Thin-layer Chromatographic Separation Research Articles

Effect of tibolone on turnover of cholesterol to bile acids in ovariectomized rats

Tibolone is used in breast cancer therapy in women. The most important aspect of this therapy concerns its influence on bone and lipid metabolism. Several studies have reported the effect of tibolone on plasma lipids. Approximately 40% of total cholesterol removal occurs by its conversion to bile acids, a process that takes place in the liver. The aim of the study was to evaluate the influence of tibolone on the conversion of cholesterol to bile acids in estrogen-deficient rats. Thirty female Wistar rats were divided into three groups: sham-operated controls, ovariectomized controls, and ovariectomized rats treated with tibolone. After 42 days of drug administration, bile was collected under anesthesia after administration of radioactive 4-C cholesterol. Bile was assayed for total C radioactivity; C bile acids were determined after thin-layer chromatography separation by using the isotopic technique. The ovariectomy decreased the concentrations of C-radioactive bile, taurocholic acid, and cholic acid. The administration of tibolone significantly increased the excretion of C-radioactive bile and C-radioactive bile acids as compared with ovariectomized controls. Moreover, the therapy with tibolone significantly increased the concentrations of C taurocholic acid, C glycocholic acid, cholic acid, and taurochenodeoxycholic plus taurodeoxycholic acids. The results of the present study suggest that tibolone may increase the concentrations of cholesterol and trihydroxy bile acids in bile. These changes may be associated with an increased risk of gallstone generation.

402 Cholesterol Synthesis in vivo in Premature Infants from Deuterated Water (D2O)

Background: Cholesterol is one of the main components of cell membranes and is required as precursor for many metabolically active compounds. To date cholesterol metabolism is uncompletely defined in premature infants. Using a 12 h i.v. constant infusion of 13C acetate, Renfurm (Pediatr Res 2004) studied de novo cholesterol synthesis in premature infants. This method required indwelling lines. Objective: To assess if cholesterol synthesis can be measured with a less invasive method based on D2O as precursor (Matthan Lipids 2000). Material and Methods: Cholesterol synthesis was studied in 15 newborns (BW<1500 gr, age<48 h) on parenteral nutrition without lipids. Subjects received orally a bolus of D2O (0.1 gr/kg), followed by 0.000625 gr/kg every 12 for 48 h. Urine and plasma samples (200 ul) were collected before study start and every 12 h thereafter till 48 h. Unesterified cholesterol was obtained from plasma lipid extraction and thin layer chromatography separation, and cholesterol deuterium enrichments measured by Gas-Chromatography-Isotope Ratio- Mass Spectrometry (GC-IRMS). Deuterium enrichment of urine samples were measured by GC-IRMS and regarded as the precursor enrichment pool. Cholesterol Fractional Synthesis Rate (FSR) and secretion time (ST) were measured as previously described (Matthan Lipids 2000). Data are given as mean±SD. Results: Study weight was 1084±546 gr, gestational age 28±2 wks, mean age 23±19 h. Energy Intake was 30±8 kcal/kg/day. Cholesterol FSR was 16±12% and ST 11±11 h. Conclusion: This method has the advantage that tracer can be given orally, steady state of precursor is easily achieved and very small amount of blood is needed. We measured cholesterol synthesis from D2O in vivo in premature infants and the calculated rate was approximately that found in adults (Jones J Lipid Res 1994). Given the high variability between patients larger study will better define factors related to endogenous cholesterol synthesis early in life.

Standardization of Crude Extract of Neem Seed Kernels (Azadirachta indica A. Juss) and Commercial Neem Based Formulations Using HPTLC and Extended Length Packed-Columns SFC Method

Two chromatographic techniques are described for the separation and quantitative determination of azadirachtin A and B, salannin, and nimbin present in the crude extract of neem seed kernels and commercial neem based formulations. The high performance thin-layer chromatography (HPTLC) separation of markers was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using ethylacetate-benzene (7.0: 3.0, v/v) as mobile phase. The other technique was based on extended length packed column supercritical fluid chromatographic (PC-SFC) separation of the markers using Cyano column (250 mm × 4.6 mm I.D, S-5.0 μ) and Kromasil 100 NH2 column (250 mm × 4.6 mm I.D, 5.0 μ) connected in series. The detection was carried out using photodiode array detector at 338 K using methanol-modified carbon dioxide (10%) as the mobile phase at flow rate of 2.0 mL min−1. The current study assesses the effect of extending column length during PC-SFC experiment to obtain maximum resolution between a number of unknown components and known markers present in neem seed extracts. Both the chromatographic methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of quantitative determination of markers by means of HPTLC and PC-SFC method.

