Ultrathin-layer chromatography

The development of ultrathin-layer silica gel plates with a monolithic structure opens up a new dimension in thin-layer chromatography (TLC). The very small layer thickness of approximately 10 microm and the absence of any kind of binder in combination with the framework of this stationary phase lead to new and improved properties of these ultrathin-layer chromatographic (UTLC) silica-gel plates compared with conventional TLC and high-performance TLC (HPTLC) precoated layers. First of all, the advantages of the UTLC plates are the very short migration distances and, in combination with this, the short development times as well as the very low consumption of solvents as the mobile phase in connection with high sensitivity. The separations of amino acids, pesticides, pharmaceutically active ingredients, phenols, and plasticizers effectively demonstrate the possibilities of the new ultrathin-layer silica-gel plates. Furthermore, a comparison of UTLC, HPTLC, and TLC concerning retention behavior, efficiency, detection limits, migration times, and solvent consumption is performed effectively by the separation of caffeine and paracetamol.     

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.     

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.     

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.     

Inkjet application, chromatography, and mass spectrometry of sugars on nanostructured thin films

Ultrathin-layer chromatography (UTLC) potentially offers faster analysis, reduced solvent and sample volumes, and lower costs. One novel technique for producing UTLC plates has been glancing angle deposition (GLAD), a physical vapor deposition technique capable of aligning macropores to produce interesting separation properties. To date, however, GLAD-UTLC plates have been restricted to model dye systems, rather than realistic analytes. This study demonstrates the transfer of high-performance thin-layer chromatography (HPTLC) sugar analysis methods to GLAD-UTLC plates using the office chromatography framework. A consumer inkjet printer was used to apply very sharp low volume (3–30 nL) bands of water-soluble analytes (lactose, sucrose, and fructose). Analytic performance measurements extrapolated the limits of detection to be 3–5 ng/zone, which was experimentally proven down to 60–70 ng/band, depending on the sugar. This qualitative analysis of sugars in a commercially available chocolate sample is the first reported application of GLAD-UTLC to food samples. The potential utility of GLAD-UTLC is further exemplified by successful coupling with electrospray ionization mass spectrometry for the first time to characterize underivatized sugars.

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.     

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.     

Comparison of log k′ — solvent composition relationships in thinlayer and column chromatography for systems of the type: Silanized silica — water + methanol

Relationships between log k' values of polynuclear hydrocarbons and composition of water/methanol mixtures were determined for HPTLC on RP-18 silica and for HPLC using RP-2 silica. In spite of differences in the adsorbents used, a good correlation between HPLC and TLC parameters was found. In the TLC experiments, a sandwich tank with a modified solvent distributor was used. It has been reported that thin-layer chromatography is a good technique for the preliminary optimization of column chromatographic separations [1–5]. So far, the comparison of TLC and HPLC has been mainly restricted to silica as the adsorbent. Since TLC plates precoated with silanized adsorbents recently became commercially available, it seemed interesting to compare the chromatographic parameters obtained for the “reversed phase” systems with the two techniques (see also ref. 6).     

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.     

Simultaneous Densitometric Determination of Rifampicin and Isoniazid by High-Performance Thin-Layer Chromatography

JPC – Journal of Planar Chromatography – Modern TLC - High-performance thin-layer chromatography (HPTLC) on silica gel 60 F254 TLC plates, with ethyl acetate-methanol-acetone-acetic...     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

HPTLC Method for Determination of 20-Hydroxyecdysone in Sida rhombifolia L. and Dietary Supplements

Naturally occurring 20-hydroxyecdysone is an important anabolic ecdysteroid. A simple thin-layer chromatography method to quantitate 20-hydroxyecdysone in methanolic extract of the whole plant material of Sida rhombifolia L. was developed. This method was successfully applied for quantitative evaluation of dietary supplements. The separation was achieved on glass TLC plates coated with silica gel 60F254, using chloroform: methanol (8:2 v/v) as developing solvent. Densitometric evaluation of 20-hydroxyecdysone was performed at 250 nm in reflectance/absorbance mode. The calibration was in the range of 200–1,000 ng spot^?1 and correlation coefficient for the calibration curve was >0.999. In addition, for six different Sida species unique fingerprints were obtained on the HPTLC plate.     

