976
ANALYTICAL CHEMISTRY, VOL. 51,
NO. 7,
JUNE 1979
Retention Behavior of Carboxylic Acids and Their Quaternary Ammonium Ion Pairs in Reversed Phase Chromatography with Acetonitrile as Organic Modifier in the Mobile Phase A. Tilly-Melin and Y. Askemark Analytical Control, ASTRA Pharmaceuticals AB, S- 15 1 85 Sodertalje, Sweden
K.-G. Wahlund and Goran Schill" Department of Analytical Pharmaceutical Chemistry, Biomedical Center, University of Uppsala, Box 574, S-75 1 23 Uppsala, Sweden
The retention behavior of some carboxylic acids (benzoic acid, salicylic acid, acetylsalicylic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid) in acidic form and as ion pairs has been studied in a reversed phase chromatographic system using LiChrosorb RP-8 (10 pm) as support and mixtures of acetonitrile and aqueous phosphate buffers as mobile phases. The influence of pH, acetonitrile content, and the quaternary alkylammonium counterions on retention has been Investigated. Equilibrium constants based on models for distribution of acids and ion pairs to a hydrophobic stationary phase are presented. Considerable adsorption of acetonitrile to the solid phase has been observed and found to increase with increaslng percentage of acetonitrile in the mobile phase resulting in a decrease of the void volume.
Organic acids can be separated in reversed phase chromatographic systems both in acidic form and as ion pairs, which opens several possibilities to regulation of retention and separation selectivity. Systems for reversed phase ion pair chromatography of hydrophilic carboxylic acids of widely different structures as well as sulfonamides and barbiturates have been developed by Wahlund e t al. (1-4) using mobile aqueous phases and a liquid stationary phase of I-pentanol or butyronitrile dynamically coated on an alkyl-bonded support. Naphthalene acetic acid derivatives have been separated as ion pairs and as acids in reversed phase chromatographic systems using aqueous mobile phases containing methanol or acetonitrile ( 5 ) . Separation of anionic solutes by similar systems after addition of quaternary ammonium compounds such as tetrapropylammonium, tetrabutylammonium, or tetrapentylammonium (6, 7) or cetrimide (8) has been reported. This paper reports on reversed phase chromatographic systems for separation of carboxylic acids in acidic form and as ion pairs using acetonitrile as organic component in the mobile phase and LiChrosorb RP-8 (10-pm particles) as hydrophobic solid phase. The influence of pH, the acetonitrile content and the counterion on the retention has been studied and models for the retention have been proposed that include distribution of acids as well as ion pairs to a hydrophobic stationary phase.
EXPERIMENTAL Apparatus. An LDC 711-47 solvent delivery system (Milton Roy Minipump with pulse dampener) was used. The detectors were LDC UV I11 (1203) Monitor measuring at 254 nm wavelength and Optilab Multiref 902, measuring cell 1.0 mm (23.0 "C). The time constant for the UV-detector was set to 0.5 s. The injector was a Valco valve injector with a sample loop of 25 pL. The pH measurements were performed with an Orion 801 Research pH meter equipped with an Ingold combined electrode. A Hew0003-2700/79/0351-0976$01 .OO/O
lett-Packard 5840 gas chromatograph equipped with a flame ionization detector was used for the determination of acetonitrile. A waterbath, HETO 02 P T 923, Birkerod, Denmark, was used to thermostat the chrometographic equipment. Chemicals and Reagents. Tetrabutylammonium iodide was from AB Hassle, Molndal, Sweden, and tetrapentylammonium bromide was from Eastman Kodak (Rochester, N.Y.). They were both transformed to hydroxide by shaking with silver oxide (9). The solutions were adjusted to the desired pH with phosphoric acid and diluted to a suitable concentration by addition of phosphate buffer of the same pH. Benzoic acid (BA),acetylsalicylicacid (ASA), salicylic acid (SA), and 4-hydroxybenzoicacid (PHBA) were of pharmacopoeial grade and supplied by Astra Pharmaceuticals AB, Sodertalje, Sweden. 3-Hydroxybenzoicacid (MHBA) from Fluka (Buchs, Switzerland) was used as supplied by the manufacturer. Acetonitrile was of Grade S quality (Rathburn Chemicals, Walkerburn Ltd., Peeblesshire, Scotland). All other reagents were of analytical or equivalent grade and used without further purification. Column Preparation. The columns were of 316 stainless steel with a polished inner surface, equipped with modified Swagelok connections and Altex stainless steel frits (2 pm). The length was 200 mm and the inner diameter 3.2 mm. LiChrosorb RP-8, mean particle size 10 fim (E. Merck, Darmstadt) was used as packing material (solid phase). According to the manufacturer it has a specific surface area of 250 m2/g and is prepared from LiChrosorb SI 100 which has a mean pore diameter of 100 %, and a specific surface area of 300 m2/g. The columns were