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Detection methods in HP-TLC (Part I)

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TB Thite, Sr Manager (Lab), Anchrom Enterprises India, gives an insight about the features of HP-TLC

Traditionally, qualitative and quantitative detection and evaluation in High Performance Thin Layer Chromatography (HP-TLC) is done by evaluating the chromatographed plate under UV (absorbance) or fluorescence (emission). Visible light was used to evaluate coloured fractions, usually after post-chromatography derivatisation (PCD). In Part I of the series, the above technique will be described while in Part II discussions will be held on HP-TLC/MS-IR-NMR and Effect Directed Analysis.

‘Chromatogram’ Type 1 plate with separated samples
‘Chromatogram’ Type 1 plate with separated samples

At the outset, it must be noted that in HP-TLC, after separation, the ‘chromatogram’ remains on the plate and does not go to waste. Only in HP-TLC, the term ‘chromatogram’ can have three meanings viz. a) separated samples embedded on the plate b) its photographic image(s) c) its ‘scans.’ Therefore, HP-TLC ‘chromatogram’ can be evaluated by the eye as well as by instrumental means. The ‘scan’ data is similar to GC-LC output, in form of peaks.

Two things to unlearn here are that one has to ‘forget practice of TLC’ because it is not instrumental and has no universally agreed methodology. LC users must get used to parallel processing of multiple and different samples and ‘stored’ chromatograms.

‘Chromatogram’ Type 2 Image of samples on plate
‘Chromatogram’ Type 2 Image of samples on plate

The ‘plate’ i.e. separating medium in HP-TLC is 20 x 10cm in size, coated with a 0.2mm thick layer of 5µm adsorbent on a flat support. Silica gel with fluorescent indicator F254 is the adsorbent of choice, unless proved unsuitable.

Once the chromatographic development is over and the plate dried, the HP-TLC ‘chromatogram’ on plate is visually evaluated under UV 366nm for native fluorescence and then UV 254nm for substances absorbing around that region against a fluorescent green background. Next, the ‘chromatogram’ is image documented in a table top dark room, again under UV 254nm and 366nm and also white light. This documentation under GLP, when handled through HP-TLC software, enables comparative evaluation of reference and sample, qualitatively (identification) and semi-quantitatively (dilution), as per regulatory requirements.

‘Chromatogram Type 3 plate Scan of samples on plate
‘Chromatogram Type 3 plate Scan of samples on plate

Next the same ‘plate chromatogram’ is scanned in a scanner, where a monochromatic beam of chosen wavelength (between 190-800nm) travels over each sample track, detecting absorbance and, generating ‘scan chromatograms.’ UV absorbance spectra of selected or all peaks are also recorded. Then the ‘plate chromatogram’ is scanned similarly for fluorescence with 366nm as incident light and a visible light transmitting filter placed before the photomultiplier detector. In HP-TLC scanner, since the photomultiplier detector is an angle to the light source the emission can be selectively measured, without interference from the light source. No separate fluorescence detector is required. A big advantage! All the above are non-destructive methods of detection that leave the ‘chromatograms’ intact! Another big advantage!!

A chromatographed sample (on multiple tracks) can be derivatised i.e. reacted on the plate with one or more derivatisation reagents in-situ to get a treasure of additional specific and high sensitivity information about the sample; PCD is very simple, applicable to every sample and needs no special accessories in HP-TLC.

Modern PCD reagent spraying machines require only 2-4ml reagent and generate an uniform mist, leading to high quality derivatisation. PCD generates more chromatograms! Where a HP-TLC scanner is available, visualisation may not be necessary unless the fraction(s) of interest do not absorb UV down to 190nm.
In research labs, it is common to apply same sample on same plate on multiple ‘tracks’ and then evaluate all tracks with same detection technique as well as each ‘track’ by a different detection mode!! All this without repeating chromatography!

No wonder HP-TLC is called the Cindrella of Chromatography. HP-TLC proves to be a simple technique with many unique advantages, particularly with respect to data obtainable from samples, high throughput and low cost of analysis. Further information can be obtained from the same plate, using MS, IR, NMR and Effect Directed Analysis, which will be described in Part II.

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