High Performance Liquid Chromatography

Question 1

Definition

Answer 1

Separation of a compound from analyse, based on its affinity for either a stationary or mobile phase. Using polarity to determine affinity between two immiscible phases. Potential for collection or quantitation.

Question 2

Components

Answer 2

Solvent
Analyte
Column
Detector/recorder

Question 3

Normal phase

Answer 3

Stationary phase is polar

mobile phase is non polar

Question 4

Reverse phase

Answer 4

Stationary phase is non polar
mobile phase is polar
non polar compounds are dissolved in the mobile phase and pass through the column. polar components are retained in the non polar column until the polarity is altered

Question 5

Output methods

Answer 5

Fluorescence
UV Spectrophotometry
Electrochemical

Question 6

Output chromatogram

Answer 6

Retention time to identify the compound
internal standard to calculate peak heigh ratio
shoulder on peak two compounds too close together alter mobile phase to resolve

Question 7

Quantification

Answer 7

Calibration curve of known conc. of analyte
Using peak heigh ratio, internal standard ensures any differences in evaporation, injection volume or subtle differences is accounted for

Question 8

Application

Answer 8

Adrenaline/Noradrenaline
HbA1C
Vitamins
Therapeutic drug monitoring
Haemoglobiniopathies

Question 9

Advantages

Answer 9

Speed
Versatile
Crude sample types can be used
Reproducibility
Sensitive

Question 10

Disadvantages

Answer 10

There may be cheaper methods
Optimisation requires skill
Solvent disposal