Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Normal Phase TLC Separation of Enantiomers Using Chiral Ion Interaction Agents

Abstract

A method for the thin layer chromatographic (TLC) separation of enantiomers and diastereomers involving the use of chiral ion interaction agents is described. Several aromatic amino alcohols were resolved by TLC on diol and/or high performance silica gel plates using a mobile phase containing (1R)-(-)- ammonium-10-camphorsulfonate or N-benzoxycarbonyl-glycyl-L-proline (ZGP). Many of these chiral aromatic amino alcohols are of pharmacological importance as α- and β-adrenergic blockers, adrenergic compounds, and anti-glaucoma agents. A comparison was made between various N-CBZ-amino acid derivatives as chiral counter ions/chiral mobile phase additives (CMAs). These separations could not be achieved on other normal phase TLC stationary phases including microcrystalline cellulose, alumina and ordinary silica gel plates.     

Chromatography of Trichothecene Mycotoxins

Abstract

Trichothecene mycotoxins occur in agricultural commodities and can cause problems from feed refusal to death in animals. This paper describes chromatographic methods for selective analysis for trichothecene mycotoxins. These methods include gas chromatography (GC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The trichothecene analysis methods by GC and TLC are shown to have a greater sensitivity than in HPLC for the underivatized mycotoxins.     

Impregnated silica-based layers in thin layer chromatography

Abstract

Impregnated thin-layer chromatography (TLC) layers based on silica gel are presented. Impregnating agents such as metal cations, inorganic ions, chelating agents, chiral selectors, surfactants, ion-pairing reagents, and ionic liquids are discussed. The role of impregnated TLC layers in medicinal chemistry is highlighted. The historical overview of TLC separations on physically coated layers is given and some future prospects ahead of this technique are discussed.     

The Use of Thin-Layer Chromatography in Experimental Xenobiology

Abstract

Studies of the metabolism and disposition of most drugs or other xenobiotics share one common feature: the need for an analytical method to measure the xenobiotic and/or its biotransformation products in biosamples. Although chromatographic methods such as GC and HPLC are important and preferred by some laboratories, initial investigations of new xenobiotic entities are often conducted using radiolabeled compounds, and, in such instances, TLC assay methods are frequently selected.

TLC is utilized in xenobiotic metabolism and disposition studies to evaluate the radiochemical purity of labeled xenobiotics, the extent of metabolism, the profile of metabolites in blood and excreta, the concentrations of the xenobiotic and specific metabolites in blood and other biosamples, the stability of the xenobiotic and its metabolites in biosamples, and the relative behavior of metabolites and reference compounds. Preparative TLC is frequently used to isolate specific metabolites. Thin-layer radiochromatography (TLRC) has been utilized to study the pharma-cokinetics of drugs and other xenobiotics in man and in animals. Especially where radiolabeled compounds are used, TLC provides an attractive chromatographic alternative to GC and HPLC in research in experimental xenobiology.     

(R)‐N,N,N‐Trimethyl‐2‐Aminobutanol‐Bis(Trifluoromethane‐Sulfon)Imidate Chiral Ionic Liquid Used as Chiral Selector in HPCE,HPLC, and CGC

Abstract

In this work, the (R)‐N,N,N‐trimethyl‐2‐aminobutanol‐bis(trifluoromethanesulfon)imidate chiral ionic liquid was first used in chromatography and exhibited a excellent chiral recognition ability in high performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), and capillary gas chromatography (CGC), which also showed for the first time that chrial ionic liquid was an effective chiral selector in HPCE and HPLC. The compounds that have been separated using this chiral ionic liquid at least included alcohol, amine, acid, and amino acid, et al. enantiomers. As the chiral ionic liquid can be easily synthesized from relatively inexpensive materials, it should have a great potentiality for chiral separation in chromatographic science.     

Methods and approaches for determination and enantioseparation of (RS)‐propranolol

Propranolol, a β‐adrenergic receptor antagonist, is a chiral compound that is marketed as a racemate, but only the (S)‐(?)‐enantiomer is responsible for the β‐adrenoceptor blocking activity. Different chromatographic methods have been applied for separation and determination of enantiomers of (RS)‐propranolol. In this article a review is presented on different liquid chromatographic methods used for enantioseparation of (RS)‐propranolol, using both HPLC and TLC. In addition, some aspects of enantioseparation under achiral phases of liquid chromatography have been briefly mentioned.     

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.     

The Application of Cross-Scanning to Conventional Thin-Layer Chromatography

Abstract

The technique of cross-scanning, the scanning of thin-layer chromatographic (TLC) plates perpendicular to the direction of development, is applicable to conventional quantitative TLC where significant advantages over normal scanning are realized by its use. Several factors are discussed which influence the ability to cross-scan, such as application of the sample, solvent development and instrumentation.     

Application of bromoacetate‐substituted β‐CD‐bonded silica particles as chiral stationary phase for HPLC

Bromoacetate‐substituted [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy]propylsilyl‐appended silica particles (BACD‐HPS), an important and useful synthetic intermediate for preparation of novel types of macrocycles‐capped β‐CD‐bonded silica particles including crown ether/cyclam/calix[4]arene‐capped β‐CD‐bonded silica particles, have been prepared and used as chiral stationary phase for HPLC. This synthetic stationary phase is characterized by means of elemental analysis. For the first time, the chromatographic behavior of BACD‐HPS was systematically evaluated with several disubstituted benzenes and some chiral drug compounds under both normal and RP conditions in HPLC. The results show that BACD‐HPS has excellent selectivity for the separation of aromatic positional isomers and chiral isomers of some drug compounds when used as stationary phase in HPLC.     

