Getting Started in HPLC
Section 4F. Putting it all Together
So far, we
have have discussed various manipulations of the sample (weighing,
dilution, etc.) and related calculations. Often two or
more of these operations are combined into a single assay
procedure. Here we will look at the calculation of a
final assay result in such cases.


We can
summarize our approach to various problems as follows:


In our first
example, consider the analysis of a liquid formulation
for phthalic acid using a singlepoint calibration. We
begin by preparing our calibrator; i.e., weighing out
phthalic acid into a volumetric flask and filling to mark
with solvent. The procedure for this method calls for 0.5
grams of phthalic acid to be dissolved in 50 mL of
deionized water:


Our analytical
procedure calls for first diluting the 1 mL of the liquid
sample to 25 mL, followed by injecting the diluted sample
into the LC system. The dilution factor for the sample is
calculated from:


Next we run our calibrator
and obtain a peak at 5.4 minutes with an area of 20,000
counts. Because we are using a singlepoint calibration,
we must calculate the sensitivity factor:

Chromatogram of phthalic acid calibrator solution (10 mg/mL). The area of the peak at 5.4 minutes was measured as 20,000 counts.


When we run the diluted
sample, we obtain a peak at 5.4 minutes with an area of
30,000 counts. Now we can calculate the concentration of
diluted sample:

Chromatogram of sample containing phthalic acid. The area of the peak at 5.4 mnutes was measured at 30,000 counts. 

What is the
concentration of phthalic acid in the original (undiluted)
sample? We obtain this from the concentration of the
dilute sample and the dilution factor:


In a second example, consider the analysis of a solid sample for dimethylchickenwire (DMCW). We begin by weighing out a certain quantity of DMCW and dissolving it in a certain volume of solvent. Let's assume the following values for calibrator and samples 1 and 2:


Now, three chromatograms
are run with the following results:


We first
calculate the sensitivity factor for the DMCW peak in the
calibrator:


Next we
calculate the concentration of DMCW in diluted sample #1:
The concentration of the total sample in the solution is 42.0 mg / mL, so the fraction of DMCS in the original solid sample #1 is:


Sample #2 was
offscale, so it must be diluted and rerun. Five mL of
sample was diluted into a 25 mL flask. The dilution
factor is:


The diluted sample was
reinjected. The resulting peak at 4.68 minutes had a peak
area of 36,000. The concentration of DMCW in this diluted sample of #2 was then:


The
concentration of DMCW in the original (undiluted) sample
is given by the concentration in the diluted sample and
the dilution factor:
The concentration of the total sample in the solution is 39.80 mg / mL, so the fraction of DMCS in the original solid sample #1 is:


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