HighPerformance Liquid Chromatography I Basic Concepts Meas

High-Performance Liquid Chromatography I- Basic Concepts & Measurements 1. Describe the function of the basic components of a HPLC system? Name at least four. 2. What is isocratic vs. gradient elution? What is a comparable technique to gradient elution that is used in GC separations? What are three benefits of gradient elution? 3. What is meant by reversed phase? Define the retention mechanism. 4. What is the purpose of a guard column? 5. What is meant by solvent strength? How would changing this affect the chromatogram? 6. How would you make peaks appear broader (take up more area)? 7. If you used cut-and-weigh or peak height for quantitative analysis, why is it important to use the same scale? 8. Name three HPLC detectors and explain their operation.

Solution

1) Four basic components HPLC and their functions:

a)The pump: This part pushes the liquid or the mobile phase through the chromatography column under normal pressure range of 2000 to 9000 psi. to high pressure range upto 18000psi at some specific flow rate typically 1-2 ml/minute.

The pump are of various types based on their specificity like constant pressure pumps or constant flow pumps .

b) The injector: It injects the liquid into the flowing mobile phase.It forces the liquid beyond the flowrate of the mobile phase.

c)The column:This is the major part packed with porous material of minute diameter(the stationary phase).This material chemically bond or interact with the sample or liquid components and separate them

d)Detector:As the components come out of the column ,UV light is made incident on it,a part of which is absorbed by the components.The reflected light frequency and also the retention time of the components are picked by the detector .This output is then put to further analysis based on the graphed information of retention time and intensity of light absorbed .

b)