Comments on Reverse-Phase Ion-Pair Partition Chromatography Sir: Over the past several years high-performance reverse-phase liquid chromatography with chemically bonded alkyl chains (especially c18) has become extremely popular. One of the most attractive features of this technique is the possibility of using the same stationary phase to separate a wide variety of molecular classes including acidic, neutral, basic, and zwitterionic species. From a practical standpoint the microparticle packings have the advantage of being relatively easy to manufacture in a reproducible manner. They also tend to be more stable than chemically bonded ion exchangers. The chemical stability of the CIS phase permits the use of a wide range of mobile phases enabling great flexibility in the design of separations. In addition, it is often possible to restore the properties of a column by using various solvents (e.g., pure methanol or acetonitrile) to remove strongly retained material. Our interest in phases stems from their great utility in the separation of the metabolites of tyrosine ( I ) , tryptophan (Z),and the purine and pyrimidine bases (3). Because the mobile phases used with reverse-phase chromatography often consist of a mixture of a polar organic solvent (especially methanol or acetonitrile) with an aqueous buffer, the system is ideally suited for amperometric detection which affords both selective and sensitive detection of phenolic metabolites. Recently we have developed clinical procedures for the metanephrines ( 4 ) and the catecholamines (5) using this approach. The use of normal (polar)-phase ion-pair partition chromatography with microparticle silica has been shown to have excellent selectivity and efficiency for biogenic amines and their metabolites (6). In this case ion pairs of the analyte molecules are formed in the hydrophobic mobile phase. Over the past year or two a great deal of attention has been given to reverse-phase ion-pair partition chromatography. Particularly noteworthy are the publications of Haney et d. (7, 8) and Waters Associates, Inc. (9). The original concept follows that of classical ion-pair liquid-liquid extraction long popular in the pharmaceutical industry. A charged solute in the aqueous phase is paired with a lipophilic counterion and the neutral complex partitions into the organic solvent (or c18 stationary phase). We believe that this conception of the process, while useful, is not generally correct. Several experimental observations suggest that another mechanism may be far more important. A typical reverse-phase separation of ionic compounds incorporates a mobile phase consisting of a mixture of an organic solvent (usually acetonitrile or methanol) and an aqueous buffer (where both the pH and ionic strength are important). The addition of very low concentrations of an “ion-pairing reagent” to the mobile phase (typically 51 mM) will dramatically influence the capacity factors for the sample components. Reagents such as alkyl sulfates or sulfonates will increase the capacity factors for cations, whereas tetraalkylammonium salts will do likewise for anions. If the process were dominated by a classical ion-pair partition phenomenon one would anticipate (1)that the full impact of the reagent would be felt after one void volume of the column were displaced, and (2) that reagents and samples of the same charge would not react significantly (the mole fractions of both are extremely small). Experimentally one observes that many void volumes must be displaced before the full effects of the reagent are established at a steady-state condition. Furthermore, the reagents can reduce capacity factors for ions of like sign. These two observations are
sufficient to suggest that in many (if not all) cases the ion-pair reagents are modifying the stationary phase and that the process of 1:l ion-pair partition is insignificant. When the reagent is added to the mobile phase, it strongly partitions onto the stationary phase modifying its surface charge. The large number of void volumes required for the reagent front to “break through” the packed bed clearly supports this idea as does the fact that the resulting surface charge repels ions of the same sign. Because the process of adsorption onto the stationary phase is complicated by nonelectrostatic factors, it is not generally possible a t this time to predict the extent to which such reagents will influence the capacity factor for an individual molecule. It would appear that normal-phase ion-pair partition chromatography can be thought of in the classical manner whereas the reverse-phase technique is not likely to be perfectly analogous in many cases. Furthermore, it is not entirely sound to view this new technique as an “alternate to ion exchange” when, in fact, it may simply afford another means of preparing an ion exchange column. A sulfonated ion-pair reagent may be thought of as a modifier used to convert a Cls column into a strong cation exchange column with properties not unlike those of chemically bonded alkyl sulfonates. The purpose of this communication is to urge that this new and very flexible separation technique not be viewed as being a well-understood extension of a very classical idea. The fundamentals are clearly far more complex than has been presumed in a number of recent publications and verbal presentations. The excellent fundamental and practical work by Knox and co-workers on “soap chromatography” reveals that the reverse-phase ion-pair idea has definitely come of age (IO, 11). Apparently Knox and Haney developed this concept independently. Hopefully further developments will not be hindered by semantics.
ACKNOWLEDGMENT The author is grateful to J. F. K. Huber, Barry L. Karger, J. J. Kirkland, James N. Little, and Hector J. Rodriguez for illuminating discussions. LITERATURE CITED (1) I. Molnar and C. Horvath. Clin. Chem. ( Winston-Salem. N.C.). 22. 1497 (1976). (2) A. P. Graffeo and B. L. Karger, Clln. Chem. ( Winston-Salem, N.C.), 22, 184 (1976). (3) F. C. Senftleber, A. G. Halline, H. Veening, and D. A. Dayton, Clln. Chem. ( Winston-Salem, N.C.), 22, 1522 (1976). (4) R . E. Shoup and P. T. Kissinger, Clin. Chem. ( Winston-Salem, N.C.), submitted.
(5) R . M. Riggin and P. T. Kissinger, Clin. Chem. (Winston-Salem, N.C.), submitted.
(6) B. A. Persson and B. L. Karger, J. Chromatogr. Scl., 12, 521 (1974). (7) D. P. Wittmer, N. 0. Nuessle, and W. G. Haney, Jr., Anal. Chem., 47. 1422 (1975).
S.P. Sood, L. E. Sartori, D. P. Wittmer, and W. G. Haney, Anal. Chem., 48, 796 (1976). (9) “Paired-Ion Chromatography, an Alternate lo Ion Exchange”, Waters Associates Inc., December 1975. (10) J. H. Knox and G. R. Laird, J . Chromatogr., 122. 17 (1976). (11) J. H. Knox and J. Jurand, J. Chromatogr., 125, 39 (1976). (8)
Peter T.Kissinger Department of Chemistry Purdue University West Lafayette, Indiana 47907
RECEIVED for review December 13,1976. Accepted February 7, 1977. ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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