Solvents and Separation Efficiency

Nurnberg [41] uses n-propanol-water (50 -f 60) and phenol-water (100 -f 40) for two-dimensional chromatograms. 

According to Jost, Rtoinobr and Sorm [42], phenol-water (90 -f 30) is suitable for separation of tyrosine, N-methylt3n osine and 0-methyltyrosine. 

The combination of solvents C and D (Fig. 203, [7]) and, in particular, the combination of H and D (Fig. 206, [9]) is suitable for two-dimensional chromatography. The latter enables all protein amino acids + jS-alanine 

It is striking that the jK/-values generally do not exceed 0.7 in onedimensional thin-layer chromatography of amino acids. Only about 2/3 of the distance from start ta front is thus utilised in separation in all solvents investigated by us. Increasing the water content of the solvent raises small J /-values more than larger, so that the effective separation path is diminished. 

6 [xg of each amino acid applied in a total of 0.5 pd O.IN hydrochloric acid. Ibe layer is aerated 20 min in a fume cupboard after the first run. The methionine spot (dotted) appears only when the performic acid oxidation is not carried out. Visualisation with ninhydrin (Rgt. No. 178) 

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The identification of separated componnds is primarily based on their mobility in a snitable solvent, which is described by the Rp valne of each compormd. Kowalska et al. have nicely discnssed in greater detail the theory of planar chromatography and separation efficiency parameters in Chapter 2 of the third edition of the Handbook of Thin-Layer Chromatography, published in 2003. 

The photometric determination of mixtures of aniline, p-nitroaniline and o-nitroaniline was described. Distribution coefficients and separation efficiency of these compounds by LLE in various solvents were compared517. Substituted nitroanilines such as 2-chloro-4-nitroaniline and 2,4-dinitroaniline are intermediates in the manufacture of the dye D C Red No. 36 and were identified as impurities by RP-LC518. A spectrophotometric method was developed for the determination of aniline and m-nitroaniline in a mixture of aniline and nitroaniline isomers by derivatization with 5,7-dichloro-4,6-dinitrobenzofuroxan (244). The relative error of the determination is <5%519. See also Section IV.D.3.b for similar derivatives. 

Over-pressure TLC (OPTLC) was introduced by Tyihak et al. in 1979 (13) m an attempt to combine the advantages of conventional TLC and HPLC. This technique employs the use of a pressurized circular ultra microchamber (PUM chamber), which houses a TLC plate and inlets for the introduction of sample and solvent onto the sorbent. The thin sorbent layer is covered by a membrane kept under external pressure so that the vapor phase above the sorbent is nearly eliminated. A substantially shorter time is required for separation than in conventional TLC and classical column chromatography (CC) and greater resolution and separation efficiency is achieved. The rate at which solvent migrates IS as stable as HPLC and consequently the technique can be used to model CC... 

In a sequence of operations of the above kind the losses of the desired component A can be considerable, and the final amount of pure crystals may easily be a minute fraction of the starting mixture AB. This question of yield from recrystallization processes is of paramount importance, and many schemes have been designed with the object of increasing both yield and separation efficiency. The choice of solvent depends on the nature of the required substance A and the impurity B. Ideally, B should be very soluble in the solvent at the lowest temperature employed, and A should have a high temperature coefficient of solubility so that high yields of A can be obtained from operation within a small temperature range. Some of the factors affecting the choice of a solvent are discussed in section 3.2. 

Concentration (or isolation) tasks are usually necessary wifli streams where the product concentration is very low (dilute). Adsorption and extraction are typical technologies utilized here. Energy considerations dominate the technology selection subject to product contamination and stability constraints. Thus, the base technology depends on the availability of the Mass Separating Agent (e.g., adsorbent or solvent) and its efficient recycle. Supercritical extraction of organic compounds from aqueous streams is a typical example of isolation step. 

The ester and catalj st are usually employed in equimoleciilar amounts. With R =CjHs (phenyl propionate), the products are o- and p-propiophenol with R = CH3 (phenyl acetate), o- and p-hydroxyacetophenone are formed. The nature of the product is influenced by the structure of the ester, by the temperature, the solvent and the amount of aluminium chloride used generally, low reaction temperatures favour the formation of p-hydroxy ketones. It is usually possible to separate the two hydroxy ketones by fractional distillation under diminished pressure through an efficient fractionating column or by steam distillation the ortho compounds, being chelated, are more volatile in steam It may be mentioned that Clemmensen reduction (compare Section IV,6) of the hj droxy ketones affords an excellent route to the substituted phenols. 

The efficiency of separation of solvent from solute varies with their nature and the rate of flow of liquid from the HPLC into the interface. Volatile solvents like hexane can be evaporated quickly and tend not to form large clusters, and therefore rates of flow of about 1 ml/min can be accepted from the HPLC apparatus. For less-volatile solvents like water, evaporation is slower, clusters are less easily broken down, and maximum flow rates are about 0.1-0.5 ml/min. Because separation of solvent from solute depends on relative volatilities and rates of diffusion, the greater the molecular mass difference between them, the better is the efficiency of separation. Generally, HPLC is used for substances that are nonvolatile or are thermally labile, as they would otherwise be analyzed by the practically simpler GC method the nonvolatile substances usually have molecular masses considerably larger than those of commonly used HPLC solvents, so separation is good. 

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