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Wong Wai Khet



Thin Layer Chromatography(TLC) Analysis
High Performance Liquid Chromatography(HPLC) Analysis
This paper will use the information in “Qualitative and Quantitative Estimation of Hydroquinone in
Skin Whitening Cosmetics” as the example of using this two method to determine hydroquinone in

Thin Layer Chromatography(TLC) Analysis
Sample preparation     

Weigh 2 grams of sample in a 25 ml beaker and add 15 ml of 96% (V/V) ethanol.
Homogenized the mixture on water bath at 60°C for 10 min
Cooled in an ice bath till the separation of fats occurred.
Use TLC analysis to filtered and filtrate.
Repeat the same procedure for all samples.

Preparation of Reference Solution 
Dissolving 0.05 g of hydroquinone in small amount of ethanol 96% (V/V) in 25 ml volumetric flask
and finally making up the volume up to the mark.
Note: The standard solution should be freshly prepared as it is stable for less than one day at room
TLC Procedure    

Prepare TLC plates (20 × 20 cm) of 0.25 mm thickness using silica gel (90 g) and water (180 ml).
Air dry the plates and activate by heating in an oven at 105°C for an hour.
The solvent system: n- Hexane/Acetone, ratio at 3:2.
Deposit twenty micro liters of each sample and standard solution on plates and develop them at
room temperature in a vertical separating chamber to the height of approximately 16 cm from the
Previously saturated the chamber with the appropriate mobile phase (saturation time was 1 hour).
After drying, performed visualization in two ways: 
In short UV light (254 nm) 
Spraying with 0.2% ethanolic dichlorofluorescein, interprete chromatograms in long wave
UV light (366 nm). 
Calculate the Rf values for each spot.

(2012). Dermatological Sciences and Dissolve it in small amount of mobile phase and made up volume to the mark. Diluted it and made up volume to the mark by mobile phase.High Performance Liquid Chromatography(HPLC) Analysis Sample preparation       Weight 2 ± 0.4236/jcdsa.23042 . Journal of Cosmetics. It is homogenize in a water bath at 60°C for 10 min.05 g of hydroquinone to a 50 ml volumetric flask. Mix 25 ml of mobile phase (Water : Methanol mixture 45:55) and add into the beaker. Filter the mixture and transfer to 100 ml volumetric flask. Pipetted 5 ml of this solution into a 50 ml volumetric flask.1 wref = Weight of hydroquinone in reference solution wspl = Weight of hydroquinone in sample solution Reference Siddique. http://doi.2012. The mobile phase is a mixture of water and ethanol (45:55) with a flow rate of 1. The volume is made up to the mark by mobile phase.6 mm). Measure and compare peak area for each sample between reference and sample solutions peaks. Preparation of Reference Solution     Weight and transfer 0. HPLC Procedure       Use a Schimadzu LC-9 with UV detector set at 295 nm having ODS column (25 cm × 4.5 ml/min. Inject 20 µL of each sample solution and recode the chromatogram. Adjust twenty at 35°C ± 1°C.1 g of a sample accurately into a beaker. Then cool it in an ice bath until the separation of fats and waxes occurred. Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening Cosmetics. S. Calculate the amount of hydroquinone as percentage by mass using the formula A %age of Hydroquinone = bi/pi ൈ wref/wspl ൈ d ൈ 100 Where bi = Peak area of hydroquinone in sample solution pi = Peak area of hydroquinone in reference solution d (dilution factor) = 0.