Elution, in TLC

Gradient development. Gradient developments have been used to achieve separation of complex mixtures such as plant extracts, dyes, etc. However, their application to lipid separation is not very common. Golkiewicz (1996) discusses in detail stationary phase gradients and mobile phase gradients, the theory behind solvent selection, automated techniques and applications of gradient elution in TLC. 

Figure 3 Devices for gradient elution in TLC. (Reprinted from Ref. 7 with permission.)...

HETP of a TLC plate is taken as the ratio of the distance traveled by the spot to the plate efficiency. The same three processes cause spot dispersion in TLC as do cause band dispersion in GC and LC. Namely, they are multipath dispersion, longitudinal diffusion and resistance to mass transfer between the two phases. Due to the aforementioned solvent frontal analysis, however, neither the capacity ratio, the solute diffusivity or the solvent velocity are constant throughout the elution of the solute along the plate and thus the conventional dispersion equations used in GC and LC have no pertinence to the thin layer plate. 

All the advantages of these methods for the optimization of the mobile phase by means of preassays in TLC can be exploited at the preparative scale. Finally, the separated zones may be easily removed from PLC plate and eluted in order to isolate quantities of the expected compounds. In PLC selection of the mobile phase, the subsequent recovery of the separated zone should be taken into consideration also. 

The problem of the separation of samples containing components of widely different polarities is difficult because of general elution. This can be solved by use of gradient elution. As has been observed, in TLC separation of plant extracts, gradient elution markedly improves the separation of spots owing to stronger displacement effects... 

Selectivity of the separation in TLC is achieved by various of the aforementioned techniques (e.g. multiple development, gradient elution, sequence TLC, AMD, HPPLC or OPLC). Multidimensional TLC methods are described in Section 7.4.4. 

A series of 200-mL tractions was collected during flash chromatography. The product was eluted in fractions 3-8 as indicated by TLC analysis using 4% ethanolic phosphomolybdic acid stain. 

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. 

The choice of solvent or a mixture of solvents used in TLC is solely guided by two important factors (a) the nature of the constituent to be separated i.e., whether it is polar or non-polar and (b) the nature of the process involved i.e., whether it is a case of adsorption or partition chromatography . It has been observed that the rate of migration of a substance on a given adsorbent depends upon the solvent used therefore, the latter may be arranged in order of the elutive power, usually termed as the elutropic series as shown in the following Table 28.1. 

The crude residue is applied to the column head using a minimum of dichloromethane. The submitters use flash-grade (230-400 mesh) silica gel purchased from E. Merck and a column 10 cm in diameter. TLC values for 2 are Rf = 0.18 (hexane acetone = 3 1) and for 3 (see Note 10) are Rf = 0.08 (hexane acetone = 3 1), employing Whatman K6F silica gel TLC plates 60 A. In a typical purification, collecting 200-mL fractions, 2 would elute in fractions 9-18 and 3 in fractions 20-30. The checkers found that immediate purification of the crude residue was necessary. Yields decreased dramatically with time between isolation and purification. Furthermore, the activity (related to the degree of dehydration) of the silica gel greatly affected yields. Only half the amount of silica mentioned above was used by the checkers to get the reported yields. When the full amount was used, the yield decreased to 40-50%. 

The crude product (10 g) is diluted with 4 mL of a solvent mixture (ethyl acetate/cyclohexane = 9 1). This solution is poured onto a column (75-mm diameter) filled with 120 g of silica gel (Merck 230-400 mesh) for flash chromatography. Elution is performed under gravity and requires 200 mL of the above solvent system, followed by 200 mL of ethyl acetate. 2-Phenyl-2-propanol mixed with methyl p-tolyl sulfide is eluted in the first fraction ( 150 mL, monitored by TLC). The subsequent fractions are collected ( 300 mL)... 

The checkers eluted the columns with a slight positive air pressure on the solvent reservoir to prevent formation of gas bubbles and cracks in the chromatographic medium. Fractions were collected in 25-mL test tubes (Note 1), analyzed by TLC on silica gel, eluting with the column solvent, and visualized with a phosphomolybdic acid solution. The checkers observed a nonvolatile hydrocarbon material (not substrate related) which was eluted in the fractions just prior to the products, which are quite nonpolar themselves and are eluted in the early fractions, ahead of any unreacted ester. Colored, metal-containing components usually remain near the top of the column, although some colored material may accompany the... 

Clayfen (1.0 g) is thoroughly mixed with the sulfide (2 mmol) in a test tube. The reaction mixture is placed in an alumina bath inside the microwave oven and is irradiated for the stipulated time. On completion of the reaction, monitored by TLC, the products were extracted with CH2C12 (3x10 mL). The solvent was removed on a rotary evaporator and the crude product, thus obtained, was charged on a silica gel column. The fractions eluted by chloroform-hexane (1 1) provided sulfone and final elution in chloroform afforded pure sulfoxide. 

Only three new amino acids were found (by automatic amino acid analysis) in poly-D,L-alanine after irradiation in 0.1% solution in the absence of O2 with doses to 5 Mrads. All were in yields less than G = 0.02. The first was eluted before aspartic acid and was therefore acidic the second was eluted in the position of aspartic acid, and the third in the position of glycine. We have not been able to confirm the identities of these products by TLC because of the low yields. The products found by amino acid analysis could not account for the discrepancy between amide-like ammonia formed (G = 0.66) and alanine destroyed (G = 1.9). Aspartic acid is formed when alanine is irradiated in solution 26), and it is likely that the carboxylation reaction proposed by these authors also accounts for the observed aspartic acid formation in PDLA. 

The analysis of the purified monophosphoryl lipid A by reverse-phase HPLC revealed the degree of purity of the fractions TLC-1, -3, -5, and -7 (23). TLC-1 and -3 each gave one major peak (85%), and these peaks had identical elution times. TLC-5 showed one major (72%) and one minor (18%) peak. TLC-7 resolved into one major (48%) and two minor (21 and 16%) peaks. One of the minor peaks in TLC-7 (16%) was the overlapping contaminant of the minor components in TLC-5. Representative results of the HPLC analysis of C-labeled TLC-3 and -5 are shown in Figure 2. 

Pure solvents of Analab grade should be used in TLC. The various solvents possess different eluting properties. Further, because of the differences in polarity of the organic insecticides and variations in their water solubility, several solvent systems are tested for separation on the TLC plate. The large number of solvent systems listed in Table II for organophosphorus-organochlorine and carbamate insecticides provide satisfactory alternative systems for the separation of the group of insecticides under consideration. 

Migration in TLC and PC is not measured by the time of elution since component zones are generally not eluted from the chromatographic bed migration is measured instead by the Rf value. The factor Rf is the ratio of the distance X - X0 migrated by the zone to the distance Xf - X0 advanced by the liquid front in the same time interval... 

Up to this point it may have appeared that the terms hand, peak, and zone have been used synonymously, but let us take a closer look. As commonly used, all three terms describe the distribution of analyte molecules in space (a concentration profile), but band represents this distribution while the analyte is still in (or on) the system, while peak refers to a distribution of analyte that has eluted from the system. The term zone is more general and includes both bands and peaks it is used in those cases when we do not wish to be more specific. In the context of our current discussion, this means that the zones in TLC and PC are called bands and those in column LC and in GC are called peaks. 

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