Rf values

During gradient elution, the plate is vertically developed with a certain mobile phase. Capillary forces cause mobile phase flow. After the elution is completed, the plate is removed from the chamber and the mobile phase evaporates. After this, the plate is placed in a new chamber with a mobile phase having a different elution strength. These steps can be repeated several times and can also be automated. Gradient elution is typically used in those cases where the range of polarity between the analytes is large. 

The larger the value for Rf, the lower the interaction between the compound and the stationary phase. Rf values range from 0 (no elution) to 1 (no affinity) and the value for Rf is specific for a compound when the chromatographic conditions are kept constant (same mobile phase, same plate). 

The relationships between the Rf value and the retention factor (k) and between the Rf value and the efficiency (N) are given by 

After development, the mobile phase is removed through evaporation. The separated compounds can be detected by the eye either by their (natural) color or by the fluorescence (after irradiation with UV). Comparing the size and intensity of spots of the sample with the size and intensity of spots of standards, a good estimate of the concentration can be made. 

Even in the absence of natural color compounds can be detected using iodine vapor and concentrated sulfuric acid. With iodine vapor, aromatic compounds form bro vn iodine complexes. Spraying the plate with concentrated sulfuric acid with subsequent placement in an oven holding 250°C, oxidation products of the compounds, formed during the heating in the presence of sulfuric acid, are detected as colored spots. Most of involatile organic compounds can be detected in this way. 

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On the basis of these observations, criticize or defend the following proposition The fact that the separate spots fuse into a single spot of intermediate Rf value proves that block copolymers form between the two species within the blend upon heating. 

An important publication by Kost et al. (63JGU525) on thin-layer chromatography (TLC) of pyrazoles contains a large collection of Rf values for 1 1 mixtures of petroleum ether-chloroform or benzene-chloroform as eluents and alumina as stationary phase. 1,3- and 1,5-disubstituted pyrazoles can be separated and identified by TLC (Rf l,3>i y 1,5). For another publication by the same authors on the chromatographic separation of the aminopyrazoles, see (63JGU2519). A-Unsubstituted pyrazoles move with difficulty and it is necessary to add acetone or methanol to the eluent mixture. Other convenient conditions for AH pyrazoles utilize silica gel and ethyl acetate saturated with water (a pentacyanoamine ferroate ammonium disodium salt solution can be used to visualize the pyrazoles). 

The emission of the indicator is reduced in places where there are substance zones that absorb at 2 = 254 nm present in the chromatogram. This produces dark zones (Fig 4A), whose intensity (or rather lack of it) is dependent on the amount of substance applied. If the plate background is set to 100% emission the phosphorescence is reduced appropriately in the region of the substance zones. When the chromatogram is scanned peaks are produced, whose position with respect to the start can be used to calculate Rf values and whose area or height can be used to construct cahbration curves as a function of the amount applied (Fig. 25). 

The separation is already complete when detection is undertaken The solvent has been evaporated off, the substance is present finely distributed in the adsorbent For a given amount of substance the smaller the chromatogram zone the greater is the concentration and, hence, the detection sensitivity For this reason substances with low Rf values are more intensely colored than those present in the same quantity which migrate further... 

Fig. 42 Chromatogram of polycyclic aromatic hydrocarbons on caffeine-impregnated precoated silica gel 60 HPTLC plates with concentrating zone (Merck). The following can be recognized in increasing Rf value. — 1. benzo(ghi)perylene, 2. indeno(l,2,3-cd)pyrene, 3 benzo(a)pyrene, 4. benzo(b)fluoranthene, 5. benzo(k)fluoranthene, 6. fluoranthene.

Only particular solvents are suitable for certain purposes. The choice depending, for instance, on their residual water content or their acid-base nature if Rf values are to be reproduced [1, 2]. Halogen-containing solvents may not be employed for the determination of chlorinated pesticides. Similar considerations apply to PAH analyses. Pro analyst grades are no longer adequate for these purposes. It is true that it would be possible to manufacture universally pure solvents that were adequate for all analytical purposes, but they would then be too expensive for the final user [3, 4]. 

The most important thing to pay attention to in the case of RP phases is the chain length. It is often forgotten, however, that RP phases are available with differing degrees of surface modification and which also differ in their hydrophobicity and wettability and separation behavior (Rf values, development times). These details should, therefore, also be documented. 

Fig. 60 Chromatogram of a 6 dyestuff mixture made up (according to falling Rf values) of Sudan red 7B, Sudan orange G, Sudan black B, Sudan yellow, Artisil blue 2RP and Sudan black B (2 components) under different humidity conditions. From left to right 72, 65, 47, 42, 32,18, 14 and 9% relative humidity layer silica gel 60 mobile phase toluene.

