Application of the relation between solvent composition and discontinuous gradient front in liquid phase chromatography

Studies on relationships between thin-layer chromatography and column chromatography. IV

Abstract

The relations between the migration rate (K_β or K _γ value) of the front formed in demixing process and the concentration of the more polar component in a developer were investigated in thin-layer (TLC) and dry column chromatography (DCC). These relations were applied to the choice of a more suitable composition of the developer.
The following sets of adsorbent and developer were used in both TLC and DCC : 1. for separation of metallic ions (Cu²⁺, Ni²⁺ and Co²⁺) ; adsorbent, purified silica gel; developer, acetone-3N HCl system (acetone system) ; 2. for separation of Sudan IV, 2, 4-dinitroaniline, and quinine; adsorbent, silica gel H; developer, chloroform-1-butanol-diethylamine systems [chloroform system A. (98-M) : M : 2, and chloroform system B. (88- N) : 12 : N]. In TLC the sample solution was spotted on the thin-layer (thickness : 0.25 mm) at a distance (Y) of 2.5 cm from the edge of the plate and developed ascendingly. The distance (X) between the immersion line and the edge of the plate and that (Z) between the solvent front (α-front) and the edge of the plate were 1 cm and 12.5 cm, respectively. A column of 1 cm (inner diameter) × 10 or 12 cm (height) was used in DCC. After the development the spots or zones of the sample and the demixing lines were detected by ultra-violet ray (3600 A) or by applying appropriate reagents. Each component of the eluate in DCC with the acetone system was determined by using acid-base titration and iodometry (Fig. 2).
Co²⁺ traveled always nearer to the β-front, Cu²⁺ in the α-zone, and Ni²⁺ in the β-zone. 2, 4-Dinitroaniline and quinine traveled on the β- and γ-front, respectively, and Sudan IV in the α-zone. Because of the phenomenon of chromatographic demixing of developer, two fronts (α- and β-front) were observed in the acetone system, and three fronts (α-, β- and γ-front) in the chloroform systems. In both TLC and DCC, the logarithmic relations between K_β (or K_γ) value and the concentration (mole per cent, C_M) of the more polar component in the developer were shown, and the equation based the adsorption isotherm of Freundlich [log (1/K-1) =κ-ξ log C_M… ( 1 ), where κ and ξ are constants] was held for K=K_β and K=K_γ (Fig. 1, 4, 5, 6). The constants of equation ( 1 ) were determined experimentally; in TLC, the following constants were obtained : κ=-0.03, ξ=0.68 (acetone system) ; κ=0.92, ξ=1.16 (chloroform system A) ; and κ=0. 75, ξ=0.98 (chloroform system B) (Fig. 1, 4, 5). While in DCC with the chloroform system B, κ=0. 70 and ξ=1.00 (Fig. 6).
The optimum composition of the developer for separation of the samples (Cu²⁺-Co²⁺, Sudan IV-2, 4-dinitroaniline, 2, 4-dinitroaniline-quinine) was calculated by using the chromatographic data of each sample and equation ( 1 ) as follows. The optimum K_β (K_γ) value in order to separate the two samples was calculated by means of equation ( 2 ) :
K_{β(or γ)}=(R_f±ΔR_f)(Z-Y)+(Y-X)/(Z-X)…(2)
where R_f is the R_f value of the sample on the β- or γ-front, and ΔR_f the difference of R_f value which is needed in order to separate the two samples.