Application of TLC for Confirmation and Screening of Pesticide Residues in Fruits, Vegetables, and Cereal Grains: Part 2. Repeatability and Reproducibility of Rf and MDQ Values

This paper illustrates the effect of major factors influencing the reproducibility of thin layer chromatography (TLC) separation and detection under largely differing environmental and laboratory conditions. The optimum conditions for reproducibility and detection sensitivity was obtained on 20 × 20 cm layer in the retention factor (Rf) range of 0.2–0.7 by applying the sample in spots of 3–4 mm diameter at 2 cm from the edge of the plate. The reproducibility rapidly decreased below Rf = 0.2. Above Rf = 0.2 the within-laboratory reproducibility of 219 pesticides obtained in ethyl acetate silica gel elution system by several laboratories was typically below 10%. The among-laboratories reproducibility of the average retention factors was generally below 12%. The minimum detectable quantities (MDQ) of 219 pesticide residues were determined with nine detection methods. The MDQ values largely varied depending on the mode of detection. Bioassay methods enabled the detection down to 0.1–10 ng, while 20–100 ng could be achieved with the chemical reagents. Higher MDQ values are also reported in order to assist the identification of compounds potentially present. The between-laboratories reproducibility of MDQ values was typically 1–5*MDQmin.

Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin and bisdemethoxycurcumin

The objective of this work was to separate and determine the physico-chemical and color characteristics of isolated curcuminoid pigments. Thin-layer chromatographic separation of curcuminoid pigments was possible on silica gel 60G plates using dichloromethane:methanol, 99:1. The preparative separation of curcuminoid pigments was accomplished by crystallization of curcumin in methanol:water and further separation by column chromatography using silica gel 60G impregnated with sodium hydrogen phosphate and dichloromethane as the eluent. The purity of each curcuminoid pigment was confirmed by high performance liquid chromatography and determination of the melting point. The isolated pigments were characterized with respect to ultraviolet, visible, infrared, nuclear magnetic resonance and color characteristics. The molar absorptivity of each pigment was determined. This data can be used for the identification and quantification of individual curcuminoid pigments.

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.

A Remarkable Ring Contraction En Route to the Chartelline Alkaloids

General procedures. All reactions were carried out under an inert nitrogen atmosphere with dry solvents under anhydrous conditions unless otherwise stated. Dry tetrahydrofuran (THF), toluene, benzene, dichloromethane (CH2Cl2), methanol (MeOH), N,N-dimethylformamide (DMF), and triethylamine (Et3N) were obtained by passing these previously degassed solvents through activated alumina columns. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Copper(I) iodide was freshly purified from refluxing saturated aqueous NaI solution (aqueous). Yields refer to chromatographically and spectroscopically (H NMR) homogeneous materials, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as the visualizing agent and an acidic mixture of anisaldehyde or phosphomolybdic acid or basic aqueous potassium permangante (KMnO4) and heat as developing agents. E. Merck silica gel (60, particle size 0.043–0.063 mm) was used for flash column chromatography. Preparative thin layer chromatography (PTLC) separations were carried out on 0.25 or 0.5 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Bruker DRX-600, DRX-500, and AMX-400 or Varian Inova-400 instruments and calibrated using residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. High-resolution mass spectra (HRMS) were recorded on Agilent LC/MSD TOF time-of-flight mass spectrometer by electrospray ionization time of flight reflectron experiments. IR spectra were recorded on a Perkin Elmer Spectrum BX FTIR spectrometer. Melting points were recorded on a Fisher-Johns 12-144 melting point apparatus.

