Calibration method for HPLC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

5 posts Page 1 of 1
Hi,

Can anyone suggest how to validate a HPLC calibration procedure.The following are the reasons:

1) procedures are vendor specific
2) Certain universal procedures recommends restrictor coil instead of column
3) Traditional HPLC vs UHPLC

If we are planning an harmonised approach, what can be the better validation methodology?

Regards
Ratan
Hello

What is the opinion on this topic - calibrating a hplc with or without column? Is there scientific advantage on this

Regards
Ratan
First of all, a bit of pedantry: "calibration" essentially means determining the relationship between system response and analyte amount (i.e., the response factor). As such, it applies to a *method* not to an *instrument*. Closely tied to this is the concept of "system suitability". In a regulated environment (GLP or cGMP), any validated method should have a set of system suitability criteria. These will typically include things like retention time(s), plate numbers, RSDs, etc. These obviously require the use of a column.

From context, I think you are referring to "qualification" (specifically, "performance qualification" or "PQ"), which is verifying that the instrument if functioning properly within it's specifications. In a regulated environment this is typically done at least annually. Complete PQ on an HPLC system involves verifying things like:
Pump:
- intake line integrity
- check valve / seal integrity
- flow accuracy
- proportioning accuracy / dwell volume (gradient systems)
Injector:
- reproducibility & linearity
Detector:
- noise & drift
- wavelength accuracy
Some of these tests involve the pump alone; some (proportioning or dwell) require a restrictor between injector and detector; some require a column (injector tests) and for some (detector) either way will work.

That was the long answer (sorry!). The short answer is that there is no "one size fits all".
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Tom, what you describe is what I would call an OQ.
In my world, PQ is performed for a specific use. F.x. an analytical method run on the particular system. If more than one methods are used on the particular system, they have to be taken into account. One could argue though that the most demanding method e.g. involving mobile phase gradient, wavelenght shift etc. would be an adequate documentation for all the methods run on the system.
Wouldn't you agree?

Regarding the original question, I believe it's the range/linearity one wants to test - perhaps using different concentrations or injection volumes. In that case no column is required.
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Dancho Dikov
"Calibration", as a term is not applied to an HPLC system. You can calibrate for a wavelength, but we do not calibrate HPLC systems at all. We "Qualify" them to see if they meet a specification (usually the manufacturer's); Example: An Instrument Qualification (IQ) or Operational Qualification (OQ). These check each of the component modules to see if they meet specific specifications established by the manufacturer. Pipettes are calibrated. Balances are calibrated. HPLC's are not calibrated.

Lose terms such as Performance Verification (PV) usually are applied to an SOP which tests a specific metric, such as injection RSD or gradient composition. It can be almost anything that you properly define.

You Asked; "What is the opinion on this topic - calibrating a hplc with or without column? Is there scientific advantage on this".
    As mentioned, we do not calibrate an HPLC. We do run many tests though and most do NOT use a column (because in most cases, you are not trying the qualify the column, you are qualifying a specific module). Please refer to this short article, "The HPLC Restriction Capillary; Troubleshooting, Qualification and Running Without A Column"; https://hplctips.blogspot.com/2018/04/t ... llary.html
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