Ethanol Detection Assay
Project description
Lab Report
“ Full Report included (Abstract, introduction, Materials and Reagents, Experimental procedure, Results and observations, Discussion of the results, Conclusion, works cited), also need to answer the questions at the end of Lab Menu
Basic laboratory technique “ lab 05: Ethanol detection assay
Introduction
Ethanol test is frequently performed in Hong Kong in medicolegal cases. Concentration of ethanol in unknown sample can be determined through enzymatic method. Followed by the following chemical reaction, the concentration of ethanol could be detected indirectly with the help of NADH, a coloured chemical with absorbance at 340nm.
Principle of assay:
Ethanol is oxidized by the enzyme alcohol dehydrogenase (ADH) to form acetaldehyde with concomitant reduction of NAD+ to NADH:
Ethanol + NAD+ADH ? Acetaldehyde + NADH
As the amount of NADH generated is dependent on substrate concentration, the observed colour change can be used to estimate the substrate concentration. The increase in absorbance at 340 nm should reflect the amount of ethanol in the sample at the beginning of the reaction.
Materials
“ 0.1 M glycine-NaOH, pH 9.0 containing 15 units/ml Yeast ADH and 6.25 mM NAD+
“ Serum Sample œA
“ Serum Sample œB
“ Ethanol Standards Set (ethanol concentrations of 0.05, 0.1 and 0.3%)
Methods
“ Label cuvettes for Blank, Standards “ Ethanol (0.05%, 0.1% and 0.5%) and Samples (A and B)
“ Add 1.5 ml 0.1 M glycine-NaOH to each of the cuvettes
“ For œBlank, add 5 µl distilled water
“ For the œStandards or œSamples, add 5 µl Ethanol Standards or Samples
(Working from Low Concentration to High Concentration 0.05% > 0.10% > 0.5%)
“ Cover the cuvette immediately and mix by gentle inversion
“ Allow solutions to incubate for 10 minutes at room temperature
“ Measure absorbance of œStandards and œSamples at 340 nm vs œBlank
Data record
Sample Ethanol Standard (% w/v) Sample
0 0.05 0.10 0.50 A B
Abs. at
340 nm 0 0.053A 0.055A 0.245A 0.052A 0.138A
Comment on the result
Our Lab Step: (Standard: 1% Ethanol)
Step 1: Dilute 0.1M Glycine “ NaoH (Adding 4ml Glycine + 8ml Distill Water)
Step 2: Label cuvettes for blank, Standards (0.05%, 0.1% and 0.5%), Sample (A & B)
Step 3: Add 1.5ml of 0.1M Glycine “ NaOH to each of the cuvettes, then wait for 10 mins,
Step 4: Measure them in Abs. at 340nm
