Use of TLC and UV–Visible Spectrometry for Fingerprinting of Dietary Supplements

We have studied dietary supplements containing sea-buckthorn (Hippophae rhamnoides), bilberry (Vaccinium myrtillus), and cranberry (Vaccinium vitis-idaea) by use of thin-layer chromatography (TLC), image analysis, and UV–visible spectroscopy. The results were subjected to multivariate techniques, for example cluster analysis (CA), principal-components analysis (PCA), and linear discriminant analysis (LDA). Evaluation of the discriminating power of TLC coupled with digital image analysis for characterization of the supplements was performed by use of chemometrics. Zero-order, first-order, and normalized zero-order UV–visible spectra of extracts of fruit and dietary supplements were digitized and used in the chemometric analysis. To increase the relevance of the study, the dietary supplement extracts were compared with extracts of the natural fruit. Results obtained by application of CA, PCA, and PCA–LDA to the chromatograms suggest the possibility of discrimination among dietary supplements on the basis of the raw material used for their preparation.

     

Application of image analysis technique for the determination of thiophanate methyl by thin-layer chromatography

A simple and convenient thin-layer chromatography (TLC) method combined with image analysis technique was developed to determine thiophanate methyl. The detection of pesticide was based on iodine–azide reaction. Digital images of TLC plate chromatograms were analysed using TLSee software, and quantitative analysis was conducted. The linearity (0.3–3.0 µg per spot), sensitivity, accuracy and precision of the system were investigated.     

Use of SPE—TLC for Quality Control of Rhodiola rosea Extracts

SPE and TLC have been used for qualitative and quantitative analysis of salidroside, rosavin, rosarin, and rosin in commercially available dry extracts from Rhodiola rosea roots. The best separation of all the compounds was achieved on silica gel TLC plates with ethyl acetate—methanol—water, 77 + 13 + 10 (v/v), as mobile phase. UV detection was performed at λ = 215 nm for salidroside and at λ= 245 nm for the rosavins (rosavin, rosarin, and rosin). Detection limits for salidroside and the rosavins were 90 ng and 60 ng per spot, respectively. Results from quantitative analysis confirmed the manufacturer’s declaration of the amounts of salidroside and the rosavins in the extracts.

     

Application of thin-layer chromatography image analysis technique in quantitative determination of sphingomyelin

A sensitive and convenient method for sphingomyelin determination was developed based on thinlayer chromatography (TLC) and image-processing analysis. The mobile phase composition, detection and quantification conditions were systematically investigated through several trials. The molybdenum blue reaction allowed specific detection of the phospholipid with a high sensitivity and a wide linear range. Digital images of TLC plate chromatograms were captured with flatbed scanner and converted into peak chromatograms using TLSee® software and quantitative analysis was conducted. The linearity, sensitivity, accuracy and precision of the system were evaluated. The limits of detection and quantification were 0.5 and 1.7 μg/spot, respectively. Separation of the mixture consisting of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine was also carried out.     

Validated TLC-Image Analysis Method for Simultaneous Quantification of Curcuminoids in Curcuma longa

A simple and rapid thin layer chromatographic (TLC)-image analysis method was developed for simultaneous quantification of three curcuminoids; curcumin (CUR), desmethoxycurcumin (DES) and bisdesmethoxycurcumin (BIS), in Curcuma longa (turmeric). Chromatographic separation of the curcuminoids was achieved on silica gel 60 F254 TLC plates, using chloroform–hexane–methanol (1:1:0.1, v/v/v) as the mobile phase. Image analysis of the scanned TLC plate was performed by Photoshop 7.0 to quantify the amount of each curcuminoid. The method was validated and found to be accurate, reliable and convenient for the analysis of CUR, DES and BIS in turmeric.

