3. Objectives
At the end of this presentation, listeners will be able to
Identify the components of chromatogram
List and explain the chromatographic parameters with respect to HPLC
Explain the factors affecting resolution
PSG COLLEGE OF PHARMACY 3
5. Chromatographic parameters
Retention time
Adjusted Retention time
Retention volume
Retention or factor
Selectivity factor
Theoretical plate
Column efficiency
Asymmetry peaks
Resolution
PSG COLLEGE OF PHARMACY 5
6. Retention time
Difference in time between the sample injection and appearance of peak maxima. The
retention time is given by the symbol tR
Different compounds have different retention times.
Retention time may vary depending on:
i. Pressure used
ii. Nature of the stationary phase
iii. composition of the mobile phase
iv. Temperature of the column
PSG COLLEGE OF PHARMACY 6
7. Adjusted retention time
It is a measurement between retention time(𝑡 𝑅) and unretained time (𝑡0)
𝑡 𝑅
′
= 𝑡 𝑅 − 𝑡0
PSG COLLEGE OF PHARMACY 7
8. Adjusted retention time
Example:
Retention time of a compound (𝑡 𝑅) = 5 min
Unretained time (𝑡0)= 1 min
Then, the adjusted retention time(𝑡 𝑅
′
) will be (𝑡 𝑅−𝑡0)= 4 min
jump
PSG COLLEGE OF PHARMACY 8
9. Retention volume
The volume of mobile phase that passed through the column from the point
of injection to the detector is called as retention volume.
𝑉𝑅 = 𝑡 𝑅 × 𝐹
PSG COLLEGE OF PHARMACY 9
10. Retention factor
It is defined as the ratio of adjusted retention time (𝑡 𝑅′) and unretained time (𝑡0)
𝐾 =
𝑡 𝑅− 𝑡0
𝑡0
Example:
Adjusted retention time (𝑡 𝑅′) = 8 min
unretained time (𝑡0) = 2 min
Then, Retention factor (K) =
8−2
2
=
6
2
= 3
Higher the K value, greater the resolution.
PSG COLLEGE OF PHARMACY 10
11. Retention factor
PSG COLLEGE OF PHARMACY 11
K value
1-10. Good separation
<1.0 Poorly retained
>10 Too long to separate and
broaden the peaks
12. Selectivity factor (Separation factor)
Separation factor is defined as the ratio of the partition coefficient of two components to be
separated OR ratio of adjusted retention time of two components
∝=
𝐾 𝐵
𝐾𝐴
=
(𝑡 𝑅(𝐵)−𝑡0)
(𝑡 𝑅(𝐴)−𝑡0)
KB, KA= partition coefficient or distribution constant of substance B and A
𝑡 𝑅(𝐵) , 𝑡 𝑅(𝐴) = retention time of the substance B and A
𝑡0 = unretained time
Higher the selectivity factor, greater the resolution
PSG COLLEGE OF PHARMACY 12
13. Theoretical plate
Theoretical plate is an imaginary or hypothetical units of column where distribution of
analytes between stationary phase and mobile phase has attained equilibrium.
It is otherwise called as functional unit of the column
𝑛 = 16
(𝑡 𝑅)2
𝑊2
Ideal column has theoretical value more than 2000
Higher the theoretical plates, higher the efficiency of column
PSG COLLEGE OF PHARMACY 13
14. Column efficiency
It is also known as plate count, or number of theoretical plates.
Narrow peaks take up less space in the chromatogram and thus allow more
peaks to be separated
PSG COLLEGE OF PHARMACY 14
Retention time Peak width efficiency
15. Column efficiency
High equivalent theoretical plate (HETP)
HETP =
𝑳
𝑵
=
𝑳𝒆𝒏𝒈𝒕𝒉 𝒐𝒇 𝒕𝒉𝒆 𝒄𝒐𝒍𝒖𝒎𝒏
𝒏𝒐 𝒐𝒇 𝒕𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍 𝒑𝒍𝒂𝒕𝒆𝒔
Higher the column efficiency, higher the resolution
PSG COLLEGE OF PHARMACY 15
HETP Column efficiency
16. Peak asymmetry
Ideal chromatographic peaks are symmetrical, if asymmetrical peaks are
obtained then it is said to be inappropriate
There are two types of asymmetrical peaks, fronting and tailing
PSG COLLEGE OF PHARMACY 16
Tailing is due to saturation of stationary phase
Fronting is due to more active adsorptive site in
the stationary phase
17. Asymmetric factor(Af)
This is measured to find the symmetry of the peak.
If the peak is symmetrical then the value will be 1.
But practically it is very difficult to get.
Ideal Af value according to IP is 0.95-1.05
𝐴𝑓 =
𝑏
𝑎
PSG COLLEGE OF PHARMACY 17
18. Resolution
Resolution is defined as the difference in retention times between the two
components divided by the combined widths of the elution peaks.
It is otherwise defined as the measure of the extent of separation of two components
and the baseline separation achieved
𝑅 𝑠 =
2(𝑡 𝑅 𝐵 − 𝑡 𝑅(𝐴))
𝑤 𝑎+ 𝑤 𝑏
Ideal resolution value should be greater than 2
PSG COLLEGE OF PHARMACY 18
19. Factors affecting resolution
Resolution is affected by three important parameters, they are
1. Selectivity (separation factor)
2. Efficiency
3. Retention (capacity factor)
PSG COLLEGE OF PHARMACY 19
20. Current FDA values for the validation of
chromatographic methods
PSG COLLEGE OF PHARMACY 20
PARAMETER LIMIT
Retention factor K ≥ 2
Resolution value 𝑅 𝑠 > 2
Tailing factor T ≤ 2
Theoretical plate (efficiency) N > 2000