Ultrathin-Layer Chromatography (UTLC)

Ultrathin-layer chromatography (UTLC) differs from high-performance thin-layer chromatography (HPTLC) and from thin-layer chromatography (TLC) in two basis things: the layer thickness, and the migration distances of the analytes. UTLC has a monolithic or a nanostructured stationary silica gel phase bound directly to the glass plates. Layer thickness in UTLC is 10 μm, instead of 100–250 μm in HPTLC. Migration distances are in the range of 1–3 cm for UTLC, instead of 8–10 cm for HPTLC. Therefore, the major advantages of UTLC over HPTLC and TLC are the shorter development times and higher separation efficiency and sensitivity. Moreover, separations on UTLC plates require smaller reagent and sample volumes. However, the UTLC plates are very difficult to manage with the TLC and HPTLC equipment currently available. Therefore, the next challenge in this area is the development of an inexpensive solution with appropriate instrumentation (sensitive optical scanners and sample application systems). UTLC had been used for separations of many compounds, e.g., pharmaceutically active ingredients, pesticides, plasticisers, natural products, and other chemical substances.     

Morphological modification of nanostructured ultrathin-layer chromatography stationary phases

Ultrathin-layer chromatography (UTLC) provides the high sensitivities and rapid separations over short distances desirable in many analytical applications. The dependence of these performance benefits on UTLC layer microstructure motivates continued stationary phase engineering efforts. A new method of modifying the elution behaviours of nanostructured thin film UTLC stationary phases is investigated in this report. Macroporous normal phase silica thin films ～5 μm thick were fabricated using glancing angle deposition (GLAD). Reactive ion etching (RIE) and a subsequent annealing treatment modified stationary phase morphology to tune migration velocity, analyte retention, and overall separation performance. Combining this technique with a RIE shadow mask enabled fabrication of adjacent concentration and separation zones with markedly different elution properties. Although produced using an entirely new approach, GLAD UTLC concentration zone media behaved in a manner consistent with traditional thin-layer chromatography (TLC) and high-performance TLC (HPTLC) concentration zone plates. In particular, these new media focused large volume, low concentration dye mixture spots into narrow bands to achieve high-quality separations. The described approach to modifying the morphology and resultant elution behaviours of nanostructured stationary phases expands the capabilities of the GLAD UTLC technique.     

Comparison of TLC and Different Micro TLC Techniques in Analysis of Tropane Alkaloids and Their Derivatives Mixture from Datura Inoxia Mill. Extract

Planar chromatography is a very useful tool for analysis of wide range of different mixtures. Thanks to its possibility for rapid separation of large number of samples simultaneously, low solvent consumption and ability to analyse rough material allow to receive precise and reliable results in short time and low cost. Miniaturization of planar techniques brings a lot of advantages, such as shortening distance and time of chromatogram development, and further lowering of solvent consumption. Besides, it often allows to improve separation parameters and raise efficiency of chromatographic system. In this paper, ability of analysis of tropane alkaloids mixture from Datura Inoxia Mill. extract using conventional TLC technique with five micro TLC techniques (short distance TLC, HPTLC, UTLC, OPLC and ETLC) in maximally closed chromatographic conditions was compared in order to present abilities of micro TLC techniques in plant material analysis.     

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.     

Analyte migration in anisotropic nanostructured ultrathin-layer chromatography media

We investigate the performance of highly anisotropic nanostructured thin film ultrathin-layer chromatography (UTLC) media with porosity and architecture engineered using the glancing-angle deposition (GLAD) process. Our anisotropic structures resemble nanoblades, producing channel-like features that partially decouple analyte migration from development direction, offering new separation behaviours. Here we study GLAD-UTLC plate performance in terms of migration distance, plate number, retention factor and a figure of merit specific to GLAD-UTLC, track deviation angle. Migration distances increase with porosity by a factor of two for all feature orientations (up to a maximum of 22 mm) over the range of porosities considered in this study. Plate numbers approaching 1100 are observed for GLAD-UTLC plates when the nanoblade features are aligned with the development direction. We present a theoretical model describing mobile phase flow in anisotropic GLAD-UTLC media, and find good agreement with experimental results. Our plates provide channel features that reduce transverse spot broadening while providing the wide pores required for rapid migration and high separation performance. These improvements may enable a greater number of parallel separations on miniaturized GLAD-UTLC plate formats. Their small sizes should also make them compatible with the Office Chromatography concept in which office peripherals (inkjet printers and flatbed scanners) replace conventional TLC instruments. Equipped with a better understanding of the unique GLAD-UTLC elution behaviours, we expect to further improve performance in the future.     

