Figure 6 Experimental designs of policosanol and long chain aldehyde analysis in Kokuto.
1. Wax compositional analysis by TLC and HPLC-ELSD
a. Standard preparation
Several compounds were used as standards of wax components: triacontane for hydrocarbon, synthesized octacosanal aldehyde,
cholesteryl oleate sterol ester, octacosanol alcohol, triolein triacylglycerol, lignoceric acid acid, methyl palmitate methyl ester
and stigma sterol sterol. Those standards were prepared in toluene for TLC analysis and in chloroform with different concentrations 0.05
–
0.50 mgml covering the levels of sample components for quantification of
each wax component by HPLC analysis.
Kokuto
Factory A
Kokuto A
Extraction methods variation
Policosanol and long chain
aldehyde analysis Extration times
variation Policosanol and
long chain aldehyde analysis
Cane juice and Kokuto
A
Policosanol and long chain
aldehyde analysis
Factory Non-A
Cane clear juices and
Kokuto Non-A
Policosanol and long chain
aldehyde analysis
Kokuto B-G
Policosanol and long chain
aldehyde analysis
b. Sugarcane wax extraction
Briefly, 10 g of freeze-dried rind of sugarcane were placed in Advantec No. 84 thimble filter and extracted using soxhlet for 4 h with
about 150 ml of hexane, methanol 20:1 vv. The solvent solution was removed from the extract with rotary-evaporator under vacuum condition
at 40°C. The amount of dry wax extract was weighed and diluted with toluene for TLC analysis and with chloroform down to 1
–
2 mgml to the reach detection level of ELSD used in HPLC analysis.
c. TLC Analysis
Thin-layer chromatography TLC of waxy materials extracted from sugarcane rind samples using a silica gel 60, 20 cm × 10 cm × 250
µm TLC plate. Cholesteryl oleate, methyl palmitate, synthesized octacosanal, triolein, lignoceric acid, octacosanol and stigma sterol were
used as standards. Samples and standards were eluted with two steps of developing solvents. The first solvent mixture, comprising hexane,
diethyl ether, acetic acid 95:5:1 vvv, was allowed to travel 10 cm before the plate was removed and the solvent allowed to evaporate.
Once dried, the plate was redeveloped in hexane, diethyl ether, acetic acid 80:20:1 vvv to the top of the plate. Developed bands were
visualized by spraying the plate with 10 cupric sulfate solution containing 8 phosphoric acid. Then the TLC plate was dried in heater
until the developed bands were charred.
d. HPLC-ELSD analysis
Kategori