7. Theory of HPLC

Question 1

What is t0 in regards to HPLC?

Answer 1

The “dead” time between injection and measurement.

Question 2

What causes band broadening?

Answer 2

• Biggest contributor to band broadening (and hence low efficiency) is usually the column itself. The quality of the column packing, the particle size, the column dimensions, and any voids that may have formed within the column all play a part in determining the column efficiency.

Other factors:
 Injection volume
 Dead volume (such as detector cell, tubing and connections etc.)
 Flow rate

Question 3

What does it mean to have baseline seperation?

Answer 3

The peaks are seperated all the way down to the baseline (as they should prefferably be)

Question 4

What is tailing and fronting?

Answer 4

Tailing describes a peak whose tail portion is wider than the front portion and fronting is the reverse.

Question 5

What is “Theoretical Plates”?

Answer 5

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column. Therefore, the more theoretical plates available within a column, the more equilibrations are possible, and the better quality the separation.

Question 6

What is peak capacity?

Answer 6

Peak capacity is used to describe the separation efficiency for gradient elution. Peak capacity describes the maximum theoretical number of components that can be successfully separated with a given column and set of analytical conditions with Rs =1 (Figure 1 and Equation 1).