Ultrathin-Layer Chromatography (UTLC)

Ultrathin-layer chromatography (UTLC) differs from high-performance thin-layer chromatography (HPTLC) and from thin-layer chromatography (TLC) in two basis things: the layer thickness, and the migration distances of the analytes. UTLC has a monolithic or a nanostructured stationary silica gel phase bound directly to the glass plates. Layer thickness in UTLC is 10 μm, instead of 100–250 μm in HPTLC. Migration distances are in the range of 1–3 cm for UTLC, instead of 8–10 cm for HPTLC. Therefore, the major advantages of UTLC over HPTLC and TLC are the shorter development times and higher separation efficiency and sensitivity. Moreover, separations on UTLC plates require smaller reagent and sample volumes. However, the UTLC plates are very difficult to manage with the TLC and HPTLC equipment currently available. Therefore, the next challenge in this area is the development of an inexpensive solution with appropriate instrumentation (sensitive optical scanners and sample application systems). UTLC had been used for separations of many compounds, e.g., pharmaceutically active ingredients, pesticides, plasticisers, natural products, and other chemical substances.     

New method for caffeine quantification by planar chromatography coupled with electropray ionization mass spectrometry using stable isotope dilution analysis

A new high-performance thin-layer chromatography/electrospray ionization mass spectrometry (HPTLC/ESI-MS) method for the quantification of caffeine in pharmaceutical and energy drink samples was developed using stable isotope dilution analysis (SIDA). After sample preparation, samples and caffeine standard were applied on silica gel 60 F254 HPTLC plates and over-spotted with caffeine-d3 used for correction of the plunger positioning. After chromatography, densitometric detection was performed by UV absorption at 274 nm. The bands were then eluted by means of a plunger-based extractor into the ESI interface of a single-quadrupole mass spectrometer. For quantification by MS the [M+H]+ ions of caffeine and caffeine-d3 were recorded in the positive ion single ion monitoring (SIM) mode at m/z 195 and 198, respectively. The calibration showed a linear regression with a determination coefficient (R2) of 0.9998. The repeatability (RSD, n=6) in matrix was相似文献   

On-line high-performance liquid chromatography for monitoring fermentation processes for penicillin production

The control and regulation of a fermentation process requires accurate and reliable on-line measurements of media components, often over long periods of time. As an alternative to wet chemical and enzymatic methods, high-performance liquid chromatography (h.p.l.c.) can be used. In addition to the product, penicillin V, important by-products and degradation products, as well as the precursor, phenoxyacetic acid, are determined. This makes it possible to control the precursor feed and to establish the optimal harvest time. Moreover, contamination can be detected at an early stage. Control of the precursor feed is desirable because the phenoxyacetic acid represents about 10% of the production costs and because higher concentrations could be toxic. The sterile sample flow is removed from the reactor by a microfiltration probe and drawn through the injection loop of an h.p.l.c. valve. Three analyses per hour can be done, and selected data from a computing integrator are fed to a process computer. The system works reliably for 300 h without requiring further calibration.     

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.     

Determination of 2-propanol in surface cleaning solutions used for copper continuous casting process by flow injection-spectrophotometric detection with on-line column separation.

A flow-injection system has been developed for the determination of 2-propanol in the surface cleaning solutions used in the copper continuous cast rod making system. Adsorption chromatography in nitric acid medium was used for the on-line separation of oily substances in the sample solution. Cerium(IV) diammonium nitrate was utilized as the chromogenic reagent for the spectrophotometric detection of 2-propanol. The system permits a throughput of one sample per hour for the oily sample, and of 12 samples per hour for the none-oily sample. The reproducibility has been proven to be satisfactory with a relative standard deviation of less than 6.0% (2.2%(V/V) 2-propanol level, n = 23). The detection limit is 0.01% (V/V).     

Effect of Temperature on HPLC Separations of Penicillins

Abstract

Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate in the same sample the following penicillins: amoxicillin, ampicillin, piperacillin, penicillin G, penicillin V and cloxacillin. After the chromatographic separation in isocratic elution conditions of the penicillins at ambient temperature, the effect of the column temperature on resolution was analyzed. The capacity factor of each penicillin was observed to increase with decreasing temperature, a linear relationship was obtained for a plot of ln k' versus 1/T. The results also showed changes in resolution between adjacent peaks being associated with differences in the selectivity factor (α).     

Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol,chloramphenicol, furosemide,glibenclamide, penicillin V potassium,and praziquantel

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods was performed following a previously published model process. The developed and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, relatively nontoxic, readily available solvents and other reagents; silica gel 60?F₂₅₄ plates; automated bandwise sample and standard solution application; ascending mobile phase development of plates in a chamber; and automated slit scanning densitometry for detection, identification, and quantification. Validation data for methods developed in an early version of the transfer model process that did not include standard addition validation are reported for pharmaceutical products containing amitriptyline HCl, amodiaquine, diphenhydramine HCl, and mebendazole.     

Combination of different liquid chromatography/mass spectrometry technologies for the identification of transformation products of rhodamine B in groundwater

Rhodamine B and its five de‐ethylated transformation products could be identified in a groundwater sample. Using high‐performance thin‐layer chromatography (HPTLC) six fluorescent zones were detected in the sample. In order to identify the compounds in the zones by exact mass mass spectrometry (MS) measurements and tandem mass spectrometry (MS/MS), they were extracted from the HPTLC plate for subsequent analysis by nano‐chip high‐performance liquid chromatography quadrupole‐time‐of‐flight mass spectrometry (nano‐chip HPLC/QTOFMS). In addition, chemical derivatisation experiments on HPTLC plates were applied to detect the presence of a primary amino group in the transformation products. From the combined analytical results it was possible to allocate rhodamine B and its five de‐ethylated transformation products to the six different HPTLC zones. The quantification of rhodamine B in different groundwater samples was carried out by a high‐performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/MS/MS). The maximum detected concentration of rhodamine B was 83 µg L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of flavonol aglycones and terpenelactones in Ginkgo biloba extract: A comparison of high-performance thin-layer chromatography and column high-performance liquid chromatography

Advancements in automated high-performance thin-layer chromatography (HPTLC) have made it feasible to assess its use for the quantitative analysis of marker compounds in botanical preparations. We report here the findings of method comparisons for the terpenelactones and flavonol aglycones by column high-performance liquid chromatography (HPLC) with evaporative light scattering and UV detection, and HPTLC with a scanning densitometer. For the HPTLC assay of terpenelactones, total bilobalide, ginkgolide A, and ginkgolide B consistently achieved <70% of the total determined using HPLC, regardless of variations to postchromatographic derivatization time and temperature. Accuracy testing showed the possibility of a matrix interference. In contrast, a good relationship (95%) was determined between HPTLC and HPLC for determination of total flavonol glycosides (calculated from combined quercetin, kaempferol, and isorhamnetin) from an acid-hydrolyzed Ginkgo biloba L. (GBE) sample. The HPTLC flavonol aglycone method also performed well in terms of accuracy (overall average of 96% recovery for the 3 aglycones) and consecutive plate repeatability (overall percent relative standard deviation of 4.4). It is demonstrated that HPTLC can be a time-saving complement to HPLC for routine analysis of the flavonol glycosides in GBE.     

Determination of Benzylpenicillin In Pharmaceuticals By Capillary Zone Electrophoresis

Abstract

A rapid and direct method is described for the determination of benzylpenicillin (penicillin G) in pharmaceutical preparations. the method involves very little sample preparation and total analysis time for duplicate results is less than 30 minutes per sample.

The method takes advantage of the speed and separating power of capillary zone electrophoresis (CZE). Detection of penicillin is by absorption at 228 nm. an internal standard is employed to reduce sample injection error. the method was applied successfully to both tablets and injectable preparations.

larger on longer aging. It was the authors' hypothesis that this peak was due to formation of penicilloate. When this degradation product was synthesized according to an established procedure¹⁴, CZE retention time of the synthesized product was the same as that final peak in the aged penicillin samples. UV spectra on both solutions was further evidence we had properly identified the degradation product. Thus the relative amount of active and degraded compound in a sample can be estimated readily. Also, this indicates fresh solutions should be used for analytical work.

In conclusion, CZE of penicillin G was shown to not only be possible, but useful in a practical, rapid assay for the level of the antibiotic in pharmaceutical samples. Extension of this work to other types of penicillin and to the variety of samples in which the antibiotic is found in the next logical step.     

