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Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1632-1636, March 1980 Medical Sciences Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition (lipophilicity/separation techniques) JAMES L. MEEK Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Communicated by Bruce Merrifield, December 17,1979 ABSTRACT Analysis of peptides by reverse-phase high- on octadecylsilyl silica gel is a quite different process from oc- pressure liquid chromatography would be simplified if retention tanol/water partition and from the unavailability of hydro- times could be predicted by summing the contribution to re- groups of the peptides. It is the tention of each of the peptide's amino acid side chains. This phobicity data for terminal paper describes the derivation of values ("retention coeffi- purpose of this paper to show that "retention coefficients" can cients") that represent the contribution to retention of each of be derived directly from HPLC data for all amino acids and the common amino acids and end groups. Peptide retention end groups such that the retention time of a peptide can be times were determined on a Bio-Rad "ODS" column at room predicted from the sum of the retention coefficients for each temperature with a linear gradient from 0.1 M NaCIO4, pH 7.4 amino acid and end group. or 2.1, at 0 min to 60% acetonitrile/0.1 M NaCIO4 at 80 min. The NaClO4, a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients MATERIALS AND METHODS for the amino acids were computed by using a Hewlett-Packard Peptides were obtained from Sigma and Peninsula Laboratories 9815A calculator programmed to change the retention coeffi- cients for all amino acids sequentially to obtain a maximum (San Carlos, CA). HPLC grade acetonitrile was obtained from correlation between actual and predicted retention times. Cor- Fisher. The HPLC system was assembled from modular com- relations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained ponents: a one-chamber glass gradient maker, a pump (Milton for 25 peptides including glucagon, oxytocin, [Metlenkephalin, Roy, Riviera Beach, FL), and a sample valve (Rheodyne, neurotensin, and somatostatin. This high degree of correlation Berkeley, CA). The column effluent was passed in series suggests that, for peptides containing up to 20 residues, retention through a variable wavelength photometer (Altex, Berkeley, is primarily due to partition processes that involve all the resi- dues. Although steric or conformational factors do have some CA) and a filter fluorometer (Farrand, Valhalla, NY). The effect on retention, the data suggest that under the above mobile phase gradient normally used was from 0.1 M chromatographic conditions the retention of peptides con- NaCIO4/0% acetonitrile at 0 min after injection to 0.1 M taining up to 20 residues can be predicted solely on the basis of NaCl04/60% acetonitrile (vol/vol) at 80 min. For separations their amino acid composition. This possibility was tested by at pH 7.4, the starting buffer contained 5 mM phosphate buffer, using data taken from the literature. pH 7.4. For operation at pH 2.1, both starting and final buffers contained 0.1% phosphoric acid (3). The possibilities for separating and isolating small peptides have Linear gradients from 100% A to 100% B are generated if a been markedly improved by the introduction of reverse-phase solution A is placed in a mixing chamber and then B is added high-pressure liquid chromatography (HPLC) (1-5). This at 1/2 the rate at which the mixture is withdrawn (6). The technique depends upon the hydrophobic interactions between of a 3 X 13 cm with two a hydrocarbonaceous column and the peptides to be separated: gradient maker consisted glass cylinder the more hydrophobic (lipophilic) the compound, the stronger Teflon three-way stopcocks and a magnetic stirrer. To generate its retention by the column. To elute strongly retained com- the gradient, the chamber was filled with 40 ml of starting pounds, aqueous solutions (the "mobile phase") containing a buffer. Final buffer was then added to the chamber at 0.5 large amount of organic solvent must be pumped through the ml/min, while the mixture was pumped into the column at 1.0 column. Choice of the optimum mobile phase and chromato- ml/min. trial and Peptides were detected by absorbance'at 200 or 220 nm or graphic conditions for given peptides can be found by by fluorescence [after the primary amino groups of the peptides error after qualitatively examining the balance of hydrophobic had reacted with fluorescamine (7)]. With a mobile phase of and hydrophilic amino acids present in the peptide. However, pH 7.4, the column effluent could react directly with fluores- as noted by Molna'r and Horvath (1), it should be possible to camine, because the pH optimum for many peptides is near obtain quantitative estimates of the hydrophobicity of the