Subject Paper No and Title Module No and Title Module Tag PAPER: 5, Forensic Chemistry & Explosives MODULE: M12, Blood alcohol estimation FS_P5_M12 TABLE OF CONTENTS 1. Learning Outcomes 2. Sample Collection 3. Extraction of Ethyl Alcohol from Biological Materials 4. Chemical Analysis of Ethyl Alcohol 4.1 Iodoform Test 4.2 Sulphomolybdic Acid Test 4.3 Ethyl Benzoate Test 5. Determination of Ethyl Alcohol in Blood/Urine 5.1.1 Kozelka and Hine/Cavett method 5.1.2 Widmark s Formula 6. Instrumental Technique of Analysis of Ethyl Alcohol 6.1 Gas Chromatography 7. Summary
1. Learning Outcomes After studying this module, you shall be able to know How to Collect different biological samples for the analysis of ethyl alcohol? How to extract ethyl alcohol from biological materials? What are the different tests that can be used for its chemical analysis?, and About the different instrumental techniques that are generally employed in the analysis of ethyl alcohol 2. Sample Collection In the cases of suspected deaths due to alcoholic intoxication, viscera is collected and preserved in saturated saline. Special attention must be paid in the preservation of these samples; otherwise it would put the whole examination in vain. In cases of suspected intoxication, samples of blood and urine are generally collected. Before the suspect s skin is punctured with a sterile needle, it must be cleaned up by the swab of a non-alcoholic disinfectant like aqueous mercuric chloride, aqueous benzalkonium chloride (Zepiran), etc. The use of an alcoholic disinfectant may either give false positive results or may contribute to falsely high alcohol contents of blood. About 5-10 ml of blood sample must be collected in a test tube and must be stored in a refrigerator usually at 4 0 C after adding the anticoagulant (like EDTA, potassium oxalate, etc.) and preservative (like NaF). The addition of anticoagulant will prevent clotting and the preservative will inhibit the growth of micro-organisms which are capable of destroying alcohol. The urine sample is also collected in the usual manner and preserved with 30 mg of phenyl mercuric nitrate for every 10 ml of urine. 3. Extraction of Ethyl Alcohol from Biological Materials Ethyl alcohol is isolated from biological materials by acid distillation. Viscera, vomit, stomach contents and other materials should be analyzed separately. 50-100 grams of viscera-finely minced should be brought to the consistency of a thin gruel by adding 3-5 times of distilled water and acidified with sulphuric acid and submitted to steam distillation. The flask containing the material should preferably be heated on the water bath. The condenser and the receiving flask should be well cooled with ice especially in the hot season, the outlet of the condenser being dipped in little water or NaOH solution. A few pieces of pumice stone may be taken in the flask to prevent bumping. It is better to collect the distillate in 4-5 fractions, out of which the first one
should not exceed 20 ml and the remaining fractions should be 50 ml each. The distillate contains alcohol and other volatile acids, etc. 4. Chemical Analysis of Ethyl Alcohol The following tests are to be carried out for the detection of ethyl alcohol in the exhibits: 4.1 Iodoform Test Take 1 ml of the distillate and add a few drops of 10% NaOH to it. This is followed by drop wise addition till the solution becomes brown. The mixture is warmed on low flame/water bath for few minutes. A few drops of I 2 solution are added to change the color of the solution from brown to yellow. The mixture is again heated on low flame/water bath, yellow colour precipitate is formed on standing. The precipitate is observed under microscope. Characteristics hexagonal crystals of iodoform are seen (also positive in presence of ethanol, acetaldehyde, iso-propanol) which on standing for long time breaks into flower like structure as indicated below. 4.1.1 Mechanism Magnified Iodoform Crystals This test initially involves oxidation followed by substitution and hydrolysis.
