6.1: Determination of Alcohol in Serum by Headspace GC

D1: Determination of Alcohol in Serum by Headspace GC

The alcohols used in this experiment pose little hazard provided that routine laboratory precautions are taken to avoid ingestion or undue skin contact. Take care with the injection syringe to avoid puncturing the skin.

Note that the GC injection port is heated and could cause burns. Do not touch electrical connections. Ensure that the detector flame is ignited if hydrogen gas is flowing.

The serum samples must be treated as biological hazards and all work must be carried out using gloves to avoid any contact. All biological wastes and contaminated glassware must be placed in the available Biohazard containers for sterilization/disposal.

Introduction

The use of an internal standard (where you measure the ethanol peak area relative to the internal standard peak area (in this case propan-1-ol)) minimizes errors due to variation in injection volume. Temperature of the vapor sampled should be kept as constant as possible.

Apparatus

Gas chromatograph and capillary column Integrator/printer Micropipettes 500 !l headspace syringes Headspace vials with caps Reagents Stock solution of ethanol: 5% w/v (5g in 100-ml de-ionised water) Solution of propan-1-ol: 0.1% (internal standard)

Preparation of Samples and Standards

1. Prepare a series of standards containing 50, 100, 150 and 200-mg ethanol per 100 ml of water by diluting the stock ethanol solution.
2. Using a micropipette, transfer 250 !l of the propan-1-ol solution into each of eight headspace vials.
3. Transfer 250 !l aliquots of the four prepared ethanol standards into the first four vials, two 250 !l aliquots of the serum sample into vials 5 and 6 and two 250 !l aliquots of deionized water into vials 7 & 8 to act as a blank.
4. Label all vials clearly.
5. Seal each vial with septum and screw cap and place all the vials in the GC oven on the left bench as you enter the GC lab. This should be set to 40°C.
6. Leave the vials to equilibrate for about 15 min.

GC Analysis

1. The 500 µl headspace syringes must be prepared by pre-heating in oven and not washed with solvent.
2. Inject 500 µl from vial 7 (the blank) in a rapid and smooth motion into the GC and simultaneously press the ‘Inj. A’ button on the integrator.
3. Only one peak from the internal standard (the propan-1-ol) should be observed. If more than one peak is observed then repeat using Vial 8 (blank).
4. Repeat the procedure for all the standards and samples (vials 1-6).
5. From the integrator printout, you will see that each standard and sample has two peaks. The first (lower retention time) is ethanol and the second is propan-1-ol.
6. Calculate the ethanol: propan-1-ol peak area ratio for each standard.
7. Plot a calibration graph of \[\frac{\text { Peak Area of ethanol }}{\text { Peak Area of propan }-1-\text { ol }} \text { vs Concentration of ethanol. }\]
8. Determine the ethanol concentration in the plasma sample.

Thinking about Making Valid Measurements

At what sample concentration would you be confident (e.g. 95%) that the amount measured exceeds 80 mg per 100 ml. What experiment could you perform to check your prediction?

Improving Your Practical Technique

• Work in a clean environment to avoid contamination.

• Alcohol is a volatile solvent – keep solutions stoppered when not in use to prevent evaporation.

• Syringe technique can make significant difference to the volume injected into the GC so replicate injection should be carried out until good reproducibility is achieved.

• Remember that volumetric flasks are calibrated for solutions at 20 ºC when making up solutions to the mark.

• Chromatographs should be labeled and dated as they are produced.

• Always establish a good flat baseline with reagent blank.

• Run standards at the beginning and end of the run.

• Check calibration using one of the standards.

• Consider running the sample in duplicate or triplicate.