Thin-layer Chromatographic Procedure Research Articles

Neutral lipid classes of black cumin (Nigella sativa L.) seed oils

n-Hexane (H) and chloroform:methanol (CM) were used to extract the oils of Nigella sativa L. seeds. A combination of column and thin-layer chromatography procedures on silica gel were performed to fractionate the main neutral lipid classes of seed oils. The fatty acid pattern of neutral lipid (NL) fractions, triacylglycerol (TAG) and sterol content were determined. NLs constituted 97.2% and 96.1% of total H and CM extract, respectively. TAGs were the major neutral lipid class (80.8–83.1% of the total NLs), while the NL profile was characterized by high levels of free fatty acids (14.3–16.2% of the total NLs). Linoleic acid (C18:2) was the predominant fatty acid followed by oleic (C18:1) and palmitic (C16:0) acids in all examined classes. A good resolution of the TAG critical pairs was accomplished employing a gas liquid chromatograph equipped with a flame ionization detector. The separation of the TAG critical pairs C48:0, C50:1, C52:2 and C54:3 [equivalent carbon number (ECN)=48] as well as C42:0, C48:3 and C54:6 (ECN=42) was accomplished without the aid of any interacting ion such as silver. Six TAG species were determined, but two of them, C54:3 (ECN=48) and C54:6 (ECN=42) were present to the extent of 74% or above of the total TAG content. The 4-desmethylsterols isolated from the unsaponifiable fractions were β-sitosterol (1135–1182 µg/g oil) as the main component followed by Δ5-avenasterol (925–1025 µg/g oil), and Δ7-avenasterol (615–809 µg/g oil). Stigmasterol, campesterol, and lanosterol were detected in small amounts.

A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in Bdellovibrio stolpii.

Members of the bacterial genus Bdellovibrio include strains that are free-living, whereas others are known to invade and parasitize larger Gram-negative bacteria. The bacterium can synthesize several sphingophospholipid compounds including those with phosphoryl bonds as well as phosphonyl bonds. In the present study, the dominant sphingophosphonolipid component was isolated by column chromatography, and the long-chain bases, fatty acids, and polar head groups were identified by thin-layer and gas-liquid chromatographic procedures. The definitive structural identity of the sphingolipid was established by nuclear magnetic resonance and mass spectrometry of hydrolysis products and the intact compound. The compound was identified as N-2'-hydroxypentadecanoyl-2-amino-3,4-dihydroxyheptadecan-1-phosphono-(1-hydroxy-2-aminoethane).

Roasting influences on molecular species of triacylglycerols in sunflower seeds ( Helianthus annuus L.)

Sunflower seeds ( Helianthus annuus L.) were exposed to microwaves for 6, 12, 20 or 30 min at a frequency of 2450 MHz. The nucleoluses were separated from the seeds roasted in a domestic microwave oven. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from the total lipids in the nucleoluses were analysed by a combination of argentation thin-layer chromatography (TLC) and gas–liquid chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 14 different groups of TAGs, based on both the degree of unsaturation and the total chain-length of fatty acid groups. Dilinoleolein (25–26.2%), trilinolein (14.4–17.7%), dilinoleostearin (15.1–15.8%), dioleolinolein (12.2–12.4%) and triolein (10.5–11.3%) were the main components during microwave roasting. Roasting for 20 min caused a significant decrease ( P<0.05) not only in molecular species containing more than four double bonds, but also in the amount of diene species present in TAGs.

An antibiotic complex produced byStreptomyces werraensis 1365T

An antibiotic complex comprising four components (A, B, C, and X) was extracted from a native solution and mycelium of Streptomyces werraensis 1365T. The components were purified by column and thin-layer (TLC) chromatographic procedures to study their physicochemical and biological properties. The results were used to identify the substances isolated. The preliminary data allowed us to identify the components X, A, and B as the previously described compounds undecylprodigiosin, anisomycin, and copiamycin, respectively, whereas component C is a natural compound, which probably has never been described.

