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# CONFIDENTIAL

AS/JAN 2013/CHM510

## ANALYTICAL SEPARATION METHODS CHM510 JANUARY 2013 2 HOURS

INSTRUCTIONS TO CANDIDATES 1. 2. 3. This question paper consists of five (5) questions. Answer ALL questions in the Answer Booklet. Start each answer on a new page. Do not bring any material into the examination room unless permission is given by the invigilator. Please check to make sure that this examination pack consists of: i) ii) the Question Paper an Answer Booklet - provided by the Faculty

## DO NOT TURN THIS PAGE UNTIL YOU ARE TOLD TO DO SO

This examination paper consists of 5 printed pages
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QUESTION 1 a) Explain why flow rate need to be optimized in order to improve the efficiency of a column. (3 marks) In a gas chromatography (GC) analysis of three compounds using isothermal run at 120 C on a 40-cm column, the air peak was observed at 1.2 minutes while the first peak (peak 1) was observed at 3 minutes. i) Calculate the average linear velocity of the mobile phase, (2 marks) ii) Calculate the capacity factor of peak 1. (2 marks) i) The resolution, Rs for peak 1 and 2 is 0.9 while the resolution for peak 2 and 3 is 10.0. Discuss on the efficiency of the separation. (4 marks) iv) Suggest an approach to improve the above separation. Explain. (4 marks)

b)

c)

i) Figure 1 shows the separation of hydrocarbons achieved using the following GC conditions; Column: polydimethyl siloxane (PDMS), initial temperature 100 C at rate 15 C min"1. Name the phenomena observed at retention time above 20 minutes and briefly discuss the cause of the phenomena. (3 marks)
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Figure 1

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ii)

Figure 2 shows a typical chromatogram observed for the separation of polar analytes using PDMS column. Explain the cause of the observed peak tailing. Suggest an approach to avoid this problem. (3 marks)

Figure 2 d) The analysis of drug in urine was analyzed using a gas chromatograph with split/splitless injector. The drug was extracted using solid phase extraction and elution was achieved using methanol. Suggest a suitable injection mode for this analysis. Explain. (3 marks)

QUESTION 2 a) The van Deemter equation is often used to approximate the efficiency of a chromatographic column. In HPLC using a packed column, which term in the equation have the least and the greatest influence to the efficiency of the column? Explain your choice. (4 marks) Component A has a longer retention time than component B when separated using high performance liquid chromatographic (HPLC) methods stated below. For each of the methods stated, explain the reason for this observation. (9 marks) i) reversed phase chromatography ii) ion exchange chromatography iii) gel permeation chromatography Explain the purpose of each of the following components in liquid chromatography analysis. i) A gas sparging system in HPLC (2 marks) ii) A suppressor column in ion exchange chromatography (3 marks)

b)

c)

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d)

Explain the similarity and differences between chromatography and adsorption chromatography.

normal

phase

partition (3 marks)

QUESTION 3 a) Give two characteristics of a supercritical fluid which can enhanced the separation of analytes compared to gas. (2 marks) The efficiency of separation in supercritical fluid chromatography (SFC) is better than GC and HPLC. Explain. (4 marks) Carbon dioxide has a limitation to extract polar compounds. Explain. (3 marks) d) SFC can separate a mixture of volatile and non volatile compounds efficiently. Explain. (4 marks) Discuss two advantages of supercritical fluid extraction (SFE) compare to solvent extraction. (4 marks)

b)

c)

e)

QUESTION 4 a) Capillary electrophoresis (CE) is a powerful separation technique. In CE, analytes migrate in a buffer solution through a capillary based on two principles: electrophoretic flow and electroosmotic flow (EOF). i) Explain the differences between electrophoretic flow and EOF. (4 marks) ii) EOF is pH dependent. Explain why and how this will affect the migration rate of cations. (3 marks) Explain how EOF can be manipulated to separate anions efficiently. (4 marks) b) A separation of neutral compounds in micellar electrokinetic chromatography (MEKC) is based on varying hydrophobicities. Explain. capillary (4 marks)

iii)

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c)

Capillary gel electrophoresis (CGE) is commonly used to analyze DNA fragments. Explain. (4 marks)

QUESTION 5 You are required to analyze colorant and fragrance compounds from a selected plant sample. Several extraction methods such as Soxhiet, liquid-liquid extraction (LLE), solid phase extraction (SPE), solid phase microextraction (SPME), microwave assisted extraction (MAE) and pressurized liquid extraction (PLE) can be utilized prior to chromatographic analysis. However, only n-hexane is available in the laboratory. a) Suggest the most efficient and rapid method suitable for the extraction of non volatile and low polarity colorant compounds. Justify your answer by discussing why the other methods are not selected. (6 marks) These colorants are used in textile dyeing and the wastes are disposed into natural water. By using SPE method with pre-conditioned C18 sorbent, you can perform an extraction method to determine low level of colorants in the effluent. Discuss the steps involve in this process. (9 marks) Suggest a suitable method for the analysis of fragrance compounds. Justify your answer. (4 marks)

b)

c)

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