Wong Wai Khet

SKA6014
ASSIGNMENT 4

PROCEDURE TO DETERMINE
HYDROQUINONE IN COSMETICS

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
cosmetics.

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 60C 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
temperature.
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 105C 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
start.

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.

High Performance Liquid Chromatography(HPLC) Analysis

Sample preparation

Weight 2 0.1 g of a sample accurately into a beaker.

Mix 25 ml of mobile phase (Water : Methanol mixture 45:55) and add into the beaker.
It is homogenize in a water bath at 60C for 10 min.
Then cool it in an ice bath until the separation of fats and waxes occurred.
Filter the mixture and transfer to 100 ml volumetric flask.
The volume is made up to the mark by mobile phase.

Preparation of Reference Solution

Weight and transfer 0.05 g of hydroquinone to a 50 ml volumetric flask.

Dissolve it in small amount of mobile phase and made up volume to the mark.
Pipetted 5 ml of this solution into a 50 ml volumetric flask.
Diluted it and made up volume to the mark by mobile phase.

HPLC Procedure

Use a Schimadzu LC-9 with UV detector set at 295 nm having ODS column (25 cm 4.6 mm).
Adjust twenty at 35C 1C.
The mobile phase is a mixture of water and ethanol (45:55) with a flow rate of 1.5 ml/min.
Inject 20 L of each sample solution and recode the chromatogram.
Measure and compare peak area for each sample between reference and sample solutions peaks.
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) = 0.1
wref = Weight of hydroquinone in reference solution
wspl = Weight of hydroquinone in sample solution

Reference
Siddique, S. (2012). Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening
Cosmetics. Journal of Cosmetics, Dermatological Sciences and Applications.
http://doi.org/10.4236/jcdsa.2012.23042