Optimization of stepwise gradient elution in reversed-phase chromatography

Summary Equations describing multi-step gradient elution with a mobile phase of constant composition in each step were derived. These equations useful for calculating the retention volumes in both gradient HPLC and TLC were derived on the basis of the relationship between the isocratic capacity factor and the volume fraction of the organic modifier. The validity of the equations was experimentally verified in a LiChrosorbRP-18-water/methanol system for 11 methyl- and chlorobenzenes and phenols. A satisfactory agreement between the theoretical and experimental k′ values was found.     

Micro-HPLC/TLC/FTIR

Identification problems often encountered in high performance liquid chromatography (HPLC)/Fourier transform infrared spectrometry (FTIR) can be circumvented through the use of a thin-layer chromatographic (TLC) plate as deposition and infra-red sampling medium. The combination of complementary separation modes is shown to demonstrate increased resolution of the components of complex mixtures. In this particular work, the effluent from a reversed-phase microcolumn is continuously deposited on a TLC plate with alumina stationary phase. The solute remains on the plate as a continuous record of the HPLC separation, which is then analyzed by diffuse reflectance FTIR. When the HPLC separation is inadequate for full separation of the components, the immobilized HPLC chromatogram serves as a starting point for subsequent TLC separation. A number of FTIR reconstructed chromatograms and spectra which are derived from the TLC plate aid in the interpretation.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

TLC as a pilot technique for the optimization of gradient HPLC II. Use of data obtained from RP-18 plates for the prediction of gradient programs in reversed-phase liquid chromatography

Summary A method is described and experimentally verified permitting the determination of the optimal gradient programs in HPLC on the basis of isocratic TLC data. The proposed method has many advantages and can also be used in reversed-phase systems. A satisfactory agreement between the theoretical and experimental of V_R/V_O values has been confirmed. Some discrepancies for more strongly retained solutes are, probably due to the respective equilibrium and nonequilibrium state of the column in isocratic and gradient elution.Part I: Ref. [1].     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.     

Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfey's reagent and its variants

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3-dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP-Ala-NH(2)) and two of its structural variants, FDNP-Phe-NH(2) and FDNP-Val-NH(2). The binary mobile phase of phenol-water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed-phase TLC, respectively. The diastereomers were also resolved on a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l-penicillamine in d-penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d-penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Over-pressure TLC variant on a plate with an enclosed sorbent layer

An over-pressure TLC variant with an enclosed sorbent layer and forced flow of the mobile phase was suggested. A simple new type device for its realization was developed.Translated from Izvestiya Akademioi Nauk. Seriya Khimicheskaya, No. 3, pp.771–773, March, 1996.     

梯度液相色谱保留时间公式在梯形梯度洗脱研究中的应用

根据前期得到的梯度液相色谱保留时间计算公式,在不指定溶剂强度模型形式的前提下,探讨了梯形梯度洗脱的一些特点。对于溶质在梯形梯度坡度上流出时的情形,推导得到溶质流出色谱柱所对应的流动相组成(φ_R)随梯度斜率(B)变化的表达式。该公式表明,在该情形中φ_R将会随着B值的增加而增加。对于溶质在梯形梯度最后一个等度区间流出时的情形,如果初始和终止流动相组成保持不变而仅有梯度的斜率发生变化时,从理论上证明了溶质保留时间(t_R)与梯度斜率的倒数(1/B)之间呈线性关系。实验中以C18色谱柱为固定相,甲醇-水为流动相,联苯为样品,测定了不同流动相组成以及梯形梯度条件下的保留时间,所得到的实验值与理论值吻合,从而验证了理论方法的正确性。     

Fundamental equation of the dual flow rate-solvent gradient elution in liquid chromatography

An alternative expression of the fundamental equation of multi-mode gradient elution involving simultaneous changes in mobile phase composition and flow rate is derived using simple kinetic arguments and graphic interpretation. The new expression consists of a system of two integral equations and provides an easy and direct way of predicting retention times under dual-mode gradient conditions.     

A Simple Solvent Programmer for Liquid Chromatography. I

Abstract

Equations have been derived from which the dimensions of a solvent gradient generator, coupled to open tubular, micro-packed, semi-micro packed or conventional HPLC columns, may be calculated for a desired gradient volume. Packed and open-tubular generators have been considered. Calculations, using the derived equations, predict that a generator of particular dimensions is needed for each column type. These dimensions are practically feasible for all column types except the conventional column.     

