High‐Performance Thin‐Layer Chromatography

Abstract

The instrumental modularity of its basic steps, that is, sample application, chromatographic development, and detection, confers HPTLC a high degree of flexibility. Automated and computer‐controlled instruments with different levels of sophistication for each of these steps have been developed, for example, spray‐on sample applicators and chambers including the possibility of gradient separation. Detection techniques such as scanning densitometers, electronic documentation systems, bioactivity detection methodologies, and online coupling with mass spectrometry have come to be easily and firmly established in laboratories. Particularly, the introduction of an elution‐based TLC – MS interface has not only allowed original and efficient hyphenated systems to be designed but also opened the possibility of expanding the connection with other detectors. Thanks to this instrumental development, HPTLC has become a reliable, accurate, and sensitive analytical technique.

Planar chromatography and immunodetection of hydrocarbons on polyvinylidene difluoride membranes

“…Squalene, however, has a polarity that is relatively close to squalane, which makes their separation difficult (e.g., no narrow signals) under the studied conditions (Figure 1). The resolving power of PMC may also be affected by matrix effects, evidenced by signal‐broadening of phytol and squalene when chromatographed as a sample mixture, a phenomenon known in TLC and other chromatographic approaches [2]. Increasing of the migration distance (over 40 mm) can potentially improve the separation, however, it also increases the diffusion of the analyte on the membrane, affecting the signal resolution (Figure S3).…”

“…Here, we showed that PVDF membranes can also be used as the stationary phase in planar chromatographic assays of lipids and hydrocarbons of diverse origin, an application that, to our knowledge, has not been yet reported. Consequently, similar TLC applications and advantages are shared by PMC, such as the possibility of studying all nonvolatile analytes in a sample, applying different detection techniques and effect‐directed analysis to the same chromatographic run, reduced cost, high sample throughput, and overall simplicity compared to column chromatographic methods [2,4]. We showed that is possible to separate hydrocarbons and lipids from complex samples by PMC.…”

“…The PMC procedure follows the same operational format as paper chromatography and TLC [1,2]. Samples were loaded using a 10-μL glass syringe (Hamilton, Reno, NV, USA) as spots of 1 μL (∼0.07 mm 2 ) aligned 8 mm away from the bottom edge of a 40 × 50 mm PVDF membrane (0.2 μm pore size, 175−225 μm thickness; Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Analytical separation using TLC provides a relatively simple, cost-effective, and reliable high-throughput method for the analysis of diverse lipids and hydrocarbons. The capability of analyzing all sample components, without the need for sample pretreatment, using different detection Article Related Abbreviations: PMC, PVDF membrane chromatography; PVDF, polyvinylidene difluoride; SARA, saturate, aromatic, resin, and asphaltene; TBS, Tris-buffered saline methods applied to the same chromatographic run, gives TLC (and its high-performance application-HPTLC) a major analytical advantage compared to LC or GC, despite its lower resolution [1,2]. Developed TLC plates serve as archives, to which intrinsic or induced optical characteristics [3], chemical staining, effect-directed analysis [4], immunostaining [5,6], and mass spectrometric methods [7,8] can be directly applied to identify the resolved analytes and determine their physicochemical properties, biological effects, and structure.…”

HPTLC coupled to ESI-Tandem MS for identifying phospholipids associated to membrane proteins in photosynthetic purple bacteria

“…The use of an elution-based interface to directly coupling HPTLC with ESI-MS equipments has become popular and has been applied to a wide number of analytes, including lipids [2,3]. Using this interface, a modular, simple, rapid and direct transfer of separated HPTLC bands can be achieved in the desired zones of the plate, owing to: the open HPTLC configuration; the post-chromatographic removal of the mobile phase before extraction by the interface solvent; and the previous detection of the relevant chromatographic zones by densitometry.…”