HPLC-15

Column efficiency

Column efficiency is the ability of the column to produce the good resolution and narrow peaks.

Factors affecting Column efficiency:

• Length of the column
• Linear velocity of flow in the column
• Extra column volume
• Retention factor
• Particle size of the stationary phase
• Packing strength

HPLC-14

Extra-Column band broadening

Extra-Column band broadening occurs due to more Extra-column volume or Dead volume (refer post HPLC-08). Tubing connections between column and injection system, column and detector region can cause band or peak broadening. Therefore larger the radius of connecting tube, more the extra-column peak broadening and smaller the radius of connecting tube, less the extra-column broadening.

HPLC-13

How CAD detector works?

The steps involved in CAD-Charged Aerosol Detector :

• The mobile phase from the column enters into CAD
• Pneumatic nebulization of mobile phase creates aerosol droplets
• Conditioning of aerosol occurs to remove the large droplets
• Evaporation of solvent from the formed aerosol leads to dried particles
• An Ion jet induces charge to the dried particles
• An Ion trap is used to select the particles and remove the excess ions from them
• The charge of those selected particles can be measured and detected by using Electromter

HPLC-12

Causes for Pressure Fluctuations

In HPLC, Pressure fluctuations mainly occur due to the following:

• Leaks in Pump seal
• Leaks in tubings
• Inline bubble traps in pump, column and solvent tubings

Solutions:

Bubble traps can be minimized by Degassing

Pump seal can be replaced to avoid leaks

HPLC-11

Causes for the the change of Retention time

 The molecule of analyte in a sample has to elute at 8th minute. But instead, If it gets eluted either before or after that 8th minute there is a change in retention time. It happens due to some factors like variation of column temperature, evaporation of mobile phase, Improper preparation of mobile phase. So it is necessary to maintain optimum temperature in column and prepare the mobile phase accurately.

HPLC -10

Relative Retention Time (RRT)

RRT is the ratio of retention time of peaks corresponding to the compounds other than analyte and the main peak retention time. For example, If the retention time of main peak is 8 minutes and the retention time of other impurity peak is 5 minutes. The RRT of impurity peak is 5/8=0.625 and the mainpeak RRT is 8/8=1.
RRT is mainly used to control the elution of impurity peaks from the sample.

HPLC-09

Peak Fronting and Tailing

Peak Fronting

It occurs mainly due to overloading of sample..i.e., Injection volume is too high. Even mobile phases with more viscosity can lead to fronting of peaks.

Peak Tailing
It occurs due to retention of some compounds inside the column. It is the beginning of peak doubling.