packed using the balanced density slurry technique (IO) with a mixture of 1,1,2,2-tetrabromoethane+ tetrachloroethylene (4 + 6) w/w as suspending liquid. The packing procedure was performed at a pressure of 40 MPa. After packing, the column was washed with tz-hexane, dichloromethane, ethanol, and water, before equilibration with the mobile phase. Chromatographic Technique. The mobile phases were prepared by mixing known volumes of acetonitrile with aqueous phosphate buffers or, in the case of ion pair chromatography, with solutions of symmetrical quaternary alkylammonium compounds in buffers. The volume ratios were 10 + 90, 20 + 80, 30 + 70, and 40 + 60. The ionic strength of the aqueous buffer solutions was 0.05 and sodium was used as cation if not otherwise stated. The decrease of volume on mixing the aqueous solutions and acetonitrile is between 1.1and 1.9%. The mobile phases were degassed in an ultrasonic bath and thermostated t o ambient temperature (23.0 "C) before use. All chromatographic experiments were performed with injector, column, and mobile phase reservoir immersed in a water bath, thermostated to 23.00 f 0.01 "C. All chromatographic results reported are the means of duplicate or triplicate injections. Determination of Phase Components in the Column. Acetonitrile. The column was stripped with 100 column volumes of dimethylformamide. The amount of acetonitrile in the solution was determined by gas chromatography using a 2.2-meter glass column with an inner diameter of 3 mm containing 10% Triton X-100 on Supelcoport SO/lOO, which was run a t 150 OC isothermally with a nitrogen carrier of 40 mL/min. A solution of acetonitrile in dimethylformamide was used as standard. Control 0 1979 American
Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
977
Table I. Adsorption of Acetonitrile on the Solid Phaseb amount of acetonitrile in acetonitrile mobile phase in column, mL (% v/v) 0.174 10 0.180 0.324 20 0.318 30 0.463 0.443 Column not the same as in Figure LiChrosorb RP-8,10 gm.
v, ,a
vs
9
adsorbed acetonitrile mean layer pmol/m2 thickness 8 1.1
vs+ v m ,
mL mL/g mL 1.15 0.11 0.07 0.07 1.15 1.7 0.18 14 1.05 0.12 1.17 0.11 1.16 0.99 0.17 1.16 0.26 20 2.5 0.15 1.14 0.23 18 2.2 1. Mobile phase: acetonitrile + phosphate buffer pH 8.0. Solid phase: mL 1.08
by further elution with dimethylformamide (50 column volumes) showed that no acetonitrile was left on the column. Quaternary Alkylammonium Ions. The column was stripped with 100 column volumes of methanol. The solution was diluted 15 times with phosphate buffer and the quaternary ammonium compound was determined photometrically by the picrate extraction method according to Gustavii (11). As this method is designed for aqueous samples, it was controlled so that the methanol did not interfere in the determination.
RESULTS AND DISCUSSION Equilibration of the Columns. A hydrophobic solid phase (support) can be coated with a lipophilic liquid phase by passing a mobile aqueous phase containing a lipophilic organic solvent such as 1-pentanol (2-4) or butyronitrile (3, 12) through the column. Westerlund et al. ( 5 ) and Scott e t al. (13) have found that a coating with a hydrophilic liquid like methanol can be obtained by the same technique. The coating is rather rapid. In this study, a constant retention time was obtained after the passage of about 50 column volumes of a mobile phase of aqueous buffer and acetonitrile. A somewhat larger volume (about 100 column volumes) was needed for equilibration when the mobile phase contained a quaternary alkylammonium ion. The quaternary ammonium ions were strongly adsorbed on the support and to obtain a rapid equilibration with a new mobile phase, the adsorbed ions were first washed off with a t least 50 mL of methanol. Determination of the Volume of Mobile Phase in the Column. The determination of the volume of the mobile phase, V,, is usually made by injecting a nonretained compound or a sample of mobile phase of slightly changed composition. Both kinds of procedures have been used in this investigation. The studies were performed with mobile phases of acetonitrile + aqueous phosphate buffer pH 8.0. T h e samples tested were potassium nitrate and potassium dichromate (dissolved in mobile phase) and water + acetonitrile (with the same acetonitrile content as the mobile phase). The dichromate and the water samples gave coinciding peaks on the R I detector, while nitrate was more retained. The latter was even more retained than the most hydrophilic carboxylic acids (3-hydroxybenzoic acid and 4-hydroxybenzoic acid) under the same chromatographic conditions and it is obviously unsuitable for determination of V , in these systems. The retention volumes obtained with dichromate and water were taken as V,, and dichromate was used for this determination in the experiments with the UV detector. Since i t is retained in systems containing buffer of pH