Roasting influences on molecular species of triacylglycerols in sesame seeds (Sesamum indicum)

Sesame seeds were roasted at different temperatures (180–220 °C) using a domestic electric oven. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from total lipids in the sesame seeds were analysed by a combination of argentation thin-layer chromatography (TLC) and gas–liquid chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 11 different groups of TAGs, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester analysis was performed to determine the composition of each zone. Eleven molecular species of TAGs were still detected in the sesame seeds following roasting treatment. Dilinoleolein (33.7–35.8%), palmitoleolinolein (20.3–22.8%), dioleolinolein (15.0–15.4%) and trilinolein (8.8–10.7%) were the main components during roasting. However, roasting for 10 min at 220 °C caused a significant decrease (P < 0.05) not only in molecular species containing more than four double bonds, but also in the amount of diene and triene species present in TAGs (with a few exceptions). These results suggest that no significant changes in molecular species or fatty acid distribution of TAGs would occur within 25 min of roasting at 180 °C, ensuring that a good-quality product would be attained. © 2000 Society of Chemical Industry

Roasting influences on molecular species of triacylglycerols in sesame seeds (Sesamum indicum)

Sesame seeds were roasted at different temperatures (180–220 °C) using a domestic electric oven. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from total lipids in the sesame seeds were analysed by a combination of argentation thin-layer chromatography (TLC) and gas–liquid chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 11 different groups of TAGs, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester analysis was performed to determine the composition of each zone. Eleven molecular species of TAGs were still detected in the sesame seeds following roasting treatment. Dilinoleolein (33.7–35.8%), palmitoleolinolein (20.3–22.8%), dioleolinolein (15.0–15.4%) and trilinolein (8.8–10.7%) were the main components during roasting. However, roasting for 10 min at 220 °C caused a significant decrease (P < 0.05) not only in molecular species containing more than four double bonds, but also in the amount of diene and triene species present in TAGs (with a few exceptions). These results suggest that no significant changes in molecular species or fatty acid distribution of TAGs would occur within 25 min of roasting at 180 °C, ensuring that a good-quality product would be attained. © 2000 Society of Chemical Industry

The Fatty Acid and Monosaccharide Compositions of Three Neutral and Three Phosphorylated Glycolipids Isolated from Leishmania donovani Promastigotes Grown in a Chemically Defined Medium

Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.

Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity.

Altered phospholipase A2 (PLA2) activity and its relationship to cation (K+, Ca2+) uptake and growth were investigated in mycobacteria exposed to the riminophenazine antimicrobial agents, clofazimine and B669 (0.15-2.5 mg/L). Microbial PLA2 activity was measured using a radiometric thin-layer chromatography procedure, whereas K+ and Ca2+ transport were measured using 86Rb+ or 42K+ and 45Ca2+, respectively. Short-term exposure (15-30 min) of Mycobacterium aurum A+ or the virulent and avirulent isolates of Mycobacterium tuberculosis H37R to the riminophenazines resulted in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Uptake of Ca2+ by mycobacteria was unaffected, or minimally affected, by the riminophenazines at concentrations of < or = 0.6 mg/L, whereas higher concentrations resulted in increased uptake of the cation in the setting of decreased microbial ATP concentrations. The results of kinetic studies using a fixed concentration (2.5 mg/L) of B669 demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapid and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA (0.2-7.2 g/L) and BAPTA/FURA-2 (0.2-9.5 mg/L), individually or in combination, did not prevent the effects of B669 on mycobacterial PLA2 activity or K+ transport, whereas alpha-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake and growth. These results demonstrate that the antimycobacterial activities of clofazimine and B669 are related to a Ca2+-independent increase in mycobacterial PLA2, leading to interference with microbial K+ transport.

Characterization of adulteration of “espinheira santa” (Maytenus ilicifolia and Maytenus aquifolium, Celastraceae) hydroalcoholic extracts with Sorocea bomplandii (Moraceae) by high‐performance thin layer chromatography

A high-performance thin layer chromatographic (HPTLC) procedure for the characterization of adulteration of “espinheira santa” (Maytenus ilicifolia and Maytenus aquifolium, Celastraceae) with Sorocea bomplandii (Moraceae), by monitoring their flavonoid content with the aid of diphenylboric acid-2-aminoethylester-polyethylene glycol 400 reagent, is presented. HPTLC data showed the predominance of highly glycosylated quercetin and kaempferol derivatives. The derivatized flavonoids were shown to be the best markers for distinguishing between authentic and adulterated drug samples, with several advantages over triterpene analysis. Copyright © 1998 John Wiley & Sons, Ltd.

