Walter D. Conway

High-Speed Countercurrent Chromatography

I. INTRODUCTION During the past 20 years countercurrent chromatography (CCC) has been steadily improved in both partition efficiency and separation times.1-15 The original helix CCC and the successively developed nonhelix preparative-scale CCC schemes, such as droplet CCC16,17 and locular CCC,17 required relatively long separating times; sizeable separation usually exceeded 24 hr. During the last decade, development of various flow-through centrifuge schemes, such as the flow-through coil planet centrifuge18,19 (CPC), angle rotor CPC,20 elution centrifuge,21 horizontal flow-through CPC,22-26 toroidal CPC,27,28 and nonsynchronous flow-through CPC,29-31 has substantially shortened the separation times so that efficient separations became possible with overnight runs.

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography : III. Effects of physical properties of the solvent systems and operating temperature on the distribution of two-phase solvent systems

Statistical studies were made to correlate the hydrodynamic behavior of two-phase solvent system in counter-current chromatography (CCC) to their physical properties including interfacial tension, viscosity, and the difference in density of the two phases. Settling time measured under unit gravity provided a reliable numerical index for the hydrodynamic behavior of the solvent systems in a centrifugal force field. Viscosity and settling time were strongly correlated (correlation coefficient, r = +0.88) while interfacial tension (r = -0.65) and phase density difference (r = -0.45) showed moderate and weak correlation, respectively. Studies of the effect of temperature on settling time as well as a preliminary apparatus operated at higher temperature show that raising the temperature will improve the performance of high-speed CCC.

Absorption and Elimination Profile of Isoproterenol III: The Metabolic Fate of dl-Isoproterenol-7-3H in the Dog

Intravenous injection of isoproterenol-7- 3 H in dogs, in doses ranging from 0.4 to 1.6 mcg./kg. body weight, immediately induced tachycardia which declined with a half-life of about 1min. The resulting levels of radioactivity in blood declined with a significantly longer half-life of 3–4min. Unchanged isoproterenol accounted for less than 1 percent of the total radioactivity in plasma after intravenous medication. After oral administration of 15mg. of isoproterenol-7- 3 H hydrochloride to dogs, a biphasic time course was observed for both heart rate and radioactivity in blood with maxima occurring at 0.5 and 1–2hr. after medication. The metabolic fate of isoproterenol was determined by the route of medication, the intravenous and intraportal route giving rise mainly to 3- O -methyliso-proterenol, while oral medication was converted mainly to a sulfuric acid ester which was formed either by the intestinal flora or during passage through the intestinal wall. The rapid decrease in tachycardia after intravenous medication is consistent with the rapid conversion of the drug to inactive metabolites while the more prolonged tachycardia induced by oral medication results from slow absorption.

Mass spectral identification of probenecid metabolites in rat bile.

Abstract The metabolism of probenecid (p-(di-n-propylsulfamyl)benzoic acid) by the liver has been studied in biliary cannulated rats with ligated renal pedicles. The unchanged drug, N-depropylated probenecid, and three glucuronides of metabolites of this drug readily appeared in the bile. When rats were given a single 50 mg/kg dose of [ 14 C] probenecid, 96% of the administered radioactivity was excreted in the bile of renal-ligated rats in 8 hr. Approximately 64% of the drug in this system appeared as metabolites. Four major metabolites were identified by their gas-liquid chromatograms and mass spectra. They were the N-dealkylated drug, an ester glucuronide of a side chain oxidized metabolite (metabolite B) and two ether glucuronides of side-chain hydroxylated drug (metabolites A and C). None of the previously reported acyl glucuronide of probenecid was found in rat bile and none of the metabolites included in this paper have been reported before. In the homozygotous Gunn rat, reported to be deficient in glucuronidating enzymes, the same biliary metabolites as appeared in Sprague-Dawley rats, were found. It is proposed that one may be able to very rapidly assess the importance of drug metabolism for a new agent if it is administered to renal-ligated animals, collected in bile, and then subjected to GLC-MS procedures. The overall technique appears to have several advantages over the usual methods of collecting and assaying urine for drugs and their metabolites.

Counter-current chromatography: simple process and confusing terminology.

The origin of counter-current chromatography is briefly stated, followed by a description of the mechanism of elution of solutes, which illustrates the elegance and simplicity of the technique. The CCC retention equation can be mentally derived from three facts; that a substance with a distribution coefficient of 0 elutes at the mobile phase solvent front (one mobile phase volume); and one with a distribution coefficient of 1 elutes at the column volume of mobile phase; and solutes with higher distribution coefficients elute at additional multiples of the stationary phase volume. The pattern corresponds to the classical solute retention equation for chromatography, V(R)=V(M)+K(C)V(S), K(C) not being limited to integer values. This allows the entire pattern of solute retention to be visualized on the chromatogram. The high volume fraction of stationary phase in CCC greatly enhances resolution. A survey of the names, symbols and definitions of several widely used chromatography and liquid-liquid distribution parameters in the IUPAC Gold Book and in a recent summary in LC-GC by Majors and Carr revealed numerous conflicts in both names and definitions. These will retard accurate dissemination of CCC research unless the discordance is resolved. It is proposed that the chromatography retention parameter, K(C), be called the distribution coefficient and that a new biphasic distribution parameter, K(Δ(A)), be defined for CCC and be called the species partition ratio. The definition of V(M) should be clarified. V(H) is suggested to represent the holdup volume and V(X) is suggested for the extra-column volume. H(V) and H(L) are suggested to represent the volume and length of a theoretical plate in CCC. Definitions of the phase ratio, β, conflict and should be clarified.

