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Anal. Chem. 1984, 56, 2671-2673
Thermodynamics of Retention for Small Molecules in the Polystyrene/Chloroform System Merlin K. L. Bioking Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 The nature of the separatlon mechanlsm for small molecules on polystyrene gels may be partly elucldated through the use of van't Hoff plots. For a chloroform mobile phase, a varlety of tunctlonal groups (test solutes) show only excluslon effects, while two solutes (benzolc acld and phenol) distribute into the gel. A study of several small solutes used to measure pore volume lndlcates that the retentlon of deuterated moblle phase represents the border line between exclusion and partitioning, while most other small molecules give inappropriate results. Injections of deuterated mobile phase also demonstrate that chloroform thermodynamically prefers the gel, with an enthalpy of assoclatlon of about -225 cal/mol. These data allow a new lnterpretatlon of gel-based excluslon systems. The separatlon process may be vlewed as a competition between gel-moblle phase and gel-solute interactions.
Polystyrendivinylbenzene (PS-DVB) copolymer gels have been used for many years as stationary phases for size-exclusion chromatography (SEC). When allowed to swell in certain solvents, usually less polar organics, and used as stationary phases the gels exhibit elution patterns based generally on molecular size. Applications to polymer analysis are numerous, and several excellent monographs are available (1-3). More recently separations of small molecules have been studied, using gels with smaller pores. For a homologous series, the retention pattern usually correlates with size parameters, but different series often do not overlap ( I ) , and many solutes do not elute as expected. Such nonexclusion effects have been recognized for some time, and a variety of explanations have been put forth, including adsorption, liquid-liquid partitioning, and hydrogen bonding. Nonexclusion effects have been reviewed (4, 5). Several theoretical studies have been published regarding general distribution phenomena (6-8). The relative polarity of the mobile phase and the gel has also been used to predict nonexclusion effects (91,but there are limitations to this approach (10). Recently several workers (11-14) have mentioned nonexclusion effects, but a definitive interpretation has yet to appear in the literature. While large molecular weight solutes have been studied extensively, there appear to be no systematic experimental studies of mobile-phase effects when separating small molecules, under SEC conditions, which prove when a system is exhibiting pure exclusion or when other distribution phenomena are also occurring. In particular, there have been very few reports regarding the effect of temperature on retention (15,16). This manuscript describes the use of van't Hoff plots (In h vs. as a probe for identifying nonexclusion effects for small molecules in the polystyrene/chloroform system. This system is expected to show primarily exclusion effects (IO), and the present work more clearly identifies conditions under which this prediction is verified. EXPERIMENTAL SECTION Equipment. The chromatographic system consisted of an Altex Model llOA pump, Rheodpe Type 80 rotary injection valve, and a Waters Model 440 UV detector or Model 401 refractive index detector. The columns were made of borosilicate glass
l/n
(Rainin Instruments), 15 mm x 25 cm, with adjustable bed supports. Columns also included a water jacket for temperature control by a circulating bath (Forma Scientific). Retention times were measured by using an Apple II+/Isaac 91A data acquisition system (Cyborg Corp.) using software developed in this laboratory. Curve fitting software was obtained from Interactive Microware, Inc. The flow rate for all measurements was 1.5 mL/min. Columns were packed with Styragel (Waters Assoc., 60 A,