     

Detection reagents used for on-plate identification of organic pesticides in biological samples with preliminary separation by TLC/HPTLC

Many specific and non-specific chromogenic spray reagents have been used to detect organic pesticides (organophosphorus, organochlorine, carbamates, and pyrethroids) on thin-layer chromatographic (TLC)/high-performance thin-layer chromatographic (HPTLC) plates. To realize high sensitivity and improved selectivity, several chromogenic reagents have been introduced. The physical properties of organic pesticides reported so far are also presented in tabular form. The colors produced on TLC plates due to reactions taking place between pesticides and spray reagents are illustrated in the form of chromatograms, and reaction mechanism is also presented.

     

Evaluation of a TLC Densitometric Method for Analysis of Azole Antifungal Agents

A new method for identification and quantitative determination of the azole antifungal agents; ketoconazole, bifonazole, fluconazole and itraconazole is described using TLC with densitometric detection. Validation of the method was carried out to confirm its precision (%RSD ranged between 1.31 and 3.45), recovery (99.7–102.4%) and linearity (r  = 0.99287–0.99722) within the concentration range under investigation. The experimental conditions obtained enable the method to be used for both qualitative and quantitative pharmaceutical analysis.

     

Class Fractionation of Acidic Glycolipids and Further Separation of Gangliosides by OPTLC

Abstract

The use of the OPTLC method has been extended to the separation of the acidic fraction of the total lipid extract derived from a given blood element. This newly developed method is suitable for the interclass separation of sulphatides and gangliosides and further intraclass separation of gangliosides on the same TLC plate with step gradient development. The elutions can be performed on 10 × 10 cm (or larger) HPTLC plates with 13 parallels on each one. The chromatograms were stained either with orcinol-H₂SO₄ to show class separation (in this case only a single isocratic elution was performed) or with rescrcinol-HCl reagent to visualize the ganglioside intraclass separation. The chromatograms were evaluated by spectrodensitometric scanning and the reproducibility of the separation was determined.     

Use of the Software Processing of Scanned Chromatogram Images in Quantitative Planar Chromatography

The results of quantitative TLC analysis using the software processing of scanned chromatogram images are demonstrated by the example of the separation and determination of food dyes. The chromatograms obtained were digitized by scanning with a flatbed scanner. Quantitative analysis was performed using special-purpose software. The use of the software processing of scanned images does not require expensive analytical instruments and provides an unbiased quantitative evaluation of substances in a chromatographic peak with a minimum of time expenditure.     

Enzymatic Detection of Urinary Steroids on Thin-Layer Chromatograms

Abstract

Enzymatic detection of urinary steroids on thin-layer chromatograms (TLC) is described. Steroids in urine such as neutral 3α-hydroxysteroids, 3β-hydroxysteroids, acidic 3α-hydroxysteroids and 17β-hydroxysteroids are detected on thin-layer plate using enzyme reactions.

Combination of chromatographic techniques and enzyme reactions give more specific analysis of the compounds in a body fluid. Some clinical application by the use of these enzymatic methods is also described and these enzymatic detection methods are shown to be useful for the diagnosis of some disease of abnormal steroid metabolism.     

Two-Stage,One-Dimensional Thin Layer Chromatographic Method for Separation of Lipid Classes

Abstract

A thin-layer chromatographic (TLC) technique was developed for routine analysis of lipid classes of blood, milk, tissue and egg yolk. This procedure provided rapid and reproducible separations suitable for in situ quantitation by densitometry

The spotted TLC plate was subjected to two developments in one dimension to separate, in ascending order from the origin: phospholipids, monoglycerides, free fatty acids, cholesterol, 1,2-diglycerides, 1,3-diglycerides, triglycerides, and cholesterol esters. Development 1: chloroform: methanol: acetic acid (98:2:1) to 17.0 cm. Development 2: hexane: ethyl ether: acetic acid (94:6:0.2) to the top of the plate. After air drying, the plate was dipped into a solution of 3% cupric acetate in 8% phosphoric acid for 3 seconds, and heated at 130°C for 30 minutes to char the separated lipid classes. The chromatograms were scanned at 350 nm.

