USP 621 || HPLC || Chromatographic Parameters Adjustments ||

USP general chapter 621

00:00 Introduction
01:25 Temperature of column
01:50 PH of mobile phase
02:17 Concentration of salts/buffer
03:06 Injection volume
03:34 Flow rate
04:34 Column
05:08 Mobile phase ratio
07:32 Detector wavelength

Do you know that as per USP general chapter 621 You can do adjustments in the chromatographic parameters to achieve system suitability.So there are the 8 chromatographic parameters which you can adjust to achieve system suitability.So let us discuss all one by one.Let us begin with the column temperature first:

1.The column temperature :
can be adjusted by as much as ±10°. For example is column temperature is 40* then allowed adjustments will be between 30*to 50*

2.pH of the mobile phase:
The pH of the aqueous buffer which is used in the preparation of the mobile phase can be adjusted to within ±0.2 units of the value.
For example pH of 7.6 can be adjusted from 7.4 – 7.8

3.Concentration of salts in buffer : Concentration of the salt used in the preparation of the aqueous buffer can be adjusted to within ±10%. Here you have to make sure that while doing adjustments in concentrations of buffer, pH of the buffer should also remains within the allowed adjustment limit. And allowed limits for PH I have just told you in previous point.Let us see example with respect to concentration of the buffer: 20 mM Potassium phosphate can be adjusted between 18 to 22 mM, as long as proper pH is maintained.

4.Injection volume : The injection volume can be adjusted as long as precision and detection limits are achieved. Here one one important point you have to note down that increase in injection volume is not permitted. Because increased injection volume can lead to peak broadening and which will further cause reduction in theoretical plates count and resolution failure.

5.Flow rate : Flow rate can be adjusted by ±50% of the set value. For example if for 1 ml/minute flow can vary between 0.5 to 1.5 ml/minute.

6.Column : Incase of column we can adjust three parameters those are column length, column diameter and column particle size.
Column length: can be adjusted by as much as ± 70%
Column diameter:can be adjusted if flow rate is kept constant.
Particle size:Particle size can be reduced by as much as 50% but it can’t be increased.

7.Ratio of mobile phase : Before understanding this parameters first note down two important points
(I) if mobile phase mixture is having two components then it will be called as Binary mixture
(ii) if mobile phase is having three components then it will be called as tertiary mixture.Example of binary mixture can be 50:50 ratio of ACN and Methanol And example of Tertiary mixture is ACN: Methanol : TEA in the ratio of 60:35:5
So Guyz Minor component of the mobile phase can be adjusted by ±30%. However, change in any component should not exceed ±10% of the total volume of mobile phase.

8.Detector wavelength : Detector wavelength cannot be changed.

So guys these were the 8 chromatographic parameters as per USP 621 which you can adjust to achieve system suitability results.Whatever parameters I have explained today they are applicable to isocratic run.And I hope you are aware of the fact that in isocratic run mobile phase composition remains same thorough out run.
Now the question arises what about gradient runs. How much variation is allowed in gradient runs. So guys that we cover in next video.

So this is it for today I hope you har learnt something new today. Keep learning bubbye and happy learning.

Thanks
Pharma Pill