#anchrom

Method Of Separation of Coloured Dyes 

Chromatography is the science of the separation of components from a mixture. These mixtures could be anything from food, pharma, herbal, forensic or textile samples. 

Of these e.g., textile dyes, food dyes, and ink are examples of different dyes. These dyes are separated by chromatography. 

TLC/HPTLC (High-Performance Thin Layer Chromatography) is one of the best chromatographic techniques for the effective separation of dye mixtures. 

There are many examples of dyes i.e., Solvent dyes (e.g., Sudan Red R, Sudan orange G, Sudan Blue G etc.), Disperse Dyes (e.g., Celliton Fast Blue B, Celliton Fast Pink B), Azo pigments Permanent Red FR extra, Amaplast orange ORC, basic dyes (e.g., Rhodamine B, Malachite Green etc.), Acid dyes (Metanil yellow, Ponceau R) etc. 

These dyes have applications in various industries like paper, leather, textile, polyamide fibres, inks etc. Only certain acid dyes are permitted for colouring foodstuffs and such food colourants are treated under different categories) of permissible food colours. 

High-Performance Thin Layer Chromatography (HPTLC) is a powerful analytical methodology suitable for qualitative and quantitative analysis. 

HPTLC practice is performed using highly sophisticated instruments/modules and is GLP/USP/EP-compliant chromatography. 

The unique features of HPTLC are that it is the fastest, simplest, most economical and flexible, “visible” technique which can analyse more than 100 samples in parallel. It is risk-free and multiple detections can be made without repeating chromatograms. 

After doing a single analysis, the analyst gets many data points. Data is obtained at 254 nm, 366 nm, in white light and after derivatisation in white light or at 366 nm. 

For quantitative analysis, densitometric scanning is done by means of a scanner. Spectral evaluation is also possible, as it has a wide scanning range starting from 190-900nm. 

Nowadays HPTLC can also be coupled with mass spectrometry by means of a TLC-MS interface for the identification of compounds by mass-to-charge ratio and it can also be subjected to other techniques and like IR, and  NMR for further characterization studies.

Let us take one example to understand the application of HPTLC for the separation of coloured dyes simultaneous identification and quantification of synthetic food colours in Kala khatta syrup and Halwa barfi (Indian sweet) samples.

 Synthetic colours like Carmoisine, Sunset Yellow FCF and Brilliant blue FCF are FSSAI permitted colours to be used in food products.  These colours are added to food to make it visually appealing. 

The FSSAI has set a limit of 100 ppm for the usage of these food colours. Identification and quantification of these food colours in the food products can be efficiently performed by HPTLC. 

HPTLC identification and quantification of Carmoisine, Sunset Yellow FCF and Brilliant Blue FCF was performed in Halwa Barfi (sweet) and Kala khatta (syrup) samples.

After plate development, Brilliant blue FCF, Sunset Yellow FCF, and Carmoisine are separated at Rf 0.34, 0.39, 0.45 respectively in the standard mixture (combination standard) and respective food samples. 

Carmoisine and Sunset Yellow FCF are detected in Halwa Barfi and Brilliant blue FCF is detected in Kala Khatta sample. Quantification was done at λ max values of (Carmoisine) 516 nm, (Sunset Yellow FCF ) 427 nm and (Brilliant Blue FCF) 630 nm. 

The amount of Carmoisine and Sunset yellow FCF added in Halwa Barfi was detected at 8.28ppm and 3.91ppm respectively. In Kala Khatta samples, the concentration of brilliant blue FCF detected is 5.25ppm.