UTLC: An Advanced Technique in Planar Chromatography

As part of increasing research in the field of separation science, there have been many efforts to undertake planar chromatography with more efficient separation and better resolution in the shortest period of time, together with a specificity and a capability to identify more precisely an unknown compound present in a mixture. Ultra-thin layer chromatography (UTLC) is a modern technique which gives separation within 10–30 mm and development in just 1–6 min, with the consumption of less solvent. The stationary phase of UTLC is made up of a silica gel monolithic layer of 10 μm thickness having 3- to 4-nm mesopores and 1- to 2-μm macropores. Glancing angle deposition (GLAD)-UTLC is a modification of UTLC which gives separation within 15 mm distance and in less than 2 min. Anisotropic media of GLAD UTLC gives a unique migration direction effect. UTLC atmospheric pressure–matrix-assisted laser desorption ionizer–mass spectrometery (UTLC-AP-MALDI-MS) is a choice of technique for the identification of an unknown compound in a mixture or an impure form. ULTC-AP-MALDI-MS allows the fast changing of plates, produces more intact protonated molecules, less fragmentation and less entry of chromatographic material, and yielding less complicated spectra than the vacuum condition. Thus, UTLC is a useful technique for very rapidly giving the separation and identification of new components present in mixtures. This review provides a brief overview of UTLC, the stationary phases used for UTLC, and the detection options and applications of UTLC.     

Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.     

Stationary phases for thin-layer chromatography

This paper presents a review of the literature concerning development of the stationary phases for thin-layer chromatography (TLC) in the last ten years. The silica gel remains the most important adsorbent for TLC separation. The kinetic properties of the silica-gel thin layer and the new TLC plates have been presented. Other materials used as stationary phase were alumina, zirconium oxide, Florisil, and ion-exchanger. Chemically new bonded stationary phase development is also discussed. The improvement of the separations of some organic mixtures by impregnation of silica gel, cellulose, or polyamide plates (with transition metal ions and silver salts) and their applications is presented. The impregnation of the thin layer with organic stationary phase and inclusion complexes is another method used for the enhancement of the separation efficiences. Another modality to improve the selectivity in TLC using ion-pairing as reagent of impregnation is described as well. The actual state of chiral separation by TLC is discussed with concrete references to recent advances in chiral stationary phases. The use of nonpolar chemically bonded stationary phases impregnated with transitional metal ions is presented as chiral stationary phases. The cellulose, modified cellulose, chitin, chitosan, and their derivatives are presented and their potential for the analysis of the racemates is discussed. The cyclodextrines and macrocyclic antibiotics were used with very good results for enantiomeric separation by TLC. A new separation approach with molecular imprinting polymers was reported as a chiral stationary phase in TLC. The examples provide a wide range of structural types that can be readily resolved enantiomerically by TLC.     

Ultrathin layer chromatography on nanostructured thin films

Ultrathin layer chromatography (UTLC) is a relatively new variant of thin layer chromatography, with a 10mum thick monolithic silica sorbent layer that gives faster separations with lower limits of detection and reduced analyte and solvent volumes. We have produced UTLC plates with controllable nanostructure and thickness, and show that the layer separation characteristics depends on the film nanostructure. We also show that layers made with in-plane anisotropic nanostructures will exhibit a decoupling effect, where the analyte spots do not develop in the same direction as the solvent front movement. The added layer morphology and material selection adds a degree of freedom to UTLC, and may have applications in multi-dimensional TLC.     

New TLC/HPTLC commercially prepared and laboratory prepared plates: A review

Thin-layer chromatography (TLC) had its initial growth in the 1950s when TLC sorbents and devices for making TLC plates in the analytical laboratory became available. A resurgence in TLC use occurred when commercially prepared plates became available around 1965. Their advantage was greater reproducibility because of their uniformity and convenience of use. Having just passed the 50th anniversary of this date, TLC still finds wide application as a useful analytical tool throughout the world. The introduction of high-performance TLC (HPTLC)-prepared plates was also a welcome addition to the chromatography laboratory. Today, advances in TLC instrumentation that aid in sample application, plate development, qualification, and quantification continue to evolve and improve. The TLC/HPTLC plate manufacturers have continued to add new prepared plates to meet the greater demands for higher purity or special applications for these newer devices. More recently, researchers have experimented with new sorbents or preparation techniques that have resulted in special properties for thin-layer-prepared plates, particularly for use in TLC–MS applications. This article will discuss not only some classical TLC plates but also these newer thin layers, their advantages, and some of their applications.     