Adsorption TLC with binary mobile phases

Summary An equation describing R_M values in TLC with a binary mobile phase has been derived and examined by using TLC data. Two different methods have been proposed to determine the chromatographic parameters characterizing energetic heterogeneity of the adsorption system and solute-solvent interactions. These methods differ in the estimation of the mole fractions of solvents in the surface phase. In method I an analytical equation for evaluating these mole fractions is assumeda priori. Method II utilized the excess adsorption isotherm measured for the mobile phae-adsorbent system.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Chiral Separations: A Comparison of Hplc and TLC

Abstract

Comparisons are made for separations attained in normalphase HPLC and TLC involving N-carbobenzyloxy-glycyl-L-proline and (+/?)?10-camphorsulfonic acid as chiral mobile phase additives/chiral counter ions (CMA). Possible reasons for differences in stereoselectivity of β-cyclodextrin CMA and chiral stationary phases (CSP) are discussed. In addition, differences in solubility and proposed separation mechanisms for native cyclodextrins (CD) versus derivatized CD are discussed. Inherent differences in high performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) methods are outlined. The pros and cons of using HPLC and TLC are considered.     

Direction of temperature gradient for normal-phase temperature gradient interaction chromatography in polystyrene fractionation

Temperature gradient interaction chromatography (TGIC) is a powerful technique for molecular weight fractionation of polymers, in which the interaction strength is controlled by varying the column temperature. In the present paper, the effects of the sign of the temperature dependence of the retention and the direction of the temperature gradient (raising or lowering) on TGIC in the normal-phase mode were studied for the molecular weight fractionation of polystyrene samples in organic mobile phases. It was found that a positive temperature gradient was effective in the system consisting of amino-modified silica (NH(2)) column and the eluent mixture of tetrahydrofuran and n-hexane where retention decreased with increasing temperature. A negative temperature gradient was effective for the systems consisting of a bare-silica column//chloroform/n-hexane and NH(2)-column//chloroform/n-hexane, where retention increased with increasing temperature. Increasing retention with increasing temperature has been found, so far, only for a water-soluble polymer (PEO) in an aqueous mobile phase in RP-TGIC.     

Theoretical opportunities and actual limitations of pH gradient HPLC

In a series of reports published recently by our laboratory comprehensive theory and experimental conditions were established for reversed-phase high-performance liquid chromatography (RP HPLC) employing the programmed pH gradient of mobile phase. A procedure was developed providing, rapidly and conveniently, the acidity (pK_a) of weak acids and bases and their lipophilicity (hydrophobicity) log k_w. The basis of the double-gradient RP HPLC, employing simultaneous gradients of organic modifier content and mobile phase pH, was also elaborated. The fundamentals of the approach are presented briefly and systematically and its advantages and limitations are discussed. It is demonstrated that the newly introduced pH gradient method increases the analytical versatility of RP HPLC and our understanding of its physicochemical basis.     

Separation of digitalis cardenolides by HPLC

The separation of Digitalis cardenolides has been carried out by HPLC on an adsorbent column. By choice of suitable mobile phase, isocratic elution permitted resolution of mixtures of (a) aglycones, (b) secondary glycosides, and (c) primary glycosides, while gradient elution provided a means of resolving more complex mixtures of these cardiac steroids. HPLC could therefore be used in the quality control of cardiotonic drugs replacing the TLC tests for related compounds currently used, and by suitable calibration could replace the colorimetric assay procedure normally used for such drugs.     

Thin-layer adsorption chromatography with mixed mobile phases. 1. Characterization of chromatographic systems showing energetic heterogeneity of adsorbent surfaces with regard to admolecules of solvents and solutes

Thin-layer adsorption chromatography with a multicomponent mobile phase is discussed. A new equation for predicting R_M-values in TLC with mixed mobile phases using the R_M-ValUeS obtained for pure solvents is proposed. This equation takes into account effects of energetic heterogeneity of the adsorbent surface with regard to adsorbed molecules of solvents and solutes. Experimental verification of this equation is presented for R_M-data obtained by TLC using a binary mobile phase.     

Computer-aided optimization of stepwise gradient and multiple-development thin-layer chromatography

Summary Equations for the final R_F values for mobile phase gradient development and gradient multiple development are presented. Computer simulations of gradient development for both modes of thin-layer chromatography are discussed for the preliminary optimization of gradient programs.     

A comparative study of HPLC and TLC separation of amino acids using Cu(II) ion

Separation of amino acids using Cu(II) by RP-HPLC and impregnated TLC is reported. For HPLC the sample mixture of amino acids containing copper (II) was injected into the column while for TLC the silica gel plates were impregnated with Cu(II). The mobile phase used in HPLC was acetate buffer (0.3 M , pH 6.0)-acetonitrile (9:1, v/v), and that in TLC was acetate buffer (0.3 M , pH 6.0):acetonitrile-n-butanol (12:5:10, by vol.). The results have been compared and discussed.