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 “A”

– Serum Sample “B”

– 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 “Blank”, add 5 µl distilled water

– For the “Standards” or “Samples”, 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 “Standards” and “Samples” at 340 nm vs “Blank”

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

 

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