Internal and external standard method of

calibration in Chromatography
 Contents

Internal Pros and Equation Pros and

Response External
Standard Cons for Cons
Factor Standard
Calibration Calibratio
Equation Calibration
n Factor

2
 Internal Standard Calibration
Introduction

Internal standard calibration involves the comparison of

the responses of reference standards added to the sample
or sample extract before injection to the instrument
responses from the target compounds in the sample.

3
 Internal Standard Calibration
Introduction

The response of the target compound is normalized to

the response of the reference standard. This standard is
called
an internal standard as it is contained
within the aliquot of the sample or sample
extract that is added into the
instrumentation.

4
 Response Factor Equation
The ratio of the peak area of the target compound in the sample extract
to the peak area of the internal standard in the sample extract is
compared to a similar ratio obtained for each calibration standard.

This ratio is referred as the relative response factor or for short RRF, therefore
showing that the target compound response is found relative to that of the
internal standard.
 Response Factor equation

RF = ((Ax)(Cis))/((Ais)(Cx))

Where: Ax = Area of compound

Cx = Concentration of compound
Ais = Area of internal standard
Cis = Concentration of internal
standard

6
 Advantages
✘ It is the reason for routine variation in the response of
the chromatographic system.
✘ It also accounts for the variations in the exact volume
of sample or sample extract introduced in the
chromatographic system.
✘ The retention times of the target compound and the
internal standard can be used to find out the relative
retention time (RRT) of the target compound and
after that it may be used to compensate for small
retention time shifts.

7
 Disadvantages
✘ The main disadvantage of internal standard
calibration is that the internal standards must
be compounds that aren’t found in the
samples to be analyzed and also they must
produce an unambiguous response on the
chromatographic detector system.

8
 External Standard Calibration
Introduction

For an external standard quantitation, unknown data

from the sample and known data from a calibration
standard are combined to generate a quantitative report.
This is known as external standard because the standard
or known material is external or separate to the
unknown material.

9
 External Standard Calibration
Introduction

External standard calibration is one of the most

commonly used approaches to calibrations. It requires a
simple comparison of instrument responses from the
sample to the responses from the target compounds in
the calibration standards.

10
Calibration Factor Equation

What is Calibration
Factor?
The ratio of the detector response to the
mass of analyte in the calibration standard is
termed as the calibration factor or CF.

11
Equation for Calibration Factor
(External Standard Curve)

CF = (Ax)/(Cx)

Where: Ax = Area of compound

Cx = Concentration of compound
 Advantages
✘ One advantage of external standard
calibration is that it is very simple to
perform this type of calibration.
✘ Also, this calibration can be applied to a
wide variety of methods.

13
 Disadvantages
✘ The most prominent disadvantage is that it is
considerably affected by the stability of the
chromatographic detector system and the
presence of chromatographic interferences
in a sample or sample extract.

14
 References

www.Wikipedia.com
www.researchgate.net
www.chromatographyonline.com
www.analyticalsciencejournalsonline.com

This presentation is made by Hanzala Labeeb CHE-13B