Characterization of adulteration of “espinheira santa” (Maytenus ilicifolia andMaytenus aquifolium, Celastraceae) hydroalcoholic extracts withSorocea bomplandii (Moraceae) by high-performance thin layer chromatography

A high-performan ce thin layer chromatographic (HPTLC) procedure for the Characterization of adulteration of espinheira santa (Maytenus Ilicifolia and Maytenus aquifolium, Celastraceae) with Sorocea bomplandii (Moraceae), by monitoring their flavonoid content with the aid of diphenylboric acid-2aminoethylester-polyethylene glycol 400 reagent, is presented. HPTLC data showed the predominance of highly glycosylated quercetin and kaempferol derivatives. The derivatized flavonoids were shown to be the best markers for distinguishing between authentic and adulterated drug samples, with several advantages over triterpene analysis. © 1998 John Wiley & Sons, Ltd. Phytochem. Anal. 9, 263-266, 1998

High-performance liquid chromatographic determination and standardization of agmatine

In order to standardize the reversed-phase high-performance liquid chromatographic (RP-HPLC) determination of natural polyamines, particularly of agmatine, which is important in plant physiology, a simple dansylation and thin-layer chromatography (TLC) procedure was developed for the separation and purification of dansyl-agmatine. The monodansyl structure of the agmatine derivative was verified by mass spectrometry. Dansyl-agmatine was used as a reliable reference compound for the validation of an improved RP-HPLC method and to the quantitation of plant polyamines. In two varieties of wheat that had been stressed by cold treatment, significant changes were demonstrated in the agmatine content of plant seedlings.

Analysis of domoic acid in shellfish by thin-layer chromatography.

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol-acetic acid-water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 microg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay.

High-Resolution Separation and Quantification of Neutral Lipid and Phospholipid Species in Mammalian Cells and Sera by Multi-One-Dimensional Thin-Layer Chromatography

An improvement of current methods is needed for simple, rapid, and precise quantification of cellular lipids, including rare species of biologically active cellular lipids, such as phosphatidic acid (PA) and diradylglycerol (DG). In addition, further analysis of hydrolyzed acyl chains from these species by methods such as gas chromatography requires complete separations. Methods have been developed for the quantification of neutral lipids and several phospholipids extracted from mammalian cells and sera. Lipid masses were determined for the major classes of the neutral, nonpolar lipids, and of the phospholipids. The lipid classes were separated by a multistep thin-layer chromatography (TLC) procedure in different solvent systems, a method which we have designated as multi-one-dimensional thin-layer chromatography (MOD-TLC). Resolved lipid bands were visualized by the lipophilic dye primulin (direct yellow 59) and scanned by an automated laser-fluorescence detector. The mass of each band was determined by comparing band intensities of unknown samples to dilution curves of authentic standards. With modifications in solvent mixtures and length of separation times, the majority of biological lipids could be resolved and quantified with MOD-TLC methods. Since the detection method is nondestructive, purified lipids could then be recovered by scraping the visualized bands and extracting the lipids from the silica. The structural identities of the recovered lipids were confirmed by fast-atom bombardment and electrospray mass spectrometry. Extracted lipids were also hydrolyzed to release acyl chains and acyl chain species were determined in comparison to authentic standards by gas chromatography. PA and DG levels in ECV.304 cells were found to be 4.6 and 3.3%, respectively, of PC levels, with a PA/DG ratio of 1.4, which is in accord with published experience using other methods and different cell types. PA in human serum was detected at 0.6% of PC, indicating the sensitivity of the technique. In contrast to two-dimensional thin-layer chromatography, which allows for good resolution of some lipid species, but cannot be used to analyze more than a single experimental point per plate, MOD-TLC allows for direct comparative analysis of multiple samples on a single TLC plate, while still providing good resolution for the quantification of most major classes of lipid species.

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1- 14C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1- 14C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow ( R f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1- 14C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.

Mycotoxins of Alternaria alternata produced on ceiling tiles.

The production of mycotoxins by Alternaria alternata in cellulosic ceiling tiles was examined with thin-layer chromatography and high-performance liquid chromatography procedures. Alternariol and alternariol monomethyl ether were found in ceiling tile extracts, whereas extracts of control rice cultures of all three isolates produced these mycotoxins plus altenuene and altertoxin I. Extensive fungal growth and mycotoxin production occurred in the ceiling tiles at relative humidities of 84-89% and 97%.

Factors Influencing Quantity of Cantharidin Transferred to Alfalfa from Epicauta occidentalis Werner (Coleoptera: Meloidae)

