Overview of methods for the determination of trans fatty acids by gas chromatography, silver-ion thin-layer chromatography, silver-ion liquid chromatography, and gas chromatography/mass spectrometry

trans Isomers of naturally occurring cis-unsaturated fatty acids are produced when liquid vegetable oils or marine oils are partially hydrogenated to produce margarine, shortenings, and other hardened-fat products. Isomeric trans fatty acids are also formed in the intestinal tract of ruminants, and they appear in small amounts in dairy products and ruminant meat. Currently, satisfactory analyses for the fatty acid profiles of fats containing trans fatty acids are obtained by gas chromatography (GC) using capillary columns coated with highly polar cyanosilicone stationary phases. In capillary GC methods, the key limitation has been the incomplete separation of trans-monoenoic acid isomers from their cis isomers; however, recent reports have demonstrated that improvements in separation are attainable with the use of 100 m columns. In these columns, there is very little overlap of cis and trans isomers. More accurate trans fatty acid analyses can be obtained by coupling GC with either silver-nitrate thin-layer chromatography or silver-nitrate liquid chromatography.     

Two-dimensional counter-current chromatography: 1st traditional counter-current chromatography, 2nd acid-base elution counter-current chromatography

An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.     

Non-aqueous,reversed-phase,high-performance,thin-layer chromatography and column liquid chromatography of metal complexes of porphine

Summary The chromatographic mobility of 21H, 23H-porphine and its Ni(II), Cu(II), Zn(II) and Pd(II) complexes were investigated by high-performance thin-layer chromatography on an octadecyl-bonded, silica gel plate with various polar organic solvents including alcohols, acetonitrile, dimethylsulfoxide and propylenecarbonate. The mobility generally decreases according to the central metal ion of the complex as follows: Zn(II)>(free porphine)>Ni(II)>Pd(II)>Cu(II). Methanol is a good choice of solvent for the separation of these metal porphine complexes. Successful separation of porphine and the four metal complexes is accomplished within 13 min on a LiChrosorb RP-18 column with methanol eluent.     

New,unified nomenclature for chromatography

Summary The most important definitions, terms and symbols included in the new, unified Nomenclature for Chromatography issued by the International Union of Pure and Applied Chemistry (IUPAC) are outlined.     

Fast, comprehensive two-dimensional liquid chromatography

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two-dimensional liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20s for a full gradient elution run. Thus, fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 min. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

Comparison of ultra high performance supercritical fluid chromatography,ultra high performance liquid chromatography,and gas chromatography for the separation of synthetic cathinones

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite‐5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.     

Simultaneous correlation chromatography,a new technique applied to calibration in high-performance liquid chromatography

Simultaneous correlation chromatography (SCC) is introduced as a technique capable of analyzing several different samples simultaneously on the same chromatographic column. The theoretical basis of the technique is outlined and a computer simulation demonstrates the feasibility of the method. The advantages and potential of the technique are discussed. The technique is applied experimentally in a calibration procedure for high-performance liquid chromatography. During the calibration, unknown sample and calibration standards are processed under the same conditions, resulting in very accurate calibration. Other applications are outlined.     

Analysis of the anti-coccidial drug, halofunginone, in chicken feed using gas-liquid chromatography and high-performance liquid chromatography

Methods are described for the analysis of the anti-coccidial drug, halofuginone, at concentrations of 3 ppm in chicken feed, using gas-liquid chromatography and high-performance liquid chromatography. Both methods are based on ethyl acetate extraction, partition into hydrochloric acid and purification and concentration using XAD-2 column chromatography. The precision and accuracy of both methods is given.     

Temperature-responsive,polymer-modified surfaces for green chromatography

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) and its derivatives were utilized as chromatography column matrix modifiers to develop novel supports for thermoresponsive hydrophobic chromatography with aqueous mobile phase. In the column, matrix surfaces show thermoresponsive hydrophilic/hydrophobic property alterations, which alter interaction with and retention of solutes to be separated. We have also demonstrated that the electrostatic interaction of ionic solutes and charged, thermoresponsive polymer-modified surfaces can be modulated temperature changes in the aqueous mobile phase alone.     

