The separation of enantiomeric sugars by chromatographic methods

This paper has reviewed the number of chromatographic methods by which one may determine the absolute configuration of sugars. Both indirect methods (converting the enantiomeric pair into diastereomers) and direct methods (using chiral stationary phases) have been discussed. Resolving reagents for the indirect methods include chiral hydroxy compounds, chiral amines, and chiral thiols; with subsequent separation of the diastereomers either by gas-liquid chromatography or by high pressure liquid chromatography. Direct methods discussed have exclusively utilized chiral substitution of organopolysiloxane phases for the separation of enantiomeric sugars as volatile derivatives by gas-liquid chromatography.     

Gas chromatographic determination of malonaldehyde formed by lipid peroxidation

Gas chromatographic (GC) methods for the determination of malonaldehyde (MA) all require formation of a stable derivative of MA since free MA is not suitable for direct GC analysis. Most reported GC methods give a total measure of free MA and its bound forms because their assay conditions are sufficient to hydrolyze or decompose bound MA during sample preparation. In this paper, GC methods that provide a measure of total MA (free plus bound) are reviewed. A recently developed capillary GC method that allows determination of free MA apart from other forms is also discussed. The method involves derivatization of MA to N-methylpyrazole under milder reaction conditions than with other GC methods. The method represents an advantage over existing techniques for free MA determination because capillary GC offers the highest efficiency of separation among chromatographic methods thus allowing a more specific and accurate measure of MA.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Separation procedures for naturally occurring antioxidant phytochemicals

Phytochemicals in fruits, vegetables, spices and traditional herbal medicinal plants have been found to play protective roles against many human chronic diseases including cancer and cardiovascular diseases (CVD). These diseases are associated with oxidative stresses caused by excess free radicals and other reactive oxygen species. Antioxidant phytochemicals exert their effect by neutralizing these highly reactive radicals. Among the tens of thousands of phytochemicals found in our diets or traditional medicines, polyphenols and carotenoids stand out as the two most important groups of natural antioxidants. However, although collectively these phytochemicals are good antioxidants, the roles and effect of individual compounds are often not well known. Hundreds of carotenoids and thousands of polyphenols have been identified so far from various plants. A single plant could contain highly complex profiles of these compounds, which sometimes are labile to heat, air and light, and they may exist at very low concentrations in the plants. This makes the separation and detection of these antioxidant phytochemicals a challenging task. The present review focuses on the antioxidant activity, chemical types, sampling and sample processing procedures, and separation using various chromatographic and electrophoretic techniques. Detection and quantification using ultraviolet-visible-diode array and mass spectrometry will be discussed.     

Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography

We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.     

An improved method for the determination of green and black tea polyphenols in biomatrices by high-performance liquid chromatography with coulometric array detection

Tea polyphenols are strong antioxidants and are believed to have beneficial health effects. However, the blood and tissue levels of these compounds are not well characterized because of a lack of suitable analytical methods for the biological resolution of these compounds. Previously, we developed methods for the analysis of three green tea catechins. Now we report an improved method for the measurement of the levels of the different catechins and theaflavins in biological fluids and tissues. The method includes digestion of the plasma, urine, or tissue samples with beta-d-glucuronidase and sulfatase, followed by extraction with ethyl acetate and subsequent separation by reversed-phase high-performance liquid chromatography (HPLC). The polyphenols are identified on the basis of their retention times, spectral analysis, and electrochemical behavior across an array of electrodes. In a single HPLC run, it is possible to determine the major catechins and theaflavins as well as some of the catechin metabolites. The detection limits for catechins and theaflavins are from 5 to 10 ng/ml of saliva, plasma, or urine.     

alpha-1 Antitrypsin phenotypes determined by isoelectric focusing of the cysteine-antitrypsin mixed disulfide in serum.

The methods available for analysis of sugar kinase activity are usually either complicated or time consuming. Coupled assays, aside from the added cost of coupling enzymes and substrates, present problems due to the pH optima, activators, and inhibitors of the coupling enzymes. Direct separation of the product requires either ion exchange (1) or paper chromatography (2,3). The former requires constant attention and the latter usually takes either overnight for the completion of a chromatogram or a great deal of elution solvent (200 ml) for DEAE paper discs (3).Those enzymes which form phosphorylated products from nonionic substrates (hexokinases, glycerol kinase, phosphoribosyl-transferases, etc.) may be conveniently assayed by chromatograhic separation of a radioactive phosphorylated product from the radioactive nonionic substrate, where the product remains at the origin. In such assays, no interfering coupling enzymes are used and the product can be directly and sensitively measured. The only current limitation with such methods is the time required for the separation of the phosphorylated product. It would be advantageous to obtain the enzyme's activity in as short a time as possible.We present here a method of paper chromatographic separation of phosphorylated product from nonionic substrate which requires only approximately two hours, uses a large petri dish, very little chromatographic grade paper, and almost no attention.     

Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA

Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6-methyladenine and 7-methylguanine.     

Some practical aspects of measurements of betaines and their sulphur analogues by the use of HPLC

Recent interest in the analysis of glycinebetaine in algae has stimulated the search for improved methods of assaying betaines. A method based on high-performance liquid chromatographic (HPLC) separation using commercial columns and guard columns is described in detail for the newcomer. The methods is sensitive, reproducible and flexible enough to allow for minor differences among different HPLC pump systems.     

Stereoselective determination and pharmacokinetics of dihydropyridines: an updated review

All dihydropyridines, except nifedipine, have at least one chiral center, and their pharmacokinetics and clinical effects differ from one enantiomer to another. Chiral separation methods for dihydropyridines using chromatographic techniques are discussed. The stereoselective pharmacokinetics of dihydropyridine calcium antagonists were reviewed in detail in 1995. The present review article updates the methods for the stereoselective determination of dihydropyridines using chromatographic techniques and summarizes the pharmacokinetics of the dihydropyridines, including the newest drugs under development.     

Natural polyphenols-iron interaction

The iron-binding capacity of different fractions of natural polyphenols extracts was determined by chromatographic and electrophoretic methods. Their effects on iron-induced calcium homeostasis changes in liver tissue suspension showed that mate tea and green tea extracts provoke a very significant inhibition of the iron effects, whereas it is much less significant with red wine extract. The biological importance of this phenomenon is discussed.     

Determination of andrographolide,deoxyandrographolide and neoandrographolide in the Chinese herb Andrographis paniculata by micellar electrokinetic capillary chromatography

In this paper a micellar electrokinetic capillary chromatographic (MEKC) method has been developed for determining the active components (andrographolide, deoxyandrographolide and neoandrographolide) in water:ethanol extracts of the Chinese crude herb Andrographis paniculata and its preparations (Chuanxinlian and Xiaoyan Lidan tablets). The optimum separation conditions were 15 mM sodium dodecyl sulphate in 30 mM borate buffer (pH 9.5) with UV detection wavelength at 214 nm and a constant voltage of 16 kV. An HPLC method was employed in order to validate the MEKC method with respect to separation efficiency, sensitivity, linearity and repeatability. The two methods are shown to be complementary because of their different selectivity and thus are very suitable for cross-validation studies. The MEKC method is demonstrated to be more appropriate for the analysis of the active compounds in A. paniculata in that it is easier and less expensive to use and does not suffer from contamination of the chromatographic column.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Paper electrophoresis and chromatography of oligopeptides with N-terminal methionine

Several electrophoretic and chromatographic systems are described for the separation of di- and tripeptides with N-terminal methionine. The most effective methods of separation are electrophoresis in barbital-triethylamine-acetic acid, pH 8.0 and chromatography in n-butanol-acetic acid-water, 18:2:5. Increased resolution may be obtained by using two or more systems in combination. These methods were intended to separate and identify the oligopeptides formed during the initiation of eukaryotic protein synthesis, but may also be applied to the separation of small peptides generally.     

Direct chiral separation of unnatural amino acids by high-performance liquid chromatography on a ristocetin a-bonded stationary phase.

Direct high-performance liquid chromatographic chiral separation of numerous underivatized unnatural amino acids on a ristocetin A-bonded chiral stationary phase used in the reversed-phase and in the polar organic chromatographic modes is reported. The effects of different parameters such as mobile phase composition, temperature, and the structure of the analytes on the selectivity in both chromatographic modes are discussed. By variation of the parameters, the separation of the stereoisomers was optimized and, as a result, baseline resolution was achieved in most cases.     

Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

High-performance liquid chromatography of phosphatidic acid

This paper reviews existing high-performance liquid chromatographic (HPLC) methods for the analysis of phosphatidic acid (PA) in various sample matrices. In addition to the introductory background discussion on important aspects of PA in lipid biochemistry, the review provides comprehensive coverage in the areas of derivatization techniques, detection methods, and HPLC separation techniques. Conversions of PA to suitable derivatives enhance the detection sensitivity and improve the chromatographic behavior of the analytes. Detection methods include the use of state-of-the-art detectors and are discussed in terms of sensitivity, specificity, and compatibility with analytical systems. Pertinent normal-phase and reversed-phase HPLC data for PA are compiled from published methods.     

Effect of stress conditions on Escherichia coli outer and inner membranes, separated by Sephacryl S-500 chromatography

A chromatographic method for the separation of outer and inner membranes of Escherichia coli is described. This method is much faster than the generally used equilibrium centrifugation and has a greater versatility in the volumes that can be treated. Both techniques are used to examine the effects of different stress conditions on the E. coli membrane. The obtained results illustrate the equivalence of both methods.