Two-Dimensional Planar Chromatography with a Closed Adsorbent Layer

A new two-dimensional chromatography process on closed plates is proposed. A previously described device with ethanol as a mobile phase is used to experimentally study two-dimensional thin-layer chromatography (2D TLC) on plates with 5 × 5 and 10 × 10 cm sizes. This work includes the experimental study and comparison of the following TLC versions: (1) the proposed 2D TLC version with a closed adsorbent layer with 1D separation on closed plates (an ascending mobile phase flow) and (2) conventional 2D TLC with an open adsorbent layer. The ascending version of 2D TLC with a closed adsorbent layer considerably (byR~55%) shortens the chromatographic time, especially with the use of 5 × 5 cm plates in the 2D separation version compared with the 1D version on 10 × 10 cm plates.     

Analysis of Glycosaminoglycan Oligosaccharides by Combined HPTLC/MALDI-TOF MS: Reduced Silica Gel Thickness Leads to Improved Spectral Qualities and Reduced Side Reactions

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method which can be applied to virtually all natural products including oligosaccharides. Unfortunately, however, the unequivocal identification of a TLC spot is normally difficult. Fortunately, this problem can be minimized when mass spectrometry (MS) such as matrix-assisted laser desorption and ionization time-of-flight is used to identify the TLC spots. This work is dedicated to the TLC/MS analysis of oligosaccharides derived from native chondroitin sulfate and hyaluronan. We will show that the thickness of the silica gel layer (200 versus 100 µm) has a tremendous influence on the quality of the mass spectra: a reduced silica gel thickness enhances the spectral quality and, in particular, improves the achievable signal-to-noise ratio. Additionally, unwanted formylation of the GAG oligosaccharides (which occurs due to the high moiety of formic acid in the mobile phase) can also be minimized if MS-grade HPTLC plates are used.

     

HPTLC Separation and Determination of Banned Amine Isomers,with Reference to the German Ban on Azo Dyes

JPC – Journal of Planar Chromatography – Modern TLC - High-performance thin-layer chromatography (HPTLC) on silica plates, with two successive mobile phases, has been used for analysis...     

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.     

Identification of subnanomole amounts of PTH-amino acids by high performance thin-layer chromatography

Summary High performance thin-layer chromatography (HPTLC) of PTH-amino acids on 5×5 cm silica gel plates precoated with a fluorescence marker gives 10–20 fold increase in sensitivity compared to ordinary silica gel plates. Separation of PTH-Leu from PTH-Ile is easily achieved in contrast to chromatography on polyamide sheets. Only two solvent systems are required and as many as 12 samples can be chromatographed on each plate. However, if the sample is contaminated with N-phenylthiourea a third solvent system is necessary.     

Determination of Sumatriptan and Zolmitriptan in Presence of Their Corresponding Degradation Products by HPTLC Methods

Accurate, sensitive, and precise high performance thin layer chromatographic (HPTLC) methods were developed and validated for the determination of sumatriptan and zolmitriptan in presence of their degradation products. Sumatriptan was separated from its degradation products and analyzed on TLC silica gel 60 F₂₅₄ plates using chloroform–ethyl acetate–methanol–ammonia (4:3:3:0.1, v/v) as a developing system followed by densitometric measurement of the bands at 228 nm. Zolmitriptan was determined using chloroform–ethyl acetate–methanol–ammonia (3:3:3:1, v/v) as a developing system followed by densitometric measurement at 222 nm. The methods were validated over a range of 0.5–4 μg/spot for sumatriptan and 0.5–3 μg/spot for zolmitriptan. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in their pharmaceutical formulations.     

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.     

The quantitative detection of estrogens and antithyroid drugs by thin-layer and high performance thin-layer chromatography in animal tissue (author's transl)]

Methods of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) were developed for the determination of estrogens and antithyroid drugs in extracts of animal tissues. TLC proved to be suitable for quantities in the range of 200-2000 ng, with a detection limit of 50-200 ng, HPTLC in the range of 10-200 ng, showing calibration curves of good linearity even in extracts. By HPTLC better detection limits, better separation and faster ascending could be achieved than by TLC.