HPTLC on inert pre-coated layers of cellulose and of silica 50000

Summary In a manner analogous to that for surface-active silica gel, HPTLC pre-coated plates for nano TLC have also been developed from two inactive sorbents. The two materials are microcrystalline cellulose and a synthetically produced, porous silica (Silica 50000) with a very low specific surface area. The chromatographic properties of these inert sorbents and of the new HPTLC pre-coated plates prepared therefrom are examined in relation to separations of amino acid mixtures and carbohydrate mixtures and are related to the chromatographic properties of the inactive sorbents and TLC precoated plates used hitherto. The figure 50000 characterizes the type of silica. The average pore diameter of this sorbent is about 5000 nm.     

Aligned electrospun nanofibers for ultra-thin layer chromatography

The fabrication and implementation of aligned electrospun polyacrylonitrile (PAN) nanofibers as a stationary phase for ultra-thin layer chromatography (UTLC) is described. The aligned electrospun UTLC plates (AE-UTLC) were characterized to give an optimized electrospun mat consisting of high nanofiber alignment and a mat thickness of ∼25 μm. The AE-UTLC devices were used to separate a mixture of β-blockers and steroidal compounds to illustrate the properties of AE-UTLC. The AE-UTLC plates provided shorter analysis time (∼2–2.5 times faster) with improved reproducibility (as high as 2 times) as well as an improvement in efficiency (up to100 times greater) relative to non-aligned electrospun-UTLC (E-UTLC) devices.     

A review of advances in two-dimensional thin-layer chromatography

Although there are many simple thin-layer chromatography (TLC) separations, many more are complex and involve more than a few components, that means having to use special high-performance TLC (HPTLC) plates or microspotting or banding devices to increase its resolving power if developing in only one direction. However, adding a second development to perform two-dimensional TLC (2D TLC) allows even better resolution of complex samples. This is because different modes of chromatography are being invoked by the use of one stationary phase with two mobile phases, bilayer plates, graft TLC, or multidimensional TLC. This paper is a review of recent applications that have benefitted from using 2D TLC in its various forms. They were chosen for their variety of sample types as well as the unique choices of plates and/or mobile phases made by the researchers to yield improved separations.     

Carbon nanotube and carbon nanorod-filled polyacrylonitrile electrospun stationary phase for ultrathin layer chromatography

The application of carbon nanotube or nanorod/polyacrylonitrile (PAN) composite electrospun nanofibrous stationary phase for ultrathin layer chromatography (UTLC) is described herein. Multi-walled carbon nanotubes (MWCNTs) and edge-plane carbon (EPC) nanorods were prepared and electrospun with the PAN polymer solution to form composite nanofibers for use as a UTLC stationary phase. The analysis of laser dyes demonstrated the feasibility of utilizing carbon nanoparticle-filled electrospun nanofibers as a UTLC stationary phase. The contribution of MWCNT or EPC in changing selectivity of the stationary phase was studied by comparing the chromatographic behavior among MWCNT–PAN plates, EPC–PAN plates and pure PAN plates. Carbon nanoparticles in the stationary phase were able to establish strong π–π interactions with aromatic analytes. The separation of five polycyclic aromatic hydrocarbons (PAHs) demonstrated enhanced chromatographic performance of MWCNT-filled stationary phase by displaying substantially improved resolution and separation efficiency. Band broadening of the spots for MWCNT or EPC-filled UTLC stationary phases was also investigated and compared with that for pure PAN stationary phases. A 50% improvement in band dispersion was noted using the MWCNT based composite nanofibrous UTLC plates.     

Miniaturized HPTLC of Vitamin B12 Compounds in Foods

High-performance thin-layer chromatography (HPTLC) is a separation technique commonly used to identify and quantify compounds in chemical mixtures. Preliminary experiments indicated that Lichrospher silica gel 60 F254s HPTLC sheets were the most suitable for analyzing vitamin B₁₂ compounds. This study revealed the advantages of miniaturized Lichrospher HPTLC for analyzing authentic vitamin B₁₂ compounds as short migration distances (5 cm) and short development times (<45 min) in combination with high separation efficiency and sensitivity (>25 pmol at 254 nm). The practicability of using miniaturized HPTLC was demonstrated by the separation and identification of vitamin B₁₂ compounds purified from foods using an immunoaffinity column.     

Fast separation and quantification of C60 and C70 fullerenes using thermostated micro thin-layer chromatography.

