HPLC Parameters

1. HPLC Parameters,
Factors affecting
Resolution
BY DHINESHKUMAR V
IM.PHARM (PHARMACOLOGY)
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2. Contents
Chromatogram
Chromatographic parameters for HPLC
Factors affecting resolution
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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
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4. Chromatogram
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 A chromatogram is a pictorial record of the detector
response

5. Chromatographic parameters
Retention time
Adjusted Retention time
Retention volume
Retention or factor
Selectivity factor
Theoretical plate
Column efficiency
Asymmetry peaks
Resolution
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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
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7. Adjusted retention time
It is a measurement between retention time(𝑡 𝑅) and unretained time (𝑡0)
𝑡 𝑅
′
= 𝑡 𝑅 − 𝑡0
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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
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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
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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.
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11. Retention factor
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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
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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
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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
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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
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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
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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
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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
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19. Factors affecting resolution
Resolution is affected by three important parameters, they are
1. Selectivity (separation factor)
2. Efficiency
3. Retention (capacity factor)
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20. Current FDA values for the validation of
chromatographic methods
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PARAMETER LIMIT
Retention factor K ≥ 2
Resolution value 𝑅 𝑠 > 2
Tailing factor T ≤ 2
Theoretical plate (efficiency) N > 2000

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