Chlorophyll Extraction from Microalgae: A Review on the Process Engineering Aspects

Table 4

Previous studies on HPLC fractionation of chlorophylls extracted from phytoplanktons.

Study, Chromatography	Mobile phase	Stationary phase	Key results
Jeffrey [49], TLC	First dimension: 0.8% n-propanol in light petroleum (by volume)	Sucrose	In this two dimensional chromatography, there was complete separation of chlorophylls and carotenoids.
	Second dimension: 20% chloroform in light petroleum (by volume)
Jeffrey et al. [4], HPLC	90 : 10 (v/v) methanol   : acetone	3 𝜇m C18 Pecosphere	This simple isocratic protocol was able to separate only chlorophyll a from other pigments and compounds.
	for 8 min at a flow rate 1 mL/min
	Preinjection mix of sample
	3 : 1 (v/v) sample: 0.5 M ammonium acetate
Jeffrey et al. [4], HPLC	Solvent A is 80 : 20 (v/v) methanol : 0.5M ammonium acetate	3 𝜇m C18 Pecosphere	This step-isocratic protocol was found to successfully separate the three chlorophylls (a, b and c) and ten other derivative products.
	Solvent B 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 samplee
	3 : 1 (v/v) sample: 0.5 M ammonium acetat
Jeffrey et al. [4], HPLC	Solvent A is 80 : 20 (v/v) methanol:   0.5 M ammonium acetate	3 𝜇m C18 Pecosphere	This ternary gradient protocol was found to separate over 50 pigments. The resolution of this protocol is higher than that of Wright and Shearer [51]. Additionally no ion pairing reagent is required, as in Mantoura and Llewellyn [37].
	Solvent B is 90 : 10 (v/v) acetonitrile : water
	Solvent C is ethyl acetate
	Elution order:
	0–4 min: linear gradient from 100% A to 100% B
	4–18 min: linear gradient to 20% B and 80% C
	18–21 min: linear gradient to 100% B
	21–24 min: linear gradient to 100% A
	24–29 min: isocratic flow of 100% A
Lynn Co and Schanderl [52], TLC	Three different solvent systems were experimented.	Silica Gel	Two dimensional chromatography was carried out on silica gel. Eight major pigments as well as eight to ten minor derivatives were successfully separated with these solvent systems.
	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: n-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: n-propanol (8 : 2 : 0.5 v/v/v)
Madgwick [50], TLC	30 mL of 1: 1 (v/v) diethyl ether ∶ petroleum spirit	Glucose	(1) One dimensional ascending thin layer chromatography was used.
			(2) Spectrophotometric analysis was found to overestimate chlorophyll c by up to 22% and to underestimate chlorophyll b by 10–20%. Chlorophyll a was, however, correctly quantified.
Mantoura and Llewellyn [37], HPLC	Solvent P (ion pairing reagent) is made of 1.5 g tetrabutyl ammonium acetate and 7.7 g of ammonium acetate dissolved in 100 mL of water. Primary eluant is solvent P ∶ water ∶ methanol (10 : 10 : 80 v/v/v )	Four different columns:C3 Zorbax,C8 Zorbax,C18 Zorbax,Shandon Hypersil ODS	High resolution separation of chlorophylls and all major pigments were achieved. The method obtained a high recovery (over 90%) of pigments.
	Secondary eluant is 20 : 80 (v/v) acetone: methanol
	0–10 min: linear gradient from primary eluant to secondary eluant
	10–22 min: isocratic flow of secondary eluant
Riley and Wilson [53], TLC	Petroleum ether ∶ ethyl acetate ∶ diethyl amine (58 : 30 : 12 v/v/v)	Silica gel	All plant pigments were separated except for some minor components.
Sartory [48], HPLC	Solvent A is 97% methanolSolvent B is 97% acetone	Sep-PakC18 Bondapak	(1) HPLC was found to be a rapid, sensitive, and selective method that successfully separated chlorophylls and its derivatives to high resolutions. Recovery of total pigments by HPLC is greater than 96%.
	Elution order:
	0–15 min: 100% solvent A
	15–20 min: linear gradient to 77% solvent A: 23% solvent B		(2) Spectrophotometric analysis overestimated chlrophyll a and underestimated chlorophyll b.
	Then isocratic flow of 77% solvent A: 23% solvent B
	Flow rate: 1 mL/min
Wright and Shearer [51], HPLC	Linear gradient from 90% acetonitrile to ethyl acetate for 20 mins at a flow rate of 2 mL/min	C18 octadecyl silica.	Carotenes, chlorophylls, xanthophylls, and their degradation products were successfully separated with high resolutions.