Analytical methods for organic lighteners

Organic based active skin lightening compounds can be determined by several analytical techniques such as flow injection analysis, kinetic spectrophotomertry, gas chromatography mass spectrometry (GC-MS), differential pulse voltametry, and capillary electrochromatography (Doreen, 2010). Although gas chromatography is widely used and is a powerful chromatographic method, it is limited to compounds that have a significant vapour pressure at temperatures up to about 200 atmospheres. Thus compounds with high molecular weight and high polarity cannot be separated by gas chromatography (Doreen, 2010).

High-performance liquid chromatography (HPLC) is a chromatographic technique used to split a mixture of compounds in the fields of analytical chemistry, biochemistry and industrial. The main purposes for using HPLC are for identifying, quantifying and purifying the individual components of the mixture (Bassam and Rasool, 2012). The HPLC play an important and critical

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role in the field of analysis since it can be used to test the products and to detect the raw ingredient used to make them that is it can do both qualitative and quantitative analysis (Bassam and Rasool, 2012). Moreover it has the advantages of using relatively small amounts of the solvent, it is rapid and can accomplish difficult separation (Harada et al., 2001).

HPLC, a powerful tool in analysis uses the same principles as in thin layer chromatography and column chromatography. Chromatography has a stationery phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationery phase and carriers the components of the mixture with it. Different components flow at different rates based on their polarity. In thin layer chromatography, the stationary phase is a thin layer of silica gel or alumina on a glass, metal or plastic plate. Column chromatography works on a much larger scale by packing the same materials into a vertical glass column.

High performance liquid chromatography is therefore a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres making it faster and allows one to use small particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods used, making it sensitive and automated (Fifield and Kealey, 1995).

There are two types of HPLC depending on the polarity of the solvent and the stationery phase; normal HPLC and reversed HPLC. Normal is essentially the same as column chromatography.

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Although it is described as "normal", it isn't the most commonly used form of HPLC. In the normal HPLC, the column is filled with tiny silica particles, and the solvent is non-polar. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column (Fifield and Kealey, 1995).

In reversed HPLC, the column size is the same as in normal HPLC, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used where there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column hence polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC and figure 7 shows the major steps followed in HPLC (Doreen, 2010).

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After injection of the sample the time taken for a particular compound to travel through the column to the detector is called retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. For a particular compound, the retention time will vary depending on the pressure used (because that affects the flow rate of the solvent), the nature of the stationary phase (not only what material it is made of, but also particle size), exact composition of the solvent and the temperature of the column. This means that conditions have to be carefully controlled if retention times are used as a way of identifying compounds.

There are several ways of detecting when a substance has passed through the column. Common methods use ultra-violet absorption since many organic compounds absorb UV light of various wavelengths. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time. The output will be recorded as a series of peaks each one representing a compound in the mixture passing through the detector and absorbing UV light. As long as the conditions on the column are carefully controlled, retention times can be used to identify the compounds present provided, retention times for pure samples of the various compounds are measured under same identical conditions as for the mixture. Peaks can also be used as a way of measuring the quantities of the compounds present since the peak area is directly proportional to the concentration of compound of that peak (Doreen, 2010).

Achieng et al (2011) analysed using HPLC with Intertsil ODS -3v 250 mm×4.6 mm (5µm particle) column and a mobile phase of acetonitrile: buffer mixture dector was set at a(1:99 v/v) . The detector was set at a wavelength of 270 nm. With this analysis, MAP recorded a level of 1.5

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% while arbutin recorded a level of 2 %. Shou-chieh et al. (2004). In this study using HPLC, the column used was Cosmosil 5 CI8-AR-II, the mobile phase of a mixture of 0.05 M KH2PO4

buffer solution (pH 2.5) and methanol (99.1v/v) and UV detector set at 280 nm to analyze arbutin, kojic acid, ascorbyl glucoside, magnesium ascorbyl phosphate and hydroquione.

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CHAPTER THREE

3 MATERIALS AND METHODS