Stationary-phase use

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

In fact, this procedure can be used for any aliphatic series such as alcohols, amines, etc. Consequently, before dealing with a specific homologous series, the validity of using the methylene group as the reference group needs to be established. The source of retention data that will be used to demonstrate this procedure is that published by Martire and his group [5-10] at Georgetown University and are included in the thesis of many of his students. The stationary phases used were all n-alkanes and there was extensive data available from the stationary phase n-octadecane. The specific data included the specific retention volumes of the different solutes at 0°C (V r(To)) thus, (V r(T)) was calculated for any temperature (Ti) as follows. 

Fig. 6-6. Overload elution profiles of D,L-PA injected on a column (125 4 mm) packed with the L-PA imprinted stationary phase used in Fig. 6-5. Mobile phase MeCN TFA (0.01 %) FI O (2.5 %). The tendency for fronting and the increase in retention with sample load is attributed in part to saturation of the mobile phase modifier.

ODS octadecyl silyl stationary phase used in high performance liquid chromatography... 

P. A., Testa, B. Solvatochromic analysis of the retention mechanism of two novel stationary phases used for measuring lipophilicity by RP-HPLC./. Liquid Chromatogr. 1992, 35, 2133-2151. 

It was known from gas chromatographic theory that efficiency could be improved if the particle size of the stationary phase materials used in lc could be reduced. High performance liquid chromatography developed steadily during the late 1960s as these high efficiency materials were produced, and as improvements in instrumentation allowed the full potential of these materials to be realised. As hplc has developed, the particle size of the stationary phase used has... 

The stationary phases used in exclusion chromatography are porous particles with a closely controlled pore size. Unlike other chromatographic modes, in exclusion chromatography there should be no interaction between the solute and the surface of the stationary phase. 

Bechalany, A. Rothlisberger, T., El Tayar, N., Testa, B. (1989) Comparison of various non-polar stationary phases used for assessing lipophilicity. J. Chromatogr. 473, 115-124. 

In principle the wide range of stationary phases used in GLC can be used in liquid-liquid partition systems, but problems can arise from mutual solubility effects which may result in the stationary phase being stripped from the column. 

Table 4.10 Some stationary phases used in gas chromatography... 

The value of ax is then compared with published values of known compounds for the appropriate temperature and stationary phase used. 

A large variety of unique column packings (stationary phases) used in HPLC provide a wide range of selectivity. 

Table 5. Properties of commercial stationary phases used in CEC [53]... 

Particular examples for the separation of optical isomers in the (pharmaceutical) industry include prazinquatel [51], 3-blockers [52], chiral epoxide [6],thia-diazin EMD5398 [18] and hetrazipine [7]. The Belgian company UCB Pharma uses a large-scale SMB from NOVASEP to perform optical isomer separation at a scale of several tons per year. Almost all of these separations are performed on cellulose-based stationary phases using organic eluents [4]. 

The great versatility of HPLC lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography. The mobile phase must be available in a pure form and usually requires degassing before use. The choice of mobile phase (Table 3.6) is influenced by several factors. 

McReynolds used the retention index of certain solutes to compare different stationary phases and to assess their selectivity compared with a reference liquid phase, squalane. Squalane is considered to be non-polar and any increase in the retention index of the selected solute on the test column compared to squalane may be considered to be due to the greater polarity of that solvent. McReynolds constants have been determined for all stationary phases using a range of solutes of varying polarity (Table 3.8) and may be used to assist in selecting an appropriate stationary phase. 

Figure 9.23 Separation of equimolar concentrations of methylglycosides by gas-liquid chromatography. The analysis was performed on an OV-1 stationary phase using a temperature gradient from 120 to 220 °C.

Cabon tetrachloride, n-hexane, chloroform, ACN, acetone, THF, pyridine, acetic acid, and their various mixtures were applied as mobile phases for adsorption TLC. Methanol, 1-propanol, ACN, acetone, THF, pyridine and dioxane served as organic modifiers for RP-TLC. Distilled water, buffers at various pH (solutions of and dipotassium hydrogen phosphate or potassium dihydrogen phosphate) and solutions of lithium chloride formed the aqueous phase. Carotenoids were extracted from a commercial paprika sample by acetone (lg paprika shaken with 3 ml of acetone for 30 min), the solution was spotted onto the plates. Development was carried out in a sandwich chamber in the dark and at ambient temperature. After development (15 cm for normal and 7cm for HPTLC plates) the plates were evaluated by a TLC scanner. The best separations were realized on impregnated diatomaceous earth stationary phases using water-acetone and water-THF-acetone mixtures as mobile phases. Some densitograms are shown in Fig.2.1. Calculations indicated that the selectivity of acetone and THF as organic modifiers in RP-TLC is different [14],... 

The retention behaviour of flavonoids has also been extensively studied on silica stationary phases using heptane, benzene or dichloromethane as weaker components of the binary mobile phase and ethyl acetate and methyl ethyl ketone as modifier. Flavones (3-hydroxy, 5-hydroxy and 7-hydroxyflavone, tectochrysin, chrysin, apigenin, genkwanin, baicalein), flavonols (galangin, pilloin, kaempferol, rhamnetin, quercetin, robinetin,... 

TLC has been applied for the control of the synthesis of new 8-C-glucosylflavones such as orientin, parkinsonin A, isoswertia-japonin, parkinsonin B, 5-methyl orientin, 7-methyl orientin and 5,7-dimethylorientin. The purity of the products were checked on a silica stationary phase using hexane-ethyl acetate (5 1 and 3 1, v/v), and acetone-ethyl acetate-water-acetic acid (25 35 5 1, v/v) as mobile phases [142],... 

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