Using
an
Internal Standard with an HPLC,
A
Simple
Calculation Method
There
are many reasons to use an internal standard with any
chromatographic system. The most
common is to eliminate variations with the injection volume. With new autosampler technologies this
reason has largely disappeared but there remain a number of
applications where
it is a good idea to subject the standard and the analyte to the same
sample
introduction methods.
Because
an internal standard method uses the ratio of responses
for standard and analyte, quantitation does not depend on the amount of
material
injected.
In
this example the internal standard is caffeine and the analyte
is aspirin.
First
we make up a standard solution containing a known
concentration of analyte and a known concentration of internal
standard.
For
the known standards of aspirin, Solve for F
Area
internal standard peak
Concentration of internal standard
--------------------------------------- =
F
------------------------------------------------------
Area of
aspirin standard peak
Concentration of aspirin standard
Note
that in the equation above we know everything but F, which is
a dimensionless constant.
Plugging
in some rounded off numbers from a recent HPLC run we get
the following:
13 4
--- = F ------
8
5
Note
that even though we have more aspirin (5 mg/ml) than caffeine
(4 mg/ml) the caffeine peak was bigger.
Solving
for F we get 2.03
Now we
run the solutions containing a known amount of internal
standard (caffeine at 4 mg/ml) and an unknown amount of analyte
(aspirin at ?)
Area
internal standard peak
Concentration of internal standard
--------------------------------------- =
F --------------------------------------------------
Area of
aspirin unknown peak
Concentration of aspirin unknown
Now we
solve for the concentration of aspirin in our unknown
sample (again these are rounded off numbers.)
13
4
--- = 2.03
------
5
X
X =
3.12 mg / ml of aspirin.
Internal standards
can be confusing but by taking the calculations one step at a time and
working carefully, they can be a valuable addition to your
chromatography skills.
This information posted by:
Kevin Olsen
Instrumentation Specialist
Chemistry and Biochemistry Support Staff
Montclair State University
Montclair, NJ, 07043
973-655-4076
http://blake.montclair.edu/~olsenk/