A chromatogram is produced by developing a TLC/HPTLC plate, but it may be necessary to employ one of the reagents descnbed to make the positions, structures and sizes of the chromatogram zones apparent so that they can be recorded If the Rf values are the same a companson of the sizes of the zones of the sample and standard substances gives an indication for estimating the amounts If, as a result of matnx effects, the Rf values of sample and standard are not the same then their... 

It should be noted that to obtain the best results in any of these quantitative TLC methods, the spots being used should have Rf values between 0.3 and 0.7 spots with R( values <0.3 tend to be too concentrated whereas those with Rf values >0.7 are too diffuse. 

Take a second prepared plate and spot three separate 5 pL of mixture M on to the origin line using a micropipette. Place the dry plate into the tank, replace the lid and allow the chromatogram to run for about 1 h. Remove the plate, mark the solvent front and dry the plate at 60 °C for about 15 min. Identify the separated components on the basis of their Rf values. 

Releasing agents 793 Replicate determinations number of, 142 reliability of, 137 Residual current 595 Resistance 504 Resistivity 519 Resonance line sources 790 Results, comparison of 139 Reverse osmosis 90 Reversible back e.m.f. 505 Rf values 234 Rotated electrode 511 R.U. powder 766, 772... 

Likewise, synthetic 2//-azepines isomerize to 3//-azepines in refluxing chloroform (2-3 h) or in tert-butyl methyl ether at room temperature.291 The isomers can be readily separated by chromatography on silica gel, as the more basic 2//-azepines30 have lower Rf values. In contrast, 7-butyl-2//-azepin-2-acetic acid (11), obtained by heating the tert-butyl ester 10 with iodotrimethylsilane, is stabilized by intramolecular hydrogen bonding and shows no tendency to rearrange to the 3//-isomer.291... 

Thin-layer chromatography was also used in an attempt further to purify fractions that had been separated by column or gas chromatography. Rather precise Rf values for several compounds were... 

Fractions may be monitored by thin-layer chromatography on silica gel, developing with 10% v/v ethyl acetate in hexane and visualizing with iodine vapor. The following Rf values were observed famesol, 0.07 farnesyl acetate, 0.35 bromohydrin acetate, 0.20. 

TLC analysis of the crude product (elution with 50 1 pentane ether, visualization with iodine) showed three non-baseline spots Rf 0.65 (cis isomer), Rf 0.52 (unknown impurity), and Rf 0.32 (trans isomer). The unknown impurity is intensely sensitive to iodine and largely coelutes with the cw-isomer in the subsequent column chromatography. However, the ll NMR spectrum of this isomer shows excellent purity despite the presence of this spot on TLC. In 100 1 pentane ether, Rf values of the cis and trans isomers are about 0.50 and 0.15, respectively. 

Besides the calculation of the different sulfonated species, it is also possible to determine them directly by chromatographic methods. Separation of the ester sulfonate and the disodium salt is achieved by thin-layer chromatography on silica gel plates. With a solvent mixture of acetone and tetrahydrofuran (90 10 v/v) the disodium salt stays at the start whereas the ester sulfonate has an R value of 0.2. With the more polar solvent 0.1 N H2S04 + methanol + chloroform the ester sulfonate and the disalt have Rf values of 0.36 and 0.14. For visualization, the plate is sprayed with pinacryptol yellow. In UV light (254 and... 

The balloon was removed briefly while aliquots were taken. The flask was flushed again with nitrogen and the balloon was then replaced. Thin-layer chromatographic analyses were carried out on glass plates coated with silica gel G which were purchased from Analtech, Newark, Delaware. With a 10 2 3 (v/v/v) solution of 1-butanol, acetic acid, and water as developing solvent, the Rf values for the product and L-cysteine hydrochloride are 0.19 and 0.25, respectively. 

TLC Analysis. Samples were examined by TLC using standard procedures. Rf values were determined and compared with those of authentic reference materials. Radioactive components were located by scanning (Vanguard Instrument Corp., North Haven, Conn., Model 885) or by autoradiography (Eastman Kodak, Rochester, N. Y., type AA film). The relative Rf value of DCDD on silica gel plates (Brinkmann Instruments, Inc., Westbury, N. Y., type For,4) when developed with n-hexane dioxane acetic acid, 90 10 4, V/V/V, was 0.90. The observed impurity had a relative Rf value of 0.40. On Brinkmann alumina plates, developed with n-hexane, DCDD had a relative Rf of 0.32. Neither system separated the chlorinated dibenzodioxin isomers. 

Note The three reagents should be applied as quickly as possible after each other. In combination with the Rf value, and with UV detection before application of the reagent sequence this procedure allows the identification of therapeutic quantities of thiazide diuretics and methyldopa in urine together with a series of other therapeutic agents. Mobile phase residues e.g. acetic acid, should be completely removed from the chromatograms before application of the reagent sequence. 

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