Radosław Ł. Gwarda

Two-dimensional high-performance thin-layer chromatography of tryptic bovine albumin digest using normal- and reverse-phase systems with silanized silica stationary phase.

Among many advantages of planar techniques, two-dimensional (2D) separation seems to be the most important for analysis of complex samples. Here we present quick, simple and efficient two-dimensional high-performance thin-layer chromatography (2D HPTLC) of bovine albumin digest using commercial HPTLC RP-18W plates (silica based stationary phase with chemically bonded octadecyl ligands of coverage density 0.5μmol/m(2) from Merck, Darmstadt). We show, that at low or high concentration of water in the mobile phase comprised methanol and some additives the chromatographic systems with the plates mentioned demonstrate normal- or reversed-phase liquid chromatography properties, respectively, for separation of peptides obtained. These two systems show quite different separation selectivity and their combination into 2D HPTLC process provides excellent separation of peptides of the bovine albumin digest.

Inversion of type of separation system in planar chromatography of peptides, using C18 silica-based adsorbents.

Our previous results show, that C18 silica-based adsorbents used in high-performance thin-layer chromatography (HPTLC), provide complex retention mechanism basing on various polar and nonpolar interactions. Here we present, that in chromatography of peptides, due to mixed-mode properties of these adsorbents, there is a simple way to obtain inversion of separation system type (from reversed-phase, RP, to normal-phase, NP, and vice versa). The results presented provide detailed information how to obtain inversion mentioned and reflect the extent (the type and concentration of organic solvent, the type and concentration of ion-pairing reagent in the mobile phase) of this phenomenon. We show, that the system type inversion results in significant change of selectivity of separation, which may be especially useful in 2D separation of complex samples of basic/amphoteric compounds such as peptides. This results from the fact, that C18 silica-based HPTLC adsorbents, may be used in hydrophilic interaction chromatography (HILIC) or RP chromatography, in dependence on mobile phase composition.

Staining of some synthetic oligopeptides using ninhydrin solution

Ninhydrin solution is a very popular reagent, which is commonly used to stain amino acids and peptides after thin-layer chromatography (TLC) separation. It is quite cheap (in comparison, e.g., with fluorescamine), and its use does not require additional equipment (as UV lamp) to visualize chromatograms. However, many authors reported about the use of ninhydrin solutions of various compositions (various solvents and ninhydrin concentration), as well as various procedures, which are not always optimal for derivatization of compounds of interest (amino acids and peptides). In this short communication, we present some practical observations and conclusions, which are related to another our paper [1] and concern staining of some synthetic oligopeptides with ninhydrin. These may help other authors to optimize a procedure of visualization of similar compounds.

Influence of carboxylic ion-pairing reagents on retention of peptides in thin-layer chromatography systems with C18 silica-based adsorbents

One of the main problems related to chromatography of peptides concerns adverse interactions of their strong basic groups with free silanol groups of the silica based stationary phase. Influence of type and concentration of ion-pairing regents on peptide retention in reversed-phase high-performance liquid chromatography (RP-HPLC) systems has been discussed before. Here we present influence of these mobile phase additives on retention of some peptide standards in high-performance thin-layer chromatography (HPTLC) systems with C18 silica-based adsorbents. We prove, that due to different characteristic of adsorbents used in both techniques (RP HPLC and HPTLC), influence of ion-pairing reagents on retention of basic and/or amphoteric compounds also may be quite different. C18 silica-based HPTLC adsorbents provide more complex mechanism of retention and should be rather considered as mixed-mode adsorbents.

The modification of pressurized planar electrochromatography chamber in respect of reduction of dead volume in the separation system

Since the first publication concerning pressurized planar electrochromatography (PPEC) in 2004 [1], this technique has been used for separation of many various groups of compounds [2, 3]. It has many advantages in comparison to thin-layer chromatography (TLC; and its high-performance version, HPTLC), including different selectivity, short separation time, possibility of long-distance electrochromatogram development, higher efficiency, better separation system equilibration (no mobile phase demixion), etc. However, one of the main difficulties concerning PPEC is the necessity of prewetting the adsorbent layer before the separation process. This may result in the dispersion of the starting zones of solutes if these are on the plate during prewetting.

