Hplc parameters, factors affecting resolution

HPLC Parameters,
Factors affecting
Resolution
BY DHINESHKUMAR V
IM.PHARM (PHARMACOLOGY)
PSG COLLEGE OF PHARMACY 1

Contents
Chromatogram
Chromatographic parameters for HPLC
Factors affecting resolution

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

Chromatogram
 A chromatogram is a pictorial record of the detector
response

Chromatographic parameters
Retention time
Adjusted Retention time
Retention volume
Retention or factor
Selectivity factor
Theoretical plate
Column efficiency
Asymmetry peaks
Resolution

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

Adjusted retention time
It is a measurement between retention time(𝑡 𝑅) and unretained time (𝑡0)
𝑡 𝑅
′
= 𝑡 𝑅 − 𝑡0

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

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

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.

Retention factor
K value
1-10. Good separation
<1.0 Poorly retained
>10 Too long to separate and
broaden the peaks

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

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

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
Retention time Peak width efficiency

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
HETP Column efficiency

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
Tailing is due to saturation of stationary phase
Fronting is due to more active adsorptive site in
the stationary phase

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

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

Factors affecting resolution
Resolution is affected by three important parameters, they are
1. Selectivity (separation factor)
2. Efficiency
3. Retention (capacity factor)

Current FDA values for the validation of
chromatographic methods
PARAMETER LIMIT
Retention factor K ≥ 2
Resolution value 𝑅 𝑠 > 2
Tailing factor T ≤ 2
Theoretical plate (efficiency) N > 2000

Hplc parameters, factors affecting resolution

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