ml of amino acids contained in a peptide, which will reflect their neutral (8). The fluorescamine (10 mg/100 acetonitrile) of was added to the effluent at 0.1 ml/min. Continuous neutrali- retention on the reverse-phase column. Estimates hydro- zation of the pH 2.1 mobile phase was achieved by adding the phobicity based on octanol/water partition coefficients exist organic base imidazole (1.0 M final concentration) to the for many but not all amino acids. By using such values, O'Hare fluorescamine/acetonitrile mixture. and Nice (2) noted that the retention order for small peptides The retention coefficients were computed by repetitive re- was generally correlated with the sum of the values for the most amino acid were hydrophobic residues of the peptides. The many deviations they gression analysis: values for each successively the changed by 0.2 min until maximum correlation between actual found between the observed order of elution and lipophil- and predicted retention times was obtained. Starting values for icity estimates presumably derive from the fact that retention the retention coefficients of the neutral and hydrophilic amino acids were initially assumed to be zero. Starting values for the The publication costs of this article were defrayed in part by page amino acids were obtained the retention charge payment. This article must therefore be hereby marked "ad- lipophilic by plotting vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: HPLC, high-pressure liquid chromatography. 1632 Downloaded by guest on September 28, 2021 Medical Sciences: Meek Proc. Natl. Acad. Sci. USA 77 (1980) 1633 times of oligomers (e.g., diphenylalanine, triphenylalanine, were only slightly retained. Small peptides with lipophilic side tetraphenylalanine, etc.) vs. the number of residues; the slope chains (e.g., triphenylalanine) and most of the larger biologi- of the plot equals the retention per residue. To compute the cally active peptides required much higher concentration of retention coefficients, a Hewlett-Packard 9815A calculator was acetonitrile for elution. Acidification of the mobile phase in- programmed to store these starting retention coefficients for creased the retention of peptides with free terminal carboxyl the 26 amino acids and end groups, to store the actual retention groups ([Metlenkephalin and angiotensin II) or with acidic times for the 25 peptides studied, and then to calculate pre- residues [gastrin-(12-15)]. Most peptides with masked carboxyl dicted retention times for these peptides by summing the re- or carboxyl and amino groups (thyrotropin-releasing factor, tention coefficients for each amino acid contained. After cal- oxytocin) had similar retentions at both pHs. Peptides con- culating the correlation coefficient between predicted and taining the basic residues lysine or arginine (luteinizing hor- actual retention times, 0.2 min was added to the retention mone-releasing factor, eledoisin-related peptide) exhibited a coefficient for an amino acid; the predicted retention times and decreased retention at lower pH due either to increased ioni- correlation coefficient were then again calculated. If the cor- zation of the amino group or to formation of an ion pair with relation had been improved by adding 0.2 min to the retention the perchlorate in the mobile phase. Fig. 1 shows the separation coefficient, the change was kept; otherwise the value was re- at pH 2.1 of various peptides. The sharpness of the peaks with turned to that previously used, and 0.2 min was added to the minimal tailing demonstrates the high resolution possible with next amino acid and so on. After checking all amino acids to see HPLC. Fig. 1 also demonstrates the difference in detector se- whether increasing the retention coefficient could increase the lectivity between measurement of absorbance at 220 nm (top correlation, 0.2 min was sequentially subtracted from each trace) and measurement of fluorescamine-induced fluorescence amino acid in turn and correlations were again calculated after (bottom trace). Compounds such as thyrotropin-releasing factor each subtraction. At the end of these two cycles, the slope of the (peak 5) without a free amino-terminal group give little or no plot of predicted vs. actual retention times was calculated be- fluorescence. Fig. 2 shows that there is an approximately linear cause the predicted and actual times should be equal, not increase in retention time for phenylalanine oligomers as merely be correlated (proportional). Therefore, if the slope was phenylalanine residues are added to diphenylalanine. This greater than 1.0, all retention coefficients were multiplied by finding indicates that, with the linear gradient used in these 0.99; if the slope was less than 1.0, the values were multiplied experiments, the addition of each phenylalanine residue adds by 1.01.

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