The influence of pH on retention and migration of peptides in systems with octadecyl silica-based adsorbent by high-performance thin-layer chromatography and pressurized planar electrochromatography techniques

In our paper we investigate the influence of pH of the mobile phase buffer, on retention of peptides in both: normal phase (NP) and reversed phase (RP) high-performance thin-layer chromatography systems (HPTLC), using C18 silica-based adsorbent. We also compare the influence of pH on retention and/or migration distance of peptides in separation systems of HPTLC and PPEC (pressurized planar electrochromatography) techniques. Our results show, that the change of pH of the mobile phase buffer can be used for two-dimensional HPTLC separation of peptides, but much stronger change of separation selectivity can be obtained by overall inversion of separation system type (NP/RP), as we have described before (Gwarda et al., 2016). In PPEC influence of the mobile phase pH on selectivity of peptide separation is very clear and strictly different than that in HPTLC (including overall inversion of direction of solutes migration), what results from the significant share of electrophoretic effect in overall mechanism of separation. Migration of peptides in two opposite directions results in better selectivity of separation by PPEC. In separation systems of both HPTLC and PPEC techniques, the change of pH clearly affects the efficiency of the separation system, so it can be used for optimization of separation conditions. Our results also show, that some modifications of PPEC equipment and materials could be useful for application of this technique in the field of peptide analysis.

The influence of metallic impurities on the free silanol activity of commercial thin-layer chromatography adsorbents demonstrated by retention changes of basic/amphoteric compounds such as peptides

In our previous papers, we have mentioned some specific disruption of peptide zones shape and chromatogram distortion, when using mobile phase containing ion-pairing acids. This problem is investigated here. It concerns not only some specific separation conditions but also various separation systems with silica-based adsorbents and water—alcohol mobile phases. We show that the problem results from significant amount of metallic impurities present in the adsorbents investigated. Our results prove that these impurities strongly affect the activity of free silanol groups and thus the retention of basic or amphoteric compounds and the quality of the results obtained. The standard method of washing adsorbent layer with methanol is not effective against the impurities. Washing chromatographic plates with a solution containing an acid significantly reduces the amount of metallic impurities in the adsorbent, resulting in the reduction/elimination of these adverse effects. However, it also leads to the increase of heterogeneity of acidic groups activity and deterioration of separation system efficiency. Therefore, removing metal ions from the adsorbent may not always be advantageous. Avoiding of use of strong ion-pairing acids is also problematic and not always possible. Thus, the production of high-purity silica of homogenous activity seems to be the best and the most reliable solution of the problem described.

The influence of addition of ion-pairing acid and organic modifier of the mobile phase on retention and migration of peptides in pressurized planar electrochromatography system with octadecyl silica-based adsorbent

In our previous papers we have investigated the influence of the mobile phase composition on mechanism of retention, selectivity and efficiency of peptide separation in various high-performance thin-layer chromatography (HPTLC) systems with commercially available silica-based adsorbents. We have also investigated the influence of pH of the mobile phase buffer on migration and separation of peptides in pressurized planar electrochromatography (PPEC). Here we investigate the influence of concentration of ion-pairing additive, and concentration and type of organic modifier of the mobile phase on migration of peptides in PPEC system with octadecyl silica-based adsorbent, and with the same set of the solutes as before. We compare our current results with the results obtained before for similar HPTLC and PPEC systems, and discuss the influence of particular variables on retention, electrophoretic mobility of solutes and electroosmotic flow of the mobile phase. We show, that the final selectivity of peptide separation results from co-influence of all the three factors mentioned. Concentration of organic modifier of the mobile phase, as well as concentration of ion-pairing additive, affect the retention, the electrophoretic mobility, and the electroosmotic flow simultaneously. This makes independent optimization of these factors rather difficult. Anyway PPEC offers much faster separation of peptides with quite different selectivity, in comparison to HPTLC, with similar adsorbents and similar mobile phase composition. However, we also present and discuss the issue of extensive tailing of peptide zones in the PPEC in comparison to similar HPTLC systems.

Reversed-phase stepwise gradient thin-layer chromatography of 19 test dye mixtures with a single void volume of the mobile phase

Satisfactory separation of a test mixture of 19 dyes with a general elution problem was obtained by reversed-phase stepwise gradient thin-layer chromatography with single void volume of the mobile phase. An effect of the supplementary mobile phase flux to the surface of the adsorbent layer, observed previously, was eliminated. This improves the flow profile of the mobile phase and the shape of spots, and increases the repeatability of the results within the same plate and between studies. The experimental values of the relative zone/spot position, Rpg, showed good correlation with the results predicted by the computing calculation.

Comparison of the separation selectivity of purine derivatives in high-performance thin-layer chromatography and pressurized planar electrochromatography systems

Our article presents the comparison of two methods: high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC), implemented for the separation of a test mixture of purine derivatives. The two methods were compared in terms of separation selectivity and separation time. Our results show that PPEC enables the separation of the mixture (azathioprine, caffeine, theobromine, theophylline and acyclovir) which could not be efficiently separated in the HPTLC system, due to the different selectivities of separation. PPEC also enables to obtain a much faster separation, performed on the longer distance, in comparison to HPTLC. This makes PPEC a technique which can be useful in the analysis of purine derivatives and other drugs.