Thermostated micro planar chromatography was applied for systematic separation studies of C60 and C70 fullerenes using n-alkanes as mobile phases on TLC and HPTLC plates coated with polyamide, silica gel, aluminum oxide as well as two types of octadecylsilica (C18) sorbents. Retention data were collected at constant temperature at 20 degrees C (+/-0.05 degrees C) using an unsaturated chamber mode with an eluent, such as n-pentane, n-hexane and n-heptane. The separation results under both saturated and unsaturated chamber modes for selected mobile/stationary phases were also examined, and several parameters, including separation factor (alpha) and resolution (R(S)), were compared with data obtained with high-performance liquid chromatography conditions. Interestingly, C60/C70 fullerenes separation performed on HPTLC plates with a developing distance of 45 mm was better for those observed on a 25 cm length analytical HPLC column under similar conditions to that on carbon coverage of the stationary phase, n-hexane as the mobile phase and separation temperature (R(S) = 1.84 and 1.68 for HPTLC, and HPLC, respectively). Moreover the advantage of the planar chromatographic separation of fullerenes studied is a short elution time of less than 6 min. Furthermore, the reported separation protocol shows a capability for the evaluation of fullerenes quantity in commercial samples.     

Critical evaluation of experimental conditions influencing the surface-enhanced Raman spectroscopic (SERS) detection of substances separated by layer liquid chromatographic techniques

Summary Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F₂₅₄ and Si60 F₂₅₄ Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms. SERS detection of ascorbigen and 1′-methylascorbigen was performed after chromatography on silica gel 60 F₂₅₄ TLC and HPTLC plates and on Si60 F₂₅₄ Raman TLC plates. Traditional development was used for the silica gel 60 F₂₅₄ TLC plates and Si60 F₂₅₄ Raman plates, and the personal OPLC technique for the silica gel 60 F₂₅₄ HPTLC plates. It was found that the SERS spectrum gave information about the indole ring only. Because bonding of the analyte to the stationary phase results in a change in molecular conformation-in contrast with the behaviour of rhodanine-the type of the plateused and the development procedure employed can significantly influence the quality of the SERS spectrum. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary September 1–3, 1999     

Performance,data and results with various chromatographic systems and various detection patterns in high-performance thin-layer chromatography

Summary A knowledge of the relationships between particle size and layer thickness on the one hand and migration distance and plate height on the other has prompted the development of the pre-coated HPTLC plate for nano-TLC. A separation performance which is very high with respect to time is achieved with a migration distance of 3–7 cm with a plate height of about 12 m and several thousand theoretical plates, thanks to the possibility of simultaneous chromatography. In HPTLC quantitative evaluation, standardised chromatographic conditions are essential. In this context the complex influences of different types of chamber and solvents on linear and circular chromatography are described. Various methods of conditioning the adsorbent by immersion impregnation, as well as detection procedures are discussed. The scope for varying TLC procedures and the advantages of TLC in general are listed.     

Thin Layer Chromatographic Method for Rapid Quantification and Identification of Trimethoprim and Sulfamethoxazole in Pharmaceutical Dosage Forms

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of sulfamethoxazole (SMA) and trimethoprim (TMP) present in combined dosage forms were described. SMA and TMP were extracted with 90% acqueous methanol and the interfering and related contaminants were removed by thin layer chromatography (TLC) or high performance TLC (HPTLC) on silicagel plates using chloroform : isopropanol : diethylamine :: 10 : 6 : 1 (v/v) as mobile phase. Assay was done at the respective absorption maxima of the drugs by in situ densitometry and by spectroscopy after extracting the drugs from TLC plates with 90% acqueous ethanol. Results obtained by both the methods agreed well with those obtained by the method prescribed by the United States Pharmacopoeia XXI edition. Total time required for HPTLC and densitometric assay of 32 samples using 4 standards was 30 min. Probable source of errors in densitometric studies and their rectification was discussed.     

Determination of chenodeoxycholic acid on 3-cyanopropyltrichlorosilane-treated high-performance thin-layer chromatographic plates

Summary Chenodeoxycholic acid in commerical drugs was analyzed by high-performance, thin-layer chromatography (HPTLC) using a cyanoalkyl chemically-bonded stationary phase, prepared by treating pre-coated silica with 3-cyanopropyltrichlorosilane (3CPTS). The 3CPTS-treated plates were used to evaluate commerical chenodeoxycholic acid in drug capsules. After development with methanol on the 3CPTS-treated plate, the chenodeoxycholic acid spot in the chromatogram was measured with a TLC densitometer equipped with a dual-wave length TLC scanner at λ=370nm. By this method, the fluorescence intensity of chenodeoxycholic acid was measured within 2.6% error over the range 30–240ng and the limit of detection was 30ng.