The Effect of Route of Administration on the Metabolic Fate of Terbutaline in the Rat

Abstract1. The metabolic fate of [3H]terbutaline has been investigated in rats after oral, subcutaneous, intraperitoneal and intraportal administration (5 mg per kg).2. About half the administered radioactivity was excreted in the urine and the remainder in faeces regardless of route of administration. Urinary excretion was essentially complete in 24 h, but an additional 10% of the dose was excreted in the 24–48 h faeces.3. Only one metabolite, a glucuronide conjugate of terbutaline, was excreted in the urine along with unchanged drug. About 3% of the dose was excreted unchanged in urine following oral administration. Ratios of terbutaline glucuronide to free drug were 1 : 1, 2 : 1 and 13 : 1 after subcutaneous, intraperitoneal or intraportal, and oral administration respectively, suggesting that the orally administered drug is extensively conjugated in the intestinal mucosa.4. Measurement of the mobility-pH profile by high-voltage paper electrophoresis was utilized to characterize the conjugate.

Resolution in Countercurrent Chromatography

Abstract Using as an example the separation of DNP-glutamic acid and DNP-alanine in a nonplanetary countercurrent chromatograph, it was demonstrated that resolution, Rs, of a solute pair can be satisfactorily predicted from knowledge of the column efficiency, N, the fraction of column volume occupied by stationary phase, SF, and the partition coefficients of the substances in the solvent system employed. Resolution is a function of the phase volume ratio and separation of rapidly eluted compounds is favored by an increase in SF. The partition coefficients, K, the separation factor, α, and the column efficiency, N, are independent of SF. Increasing SF brings about an increase in RS by increasing the capacity factor k1. For SF of 0.4, characteristic of the horizontal flow-through CCC, baseline separation (RS = 1.5) is obtained for values of K1, the first eluted substance, of about 0.5 for N = 1000 or K1 of 10 for N = 100, where α is 2. Increasing SF to 0.8, characteristic of the multilayer coil, high speed ...

Lipophilic benzamide and anilide derivatives as high-performance liquid chromatography internal standards: application to sirolimus (rapamycin) determination

Finding a suitable internal standard in reversed-phase high-performance liquid chromatography is often difficult. A general approach for selecting and synthesizing the proper internal standard is presented and applied to a validated method for quantitation of sirolimus in several biological matrices. A series of fifteen N-alkylbenzamides, N-alkyltoluamides and N-alkanoylanilines with a log P range of 3.51 to 6.68 were synthesized as internal standards; N-undecyl-o-toluamide was evaluated most extensively. Sirolimus quantitation involves a simple sample clean-up procedure followed by isocratic chromatography on a heated C18 analytical column with an 70% methanol-water mobile phase and ultraviolet detection at 278 nm. This method was linear from 2.5 to 200 ng with a limit of quantitation of 2.5 ng using a 1-ml blood sample. Sirolimus recovery was above 72.1%. The intra-day and inter-day coefficients of variation were less than 11.7%. Several drugs and sirolimus metabolites do not interfere with the analysis. This method was used to measure sirolimus in blood from rats, rabbits and humans.

Solvent Selection for Countercurrent Chromatography by Rapid Estimation of Partition Coefficients and Application to Polar Conjugates of p-Nitrophenol

Abstract A rapid and moderately precise technique to measure partition coefficients of UV-absorbing solutes in solvent systems for countercurrent chromatography is described and applied to p-nitrophenol and its conjugates with-glucose, sulfuric acid and glucuronic acid. It involves equilibration of one ml of each phase with solute in a narrow test tube, removal of the entire lower phase, dilution with methanol and calculation of the partition coefficient as the ratio of the absorbance values of each dilution at any suitable wavelength. The polar conjugates of p-nitrophenol can be separated by countercurrent chromatography using ethyl acetate as mobile phase and aqueous KH2PO4 as stationary phase.

Counter-current chromatography

Abstract The simplicity of counter-current chromatography (CCC) is sometimes overshadowed by a complex description of its mechanical and chromatographic attributes. The universal features of chromatographic theory relevant to CCC are summarized here, using a partition coefficient elution scale to present th chromatogram in a general, readily visualized, format. The principal types of CCC apparatus are summarized, along with selected applications and an indication of the type of apparatus best suited for some specific applications.