Thin-layer chromatography gradient elution

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

By appropriate choice of the type (or combination) of the organic solvent(s), selective polar dipole-dipole, proton-donor, or proton-acceptor interactions can be either enhanced or suppressed and the selectivity of separation adjusted [42]. Over a limited concentration range of methanol-water and acetonitrile-water mobile phases useful for gradient elution, semiempirical retention equation (Equation 5.7), originally introduced in thin-layer chromatography by Soczewinski and Wachtmeister [43], is used most frequently as the basis for calculations of gradient-elution data [4-11,29,30] ... 

For metabolite isolation, 1.5 liters of pooled urine were applied to a XAD-2 resin column first. The ethyl acetate extract obtained containing 85 % of the radioactivity was applied upon evaporation to semipreparative HPLC on a Zorbax RX C18 column (9.4 x 250 mm, 5 pm) using gradient elution. Fractions obtained were further separated by isocratic elution on the semipreparative column. The metabolite fractions obtained were finally purified by preparative thin-layer chromatography. Liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis was applied to the isolated metabolite fractions for structure elucidation. 

The evaluation of alkamides, as an indicator for standardization, in Echinacea preparations is commonly completed using HPLC. The method of Bauer (1999b) illustrates the most common method to evaluate alkamides using reverse-phase HPLC. In this method, the lipophilic components are separated by gradient elution using water (eluent A) and acetonitrile (eluent B) linearly from 40% to 80% eluent B at 1 ml/min. The separation was completed on a C18 reverse-phase column and detection was at 254 nm. Thin-layer chromatography using silica 60 plates with... 

The elution of a strong solvent shock of finite amplitude causes the effect known, in thin-layer chromatography, as demixion. The solvent front is eluted before the limit retention time of the strong solvent at infinite dilution and it is extremely sharp. The components eluted before the solvent front are eluted im-der isocratic conditions in the pure weak solvent. The sudden breakthrough of the strong solvent constitutes an extremely steep gradient of the strong solvent inside the column. It is accompanied with the very rapid elution of numerous components that are poorly or not resolved. Such a situation must be avoided in analytical applications. 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

To ensure high enantiomeric purity of the product there should be <0.5% 1,1 -bi-2-naphthol or its monoester in this solution. The relative amounts of binaphthol species can be accurately determined by HPLC on a reverse-phase column eluted with a water-acetonitrile gradient (50-100% over 10 min). Both 1,1 -bi-2-naphthol and its dipentanoate have equal (within 2%) extinction coefficients at 254 nm. The monopentanoate absorbs more strongly the relative extinction coefficient at 254 nm is 1.13. Alternatively, the solution composition can be estimated using thin layer chromatography silica gel eluted with 1 4 ethyl acetate/cyclohexane 1,T-bi-2-naphthol, Rf 0.39 monopentanoate, Rf 0.56 dipentanoate, Rf 0.71. 

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