Therapeutic drug monitoring by high performance thin-layer chromatography

Summary Methods for measuring concentrations of therapeutic drugs in blood serum have been devised which involve separation of the drugs by high-performance thin-layer chromatography (HPTLC) on silica gel followed by quantification by reflectance or fluorescence scanning. Aliquots of from 20 to 300 L serum are extracted with organic solvent. Portions of the extract containing approximately 1/4 of the drug present are deposited on a Contact Spotter (Clarke Analytical Systems), concentrated to a pin-point and applied to the HPTLC plate which is then developed and scanned.HPTLC permitted 12 samples and three standard solutions of drugs used to treat cardiac arrhythmias to be assyed for 6 drugs simultaneously, with scanning at a single wavelength. Results of these assays correlated well with those by EMIT^® immunoassays and by HPLC. No interference by other common drugs was observed. The total time required was two hours, making the work output esiily competitive with that of moderately rapid HPLC. Since sample preparation and quantification by scanning required only a small portion of this time, and the chromatography, which was time consuming, could be done on all the samples simultaneously, analysis of twice as many samples requires only an additional 1/2 hour. HPTLC thus offered the possibility of highly efficient mass production with relatively little capital equipment. Assay of chloramphenicol in serum, which in our laboratory requires fairly rapid turnaround of nnly a few samples at one time, required a similar length of time. Since standards and quality assurance samples could be assayed simultaneously, the analytical output was again comparable to that from a single HPLC instrument.Dedicated to Professor A. Zlatkis on the occasion of his 60th birthday.     

Screening for Antimicrobials in Mouthwashes Using HPTLC-Bioluminescence Detection

One of the tasks of food law enforcement authorities is to supervise the composition of cosmetics. In the case of mouthwashes, they are likely to contain (labeled or unlabeled) antimicrobial compounds. Conventional analyses, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) only shed light on a compound’s structure, but not on its biological function. In this study, we demonstrate that the task of detecting antimicrobials in mouthwashes can be streamlined using the luminescent bacterium Vibrio fischeri as a biodetector coupled with high-performance thin-layer chromatography (HPTLC) as a pre-separation method. The employment of subsequent conventional techniques could then be restricted to fractions with proven V. fischeri toxicity. Samples were separated in parallel on silica gel and amino layer HPTLC plates, developed with a solvent system containing tertiary butyl methyl ether and n-hexane and dried on a plate heater. After applying V. fischeri onto the HPTLC plate, zones of interest were extracted from a parallel plate and identified by HPLC–UV or GC-mass spectrometry. The reaction of V. fischeri to more than 40 standard substances which might be present in mouthwashes was determined. Based on this information, six commercially available mouthwashes were analyzed. The workflow proved to be viable for an effect-directed screening for antimicrobial compounds. The analysis of mouthwashes revealed that not only declared preservatives are used (sodium benzoate, cetylpyridinium chloride) but also other compounds, especially constituents of essential oils. Because their main purpose is flavoring of the mouthwash, they are summarized as “aroma” (anethole, carvone, menthol, thymol) which is in compliance with legal restrictions.     

Anodic Polarographic Behaviour of Hydrolyzed Penicillin V

Abstract

The electroactivity of a degradation product from penicillin V is described. This electroactive product is formed by acidic hydrolysis at pH 4.0 and heating at 90°C for 60 minutes.

This derivative has not been identified, but would seem to contain a thiol group. It gives a diffusion controlled anodic polarographic wave with a peak potential of -0.204 V versus SCE at pH 4.0.

The developed method has been applied to the analysis of penicillin V dosage forms and a recovery of 100.8 has been obtained.     

A simplified HPTLC screening method for the estimation of the PAH content in soil samples

Based on a highly significant correlation between the visual fluorescing fraction of PAH on the one hand and the total EPA-PAH₁₆ content in mineral soils on the other hand, a deliberately incomplete RP-TLC separation of these compounds into a few fingerprint-like compressed bands within a determined “PAH window” has been achieved. The resulting band-pattern does not interfere normally with the more or less non-polar phenolic compounds which are associated with natural soil humic substances. The extraction step has been extremely simplified with regard to the quantity of the soil sample and of the extractant. The accuracy of this procedure has been ascertained by means of recovery experiments with an artificial soil enriched with PAH. A single spot application mode and an evaluation scheme allow the estimation of EPA-PAH₁₆ contents of soil samples in relation to threshold values ¶(1 or 5 or 10 mg/kg). This HPTLC screening method has been compared against standard HPLC methods. The simplified extraction step and the separation by HPTLC minimizes the actual costs and the time spent per sample.     

Accurate and Reproducible Determination of Molecular Weight Distribution of Sodium Heparin U.S.P. By HPLC

Abstract

Accurate molecular weight averages and molecular weight distributions have been determined for bulk Sodium Heparin, USP. The molecular weight averages were approximately 10,000 daltons (one dalton = one molecular weight unit) and ranged from 3,000 to 17,000 daltons in a given sample. The method uses high pressure liquid chromatography (HPLC) with 5–10 micron particle size controlled porous glass of different pore diameters (40 Å, 100 Å, and 250 Å) and simultaneous detection with a differential refractometer and ultraviolet detector (254 nm). The columns were calibrated with different molecular weight fractions of Sodium Heparin and baseline resolution obtained between 6.5K daltons differences in molecular weight. Analysis time was 25 minutes per sample and the method gave excellent reproducibility for calculated molecular weight averages in a repeated series of analysis. It was determined that the column doing most of the separation in the set was the one packed with 250 Å pore diameter size material.     

