HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC

DETERMINATION OF ALCOHOLS WITH REFERENCE

TO BODY DISTRIBUTION OF METHANOL

EKB 3171 : Analytical Technique and Instrumentation

Lab

BY :
Manivannan Jayaprakasam
1103161011
BACHELOR OF CHEMICAL ENGINEERING
 Sample Preparation
• Basically this method is determination of alcohol is presented in body

• By using this method, distribution of methanol in tissue has been determined

• This method was used for the analysis of postmortem biological samples which were lung,
liver, spleen, kidney, brain and heart homogenates, blood, urine and gastric contents from
the three cases of methanol poisoning.

• Ten milliliters of blood or 10 g of homogenized tissue was subjected into distillation flask.

• The acid distillate was collected.

• Then it is subjected to HPLC analysis.

• Recovery studies were performed by adding various known amounts of methanol and
ethanol to the blood or liver and lung homogenates.

• These were subjected to distillation and finally injected onto the HPLC.
 Type of Chromatography
High Performance Liquid Chromatography (HPLC)

Specification of the Chromatography

 Mobile phase is water
 Type of column : A Zorbax ODS (CM; 4.6 mm x 25 cm stainless steel) column
 Flow rate :0.5ml/min
 UV wavelength :240-360nm
 Result and Discussion
• The chromatograms were recorded for methyl, ethyl and isopropyl alcohol for the
determination of retention times.
• Separation was achieved using pure water as the mobile phase.
• Elution was noted to be in the order of increasing hydrocarbon chain length which is methyl
alcohol (7.78), ethyl alcohol (12.43) and isopropyl alcohol (26.48).
• The detection limit was found to be 5 mg/100 ml for all three alcohols.
• Recovery studies were carried out and found to be 78-90% yield.
• Ethanol was used as a standard for these test samples.
• In the test samples the concentration of methanol has been noted to be in the order of urine >
blood > gastric contents > liver > kidney > heart > brain > lung >spleen in cases 1 and 2
whose survival was 10 and 12 h, respectively.
• As the third case shows methanol concentration to be higher in the blood than in the gastric
contents and urine .
• The period of survival was greater in this case 1 (18 h).
• The material recovered from the scene of the incident was also tested directly without
distillation and analysis for the alcohols on HPLC showed methanol in the concentration of 40
g% .
• This implied that the deceased had consumed the same material as was recovered from the
scene of the incident.
• The levels of methanol in the test blood samples were 240 mg%, 290 mg% and 150 mg%
respectively in the three cases.
• The longest survivor of the three cases had the lowest levels which is 150 mg%.