Overloading of TLC plates with sample using the spotter resulted in the appearance of distorted kidney-shaped spots. A mathematical model and general explanation for the shape of these distorted spots was described.     

Chromatographic Separation of Some Inorganic Isomers

Abstract

Isomeric simple complexes of Ti (III) and Hg(II) with p-diethylaminoanil of anthraceneglyoxal (DEAAnG) and isomeric mixed ligand complexes of Zn (II) with DEAAnG and Thiourea (TU) have been synthesized, resolved in bulk on silica gel column, and characterized on the basis of elemental analyses, molar conductance and infrared studies.

Binary mixtures of isomers have been analysed by TLC, PC and EC techniques. TLC method showing highest resolving capacity was used in the quantitative analysis of isomeric mixtures. Resolved isomers were identified using i.r. and migration rate correlations.     

Marker-based standardization of polyherbal capsule formulation by high-performance thin-layer chromatography (HPTLC)–densitometric method using andrographolide and catechin

Himalaya PartySmart capsule is a polyherbal formulation recommended for its liver-protective properties. As the formulation contains extracts of six different herbs, a large number of markers are present in the same. This research work reports the standardization of Himalaya PartySmart capsule using andrographolide and catechin as therapeutic phytoconstituents to assess its quality and efficacy. A specific, sensitive, precise, and accurate high-performance thin-layer chromatography (HPTLC) method has been developed for the quantitative estimation of andrographolide and catechin. Separation was performed on TLC silica gel 60 F₂₅₄ aluminum plates as the stationary phase using chloroform‒acetone‒formic acid (7:3:0.5, V/V) as the mobile phase with densitometric detection at 259 nm. The developed method was validated as per the recommendations of the International Council for Harmonisation (ICH) Q2(R1) guideline. Each marker phytoconstituent showed a good linear relationship with an average correlation coefficient (r²) = 0.99 in the concentration range studied. The proposed method was found to be specific, precise, and accurate with recovery within the range of 95‒105% and hence can be used for the routine analysis of PartySmart capsule formulation.

     

The use of thin-layer chromatography in the assessment of the quality of lutein-containing dietary supplements

Bivariant multiple development thin-layer chromatography technique (BMD–TLC) along with high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry (HPLC–DAD–ESI–MS) analysis was used in determination of lutein or lutein mixed with zeaxanthin in eight dietary supplements. The developed two-step TLC separation procedure combined purification, compaction of samples and separation of the analyzed compounds what significantly shortened and simplified samples preparation. Qualitative analysis was based on co-chromatography with reference substances and HPLC–DAD–ESI–MS analysis. It was revealed that three of eight dietary supplements did not contain lutein. In turn, quantitative analysis with the use of developed TLC conditions along with densitometry showed that the amount of lutein or its mixture with zeaxanthin in the others differed from that claimed by producers.

     

Mechanistic Study of Microwave-Assisted Acid Hydrolysis of Peptides on TLC Plates

Abstract

Microwave-induced acid hydrolysis was performed with isolated peptides on TLC layers. This vapor phase method was compared to solution phase techniques. A mechanistic study of peptide bond clevage was carried out by varying exposure conditions and reaction time. Acid hydrolysis products were identified by ninhydrin visualization. Also, TLC and TLC/MS were used to elucidate hydrolysis mechanisms.     

