Thin-layer chromatography with densitometric detection has been used to monitor different compounds produced by condensation of 4-methoxybenzaldehyde, 4-methylbenzaldehyde, and pyrrole in the ratios 1:3:4, 2:2:4, and 3:1:4. Qualitative results for the products obtained were converted into quantitative results by use of densitometry. The effect of changing the ratio of components on the yield of five porphyrin products was followed.
High-performance thin-layer chromatography (HPTLC) has been used for normal-phase separation of the components of hexane, chloroform, methanol, and water extracts of
roots. Computerized densitometry was used for two-dimensional spectrographic image analysis of the HPTLC plates. High performance liquid chromatography (HPLC) was also used for reversed-phase separation of these extracts. Different chromatograms of
root extracts, obtained by use of these techniques, revealed the presence of three marker indole alkaloids, ajmaline, ajmalicine, and reserpine, in all four extracts. Use of chloroform resulted in most efficient extraction of these three alkaloids. The results also showed that defatting with hexane may result in loss of the alkaloids.
Authors:Anna Gumieniczek, Tadeusz Inglot and Anna Kończak
Classical densitometry and videoscanning were compared for a new TLC method of quantitative analysis of two AT1 receptor antagonists, candesartan and losartan, in pharmaceuticals. Chromatography was performed on silica gel with 1,4-dioxane-hexane-99% formic acid 5:5:0.1 (ν/ν) as mobile phase. Classical densitometry was performed at the wavelengths of maximum absorption of candesartan (258 nm) and losartan (243 nm) whereas videoscanning was performed at 254 nm for both drugs. Compact spots were obtained for candesartan (RF 0.47 ± 0.01, mean ± SD) and losartan (RF 0.35 ± 0.01). Calibration plots were constructed in the range 0.2–1.4 μg per band for the both drugs and were linear with good correlation coefficients — 0.9997 and 0.9981 for candesartan, and 0.9986 and 0.9982 for losartan, for densitometry and videoscanning, respectively. The methods were validated for robustness, precision, accuracy, and specificity in relation to excipients present in the respective formulations. Finally, the methods were compared statistically in respect of robustness, precision and accuracy.
Salicylic acid and its derivatives, such as acetylsalicylic acid, are commonly used non-steroidal anti-inflammatory agents. In the present work, different sorbents as the stationary phases and also various manners of detection using well known and new visualizing reagents have been tested for the separation and detection of the two compounds by adsorption and also partition thin-layer chromatography (TLC) with densitometry. Densitometric and spectrodensitometric analysis was used to evaluate the detectability of the examined compounds using the newly developed TLC procedures. Of all the applied manners of detection, the most universal to distinguish acetylsalicylic acid from salicylic acid in normal- as well as in reversed-phase TLC system is the use of methanolic solution of FeCl3 and CoCl2. The densitograms obtained under these conditions show symmetric and also by adsorption well separated TLC peaks of both compounds. The shape of all absorption bands of acetylsalicylic acid (ASA) and 2-hydroxybenzoic acid (SA) recorded using a scanning densitometer in the range of 200–700 nm is regular. In the case of adsorption TLC performed on chromatographic plates precoated with silica gel 60 F254 and silica gel 60, a very effective spray reagent was also Janus blue. The satisfactory results of detection on silica gel 60 plates assure the use of 1% NaOH as spray reagent followed by heating at 90°C for 60 min. The results performed in the present work confirm the utility of TLC in combination with densitometry in the qualitative screening and identification of SA and ASA. The most efficient manners of detection described in this work can be helpful in the quality control of acetylsalicylic acid, e.g., in monitoring the synthesis reaction of acetylsalicylic acid from salicylic acid as well as in the quality control of ASA in commercially available products.
Authors:Aradhana Sharma, Kumar Gaurav, Ram Singh and Richa Srivastava
Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and Cosmetics Act of India. Its usefulness has been stated in erectile dysfunction, impotence, and premature ejaculation. Opium is the major ingredient of formulation which is highly addictive as it contains narcotic alkaloids (morphine, codeine, and thebaine). Opium is a natural product; hence, the morphine content varies from 4–21%. The aim of this study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography— densitometry method for the quantitative estimation of morphine in the tablets of the said Ayurvedic medicines. Aluminum-backed silica gel F254 (20 cm × 10 cm) was used as the stationary phase and ethyl acetate, methanol, and ammonia (85:10:5, v/v) as the mobile phase. The RF value for morphine was 0.36, and the quantitative evaluation of the bands over plates was performed in the reflectance—absorbance mode at 280 nm. The regression analysis of the calibration plot showed good linear relationship between peak area vs. morphine concentration. Linearity was found in the range of 400 to 1200 ng per band, and the amount of morphine was estimated by comparing the peak area of the standard morphine.