TLC separation of rare earths using di(2-ethylhexyl)dithiophosphoric acid as complexing reagent

The thin-layer chromatographic (TLC) separation of rare earths has been studied using di(2-ethylhexyl)dithiophosphoric acid as complexing agent in the mobile phase. Silica gel H and silica gel H impregnated with 2.5 m NH 4 NO 3 were investigated as stationary phases. The TLC behavior of rare earths on these adsorbents was compared using different mixed mobile phases. The best results were obtained by use of ethyl methyl ketone-tetrahydrofuran-di(2-ethylhexyl)dithiophosphoric acid (1 m ), 6.8 + 3.2 + 0.4 ( v/v ), as mobile phase. Double development was used to achieve better separation of consecutive rare earths.

Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods

Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM-DNA adducts were assayed by high pressure liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS/MS), TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), and (32)P-postlabeling with either thin layer ((32)P-P-TLC) or liquid chromatography ((32)P-P-HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using 'in house' TAM-DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM-DNA values were quantified using both 'in house' approaches as well as a newly synthesized [N-methyl-(3)H]TAM-DNA standard that was shared among all the participating groups. In the second study, the total TAM-DNA adduct values varied by 2-fold, while values for the dG-N(2)-TAM varied by 2.5-fold. Ratios of dG-N(2)-TAM:(E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-N(2)-N-desmethyl-TAM) in the second study were approximately 1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM-DNA adduct measurements among the diverse methods employed.

Stress degradation studies on etamsylate using stability-indicating chromatographic methods

Two sensitive and reproducible methods are described for the quantitative determination of etamsylate in the presence of its degradation products. The first method was based on high-performance liquid chromatographic (LC) separation of the drug from its degradation products on the reversed phase, kromasil column [C 18 (5-μm, 25 cm × 4.6 mm, i.d.)] at ambient temperature using a mobile phase consisting of methanol and water (50:50, v/v). Flow rate was 0.6 ml min −1 with an average operating pressure of 180 kg cm −2 and retention ( t R) time was found to be 2.93 ± 0.05 min. Quantitation was achieved with UV detection at 305 nm based on peak area with linear calibration curves at concentration range 10–100 μg ml −1. The second method was based on high-performance thin layer chromatographic (HPTLC) separation followed by densitometric measurement of spots at 305 nm. The separation was carried out on Merck HPTLC aluminium sheets of silica gel 60 F 254 using toluene:methanol:chloroform (8.0:4.5:6.0, v/v/v) as mobile phase. This system was found to give compact spots for etamsylate after double development (retention factor, R f value of 0.23 ± 0.02). The second order polynomial regression analysis data was used for the regression line in the range of 500–6000 ng spot −1. Both methods have been successively applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found. Both methods were validated in terms of precision, robustness, recovery and limits of detection and quantitation. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of etamsylate determination in dosage form by means of HPTLC and LC method. Drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment and photo-degradation. As the proposed methods could effectively separate the drug from its degradation products, they can be employed as stability indicating one. Moreover, the proposed LC method was utilized to investigate the kinetics of the acidic, alkaline and oxidative degradation processes at different temperatures and the apparent pseudo first order rate constant, half-life and activation energy was calculated. In addition the pH-rate profile of degradation of etamsylate in constant ionic strength buffer solutions with in the pH range 2–11 was studied.

Platelet Releasate: A Potential Treatment for Ocular Surface Disease?

In this brief overview various neurochemical isolation procedures that can be adopted for the analysis of several monoamine neurotransmitters/neuromodulators and their principal oxidatively deaminated metabolites are outlined. With respect to the trace amines, they can be identified and quantitated as their so-called dansyl derivatives after thin-layer chromatographic separation by mass spectrometric (MS) electron-impact (EI) ionisation followed by selected-ion monitoring (SIM) of their molecular ions. Deuterated homologues are added as internal standards at the start of the analytical procedure. The MS-EI-SIM procedure offers a tissue extract or releasate sensitivity of about 100 pg/g of tissue or fluid. In the case of tryptamine or phenylethylamine, by utilising different derivatives (N-acetylpentafluoropropionyl or N-acetylpentafluorobenzoyl), which cyclise to form perfluorinated spirocyclic compounds, it is possible using MS negative chemical ionisation techniques coupled with monitoring of the (M — HF) ions to achieve sensitivities for tissue extracts of 1 pg/g or less. Acidic and neutral metabolites (up to twelve of them can be assayed simultaneously) can be detected and quantitated in tissue extracts, releasates or biological fluids as their methyl-pentafluoropropionyl or trifluoroethyl-pentafluoropropionyl derivatives in the 100–1000 pg range using gas chromatographic-MS-SIM procedures.

