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.     

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.     

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.     

New surfaces for desorption electrospray ionization mass spectrometry: porous silicon and ultra-thin layer chromatography plates

The performance of nanoporous silicon (pSi) and ultra-thin layer chromatography (UTLC) plates as surfaces for desorption electrospray ionization (DESI) was compared with that of polymethyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE), both popular surfaces in previous DESI studies. The limits of detection (LODs) and other analytical characteristics for six different test compounds were determined using all four surfaces. The LODs for the compounds were in the fmol-pmol (pg-ng) range. The LODs with the pSi surface were further improved for each of the compounds when heat was applied to the surface during sample application which gave LODs as low as or lower than those achieved with PMMA and PTFE. The UTLC plates were successfully used as a rapid means of chromatographic separation prior to DESI-MS analysis. Another advantage achieved using the newer pSi and UTLC surfaces was increased speed of analysis, associated with drying of solution-phase samples. This took place immediately at the UTLC surface and it could be achieved rapidly by gently heating the pSi surface. The presence of salts in the sample did not cause suppression of the analyte signal with any of the surfaces.     

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.     

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.     

On-chip ultra-thin layer chromatography and surface enhanced Raman spectroscopy

We demonstrate that silver nanorod (AgNR) array substrates can be used for on-chip separation and detection of chemical mixtures by combining ultra-thin layer chromatography (UTLC) and surface enhanced Raman spectroscopy (SERS). The UTLC-SERS plate consists of an AgNR array fabricated by oblique angle deposition. The capability of the AgNR substrates to separate the different compounds in a mixture was explored using a mixture of four dyes and a mixture of melamine and Rhodamine 6G at varied concentrations with different mobile phase solvents. After UTLC separation, spatially-resolved SERS spectra were collected along the mobile phase development direction and the intensities of specific SERS peaks from each component were used to generate chromatograms. The AgNR substrates demonstrate the potential for separating the test dyes with plate heights as low as 9.6 μm. The limits of detection are between 10(-5)-10(-6) M. Furthermore, we show that the coupling of UTLC with SERS improves the SERS detection specificity, as small amounts of target analytes can be separated from the interfering background components.     

Electrospun glassy carbon ultra-thin layer chromatography devices

The development and application of electrospun glassy carbon nanofibers for ultra-thin layer chromatography (UTLC) are described. The carbon nanofiber stationary phase is created through the electrospinning and pyrolysis of SU-8 2100 photoresist. This results in glassy carbon nanofibers with diameters of ∼200–350 nm that form a mat structure with a thickness of ∼15 μm. The chromatographic properties of UTLC devices produced from pyrolyzed SU-8 heated to temperatures of 600, 800, and 1000 °C are described. Raman spectroscopy and scanning electron microscopy (SEM) are used to characterize the physical and molecular structure of the nanofibers at each temperature. A set of six laser dyes was examined to demonstrate the applicability of the devices. Analyses of the retention properties of the individual dyes as well as the separation of mixtures of three dyes were performed. A mixture of three FITC-labeled essential amino acids: lysine, threonine and phenylalanine, was examined and fully resolved on the carbon UTLC devices as well. The electrospun glassy carbon UTLC plates show tunable retention, have plate number, N, values above 10,000, and show physical and chemical robustness for a range of mobile phases.     

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.     

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.     

Coupling protocol for thin layer chromatography/matrix-assisted laser desorption ionization

Summary This paper reports the investigation and optimization of the thin layer chromatography/matrix-assisted laser desorption/ionization (TLC-MALDI) coupling protocol. The fundamental coupling parameters which influence sensitivity and lateral analyte spreading are extraction solvent selection, extraction time, and pressure. Selection of the solvent is dependent upon its extraction efficiency, which has been correlated with extraction solventR _f value. Maximum extraction efficiency occurs after two minutes for the analyte/solvent system studied. High extraction efficiency solvents cause significant lateral spreading of analyte; up to a three-fold increase in initial analyte spot size. Analyte recovery was found to be limited by the silica gel inter-partice porosity and the solvent extraction efficiency. For maintaining chromatographic resolution and maximizing signal intensity, extraction solvents withR _f values between 0.4 to 0.6 are optimal. The upper analyte recovery limit, using extraction solvents within thisR _f range, is estimated at 22%. Dedicated to Professor Werner Haerdi on the occasion of his 70^th birthday.     

