Elution and Development

TLC separations can be carried out based on several modes. The following are the most used modes. 

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A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

The moist cells were suspended in 750 parts of volume of ethanol and extracted by warming at 60°C for 1 hour. A total of 3 extractions were carried out in a similar manner and the extracts were pooled, diluted with water and further extracted three times with 1,000 parts of volume portions of n-hexane. The n-hexane layer was concentrated to dryness under reduced pressure to recover 4.12 parts of a yellow oil. This oily residue was dissolved in 6 parts by volume of benzene and passed through a column (500 parts by volume capacity) packed with Floridil (100 to 200 meshes). Elution was carried out using benzene and the eluate was collected in 10 parts by volume fractions. Each fraction was analyzed by thin-layer chromatography and color reaction and the fractions rich in ubiquinone-10 were pooled and concentrated under reduced pressure. By this procedure was obtained 0.562 part of a yellow oil. This product was dissolved in 5 parts by volume of chloroform, coated onto a thin layer plate of silica gel GF254 (silica gel with calcium sulfate) and developed with benzene. The fractions corresponding to ubiquinone-10 were extracted, whereby 0.054 part of a yellow oil was obtained. This oil was dissolved in 10 parts by volume of ethanol and allowed to cool, whereupon 0.029 part of yellow crystals of ubiquinone-10 were obtained, its melting point 4B°to 50°C. 

To determine secondary alkanesulfonates in sewage wastewaters, solid phase extraction (SPE) and a single-step procedure which combines elution and injection port derivatization for analysis with GC-MS were developed [36]. Again a tetrabutylammonium ion pair reagent was employed both to elute the secondary alkanesulfonates as their ion pairs from CI8-bonded silica disks and to derivatize sulfonate ion pairs under GC injection port conditions. Secondary alkanesulfonates were effectively recovered from samples of raw sewage (>92%) and from primary (>98%) and secondary (>85%) effluents. No... 

An area worthy of study is the development of systems of increasing sample throughput beyond the single column operation. Scott has introduced a prototype multicolumn system based on the centrifugal analyzer principle (53). In this set-up a series of LC colimns is rotated on a disc, with sample delivery at the center of the disc and elution and spectrophotometric analysis on the outside. He has suggested using affinity columns for rapid serum protein analysis by this approach. Of course, other principles, such as segmented flow, could be envisioned in an automated LC system as well. Undoubtedly, we can expect to see the availability of such systems in the next few years. 

This is the most polar group of lipids in natural lipid samples. When developed in a nonpolar solvent system, phospholipids remain at the origin and more polar solvent system should be used to elute and separate individual phospholipids. The most popular system is the Wagner system, which consists of chloroform metha-nohwater (65 25 4) [51] for the separation of common phospholipid species in natural tissue samples. 

Newark, DE) and developed using toluene-ethyl formate-formic acid [(5 4 1), 41]. Compounds were then located with ultra-violet light (UV) at 250 nm. The compounds were eluted from the silica gel with ethyl acetate-methanol (2 1) followed by filtration with Whatman 1 filter paper. The filtrate was dried in vacuo and the residue dissolved in 80 ml of methanol. 

With isocratic elution and a sample having solutes with a wide range of polarity it is sometimes not possible to achieve the desired resolution in an acceptably short time. It may be possible to improve the chromatogram using gradient elution. A practical example of the development of a gradient is discussed. 

Antia et al. [96] proposed a colorimetric method comprising a concentration step and a reaction with 2,7-dihydroxynaphthalene. This method has a detection limit of 0.1 mg/1. Methods comprising concentration by adsorption on alumina, followed by elution and colour development, yielded a detection limit of 5 ng/1 [97,98]. These methods have not been too successful in hands other than those of the original authors, possibly because of the amount of manipulation necessary in the analysis. 

The method development process with the multisorbent plate consists of three steps. In step 1, the sorbent chemistry and the pH for loading, washing, and elution are optimized. In step 2, optimization of the percentage organic for wash and elution and the pH of the buffer needed is carried out. Step 3 is validation the method developed from the results of the previous two steps is tested for linearity, limits of detection, quantitation of recovery, and matrix effects using a stable isotope-labeled analyte as an IS. 

Open format of stationary phase and evaluation of the whole sample In TLC separation, a mixture is applied to the stationary phase followed by development. It is an open system from separation to detection. In contrast to TLC, HPLC is a closed-column system in which a mixture is introduced into the mobile phase and solutes are eluted and detected in a time-dependent manner. There are times that TLC reveals new and unexpected information about the sample, while that information is lost in HPLC by retention on the column, because of strongly sorbed impurities, early elution, or lack of detection. In addition, TLC has little or less contamination with a disposable stationary phase while in HPLC the column is repeatedly used. 

For development, the plate is transferred to the n-hexane tank and developed to the first solvent limit line. The plate is removed and dried in the t.l.c. plate drier until no odour of solvent is apparent (ca. lOmin). The plate is cut in half and the lower half is placed in the toluene tank, and eluted up to the second solvent limit line. The plate is removed and dried as above. The plate is reversed by putting it back in the hexane tank with the cut edge dipping in the hexane. The plate is eluted back to the origin line, removed and dried as above. 

The method was proposed for the approximate prediction of the retention of analytes in gradient elution and for the facilitation of the development of optimal gradient elution strategy [86], This prediction and optimization procedure is similar to those discussed above, consequently, its application in the field of RP-HPLC analysis of natural pigment may be similar. 

HPTLC is a very fast and convenient assay to separate samples components and is often used in Organic Chemistry and in Synthetic approach. Unknown substances, after different display assay, were generally scraped off from the TLC/HPTLC plate, diluted into a tube and transferred into the MS system for structural elucidation and characterization. Now, a TLC-MS interface was developed by CAMAG, which can semi-automatically extract zones of interest and on-line direct them into any brand of a HPLC-MS system. The TLC-MS interface is connected by two fittings to any HPLC instrument coupled with mass spectrometer, without other system configuration adjustments or mass spectrometer modifications. By this way, the unknown substances can be directly extracted from a TLC/HPTLC plate, eluted and resolved by HPLC system and sensitive and selective mass spectrometric signals are obtained within a minute per substance zone [33],... 

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