting, and K. Kruse, Chromatographia, IO. 444 (1977). (33) G. Losse and K. Kuntze, 2. Chem., 10,22 (1970). (34) A. V. Semechkin, S. V. Rogozhin, and V. A. Davankov, J. Chromatogr., 131, 65 (1977). (35) R. J. Baczuk, G. K. Landrom, R. J. Dubois, and H. C. Dehm, J. Chromatogr., 60,351 (1971). (36) J. J. Hansen, T. Creibrokk, B. L. Currie, K. N-G. Johansson, and K. Foikers, J . Chromatogr., 135, 155 (1977). (37) C. J. 0. Morris and P. Morris, "Separation Methods in Biochemistry", John Wiiey & Sons, New York, N.Y., 1976. (38) L. L. Hoffpauir and J. D. Guthrie, Textile Res. J., 20,617 (1950). (39) S. Blackburn and P. Tetiey, Biochim. Biophys. Acta, 20,423 (1956). (40) S. Yanari, M. Voiini, and M. A. Mitz, Biochim. Siophys. Acta, 45,595 (1960). (41) F. C. Neuhaus, J . Bioi. Chem., 237,778 (1962). (42) T. Wieiand and H. Bende, Chem. Ber., 98, 504 (1965). 143) K. Noda. H. Okai. T. Kato. and N. Izumiva. Bull. Chem. SOC.Jon.. 41. 401 (1968). (44) H. Okai, N. Imamura, and N. Izumiya, Bull. Cham. SOC.Jpn., 40,2154 (1967). (1) (2) (3) (4)
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978 (45) M. Muraoka, N. Yoshida. K. Noda, and N. Izumiya, Bull. Chem. Soc. Jpn., 41, 2134 (1968). (46) M. Muraoka, H. Aoyagi, and N. Izumiya, Bull. Chem. SOC.Jpn., 44, 3391 (1971). (47) A. Niederwieser, J . Chromatogr., 61, 81 (1971). (48) H. Joshua and C. H. Deher, in "Chemistry and Biology of Peptides", Proceedings of the 3rd Am. Pept. Symposium, J. Meienhofer, Ed., Ann Arbor Science Publishers, Ann Arbor, Mich., 1972. (49) T. Sokoiowska and J. F. Biernat, J . Chromatogr., 13, 269 (1964). (50) L. Kesner, E. Muntwyler, G. E. Griffin, and J. Abrams, Anal. Chem., 35, 83 (19631. s (51) P:Hubert and E. Dellacherie, J . Chromatogr., 80, 144 (1973). (52) 2. Pravada, K. Poduska, and K. Blaha, Collect. Czech. Chem. Commun., 29. 2626 (19641. (53) A. Arendt,' A. Koiodziejczyk, and T. Sokolowska, Chromatographia, 9, 123 (1976).
1161
(54) K. Krummen and R. W.Frei, J . Chromatogr., 132, 27 (1977). (55) P. J. Elving, J. M. Markowitz, and I. Resenthal, Anal. Chem.. 28, 1179 (1956). (56) S. Moore and W.H. Stein, J . Elol. Chem., 192, 663 (1951). (57) A. Niederwieser "Chromatography", E. Heftmann, Ed., Van Nostrand, New York, N.Y., 1975, p 393. (58) C. Horvath, W.Melander, and I. Molnar, Anal. Chem., 49, 142 (1977). (59) S. Pahlman, J. Rosengren, and S. Hjerten, J. Chromatogr., 131, 99 (1977). (60) S. Hjerten, J . Chromatogr., 87, 325 (1973). (61) W. Monch and W. Dehnen, J . Chromatogr., 140, 260 (1977). (62) I. Molnar and C. Horvath, J. Chromatogr., 142, 623 (1977). (63) I. Molnar, private communication, Amsterdam, 1977.
RECEIVED for review November 28, 1977. Accepted April 14, 1978. Work supported by NIH grant No. GM-20846.