Effect of solvent composition on signal intensity in liquid chromatography-matrix-assisted laser desorption ionization experiments

The use of reversed phase liquid chromatography for the preparation of complex peptide mixtures for analysis by matrix assisted laser desorption ionization mass spectrometry has led to the observation of the critical importance of the matrix/analyte formulation in regards to the percent organic solvent in the mixture. This paper outlines the study using liquid chromatography to systematically vary the acetonitrile concentration in the formulation used for MALDI spot preparation to examine the impact the parameter has on analyte signal intensity. The results show that for five of six peptides tested across a wide mass range a formulation of approximately 75% acetonitrile is optimal for average MALDI signal intensity as determined on both time-of-flight and quadropole mass spectrometers. Examination of the individual spots shows that the organic solvent content in formulation significantly affects parameters such as crystal density and morphology.     

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.     

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.

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.     

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.     

Shpol'skii spectroscopy at the interface of two non-polar microenvironments: a novel approach for the analysis of polycylic aromatic compounds

This article presents a thorough investigation of quantitative parameters for the analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PDBFs) partitioning between an octadecyl silica extraction membrane and microliters of an n-alkane solvent spiked on the surface of the membrane. Shpol'skii spectrometry is directly performed on the surface of the membrane with the aid of a cryogenic fiber optic probe. The analyte concentration in the layer of Shpol'skii solvent follows a linear relationship with the analyte concentration in the water sample and the same is observed for the phosphorescence signal of the cryogenic probe. The accuracy and precision needed for quantitative analysis in aqueous samples is demonstrated. The analytical figures of merit show the feasibility to determine organic pollutants at the parts-per-trillion level with minimum solvent consumption.     

Fabrication of polymer microsphere particle standards containing trace explosives using an oil/water emulsion solvent extraction piezoelectric printing process

We present a methodology for fabricating polymer microspheres using inkjet printing of a biodegradable polymer containing either high explosives or high explosive simulant. Poly(dl-lactide/glycolide) 85:15 (PLGA) microsphere production is based on an oil/water emulsion solvent extraction process. The inkjet printing process allows for precise control of the microsphere diameter and the chemical composition. The microspheres can be used as calibrants or verification standards for explosives trace detection instruments. Gas chromatography/mass spectrometry analysis demonstrated that the composition of the microspheres was consistent with predicted concentrations based on the amount of analyte incorporated into the polymer solution and the inkjet operating parameters. We have demonstrated that the microspheres can be fabricated with a mass fraction of 70% of an analyte compound.     

Quantitative transfer of polar analytes on a solid surface to a liquid matrix in MALDI profiling

In profiling of a specimen by matrix‐assisted laser desorption ionization (MALDI) using a solid matrix, the solvent of the matrix solution extracts an analyte(s). A quantitative profiling cannot be achieved if the solvent evaporates before the complete extraction of the analyte. The extraction can become more quantitative when a liquid matrix dissolved in a solvent is used, which remains a liquid even after the evaporation of the solvent. To check this, radii of an analyte circle (r_A), a matrix solution drop (r_D) and a liquid matrix (r_M) remaining after the solvent evaporation were controlled. Three types of samples were prepared, case A (r_A, r_D < r_M), case B (r_D ≤ r_M < r_A) and case C (r_M < r_D < r_A). In case A, the analyte amount in the matrix layer determined by MALDI was the same as the prepared amount inside the analyte circle. In case B, the analyte amount was the same as the amount inside the matrix circle. Only the analytes in contact with the liquid matrix layer, not more and not less, are transferred to the matrix layer. In case C, the analyte amount was greater than the amount inside the matrix circle, presumably because some of the analyte outside the matrix circle was dissolved by the solvent of the matrix solution. Copyright © 2016 John Wiley & Sons, Ltd.     

Impact of column temperature and mobile phase components on selectivity of hydrophilic interaction chromatography (HILIC)

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.