Visualisation of Spots in Paper Chromatography

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UV Viewing Cabinet (Image Courtesy : www.camag.com)

Coloured spots are easily observed on developed chromatograms. However, different approaches need to be adopted when colourless components are to be observed. It is convenient to classify such methods as specific or non-specific.

Non-specific methods

Iodine chamber – the developed plate is suspended in a closed jar containing a few crystals of iodine for about a minute. In presence of iodine vapour most organic compounds appear as brown spots.

UV viewing cabinet – majority of colourless compounds can be viewed under illumination with UV light in a UV viewing cabinet. Commonly the cabinets are equipped with a long wavelength (366 nm) and short wavelength ( 254 nm) light sources. Under exposure of selected source coloured spots appear on a dark background and under exposure to light from short wavelength source dark spots are seen against a bright background. In either case the spots should be marked with a pencil while observing in the cabinet for Rf calculations.

Specific Methods

Amino acids ,primary and secondary amines –A 0.2% ninhydrin solution in water saturated with butanol is sprayed on briefly heated paper chromatogram when deep blue or purple spots begin to appear.

Alkaloids – Dragendroff’s agent gives orange or orange – red precipitates in presence of alkaloids

Phenols – Ferric chloride solution spray results in appearance of different coloured spots – read, blue, green or purple when phenols are present.

Aldehydes and Ketones – 2,4 – dinitro phenyl hydrazine in a mixture of methanol and sulphuric acid (Brady’s reagent) spray results in bright orange or yellow colour spots in presence of aldehydes and ketones.

Paper chromatography is a versatile analysis technique and has wide range of applications which will be taken up in a subsequent article

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

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