High Performance Liquid Chromatography of Some Acidic and Basic Organic Compounds on Silica Gel with Mobile Phases Containing Organic Acids James F. Lawrence* and Raymonde Leduc Food Directorate, Health Protection Branch, Tunney's Pasture, Ottawa, Ontario, K 1A OL2, Canada
The addition of small quantities of organic acids to the mobile phase significantly improved the chromatography on silica gel of both polar basic compounds, such as the hydroxylated metabolites of triazine herbicides, and acidic compounds, such as herbicidal nitrophenols and phenoxyalkanoic acids. Six hydroxy-s-triazines were separated on 5-pm silica gel with a mobile phase consisting of 10 % methanol in dichloromethane containing 0.1 M of propionic acid. Increases in acid concentration from 0.008-0.08 M did not improve efficiency but reduced retention. Without the presence of acid, the triazines chromatographed poorly or not at all. The retention of the triazines decreased with increasing molecular weight of the acids studied, while the reverse was true for the acidic compounds. Because of their respective acid strengths, the phenoxyalkanoic acids required 0.5 M acetic acid for optimum chromatography while the nitrophenols needed 0.025 M acid.
Reversed-phase ion-pair partition chromatography has become a very popular tool for separating a wide range of acidic or basic substances. The chromatographic systems usually consist of a stationary phase (aliphatic CIS)chemically bonded to a support such as a silica gel, and an aqueous mobile phase containing a suitable lipophilic counterion buffered a t a specific pH. While the mechanism involved in the chromatographic process appears to be derived from classical ion-pair liquid-liquid extraction (a molecular ion in the aqueous phase combines with a suitable counterion to form a neutral ion-pair, which then partitions into the organic phase), some doubts were brought forward by Kissinger ( I ) who has suggested that while this might hold true for normal-phase ion-pair partition chromatography, it may in fact not be the case for reversed-phase ion-pair partition chromatography with chemically bonded stationary phases. He suggests that in the latter case an ion-exchange mechanism may be in operation. Further evidence of this has appeared 0003-2700/78/0350-1161501 .OO/O
recently in the work of Kraak et al. (2) on amino acid separations. However, Scott and Kucera ( 3 ) have pointed out through their work that such an effect may only be evident under %on-wetting" chromatographic conditions. When the mobile phase "wets" the stationary phase, the ion-exchange mechanism is not in effect. This latter view has been supported in the recent work of Horvath et al. ( 4 ) . Through our work on polar metabolites of triazine herbicides as well as herbicidal phenoxy acids and phenols, we have developed a high-performance liquid chromatographic (HPLC) technique which makes use of microparticulate silica gel with mobile phases which consist of organic solvents containing traces of organi'c acids. The inclusion of acids or bases in solvents for chromatography of polar compounds such as alcohols, phenols, or carboxylic acids is not new and has been used before in thin-layer chromatography to prevent spot tailing, and also in the stationary phases in gas chromatography to improve peak shape. Acids and bases have also been utilized for the improvement in peak shape in HPLC (5-7). It has been generally accepted that the addition of acid or base to the above systems improves the chromatographic process by blocking hydrogen-bonding sites which cause slow mass transfer resulting in tailing. This work describes some of our results with several hydroxylated triazine metabolites and other herbicidal compounds such as phenoxy acids and phenols using the above-mentioned HPLC system. An interesting observation in this work is that the addition of acid to the mobile phase not only improves chromatography of the acidic herbicides as mentioned above, but also the basic triazines as well. I t also appears that the mechanisms involved are different. The use of adsorption chromatography rather than reversed-phase chromatography is of some significance to us since, by far, most pesticide methodology is based on extractions and purifications in organic solvents. Thus a relatively nonpolar organic solvent-based chromatographic system can be more easily integrated into existing pesticide methodologies than a chromatographic system based on 0 1978 American
Chemical Society