Spectrophotometric and spectrodensitometric quantification of a new antiviral combination

Accurate, simple, and selective spectrophotometric and spectrodensitometric methods were developed and adopted to quantify velpatasvir (VPS) and sofosbuvir (SFV) concurrently in their pure forms and tablets. The spectrophotometric technique was based on the first derivative of ratio spectra (¹DD) technique and developed to determine VPS and SFV simultaneously in table formulation. However, the spectrodensitometric technique was based on thin-layer chromatography (TLC) and densitometry and developed to determine VPS and SFV simultaneously in tablet dosage form. Chromatographic separation was performed using chloroform:methanol 9.5:0.5 (%, v/v) as the mobile phase on glass-coated TLC plates. Detection was achieved using a 265-nm deuterium lamp in absorbance mode. Both analytical methods were validated according to the International Conference on Harmonization (ICH)-Q2B guidelines. The linearity in the range of concentration ranges of 1–50 μg/mL and 5–80 μg/mL were obtained for VPS and SFV, respectively, using ¹DD spectrometric method. However, the linearity in the range of 5–50 and 10–70 μg/band for VPS and SFV, respectively, were recorded using TLC–densitometric method. Accuracy was recorded ˃100% for VPS and SFV using both methods. This is the first TLC–densitometry method that can separate and quantify the studied mixture of the drugs. The proposed analytical methods were found to be accurate, precise, selective, robust and sensitive for simultaneous analysis of VPS and SFV in tablet dosage forms.

     

Pressurized planar electrochromatography as a supporting tool for qualitative toxicological chemical analysis with thin-layer chromatography and UV–Vis spectrometry

ABSTRACT

Pressurized planar electrochromatography (PPEC) was applied to support qualitative toxicological chemical analysis performed with thin-layer chromatography (TLC) and UV–Vis spectrometry. Based on retention/migration distance data of substances obtained in TLC and PPEC systems and database of their wavelength maxima of remission UV–Vis spectra as well, a combined fit factor was calculated for substance identification. The involvement of PPEC, TLC, and UV–Vis spectral data together in calculation of the combined fit factor lead to its lower values for substances, which were not identical with reference, in comparison with those when the combined fit factor was calculated using TLC and spectral data only. The results evidence that involvement of PPEC data in qualitative toxicological chemical analysis performed with TLC and UV–Vis spectrometry enhances reliability of it.     

Critical evaluation of experimental conditions influencing the surface-enhanced Raman spectroscopic (SERS) detection of substances separated by layer liquid chromatographic techniques

Summary Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F₂₅₄ and Si60 F₂₅₄ Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms. SERS detection of ascorbigen and 1′-methylascorbigen was performed after chromatography on silica gel 60 F₂₅₄ TLC and HPTLC plates and on Si60 F₂₅₄ Raman TLC plates. Traditional development was used for the silica gel 60 F₂₅₄ TLC plates and Si60 F₂₅₄ Raman plates, and the personal OPLC technique for the silica gel 60 F₂₅₄ HPTLC plates. It was found that the SERS spectrum gave information about the indole ring only. Because bonding of the analyte to the stationary phase results in a change in molecular conformation-in contrast with the behaviour of rhodanine-the type of the plateused and the development procedure employed can significantly influence the quality of the SERS spectrum. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary September 1–3, 1999     

Automatic Headspace Sampling for Determination of Ethylene Dibromide Residues in Cereals

Abstract

The possibility was investigated to apply a commercially available automatic head-space sampler in trace analysis of ethylene dibromide (EDB, 1,2-dibromoethane), in cereals.

Samples of rice and wheat flour were thermostatted in closed vials at 70[ddot]C for 30min. The top gas was then automatically introduced into a gas chromatograph equipped with an electron capture detector. Quantitation was performed using external standards (untreated samples spiked with solutions of EDB in N,N′-dimethylacetamide).

The relative standard deviation of the method was 3.4% for rice and 4.5% for wheat flour, at a residue level of 0.008 mg/kg. The detection limit was 0.001 mg/kg (the official EC residue tolerance is 0.01 mg/kg).

Preliminary experiments with other fumigants were carried out as well.

The headspace technique in question has the following advantages over other methods for determining EDB residues in cereals: 1. No sample pre-treatment like extraction, steam distillation, purge and trap etc.; 2. Automated sample handling; 3. “Clean” chromatograms.     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.