a) Regarding HPLC, explain specifically why it is vital that the mobile phase (Carrier) is either held under a slight vacuum or is purged with helium gas before entering the HPLC column.

b) An analysis of fatty acids is desired. These fatty acids have boiling points in excess of 275O C. The sample is extracted with methanol to obtain a solution that only contains the fatty acids. Which of the following Methods/Cases is not possible and why, & which is the best approach & why?

Case I-The methanol extract is mixed with a compound that reacts with the fatty acids and adds a methyl group to the end of the fatty acid "chains". The result is that the resulting products have drastically lowered boiling points (between 140oC & 210oC).

This modified extract is then injected into a GC under Isothermal conditions with quantification using a Thermal Conductivity Detector.

Case II-The original methanol extract is injected into an HPLC with a water/acetonitrile mixture as the mobile phase. It is varied from 10% acetonitrile to 40% over 15 minutes and a Refractive Index Detector is used.

Case III-Similar to the first Case, except an FID detector is used with Temperature Programming.