1. Explain about HPLC?

HPLC is a separation technique that involves: The injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron (m) in diameter called the stationary phase). Where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flowthrough device (detector) that measures their amount. An output from this detector is called a liquid chromatogram.

2. Give and describe a function of basic component in HPLC? Solvent reservoir and Pump: This contains the solvent used to carry the sample through the system. The solvent should be filtered with an inlet solvent filter to remove any particles that could potentially damage the system's sensitive components. Solvent is propelled through the system by the pump. Sample injector: Equipped with a sample loop of the appropriate size for the analysis being performed, allows for the reproducible introduction of sample into the flow path. Analytical column: Allows the primary sample separation to occur. This is based on the differential attraction of the sample components for the solvent and the packing material within the column Detector: Flow cell which is place that data be detected and digitized Read Out: Helps analyze and interpret the data.

3. Define:

Stationery Phase: is a liquid supported on an inert solid. Mobile Phase: may be a liquid ( liquid liquid partition chromatography) or agas ( gas liquid chromatography, GLC)

Reverse Phase: Reverse phase liquid chromatography is the separation of molecules based upon their degree of hydrophobic matrix which is largely based on their polarity. Normal Phase: An elution that the polar solutes elute later than non-polar hypophilic.

4. Separation of mixture occurs in HPLC? Separating occur when components of compound are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

5. 4 types of HPLC? Partition Absorption Ion exchange Size exclusion or gel

6. 2 types of partition chromatography. Reverse Phase: A polar stationary phase (e.g., methanol, silica) is used with non polar mobile phase ( e.g., hexane).This favors retention of polar compounds and elution of nonpolar compounds. Normal Phase: A non - polar stationary phase (e.g., C 18; n Octyldecyl ) is used with polar mobile phase ( e.g., water, methanol).This favors retention of non - polar compounds and elution of polar compounds. 7. Separation occurs in size exclusion chromatography? Solvated molecules are separated according to their size by their ability to penetrate a sieve like structure ( the stationary phase ). 8. The factor may reduce the accuracy of result using HPLC? Internal diameter of column Pump pressure Sample size The polarity sample, solvent and column. Temperature

9. 2 types of mobile phases: Purity Detector compatibility Solubility of the sample Low viscosity Chemical inertness

10. Calculation and Procedure of HPLC. Preparation of Benzoic acid/ caffeine standards Prepare standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppmby diluting portions of the 1000 ppm solution with distilled water. Preparation of soda samples Obtain a soft drink sample. Degas the sample by placing it in a vacuum flask and connecting the flask to a vacuum pump or water aspirator. Leave it under vacuum until no more bubbles appear in the soda sample. (If no vacuum is available, allow the soda to stand open overnight.) Filter the degassed soda through #42 filter paper. After preparing the serial dilution and sample, your instructor will brief on standard operating procedure of HPLC.

11. Explain about Preparative HPLC and LC-MS Preparative HPLC: Used for the isolation and purification of valuable products in the chemical and pharmaceutical industry as well as in biotechnology and biochemistry. Sample goes from detector into fraction collector and the objective using this device is isolation and purification of compounds. LC-MS: A technique used to measure the molecular weight and determining the molecular formula of an organic molecule using liquid chromatography. 12. Main advantage of HPLC compared to GC? Non-destructed sample during operation compared to GC. Can accommodate non-volatile and thermally unstable samples. Generally applicable to inorganic ions 13. Typical application of ion exchange chromatography? Preparation of high purity water for power engineering, electronic and nuclear industries.

14. Application of HPLC?