Rf values measurement

Polyphenols were eluted with various mixtures of water-methanol (8 2, 7 3, 6 4 and 5 5 v/v). Further purification was achieved on a cellulose layer, and on an ODS SPE cartridge. The TLC conditions used for the measurements are listed in Table 2.38. The RF values measured on the HPTLC plates are compiled in Table 2.39. 

Based on the chromatographic profiles observed on both plates, suitable binary solvent mixtures are chosen. Using another TLC plate, an eluotropic series of these solvents is prepared and tested. The start and end composition of this eluotropic series depends on the Rf values measured for the strongest solvent of the mixture. 

Recently the method has been extended for a two-factor optimization in general HPTLC. The two-factor optimization system uses a two-factor selectivity rectangle concept a full 3 factorial design (Figure 4) for each of the two-factor components represents three different levels. Nine experiments are carried out and the Rf values measured. These values are then substituted into the following equation in order to establish the values of the constants ... 

Rf values measured when solvent front was at end of paper in every case... 

The Rf values for a substance measured in linear, circular and anticircular chromatograms, for which the flow conditions vary, can be related to each other by equation 7.18... 

In all TLC work, identification is confirmed by measurement of the distance travelled along the plate by the analyte compared to the solvent front, the Rf value, and by reference to standard solutions run on the same plate. Further confirmation can be obtained by using a reference sample and measuring this under the same conditions as the sample. This allows measurement of the Rx value. This is illustrated in Figure 4.1. 

Diffusion and mass transfer effects cause the dimensions of the separated spots to increase in all directions as elution proceeds, in much the same way as concentration profiles become Gaussian in column separations (p. 86). Multiple path, molecular diffusion and mass transfer effects all contribute to spreading along the direction of flow but only the first two cause lateral spreading. Consequently, the initially circular spots become progressively elliptical in the direction of flow. Efficiency and resolution are thus impaired. Elution must be halted before the solvent front reaches the opposite edge of the plate as the distance it has moved must be measured in order to calculate the retardation factors (Rf values) of separated components (p. 86). 

Most of the reactions described in the following chapters were monitored by Thin Layer Chromatography (TLC) using plastic TLC plates coated with a thin layer of Merck 60 F254 silica gel. The products were detected by using an ultraviolet lamp or the TLC plates were treated with p-anisaldehyde reagent, prepared as explained below, and then heated to 120 °C to stain the spots. After visualization and measurement, the Rf values were recorded. 

Allow the plate to dry. Observe and measure Rf values, if possible, and identify the dyes in the cough syrups. 

Test spots can be identified by comparing their migration with that of reference samples, together with additional evidence, e.g. using a specific colour reagent. The RF value (rate of flow) is a measure of the movement of a compound compared with the movement of the solvent ... 

The measurement of effluent volume is not very reliable because of the effect of the geometry and packing characteristics of any column. It is often more useful to use a reduced parameter, such as V/V0, which is not so dependent upon column characteristics and is comparable with the calculation for RF values in thin-layer chromatography. 

The R( value varies between 0 (analyte remains on the start) and 1 (analyses move with the front of the mobile phase). The reproducibility of the measurement of Rt value can be enhanced by relating it to the Rf value of a standard analyte. As the correlation between the R value of the analyte and the concentration of the stronger component in the mobile phase (C) is not linear, the RM value was introduced to linearize the relationship between the mobility of solutes and the concentration of the stronger component in the mobile phase. 

Thus, the rs is a complex term which embodies the fluorescence lifetime, rotational correlation time (0), and also r0 (r1 in the absence of depolarizing motion). The most common type of experiment involves a comparison of rs for two experimental conditions however, such a comparison of rs ignores possible changes in x, , and r0. Nevertheless, for many cases a comparison of rf values alone may be satisfactory although a more rigorous analysis requires a time-resolved measurement. A comparison of the effects of changes in common membrane properties on time-resolved fluorescence parameters is shown in Table 5.3. 

Measure the distance of the solvent and the compound travelled to obtain the Rf values. 

The independent determination of molecular masses by SDS-PAGE is impossible. To estimate the molecular mass of a protein, measure the path of that protein or calculate its Rf value (distance of the protein from origin/distance of electrophoresis front from origin) and compare these values with that of marker proteins, i.e., proteins with independently determined molecular masses. This method is successful only if the protein of interest behaves regularly in SDS-PAGE, i.e., it is totally unfolded by SDS, has a rod-like shape, and the SDS/protein ratio is the same for the unknown and the marker protein. Especially highly hydrophobic proteins and glycoproteins often deviate from these assumptions. 

More recently, Otocka measured the Rf values for several polystyrenes using dioxane-methanol and dioxane-2-propanol 0 solvents (16). The results shown in Table I indicate that despite a great variety in the... 

A number of studies have been directed specifically at the acridine nucleus. The effect of substituents in the 9-position in acridine (V-oxides on Rf values for thin-layer chromatography (TLC) is measured by ap,198 and there is also a relation between this parameter and the N—O stretching frequencies. The effect of substituents on the basicity of acridine and the possibility of amine-ketimine tautomerism in 9-aminoacridines have been evaluated, taking quinoline as a model... 

Mixing of 1 mmol of triazene 1-oxide 1 with 1 mmol of a- or -naphthol in acetone followed by evaporation gave the mixed crystals. These were finely ground in a mortar, spread on petri dishes and directly exposed to sunlight. TLC examination showed that the reactions were finished within 7-10 h. The temperature was measured as 18 to 25 °C. Chromatographic separation on silica gel with ethyl acetate-toluene (1 10) as eluent gave two zones. The first zone with the higher Rf value (at the start) afforded a multicomponent mixture, which could neither be separated nor could individual components be identified. 

Numerical measurements (retardation factors) known as Rf values can be obtained from chromatograms. An Rvalue is defined as the ratio of the distance travelled by the solute (for example P, Q or R) to the distance travelled by the solvent. 

Figure 2.136 indicates the method of measurement of RF values of each of the components of a typical chromatogram ... 

Polymerization of phenolic compounds (measurement of optical density) A log K and RF values yields of humic polymers... 

The most commonly described USP procedure for quantification is the scrap and elution approach. Low analyte recoveries can occur but can be minimized by using polar organic solvents such as methanol, ethanol, or acetone. Generally, analytes with high-Rf values can be desorbed with high recoveries by using the mobile phase. One example of this procedure is the USP assay procedure of the steroid methyl prednisolone acetate in cream formulation. This steroid is separated from its excipients by TLC, extracted from the sorbent, derivatized, and measured spectrophotometrically. 

Remove the staples from the dried chromatogram. Mark the spots of the pigments by circling with a pencil. Note the colors of the spots. Measure the distance of the center of each spot from its origin. Calculate the Rf values. 

In the present experiment you will determine the Rf values of three amino acids phenylalanine, aspartic acid, and leucine. You will also measure the Rf value of aspartame. 

In paper and thin-layer chromatography, retention is usually measured by an Rf value, similar but not equal to R. There is no simple equilibrium expression for Rfi as will be explained in Section 10.5. 

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... 

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