Thin-layer chromatography normal-phase

Thin layer chromatography, normal, reverse phase, multiple development, analgesics. 

J. W. Hofstraat, S. Griffioen, R. J. van de Nesse and U. A. Th Brinkman, Coupling of narrow-bore column liquid chromatography and thin-layer chromatography. Interface optimization and characteristics for normal-phase liquid chromatography , J. Planar Chromatogr. 1 220-226 (1988). 

There are numerous analytical techniques besides reversed-phase HPLC that can be used to analyze stress test samples. Some of the more common ones include normal-phase HPLC, thin layer chromatography (TLC), capillary electrophoresis (CE), and gas chromatography (GC). The following paragraphs contain brief discussions on the use of these techniques for analysis of stress test samples. 

The techniques of chromatography are used mainly for the separation of materials that are normally difficult to separate and are present in very small quantities in mixtures. There are various methods of chromatography, but the principles are similar. They all depend upon a material for the sample to stick to and a solvent (liquid or gas) to push the sample along. The absorbent surface can be paper or a thin layer of a suitable unreactive powder, like alumina. This is called the stationary phase. In thin layer chromatography (TLC), the material can be chosen for ease of separation. 

The mobile phases used in the development of thin-layer chromatography plates are usually combinations of organic and aqueous solvents. Ethanol, t-amyl alcohol, acetonitrile, and methanol are commonly used organic solvents. Acetic acid is the most commonly used aqueous solvent. In normal-phase partition chromatography the polar stationary phase is developed with relatively nonpolar mo-... 

Normal-Phase Thin-Layer Chromatography (NPTLC)... 

Using aminopropyl-modified silica gel plates in a normal phase system, the retention behavior of 12 acidic drugs and biologically active aromatic acids was investigated by high-performance thin layer chromatography. 

In the Martin-Synge relationship (1), C , and are molar concentration of the solute in the mobile and stationary phase, respectively, and V, and V , are the volumes of these two phases. VJV, is numerically equal to AJA , the ratio of the phase cross section normal to the direction of the solvent flow, which better describes the local conditions in thin-layer chromatography. The validity of the equation is limited because the amount of solvent on the layer decrease going toward the solvent front and, therefore, the phase ratio changes. 

The techniques of normal and reversed-phase thin-layer chromatography, 14 115 and high performance thin-layer chromatography (HPTLC)116 have also been described. 

TLC is a good technique to use when normal-phase solvents provide optimum separation. Typical thin-layer separations are performed on glass plates that are coated with a thin layer of stationary phase. The stationary phases used in TLC encompass all modes of chromatography including adsorption, normal- and reverse-phase, ion-exchange, and size-exclusion." The equipment required is simple and inexpensive. TLC is an ideal technique for the isolation of compounds because of its simplicity. However, for TLC to be successful, the impurity and/or degradant level should be at or above 1%. Any component present below this level is very difficult to isolate on a TLC plate because of higher detection limits. 

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

As an example, Urakova et al. compared the analysis of chlorogenic acid in green coffee bean extracts by thin layer chromatography on silica (i.e. normal-phase liquid chromatography) and by reversed-phase HPLC The validation data (LOD, LOQ, repeatability, and various precision parameters), the recoveries and the quantitative results were totally comparable. It can be assumed that both methods find the true value. Either method can be used, depending on the preference or instrumentation of a laboratory. 

Normal-phase SPE methods development is a straightforward process, and for this reason, many methods have been developed on silica sorbents, especially with the broad and comprehensive literature available in thin layer chromatography (TLC). The development of the silica bonded phases has introduced a new and important aspect to methods development with normal-phase SPE. 

NP-TLC Normal phase thin-layer chromatography. RP-HPLC Reverse phase high performance liquid chromatography. 

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