Determination of Ticarcillin in Serum by Reversed Phase High Performance Liquid Chromatography

Abstract

Ticarcillin is a semi-synthetic penicillin useful against several Pseudomonas species. In order to easily quantitate this drug, a new procedure was developed whereby ticarcillin in serum is converted to its free acid form by the addition of citric acid and, subsequently, extracted into ethyl acetate. The organic extract which contains the nonionized form of ticarcillin is dried under nitrogen, the sample is reconstituted with mobile phase and analyzed by high performance liquid chromatography. Elution is completed in less than five minutes. The assay is linear from 1 mg/L through 100 mg/L. The correlation coefficient of ticarcillin concentration to peak area (r) was 0. 999 over this concentration range. The small sample volume (100 yl) makes this assay particularly suitable for pediatric patients.     

Thin Layer Chromatographic Method for Rapid Quantification and Identification of Trimethoprim and Sulfamethoxazole in Pharmaceutical Dosage Forms

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of sulfamethoxazole (SMA) and trimethoprim (TMP) present in combined dosage forms were described. SMA and TMP were extracted with 90% acqueous methanol and the interfering and related contaminants were removed by thin layer chromatography (TLC) or high performance TLC (HPTLC) on silicagel plates using chloroform : isopropanol : diethylamine :: 10 : 6 : 1 (v/v) as mobile phase. Assay was done at the respective absorption maxima of the drugs by in situ densitometry and by spectroscopy after extracting the drugs from TLC plates with 90% acqueous ethanol. Results obtained by both the methods agreed well with those obtained by the method prescribed by the United States Pharmacopoeia XXI edition. Total time required for HPTLC and densitometric assay of 32 samples using 4 standards was 30 min. Probable source of errors in densitometric studies and their rectification was discussed.     

A simplified HPTLC screening method for the estimation of the PAH content in soil samples

Based on a highly significant correlation between the visual fluorescing fraction of PAH on the one hand and the total EPA-PAH16 content in mineral soils on the other hand, a deliberately incomplete RP-TLC separation of these compounds into a few fingerprint-like compressed bands within a determined "PAH window" has been achieved. The resulting band-pattern does not interfere normally with the more or less non-polar phenolic compounds which are associated with natural soil humic substances. The extraction step has been extremely simplified with regard to the quantity of the soil sample and of the extractant. The accuracy of this procedure has been ascertained by means of recovery experiments with an artificial soil enriched with PAH. A single spot application mode and an evaluation scheme allow the estimation of EPA-PAH16 contents of soil samples in relation to threshold values (1 or 5 or 10 mg/kg). This HPTLC screening method has been compared against standard HPLC methods. The simplified extraction step and the separation by HPTLC minimizes the actual costs and the time spent per sample.     

HPTLC and ATR/FTIR Characterization of Antioxidants in Different Rosemary Extracts

The effect of spontaneous fermentation by lactic acid bacteria on the extraction yield of bioactive compounds and antioxidant activity from rosemary leaf extracts was investigated using high-performance thin-layer chromatography (HPTLC). Brining and spontaneous fermentation with lactic acid bacteria more than doubled extraction of polyphenolics and antioxidants from the rosemary leaves. The results show that lactic acid fermentation enhances antioxidant activity in extracts by increasing the total phenolic content but does not increase extraction of phytosterols. Increased extraction of phenolic oxidants during fermentation assisted extraction, results from the in situ generated natural eutectic solvent from the plant sample. ATR-FTIR spectra from the bioactive bands suggests that this increased antioxidant activity is associated with increased extraction of rosmarinic acid, depolymerised lignin, abietane diterpenoids and 15-hydroxy-7-oxodehydroabietic acid.     

Liquid Chromatographic Determination of Azide as the 3,5-Dinitrobenzoyl Derivative

Abstract

A method for the determination of azide after conversion to 3,5-dinitrobenzoyl azide has been developed. The derivatization reaction is fast (3 min.), quantitative, and yields a product with strong ultraviolet absorption. The derivatization reaction mixture is separated by high performance liquid chromatography so that the azide derivative can be easily quantitated. The detection limit of the method is 10 ng NaN₃/mL. The total analysis time is 20 minutes per sample.