Characterisation of retention in micellar high-performance liquid chromatography, in micellar electrokinetic chromatography and in micellar electrokinetic chromatography with reduced flow

The retention (migration) behaviour of various barbiturates, phenylurea and triazine herbicides in micellar electrokinetic chromatography (MEKC) with uncoated fused-silica capillaries was compared with the behaviour in micellar electrokinetic chromatography with reduced electroosmotic flow (RF-MEKC) using capillaries modified with linear polyacrylamide. The error in the values of the retention factors caused by the neglection of the contribution of the electroosmotic flow in RF-MEKC was investigated and a method for correcting this error was suggested. The retention was characterised using the lipophilic and polar indices to characterise and to predict the retention as a function of the concentration of the surfactant (sodium dodecylsulphate) in the running buffer in MEKC and in RF-MEKC. Homologous series of n-alkylbenzenes and of n-alkan-2-ones were compared as the standard sets for the calibration of the retention (migration) index scale. The values of the lipophilic indices of a given solute measured in reversed-phase HPLC, MEKC and RF-MEKC are close to each other. Under ideal MEKC conditions, the values of the polarity indices are close to one for various sample solutes. However, for partially ionised compounds such as weakly acidic barbiturates, where the contribution of the electrophoretic migration is significant, the values of the polarity indices are significantly lower than one. Optimum conditions for separations of mixtures of triazine and phenylurea herbicides and of barbiturates using various techniques tested were compared.     

Determination of benomyl, diphenyl, o-phenylphenol, thiabendazole, chlorpyrifos, methidathion, and methyl parathion in oranges by solid-phase extraction, liquid chromatography, and gas chromatography

A simple and rapid method was developed for determination of benomyl, diphenyl (DP), o-phenylphenol (OPP), thiabendazole (TBZ), chlorpyrifos, methidathion, and methyl parathion in whole oranges. These compounds were extracted from a mixture of samples and anhydrous sodium acetate with ethyl acetate. The ethyl acetate extract was concentrated and cleaned up by passing through tandem solid-phase extraction columns consisting of anion-exchange and primary/secondary amine bonded silica. The eluate was concentrated and volume was adjusted with methanol for subsequent liquid chromatography (LC) and gas chromatography (GC). Benomyl (as methyl-2-benzimidazole carbamate, MBC), DP, OPP, and TBZ residues were determined by LC with fluorescence detection. Recoveries at 3 fortified levels (0.1, 1, and 10 micrograms/g) ranged from 63.9 to 97.4%, with coefficients of variation (CVs) of 1.6 to 15.5%. Limits of detection (LODs) were 0.01 microgram/g for DP, OPP, TBZ and 0.05 microgram/g for benomyl. Chlorpyrifos, methidathion, and methyl parathion residues were determined by GC with flame photometric detection. Recoveries ranged from 90.4 to 97.0%, with CVs of 2.1 to 5.9%. LODs were 0.005 microgram/g for chlorpyrifos and methyl parathion, and 0.01 microgram/g for methidathion.     

Comparison of micellar electrokinetic chromatography, liquid chromatography, and microbiologic assay for analysis of cephalexin in oral suspensions

Two well-accepted methodologies, based on a microbiologic assay (MA) and liquid chromatography (LC), and a novel methodology using micellar electrokinetic chromatography (MEKC), were compared for the determination of cephalexin in commercially available and simulated samples of oral suspensions. The MA, described in the Brazilian Pharmacopeia, was performed with a strain of Staphylococcus aureus ATCC 6538 as the test organism, following the cylinder-plate method. The LC analysis followed the European Pharmacopoeia, 3rd Ed., and was used with minor modifications. The MEKC analysis was based on a previous work of the group. Estimates of the repeatability relative standard deviations of the MA, LC, and MEKC methods in the analysis of a commercial sample were 0.34, 0.42, and 0.37%, respectively. The recovery obtained with LC was 99.90 +/- 1.11%; for MEKC, it was 100.09 +/- 0.56%. Although the 3 methodologies were statistically equivalent for the determination of cephalexin in oral suspensions, MA gave suitable repeatability despite being nonspecific and time-consuming. MEKC provided faster analysis and higher column efficiency, whereas LC presented superior sensitivity. The results indicated that MEKC can be used as an alternative method to MA and LC in routine quality control laboratories.