HPLC and Ion Chromatography Calibration Primer


Imagine the scenario, you have

almost developed a rugged

HPLC/Ion Chromatography

method, which provides

reproducible and well resolved

analyte peak/s of interest, at

reproducible retention times.


In order to continue with method validation, a reliable means of quantifying each analytical peak of interest is required.  For the purposes of method validation, with each of the mentioned calibration methods, repeat the process at least twice, to determine adequate linear regression and overall reproducibility.

Each analytical peak of interest

has quite a symmetrical peak

shape and is well integrated.



Chin up, it is not all bad news.


Step 1

Let’s decide how we wish to quantitate each integrated peak.   

Peak Height or Peak Area.  These both provide an indication of the detector signal/s to the analytical peak/s of interest. Be consistent in your choice.


You may use peak height if you are one of the rare few, where the analytical peak/s of

interest have near perfect symmetry,  


If not, then join the majority

and use peak area.


Step 2

There are four main techniques of analyte quantitation (calibration), external standard, internal standard, standard addition and normalisation. Whatever your choice, the Cecil Instruments’ PowerStream software will provide for automatic calculations.


External Standard


Aliquots of an analyte standard of known

concentrations, are injected separately from actual samples, and the acquired response factor, is used to calculate the  concentration of that same analyte within a sample/s.  


This is a simple technique, dependent on precision of injection volume, use an autosampler.  


Use when matrix effects are negligible and for samples which do not require large degrees of sample pre-treatment.  


Make up separate analytical standard solutions, using a high purity chemical.  


Detector response/s of the sample/s must fall within the range covered by the standard solutions.


A direct plot of detector response of analyte peak, to concentrations of standard, is used to create a calibration curve.

















Internal Standard


For use when a large amount of sample pre-treatment is required, as any loss of the analyte/s of interest correlates to losses of the internal standard.

 

The same amount of an internal standard is added to each standard/sample prior to pre-treatment, such as (extraction, dilution, filtration, centrifugation etc.). Standard/sample is injected.  


Make up separate analytical standard solutions, using a high purity chemical.  


Detector response/s of the sample/s must fall within the range covered by the standard solutions.


Choice of a suitable internal standard can be complex as it must behave in a similar manner to the  analyte/s of interest, but produce chromatographic peaks which are well resolved from the analyte/s of interest.  The internal standard should also have a similar linear response as the analyte of interest.


The ratio of the detector response of the analyte standard’s peak, to the internal standard’s peak, is used to create a calibration curve.  















Standard Additions


Use if sample matrix effects are problematic.


Often used when working with complex sample matrices, such as clinical, biological or food samples and when a blank sample matrix is not available.


To negate interference from components in the sample matrix, known concentrations of pure standards are added to aliquots of a single sample (spiking).  These spiked samples then become the analyte standards.

 

Pre-treat (extract, dilute, filter, centrifuge etc.) each analyte standard and actual sample and inject on the HPLC/Ion Chromatography system.

 

Make up separate analytical standard solutions, using a high purity chemical.  


Detector response/s of the sample/s must fall within the range covered by the standard solutions.


A direct plot of the detector response of each analyte peak, to concentrations of standard, is used to create a calibration curve.  There will be an intercept on the detector’s response axis.


Spiking may also be used to quickly check the identity of an analyte/s of interest’s chromatographic peak.


Normalisation


This applies to only a very limited number of analyses where the quantitative response of the detector is the same for all the eluted analytes of interest.


The percentage area of each eluted component, provides an estimate of the relative concentration of each component.  


An actual sample is injected and a chromatogram obtained.


For each peak of interest, create a normalisation calculation, based on the formula:-







A one–off response factor may be obtained for one peak.  The calculation would be:-







Should you wish to correlate the normalised quantity of peak B in a sample, the calculation would be:-













Cecil Instruments Limited  

Milton Technical Centre, Cambridge  United Kingdom

www.cecilinstruments.com      telephone  +44 (0)1223 420 821          info@cecilinstruments.com