Determination of pesticides in honey by ultrasonic solvent extraction and thin-layer chromatography

A rapid method for quantitative determination of atrazine and simazine in honey samples was investigated. The procedure was based on the extraction of pesticides by sonication with benzene:water = 1:1 (v/v) mixture, thin-layer chromatographic separation and quantification by CAMAG Video Documentation system in conjunction with the Reprostar 3. The extraction procedure was optimized with regard to the amount of solvent, duration of sonication and the number of extraction steps. The apparent recovery of pesticides from honey was 92.3 ± 2.4 for atrazine and 94.2 ± 2.8 for simazine, when they were extracted in three steps for 20 min using 20 ml of solvent. Ultrasonic solvent extraction was compared with traditional shake-flask extraction method.

Meiosis-activating sterol synthesis in rat preovulatory follicle: is it involved in resumption of meiosis?

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

Fast screening of low molecular weight compounds by thin-layer chromatography and "on-spot" MALDI-TOF mass spectrometry.

Fast screening of low-MW compounds is performed by thin-layer chromatography (TLC) followed by direct on-spot matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification with nearly "matrix-free" mass spectra using an UV-absorbing ionic liquid matrix. Owing to minimal background ions from the proton donor triethylamine/alpha-cyano-4-hydroxycinnamic acid ionic liquid matrix, three arborescidine alkaloids, the anesthesics levobupivacaine and mepivacaine, and the antibiotic tetracycline were readily characterized most frequently by the MS detection of their protonated molecules. The technique is fast and sensitive, requires little sample preparation and manipulation, and is therefore suitable for fast screening with TLC separation and MS identification of low-MW compounds, with potential applications in areas such as phytochemistry, synthetic chemistry, and product manufacturing quality monitoring.

Determination of gallic acid after thin-layer chromatographic and paper chromatographic separations

This paper describes rapid and cost-effective analytical procedures for determination of gallic acid after chromatographic separation. Optimum conditions were established for separation and assay of gallic acid, salicylic acid, and tannic acid by thin-layer and paper chromatography (PC). Detection was by means of a sensitive spectrophotometric method based on the coupled redox-complexation reactions occurring in the system Fe(III), gallic acid, and 2,2′-dipyridyl ( λ max = 522 nm). The procedure was then applied to the determination of gallic acid in a pharmaceutical preparation. Thin layer chromatographic analysis with densitometric UV detection at λ = 280 nm gave comparable results.

Suppression of 15-hydroxyprostaglandin dehydrogenase messenger RNA concentration, protein expression, and enzymatic activity during human ureteral obstruction.

Prostanoids produce significant effects in the ureter, particularly in response to obstruction. Ureteral obstruction is associated with increased prostanoid synthesis via cyclooxygenase induction; however, prostaglandin degradation mediated by 15-hydroxyprostaglandin dehydrogenase (PGDH) has not been evaluated in the ureter. The purpose of this study was to determine whether PGDH steady-state mRNA, protein, and enzyme activity are altered in the human ureter during obstruction. Human ureteral segments from patients undergoing donor nephrectomy (normal segments) or ureteral stricture repair (obstructed segments) were obtained with proper informed consent. We evaluated PGDH steady-state mRNA relative to ribosomal protein S26 reference gene by reverse transcription-polymerase chain reaction and Vistra Green fluoroimaging. We determined PGDH protein content relative to glyceraldehyde-3-phosphate dehydrogenase by immunoblotting and PGDH localization by immunohistochemistry. PGDH enzymatic activity was determined by measurement of conversion of 15-hydroxy- to 15-keto-prostaglandin using thin layer chromatography separation. We found that PGDH mRNA and protein were decreased 4- to 6-fold, and enzyme activity was decreased >3-fold in obstructed human ureter relative to normal controls. PGDH was localized to the urothelial cells, with little or no expression in smooth muscle. Our results indicate that PGDH mRNA, protein, and enzyme activity are suppressed in the human ureter during obstruction. Increased concentrations of prostanoids subsequent to ureteral obstruction seem to be due to decreased degradation as well as increased synthesis. Modulation of prostanoid degradation may have therapeutic relevance in obstructive disorders of the ureter.

