Course Work 1 Questions

Respond to these question using short answers in your own words and show all your reasoning fully when answering the calculations. 1. Explain why it is necessary to have a robust and accurate method to determine blood ethanol levels. (1 marks)

2.

Briefly describe the principles of the main technique used as cited in this paper. (3 marks)

3.

Describe what other methods have been commonly used to determine blood ethanol levels and how are they used in conjunction with each other. (2 mark) The other two methods used to determine blood ethanol levels are gas chromatography and HPLC.

4.

Other HPLC methods have been described in the paper. Why are they not suitable? (2 mark) They are not suitable because they are laborious and time-consuming, the method involves the deproteinisation of blood, and this is changing ethanol into acetaldehyde.

5. What is the method of detection based on in the experiment discussed in this paper? (2 marks) In the existence of NAD and ADH, the oxidation of ethanol creates acetaldehyde and reduced NAD. The method of detection based on the experiment is spectrophotometrically. . 6. What source does the ADH come from? How much was used in the suspension? (1 marks) The source of ADH is from and 20l was used in the suspension. 7. How would you prepare 50mM of NAD? (2 marks)

8.

How would you prepare 75mM of Na4 P2O7 ?

(2 marks)

9.

What constituents is the mobile phase produced from? (2 marks) The constituents that the mobile phase is produced from are aqueous KH2PO4 and K2HPO4.3H20 solutions and HPLC-grade acetonitrile (C).

10. What proportions of these constituents are used and how would you prepare a 5L solution from them? (2 marks) 11. What samples were used as the negative control? (1 marks) Ethyl alcohol concentrations were taken as the negative control from 14 healthy subjects. 12. What subjects were used as the positive control? (1 marks) Blood serum and the post mortem samples were used as the positive control. 13. What did the sample preparation consist of? Why was there an incubation time of 30 minutes? What is the sample being analysed? (3 marks) The sample preparation consisted of pyrophosphate buffer, 1.0ml.The sample being analysed was 40l phenyldrazine, 80l NAD and 20l of ADH suspension. 14. What were used as the standards? (1 marks)

15. During the analysis of the lower and upper limits of linearity and detection what standards were used and why? (2 marks) A series of aqueous standard solutions of low concentrations of ethyl alcohol between the ranges of 10.0 and 0.002 g/l. 16. How would you prepare a dilution series to produce these ranges? (3 marks)

17. Table 1 shows the ranges tested and the ranges found for water, serum and blood and the linearity of the method results. (3 marks) a) What do the results tell you? b) What were the limits of detection for each specimen? c) Which sample gave the most accurate result?

18. The HPLC method was compared to a gas chromatography method. What was the carrier gas, column temperature and injector and detector temperatures? What were the dimensions of the column used? (2 marks) The carrier gas used was nitrogen. The column temperature was 95C. The injector and detector temperatures were 150C. The dimensions of the column consists of the glass column (2m x mm I.D) which was packed with 60/80 mesh carbopack B coated with 5% carbowax 20M. 19. When phenylhydrazine reacts to form phenylhydrazone what 2 products are formed? What were the peak wavelengths detected in the spectral scan? (2marks) The reaction produces two syn- and anti-diastereoisomers. The peak wavelengths detected in the spectral scan was generated at 272nm and 276nm.

20. What would be a suitable wavelength to set the detector to and explain why? (2 marks) A suitable wavelength to set the detector to would be 282nm because 21. Table 2 shows samples that have been spiked with known concentrations of ethanol. What can you say about the reproducibility of the sample assays and is there a best sample type to use? (2 marks)

22 .Draw the structure for the following alcohols: marks) Methyl, n-propyl, isopropyl and n-buytyl (Any structural form acceptable)

(4

Methyl

n-propyl

Isopropyl

n-butyl

Total./45 CW1%...............