This document discusses external and internal standardization techniques for quantitative analysis using high performance liquid chromatography (HPLC). External standardization involves preparing solutions of a reference standard at known concentrations, measuring peak areas, and generating a calibration curve to determine unknown concentrations. Internal standardization adds a known amount of internal standard compound to samples and standards, and quantifies unknowns based on peak area ratios and a calibration curve plotting ratios of analytical standard to internal standard areas versus concentration ratios. The internal standardization method is commonly used for gas chromatography as it avoids needing to know exact injection amounts.

1. HPLCExternal and internal
standard
(Source: Remington, The Science
and Practice of Pharmacy )
Dr. Amit Gangwal
Smriti college of pharmaceutical education, Indore
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Amit Ratn Gangwal Jain Amit Ka PPT

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4. ExtErnal standardization
• A Pure reference standard material
corresponding to the substance to be
determined is dissolved in a solvent at a
known concentration.
• Various quantities of this solution are injected
successively.
• The areas of each the peaks produced are
plotted versus the mass of the solute injected
and a calibration curve is produced.

5. Now the unknown is injected and area of peak
determined and the concentration is found by
interpolation.

6. intErnal standardization
• This is to avoid difficulty of introducing
precisely measured quantities into the
Gas Chromatography.
• It involves two standards:
– Analytical: a pure sample of the compound to
be analyzed
– Internal: it is normally a substance that elutes
at a position close to the substance being
analysed and is well resolved, but it should
not be converted into analyte under normal
course of analysis.

7. Continue
• A series of solutions is prepared containing
varying amounts of analytical standards and
constant amounts of the internal standard.
• These are chromatographed and a calibration
curve is determined by plotting the ratio of the
areas of two peaks versus the ratios of their
concentrations.
• The unknown then is dissolved in a suitable
solvent, the same amount of internal standard is
added, and the mixture is chromatographed.

8. Continue
• The ratio of the area is calculated and by
interpolation on the calibration curve, the
amount of the unknown determined.
• This method is mostly use technique for
quantitative analysis by GC because it is not
necessary to know the exact amount of solution
injected.
• Usual practice is to prepare a solution of the
internal standard in the solvent employed to
dissolve reference standards or samples, thus
ensuring unvarying ratios of standard or sample
peaks areas to that of the internal standard.