Suppression of deleterious effects of free silanols in liquid chromatography by imidazolium tetrafluoroborate ionic liquids

Silica-based stationary phases are commonly used in liquid chromatography, but their surface acidity causes known problems, especially when separating basic compounds. Deleterious effects of free silanols are not fully removed by standard prevention procedures consisting in adding alkylamines or other amino quenchers to the eluents. We found that ionic liquids of the imidazolium tetrafluoroborate class, added to mobile phases at concentrations of 0.5–1.5% (v/v), blocked silanols and provided excellent thin-layer chromatographic separations of strongly basic drugs which were otherwise not eluted, even with neat acetonitrile as the mobile phase. The silanol suppressing potency of imidazolium tetrafluoroborates was demonstrated to markedly exceed that of the standard mobile phase additives, like triethylamine, dimethyloctylamine and ammonia. The proposed new mobile phase additives were also demonstrated to provide reliable lipophilicity parameters of base drug analytes as determined by gradient mode of high-performance liquid chromatography. By applying the readily available and environmentally friendly imidazolium tetrafluoroborate ionic liquids, simple and efficient means of improvement of liquid chromatographic analysis of organic bases were elaborated.

Indole derivatives produced by the fungus Colletotrichum acutatum causing lime anthracnose and postbloom fruit drop of citrus

Postbloom fruit drop (PFD) of citrus and Key lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.

On-line overpressure thin-layer chromatographic separation and electrospray mass spectrometric detection of glycolipids.

On-line thin-layer chromatographic separation and electrospray mass spectrometry (TLC/ESI-MS) has been accomplished by direct linking of a commercial overpressure TLC instrument, OPLC 50, and a Q-TOF mass spectrometer. Mass spectrometric detection sensitivity and chromatographic resolution achieved by this configuration were assessed using acidic glycolipids as examples. Under the optimized conditions, a sensitivity of 5 pmol of glycosphingolipid was readily demonstrated for TLC/ESI-MS and 20 pmol for TLC/ESI-MS/MS production scanning to derive the saccharide sequence and long chain base/fatty acid composition of the ceramide. Initial preconditioning of TLC plates is necessary to achieve high sensitivity detection by reducing chemical background noise. Plates can be used repeatedly (at least 10 times) for analysis, although this may result in a minor reduction in TLC resolution. Following solvent development, separated components on the TLC plates can be detected in the conventional way by nondestructive staining or UV absorption or fluorescence and can be stored for on-line TLC/ESI-MS analysis at a later stage without reduction in mass spectrometric detection sensitivity and chromatographic resolution. Aspects for further improvement of OPLC instrumentation include use of narrower TLC plate dimensions and refined design of the eluate exit system.

Thin-layer chromatography of aromatic amines with hybrid CTAB–alcohol–water mobile phase: separation of indole from diphenylamine and p-dimethylaminobenzaldehyde

Cationic and non-ionic surfactant-mediated systems have been used as mobile phases in thin-layer chromatographic separation of aromatic amines on silica gel layers. The effect of surfactant concentration below and above its critical micellar concentration on mobility of amines was examined. The influence of organic and inorganic additives such as alcohols, urea, NaCl and NaBr in micellar solutions on the mobility and separation efficiency of amines was also assessed. A mobile phase composed of 99.5:0.5 v/v water and (CTAB + methanol, 1:2 w/v) was identified as the best solvent system for rapid separation of indole from p-DAB and DPA in the presence of cations and phenols. The semiquantitative determination of p-anisidine by spot-area measurement method was also attempted. The lower limits of detection of amines have been determined.