Once recovered from microalgal biomass, the extracted chlorophyll mixture will have to be fractionated in order to separate out the different chlorophyll types and to remove unwanted components (neutral lipids, polar lipids, other pigments) that have been inevitably co-extracted. Chromatographic techniques are traditionally used to fractionate chlorophyll mixtures. The three types of chromatography that have been widely used are paper chromatography, thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC) [1, 23, 31, 43].

Paper chromatography was used extensively during the early development of chromatographic techniques (1950s and 1960s). The method was able not only to separate chlorophyll into its fractions (a, b, and c) but also to effectively fractionate other pigments, such as pheophytins and carotenes [22]. However, the inception of TLC has resulted in the decline of paper chromatography usage. This later tech­nique was preferred due to the ease in recovering pigment fractions from its adsor­bent [31,41]. Additionally, TLC requires less sample, is less laborious, and produces chromatograms with sharper resolutions [27, 41]. Organic adsorbents, such as sucrose and cellulose, were found to be the most effective stationary phases for use in two dimensional TLC. Even though the use of silica gel as a stationary phase was effective in separating all plant pigments (except for some minor components), it was found to promote the formation of chlorophyll degradation products.

HPLC is superior to TLC because it requires even less sample for analysis, is faster and can be easily coupled with an automatic detection system [48, 57]. In addition to these, HPLC is more precise and has a higher degree of sensitivity. Reverse phase HPLC is preferred to normal phase as the latter does not separate polar compounds effectively. An additional drawback to normal phase HPLC is its lack of compatibility with aqueous samples. Several HPLC configurations have been employed, each being able to separate pigments to variable extent and differ­ent resolution [24]. There are different types of detectors that may be used to mea­sure the concentrations of separated pigments as they exit the chromatographic column. The most commonly used detectors rely on fluorescence and absorbance analyses. Jeffrey et al. [24] found fluorescence detection to be more sensitive and more selective than absorbance detection especially when used to analyze chloro­phylls amongst carotenoids. Table 4 summarizes previous studies on chromato­graphic fractionation of phytoplankton pigments. It is noted that the use of chromatographic techniques to purify recovered chlorophylls, albeit very effective on a laboratory scale, is not commercially applicable due to the high installation and operating costs associated with the techniques. Investigating a cost-viable, energy — efficient purification technology that can be retrofitted to industrial-scale chloro­phyll production is a current research endeavour.