Acidic IP Reagents for the TLC of Bases

A number of applications of the use of IP-TLC for the chromatography of bases have also been described. That said, it is our experience that the TLC (both normal-phase (NP) and RP) basic compounds can often be performed without recourse to IP systems. Thus, the inclusion of ammonia in NP-TLC systems is often enough to suppress the ionization of weak bases and obtain good chromatography. In RP systems the main problem is usually spot tailing, as the basic analyte interacts with the residual silanols present on the silica surface. This effect can usually be controlled by using a competing base such as ammonia or triethylamine in the eluent. Alternatively a strong organic acid, such as trifluoroacetic acid (TFA), can be used as a mobile-phase additive (0.1% v/v). It is arguable that this is in fact an ion pairing effect and that the TFA is forming an IP with the basic group on the analyte, thus preventing silanophilic interactions. In HPLC the IP chromatography of bases is usually effected using sulfonic acids such as heptanesulfonic acid and the longer chain dodecylsulfate. Studies have been performed with these reagents on silica gel, silanized silica gel, paraffin-coated silica gel and a range of C18-bonded phases, with the best results usually obtained by coating the reagents on to the plate rather than including them in the mobile phase.

In an early study silanized silica gel was used and the reagents (sodium lauryletherosulfate or triethanol-amine dodecylbenzenesulfonate) were mixed with the stationary phase prior to the preparation of the plates. The amount of reagent employed varied from 0.025% up to 4%, with the maximum effect on the Rf of the test amines seen between 2 and 3%. Later studies showed that a range of reagents, from methyl up to dodecyl sulfonic acids, could be employed with C18-bonded plates and acetone-water (0.1 mol L 1 IP reagent) solvent system for the IP-TLC of analytes such as atropine, codeine, eupaverin and papaverine. Not surprisingly, the authors found that the trend for decreasing RF of the test solutes was correlated with increasing alkyl chain length for the reagent.

In our own studies the IP-TLC of ^-blockers was investigated using silica gel, paraffin-coated silica gel or C18 plates impregnated with sodium dodecyl sulfate. In fact it was possible to obtain good results for these analytes only on the C18-bonded materials. The largest effects on chromatographic behaviour were seen for the dodecyl sulfate reagent.

Overpressure IP-TLC Overpressure TLC (described in detail elsewhere in this Encyclopedia) is a forced flow planar chromotagraphy system whereby the TLC plate is held under pressure beneath an inert and impermeable membrane and the mobile phase is pumped through the layer. This mode of TLC has been used successfully with quaternary ammonium IP reagents using both silica gel and bonded stationary phases and with 10-camphor sulfonic acid on silica gel. When using a reagent such as cetrimide the results were shown to be only really acceptable if the plates were precoated with the reagent, at a concentration of 0.05 mol dm~3. However, if in addition to coating the plates, cetrimide (optimally 0.1 mol dm~3) was also included in the developing solvent, a noticeable improvement in spot shape was seen. Interestingly, and unlike the situation observed for normal IP-TLC, a noticeable dependence of RF on pH was noted: best results depended upon the stationary phase. So, with bare silica pH 5 gave the best separations whilst on a C18-bonded silica pH 8 was optimal. Later work extended the use of IP-overpres-sured liquid chromatography (IP-OPLC) to basic compounds with 10-camphor sulfonic acid as the IP reagent, with silica gel as the stationary phase. Interestingly, the investigators noted that this reagent was ineffective when used alone (impregnated into the silica gel by immersion, with or without the reagent also present in the solvent). However, when the plates had previously been impregnated with cetrimide, good results were obtained. This was interpreted as the formation of a double layer of some description. Presumably the cetrimide interacts with the silanols on the surface of the silica gel to form a lipophilic layer on to which the camphor sulfonic acid can be adsorbed. Optimum results are obtained if the plates are pretreated with 0.05 mol L_1 solutions of the reagents and both cetrimide and 10-camphor sulfonic acid are present in the eluent at the same concentration. pH effects were also noted in this work, with the best results seen at pH 5.5.

Chiral IP-TLC IP reagents have been used in a limited number of cases in HPLC for the resolution of enantiomers and similar work has also been performed in TLC. Here the aim is to form diastereoisomeric complexes with different chro-matographic mobilities rather than mask polar ioniz-able groups in order to moderate polarity. A number of IP methods for ^-blockers such as propranolol and alprenolol have been reported, based on the use of N-benoxycarbonyl-glycyl-l-proline (ZGP) and a diol-bonded HPTLC phase. These plates are firstly pre-washed with dichloromethane and then dipped in the mobile phase to impregnate them with the reagent. The mobile phase consists of a 5 mmol L_1 solution of ZGP and 0.4nmolL~1 ethanolamine in di-chloromethane. In one method the samples were applied to the plate followed by continuous developments to c. 4 cm for 10-20 min. The plates were then dried and the process repeated. Solvent composition for this type of IP-TLC can be critical, and in our hands the inclusion of 2% v/v ethanol to the di-chloromethane is essential in order to obtain separation of the enantiomers. With less than this amount of ethanol, no resolution is obtained whilst higher proportions result in increased RF values for the analytes and a loss of resolution. Sample loading is also an important factor: the best results are obtained with loadings of propranolol of ca. 5 |g applied as a 1 cm band.

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