4.2 Sulphomolybdic Acid Test 1 gm of molybdic acid in 25 ml of concentrated sulphuric acid constitutes the reagent. Add 2ml of hot reagent to 2 ml of distillate. A deep blue ring appears at the junction of two liquids. On shaking whole mixture becomes deep blue. This blue color indicates the presence of ethyl alcohol. This test is very sensitive and is negative with acetone, acetaldehyde and dilute solution of methyl alcohol. Strong solution of methyl alcohol gives only a light blue colour after several minutes. 4.3 Ethyl Benzoate Test Add 2 drops of Benzoyl Chloride to 2 ml of distillate. The mixture is made alkaline by adding 10% NaOH drop by drop. On warming, irritating smell of benzoyl chloride is replaced by sweet fruity odour of ethyl benzoate. Methyl alcohol also gives this test but it does not give the iodoform test. 5. Determination of Ethyl Alcohol in Blood / Urine In the cases related to drunkenness, detection of alcohol in body becomes very important. However, the best method is to note the behavioral abnormalities of the suspect. But it could only be confirmed by analyzing the breath, blood or urine. The analysis of breath can be performed on the spot with the help of breath-analyzer instruments like Alcosensor, Breathalyzer, etc. However, the alcohol content of the blood could be determined by using the modified version of Kozelka and Hine/Cavett method. 5.1 Kozelka and Hine/Cavett method The basic principle behind this method is that alcohol is easily oxidized to acetic acid in the presence of oxidizing agents like potassium dichromate and sulphuric acid. So, each ml of N/20 potassium dichromate solution that is reduced in this process is equivalent to 0.575mg of alcohol. 5.2 Widmark s Formula Widmark has given simple formulas to estimate the amount of alcohol present in the body-
a) For Blood Analysis a=cpr Here, a=total amount of alcohol absorbed in the body p=weight of the person c=concentration of alcohol in the blood r=constant namely, 0.68 for men and 0.5 in women b) For Urine Analysis a=3/4qpr Here, a=total amount of alcohol absorbed in the body p=weight of the person q=concentration of alcohol in urine r= Constant namely, 0.68 for men and 0.5 in women 6. Instrumental Technique of analysis of Ethyl Alcohol 6.1 Gas Chromatography Numerous methods have been described for the determination of ethanol in whole blood, serum, and urine, the most popular being gas chromatography (GC). The sample is injected into a heating chamber. Due to high temperature of the chamber, alcohol is converted into vapors which are carried by an inert carrier gas, usually nitrogen, through a column packed with a suitable adsorbent material. Different components of the sample get separated on the basis of their differential affinity (on the basis of partition coefficient) towards the stationary adsorbent phase, which are later on detected by a sensitive detector as shown in figure given below.
Gas Chromatography A chromatogram so obtained helps in qualitative as well as quantitative analysis. Position of the peak is representative of a particular compound and the area covered by the peak represents its amount. A Gas Chromatogram
Operating Conditions: Column: Porapack polymer bead 80-100 mesh or its equivalent, which can separate or resolve the ethanol Column Temperature: 160 0 C Carrier Gas: Nitrogen Rate of Gas Flow: 50 ml/min Detector: Flame Ionization Detector Alternative Operating Conditions: Column: 0.3% Carbowax 20 M on 80-100 mesh Carbopak C, 2m X 2mm ID or its equivalent Column Temperature: 35 0 C for 2 minutes and then programmed at 5 0 C per minute to 175 0 C and hold for atleast 8 minutes Carrier Gas: Nitrogen at 30 ml/min 7. Summary About 5-10 ml of blood sample must be collected in a test tube and must be stored in a refrigerator usually at 4 0 C after adding the anticoagulant (like EDTA, potassium oxalate, etc.) and preservative (like NaF). The urine sample is also collected in the usual manner and preserved with 30 mg of phenyl mercuric nitrate for every 10ml of urine. Ethyl alcohol is isolated from biological materials by acid distillation and confirmed by Iodoform test, Sulphomolybdic Acid test and Ethyl Benzoate test. The concentration of alcohol in blood and urine is determined by Kozelka and Hine/Cavett method which could further be confirmed by Gas Chromatography.