The work described here was carried out in the Department of Chemical Engineering, Monash University, in 2010 and has not been published elsewhere. The study describes the kinetics of chlorophyll extraction from T. suecica using acetone or methanol as an extractant. The three parameters investigated in order to optimize the extraction process were storage temperature of the biomass prior to chlorophyll extraction, level of intracellular water in the biomass during chlorophyll extraction, and average temperature during chlorophyll extraction.

3.2 Materials and Methods

3.2.1 Chemicals and Reagents

Chlorophyll a and chlorophyll b standards were purchased from Sigma-Aldrich Pty. Ltd (Australia). Organic solvents (100% acetone and 100% methanol) were analyti­cal grade.

Table 4 Previous studies on HPLC fractionation of chlorophylls extracted from phytoplankton

Study Mobile phase Stationary phase

Jeffrey [23] First dimension: 0.8% «-propanol in light petroleum (by volume) Sucrose

Second dimension: 20% chloroform in light petroleum (by volume)

Jeffrey et al. [24] 90:10 (v/v) methanol:acetone for 8 min at a flow rate 1 mL/min 3 pm C18 Pecosphere

Pre-injection mix of sample 3:1 (v/v) sample: 0.5 M ammonium acetate Jeffrey et al. [24] Solvent A is 80:20 (v/v) methanol:0.5 M ammonium acetate 3 pm C18 Pecosphere

Solvent В is 90:10 methanol: acetone

Elution order: 0-3 min: solvent A; 3-17 min: solvent B. flow rates: 1 mL/min Pre-injection mix of sample 3:1 (v/v) sample: 0.5 M ammonium acetate

Jeffrey et al. [24] Solvent A is 80:20 (v/v) methanol:0.5 M ammonium acetate 3 pm C18 Pecosphere

Solvent В is 90:10 (v/v) acetonitrile:water Solvent C is ethyl acetate Elution order:

0-4 min: linear gradient from 100% A to 100% В 4-18 min: linear gradient to 20% В and 80% C 18-21 min: linear gradient to 100% В 21-24 min: linear gradient to 100% A 24-29 min: isocratic flow of 100% A

Lynn Co and Three different solvent systems were experimented Silica Gel

Schanderl [27] Solvent system 1 (modified Bauer solvents):

First dimension is benzene: petroleum ether: acetone (10:2.5:2 v/v/v).

Second dimension is benzene: petroleum ether: acetone: methanol (10:2.5:1:0.25 v/v/v)

Solvent system 2:

First dimension is benzene: petroleum ether: acetone: methanol (10:2.5:1:0.25 v/v/v).

Second dimension is petroleum ether: acetone: «-propanol

(8:2:0.5 v/v/v)

Solvent system 3:

First dimension is benzene: petroleum ether: acetone (10:2.5:2 v/v/v).

Second dimension is petroleum ether: acetone: «-propanol

(8:2:0.5 v/v/v)