Optimization of separation protocol of tryptophan and proline in a reversed-phase system using temperature-controlled micro-thin-layer chromatography (micro-TLC)

Chromatografia cienkowarstwowa jest dogodną metodą do tzw. analiz przesiewowych (z ang. screen analysis) oraz szybkiego badania profili retencji z użyciem różnych faz ruchomych. Nasze badania dotyczą zastosowania termostatowanej mikrochromatografii cienkowarstwowej do optymalizacji procesu rozdzielania wybranych aminokwasów tryptofanu i proliny w układzie faz odwróconych (RP). Uzyskane dane retencyjne umożliwiły określenie optymalnych warunków rozdzielania przy zastosowaniu prostego parametru optymalizacji: maksymalnej wartości współczynnika rozdzielenia (αmax). Parametr ten był obliczany z dokładnością 2% stężenia binarnej fazy ruchomej w zakresie od 0 do 100%, na podstawie danych eksperymentalnych uzyskanych z rozdzielczością 20%. Wybrane kryterium optymalizacji umożliwia bezpośrednie przeniesienie uzyskanych wyników na systemy rozdzielania typu RP z użyciem kolumny zamiast płytek np. wysokosprawnej chromatografii kolumnowej (HPLC).Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) are commonly used in separation, identification, purification and quantification of different analytes present in complex biological, environmental and pharmaceutical samples. Planar chromatography can be also applied to screen analysis and fast exploration of chromatographic retention profiles using different concentrations of mobile phases components. This method does not require expensive equipment, tedious and time-consuming sample pretreatments and, what is important, allows a parallel separation of many samples at the same time. The spots or bands sprayed onto the TLC plate can be easily detected under visible and UV light (usually 254nm, 366nm) or by post-chromatographic derivatization with visualization reagents, and then digitalized using office scanners. It should be noted that the great advantage of micro-planar chromatography compared to the regular sized TLC is low consumption of the eluents (usually less than 1 mL per analytical run) as well as short analysis time, due to mobile phase migration distance less than 50 mm. Under particular cases (high temperature and low viscosity eluents like acetone, dichloro-methane or n-hexane), non-forced eluent flow micro-chromatographic analysis can be completed within less than 5 minutes. This work is focused on optimization of separation process of two amino acids including tryptophan and proline (Fig. 1) under reversed-phase micro-chromatographic conditions. Particularly, selected amino acids were chromatographed on HPTLC RP-18W (wettable with water) 25 x 50 mm cutted plates using thermostated horizontal micro-chamber and unsaturated conditions (Fig. 2). The target components were sprayed on the start line (1Μg per lane) using a semi-automatic sampler (Linomat 5, Camag). The plates were developed with n-propanol: water binary mixtures as the mobile phases. Different concentrations of alcohol ranging from 0 to 100% (v/v) with steps of 20% were applied. The temperature of chromatographic separation was set at 40oC and chromatographic runs were completed within 10-30 minutes, depending on the water concentration in the mobile phase. The amino acids were visualized by dipping the plates in ninhydrin solution (prepared for concentration 0.3% in ethanol) followed by heating at 120°C for 10 minutes. Picture acquisition was performed using a Plustek OpticPro S12 scanner with an 8-bit per channel color deep mode, 300 DPI resolution, and saved as TIFF files. Retention data were derived from the plates via densitometric scans obtained with help of ImageJ freeware (http://rsb.info.nih.gov/ij). Using the raw retention data set (RF values, retardation factor), the chromatographic profiles of amino acids were calculated with step of 2% (Fig. 3A). Based on the curves observed and relationship RM = logk, where RM and k correspond to mobility and retention factors, respectively, a simple optimization criterion expressed as a separation factor (α) was calculated (Fig. 3B). Using this plot, the best separation conditions of target components were selected taking into account the maximum value of the separation factor (αmax). Due to the equal spot shape of amino acids investigated across the whole range of the mobile phases composition, such approach involving the α values allows fast optimization of separation in planar chromatography and direct application of selected conditions into column chromatographic systems based on e.g. high-performance liquid chromatography (HPLC). It should be noted that the advantages of retention data screening using micro-TLC are very low mobile phase consumption (at level of 300 ΜL per chromatographic run), short analysis time and effective detection of UV-Vis transparent substances using simple visualization reagent