Question

List at least five variables that affect change in resolution between two compounds and their retention time when working with HPLC (High Performance Liquid Chromatography).

Answer 1

Resolution is a measure of the ability to separate two components. According to fundamental resolution equation i.e.

R_s=N/4 X (-1)/ X K/K+1

where R_s is the resolution which depends on N (which is related to column efficiency depends on plate number N), (which is related to selectivity or separation factor and depends on mobile phase and stationary phase) and K (which is related to retention factor depending on solvent strength of mobile phase.

1. N (column efficiency): for high efficiency separations i.e. better resolution, the plate number (N) will be high.

2. (selectivity factor or separation factor): is a measure of relative difference in retention. it can be varied by five parameters i.e.

(a) change in organic solvent

(b) pH of the mobile phase : have drastic effect on the selectivity of a separation, especially when acidic or basic analyte molecules are involved

(c) sovent strength and additives

(d) type of stationary phase (the hydrophobicity, polarity, and nature of the stationary phase ie. base silica plays major role.

(e) tempearature of the column:. temperature of the column and hence the mobile phase also affects resolution.

K (retention factor):measuring the retention of an analyte on the chromatographic column.The convenient way to alter the retention factor is to adjust the ‘solvent strength’ of the chromatographic mobile phase. The largest gain in resolution is achieved when the k value is between 1 and 5

Retention time: It is the amount of time a compound spends on the column after it has been injected

the retention time depends on parameters given below:

(a) the pressure used (because that affects the flow rate of the solvent). A high flow rate lowers retention times and yields a poor separation.

(b) the nature of the stationary phase (both on what material it is made of and the particle size) eg. Boiling point: If a component has a low boiling point, then it spends more time in the gas phase. Therefore its retention time will be lower.

(c) the exact composition of the solvent.

(d) the temperature of the column: high column temperature will give shorter retention times, as more components stay in the gas phase which results in poor separation.

(e) Column length: A longer column will produce longer retention times and better separation