Blister beetles, including Epicauta occidentalis Werner, contain cantharidin, which is a toxin that may cause severe irritation of the digestive tract and even death of livestock that ingest contaminated alfalfa. During harvest, alfalfa is easily contaminated by fluids and bodies of blister beetles, either by the crushing action of mechanical conditioning or by running over beetles with tractor tires when using sicklebar mowers for cutting. We developed a thin-layer chromatographic cleanup procedure followed by gas chromatographic analysis to quantify how cantharidin transfer to alfalfa is influenced by the condition and location of beetles in the crop. During the harvest process, living E. occidentalis located on top of cut alfalfa transferred 6.51% (93 µg per beetle) of their total cantharidin content to the hay. After all body parts were removed, 34 ppm of cantharidin was present in a 500-g sample of alfalfa contaminated by crushing 50 beetles onto the hay. Living beetles located below the cut alfalfa transferred less (4.44% or 63 µg per beetle) of their cantharidin to the hay. Recently killed and living beetles transferred significantly greater percentages of cantharidin to hay than beetles that were dead and dried. Male E. occidentalis contained greater amounts of cantharidin (1.28 mg per beetle or 6% body dry weight) than did female beetles (0.87 mg per beetle or 3%body dry weight). Dead beetles, stored for 9 mo at room temperature (25 ± 5°C), contained 34% less cantharidin on a body dry weight basis than did dead beetles kept at -10°C, underscoring the stability of cantharidin and the attendant long-term risk to livestock.

A two-dimensional thin-layer chromatography procedure for simultaneous separation of ceramide and diacylglycerol species

We have developed a novel, simple, and rapid, two-dimensional thin-layer chromatography method to separate 1,2-, 1,3-diacylglycerols and ceramides containing alpha-hydroxy and normal fatty acids from other neutral lipids on one 10 x 10 cm precoated silica gel plate. The three solvent systems used in succession leave the phospholipids at the origin and separate neutral lipids of interest into component species. We have applied this method to incorporation of 9,10-[3H]myristic acid into lipids of gills from sea bass and obtained results that are similar and comparable to those obtained by described methods.

Automated liquid-chromatographic analyzer used for toxicology screening in a general hospital: 12 months’ experience

We evaluated the clinical utility of an automated HPLC system (Remedi, Bio-Rad) for identification of drugs and metabolites in biological fluids. Serum or urine or both from 354 consecutive cases of poisoning were analyzed by the system and by a set of fluorescence polarization immunoassay (FPIA, Abbott) and thin-layer chromatographic (TLC) procedures. Antidepressants and most phenothiazines were recognized by the new system. Comparison of Remedi results with final clinical diagnoses yielded diagnostic specificity and sensitivity of 80% and 90%, respectively. Remedi detected 26 additional compounds that were neither reactive in the immunoassay screening tests nor detected by TLC procedures. Because the Remedi expands the range of drugs covered by the immunoassays and provides a rapid, preliminary report in emergency situations, we conclude that this system can be a useful complementary technique in the clinical toxicology laboratory. Although urine toxicological screening seemed adequate for a good toxicological report, blood analysis allows extra toxicokinetic data such as blood concentrations and half-life estimations.

High-performance liquid chromatography quantitation of limonin D-ring lactone hydrolase and limonoate dehydrogenase activities

Ion-pairing reversed-phase HPLC has been used to separate and determine different derivatives of limonin, i.e. disodium limonoate, 17-dehydrolimonoate, limonoate A-ring lactone and 17-dehydrolimonoate A-ring lactone. Consequently, it has been possible to measure, by this chromatographic technique, the activities of the two enzymes that sequentially catalize the first two steps in limonin degradation: limonin D-ring lactone hydrolase and limonoate dehydrogenase. In both cases linear responses were found when using increasing enzyme amounts. The determination of limonoate dehydrogenase by HPLC measurements of limonoid products was compared with that obtained by the spectrophotometric method, based on pyridine nucleotides reduction. A good correlation was detected by using both procedures, which allows the proposition of the chromatographic method as an alternative to pyridine nucleotides determination. Besides, the chromatographic method appears to be a better assay than thin-layer chromatography or radioactive procedures, the methods traditionally used to measure limonin D-ring lactone hydrolase activity.

Screening for cyanobacterial toxins in bloom and strain samples by thin layer chromatography

Two thin layer chromatographic (TLC) procedures were evaluated for the capacity screening of cyanobacterial peptide hepatotoxins and anatoxin-a in laboratory-cultured strain samples and in natural bloom samples. Seventy-five samples previously investigated with the reference methods, the chief of which was mouse bioassay, were analyzed blindly by TLC. Forty-three of the samples were hepatotoxic according to the reference methods, and 39 of them were found to contain peptide hepatotoxins by TLC coupled to u.v.-densitometry. Seven samples contained anatoxin-a according to the reference methods, and five of these were found to contain anatoxin-a by TLC, using derivatization with Fast Black K salt. Four of the samples which were neurotoxic by mouse bioassay also contained hepatotoxins by TLC. Twenty-four samples were nontoxic according to the reference methods, and in 23 of these no toxins were found by TLC either.