Analytical Resolution of 4-ARYL-1,2,3,4,5,6,7,8-Octahydroquinazoline-2-Thiones using a Cellulose Chiral Stationary Phase

ABSTRACT

Racemic 4-aryl-1,2,3,4,5,6,7,8-octahydroquinazoline-2-thiones were resolved into their enantiomers by HPLC on cellulose tris(4-methylbenzoate) coated on silica gel using ethanol:n-hexane (70:30) as a mobile phase. A convenient method for preparation of cellulose tris(4-methylbenzoate) chiral stationary phase was described and the enantiomers were characterized by some chromatographic data.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

Chiral Polymers and Optical Resolution: Investigation of the Mechanism of Ligand Exchange for Resolution of DL-Amino Acids

Abstract

Using novel class chiral ligand polymers which have different chemical environment in chiral cavity, we investigated their functional properties of chiral recognition for DL-amino acids and discussed ligand exchange chromatography of DL-amino acids on different chromatographic conditions in detail.     

Advantages and limitations of HPLC in environmental analysis

Summary The application of HPLC to environmental analysis is often hindered by difficulty not experienced in other areas of analysis. Usually the components being determined are at parts per million levels or less and are usually in sample matrices that can yield many interferences. In order to develop successful methodology the prime requirements for an HPLC system are column efficiency and the sensitivity and selectivity of the detection system. In this presentation, examples are given to illustrate how HPLC can be used to advantage by comparing it to gas chromatographic (GC) methods and even TLC methods. For many classes of compounds, such as halogenated hydrocarbons (pesticides, PCB’s, dioxins), the GC methods may be preferred. However, for polycyclic aromatic hydrocarbons (PAH) HPLC with fluorescence detection has proven to be excellent for trace environmental analysis. Comparisons of HPLC with TLC for aflatoxins and with bioassays for paralytic shellfish toxins are made. Novel combinations such as headspace-HPLC analyses for SO₂ and HPLC-AA for organometallic compounds are discussed.     

HPLC Detection of Sulphated Carbohydrates with a New Detection Method,that is Based on Measurement of Optical Activity by a Polarized Laser (ChiramonitorR)

ABSTRACT

The synthesis of sulphated pentasaccharides with heparin-like activity^1,2,3 proceeds through many chemical steps of protection and deprotection. As long as the pentasaccharide is fully protected with hydroxyl protecting groups such as benzylic and ester groups, the purity of intermediates can be determined by TLC. During deprotection and sulphation procedures, this determination of the purity with TLC becomes more difficult and reversed phase HPLC turns out to be a better chromatographic technique. The purification and purity control of the deprotected and sulphated pentasaccharide I (ORG31540/CY234) is performed on ion-exchange columns with a NaCl gradient as a mobile phase and UV-detection at 215 nm.     

Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicum

Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate‐based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl‐t‐butyl ether (90:10), both containing 0.1% diethylamine.     

Direct Combination of a High Performance Liquid Chromatograph and a Circular Dichroism Spectrometer for Separation and Structural Analysis of Proteins

Abstract

Basic studies of the combined system of a high performance liquid chromatograph (HPLC) and a circular dichroism (CD) spectrometer for separation and analysis of proteins are described. The HPLC-CD measurement of standard protein mixture was easily carried out by using a micro flow-cell device with a beam condenser and with a thin cell of a 1 mm-optical path. The effluent was firstly monitored at 280 nm by using an UV detector and subsequently monitored at 220 nm by using a CD spectrometer. The CD spectrum at each chromatographic peak by CD was measured in the wavelength region of 250–195 nm by a stopped flow method.     

Fluorogenic Labelling of Carbonylcompounds with 7-Hydrazine-4-nitrobenzo-2-oxa-1,3-diazole (NBD-H)

Abstract

A method for the prechromatographic fluorescence derivatization of carbonyl compounds with 7-hydrazino-4-nitrobenzo-2-oxa-1,3-diazole (NBD-H) is presented. The separation and quantitation of the hydrazones is carried out by TLC and HPLC on silica gel and RP-ma-terials. Detection limits obtained for benzaldehyde by TLC with fluorodensitometric evaluation are 5 ng/spot and by HPLC with fluorescence detection 200 pg.     

Chiral Resolution of Benzophenone Imine Derivatives of Amino Acid Esters on Chiral Stationary Phases

Abstract

A convenient way of resolving the enantiomers of amino acid esters after their derivatization using benzophenone imine is described. The enantiomers of benzophenone Schiff base derivatives of various amino acid ethyl esters are readily separated on three commercially available chiral stationary phases (CSPs). Among them, CSPs 2 and 3 afford generally the base-line enantioresolution for the analytes studied. From understanding of chromatographic results, a plausible chiral recognition mechanism is discussed.     

Direct Thin – Layer Chromatographic Separation of Enantiomers of Six selected Amino Acids Using 2-O-[(R)-2-Hydroxypropyl]-β-CD as a Mobile Phase Additive

Abstract

Compared with β-Cyclodextrin (β-CD), 2-O-[_(R)-2-hydroxypropyl]-β-CD (A) is more sensitive for chiral recognition. When (A) was used as the chiral mobile phase additive, some racemic amino acids were directly separated on silica gel TLC for the first time.