Authors:Alexandra Hunsberger, Bernard Fried and Joseph Sherma
Silica gel high-performance thin-layer chromatography (HPTLC) was used to study the effects of both Schistosoma mansoni infection and high temperatures on the neutral and polar lipid content of whole bodies of Biomphalaria glabrata snails. Neutral lipids were determined using petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase, phosphomolybdic acid detection reagent, densitometry at 610 nm, and polar lipids with chloroform-methanol-water (65:25:4) mobile phase, cupric sulfate-phosphoric acid reagent, and scanning at 370 nm. The high-temperature experiments were done at ambient (22–24°C), 28°C, and 34°C. Snails were maintained at these temperatures for 7 days prior to necropsy. Extracts of their bodies were then analyzed by HPTLC to determine changes that occurred in the lipid content as a function of temperature and to compare unexposed to exposed cultures at each temperature. At 4 weeks postinfection (PI), the 34°C exposed snails had significantly lower amounts of free sterols than the unexposed culture. At 4 weeks PI, the 34°C exposed snails also had significantly lower amounts of free sterols than the ambient and 28°C exposed snails. At 6 weeks PI, ambient exposed snails had significantly lower free fatty acids and significantly higher phosphatidylcholine than unexposed snails. The 28°C exposed snails had significantly lower amounts of free sterols and phosphatidylethanolamine than the unexposed snails. The 28°C exposed snails also had significantly higher amounts of free sterols, triacylglycerols, and phosphatidylcholine than the ambient snails and significantly lower amounts of free fatty acids than the ambient temperature snails. The ambient exposed snails had significantly lower amounts of free sterols than the 28°C and 34°C snails. The 34°C exposed snails had significantly lower amounts of triacylglycerols than the ambient temperature and 28°C exposed snails. At 8 weeks PI, the 28°C exposed snails had significantly higher amounts of phosphatidylcholine than the unexposed snails. These findings suggest that high temperature and S. mansoni infection had individual and combined deleterious effects on the lipid metabolism of the snails.
A sensitive, selective, precise, and stability-indicating high-performance thin-layer chromatographic method coupled with densitometric analysis has been developed for determination of nitrendipine both as the bulk drug and in pharmaceutical dosage forms. The active substance was extracted from tablets with methanol (recovery from 97 to 105%) and chromatographed on silica gel 60 F
HPTLC plates in horizontal chambers with
-hexane-ethyl acetate-acetone, 6 + 3 + 2 (
), as mobile phase. UV densitometric analysis of nitrendipine was performed in absorbance mode at
= 335 nm. The calibration plot was constructed in the range 0.025 to 0.150 μg μL
(corresponding to 0.5–3.0 μg spot
) with good correlation (
≥ 0.990) and expressed as a second-order calibration function. Determination of nitrendipine in tablets was characterized by good precision (1.94% <
< 6.62%) and accuracy (−3.0 <
< 5.12). The HPTLC-densitometric method was successfully used for identification of nitrendipine in the presence of its induced degradation products. Pure drug and tablet extract were subjected to acid and alkali hydrolysis, oxidation, UV degradation, and photodegradation. The degraded products were well resolved from the nitrendipine with different
A chromatographic-densitometric method has been established for identification and quantitation of selected beta-adrenergic-blocking agents in pharmaceutical preparations. Retention factors,
, and characteristic absorption spectra of 11 drugs chromatographed on silica gel 60 F
HPTLC plates with six mobile phases were used for identification. Quantitation and validation of the method was performed for atenolol, acebutolol, propranolol, and bisoprolol using chloroform-methanol-ammonia, 15 + 7 + 0.2 (
), as mobile phase. UV densitometric measurements were performed at the wavelength of maximum absorption. Pharmaceutical preparations used in medicine and from a variety of manufacturers were analyzed and the method shown to be sufficiently sensitive for analysis of these samples. The limits of detection and determination ranged from 30 to 400 ng and recovery was from 97.14 to 102.18%. The precision of the method, described by the equation
, is good and the range of linearity is wide — from 0.020 to 0.250% for individual constituents.
Authors:Krystyna Skalicka-Woźniak, Michał Hajnos and Kazimierz Głowniak
High-performance thin-layer chromatography with densitometric detection has been used for quantitative analysis of chlorogenic acid in extracts obtained by different methods from the fruits of
. Separation was achieved on silica gel HPTLC plates with ethyl acetate-formic acid-acetic acid-water 100:11:11:21 as mobile phase. Densitometric detection was performed before and after derivatization with Naturstoffreagenz A at 320 nm. Calibration plots were linear in the range 10–75 μg mL
. The results obtained were very similar to those from HPLC, so this densitometric method can be easily used for easy and economic quantitative analysis of active compounds in plant material.