Solvent A is 80 : 20 (v/v) methanol: 0.5 M ammonium acetate
3 m 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].
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)
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.
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
(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