Quantitative & Qualitative HPLC

Septiawan Adi Nugroho

Fakultas Farmasi
Institut Ilmu Kesehatan Bhakti Wiyata
 Qualitative Analysis Overview

In the former case, it might be possible to inject

standards of the pure compound, and assign the
peaks in the chromatogram based on the retention
time of the standard. Having a selective detector,
such as diode-array UV or Fluorescence detector,
which assists in identification by producing spectra
or a specific response, can assist in peak
assignment.
Qualitative Analysis Overview
Sample Spiking
The technique of
spiking a sample
involves the addition
of a known reference
material to a sample
matrix, in order to
confirm the identity of
one of the sample
component peaks.
Spectral Peak Identification
The use of selective detectors and
spectrometers can greatly increase the
confidence in the peak assignment. Detector
systems such as Diode Array UV
Spectrometers or Mass Spectrometers are
able to record unique spectra for each peak
within the sample chromatogram. The spectra
may be recorded in real time as the eluent
can be directly introduced into the detector
system.
Mass Spectrometric detectors can be configured to
produce fragmentation patterns that can be assigned
to analyte moieties, so building up a picture of the
analyte molecule.
 Quantitative Analysis Overview

After the peaks have been integrated and

identified, the next step in the analysis is
quantification. Quantification uses peak areas
or heights to determine the concentration of a
compound in the sample.
 Area %/ Height %
The Area% calculation procedure reports the area of each
peak in the chromatogram as a percentage of the total area
of all peaks. Area% does not require prior calibration and
does not depend upon the amount of sample injected within
the limits of the detector. No response factors are used. If
all components respond equally in the detector and are
eluted, then Area% provides a suitable approximation of the
relative amounts of components.
 External Standard Quantitation

The external standard (ESTD) quantitation

procedure is the basic quantification
procedure in which both calibration and
unknown samples are analysed under the
same conditions.
The results (usually peak height or peak area
measured using a data system) from the
unknown sample are then related to those of
a calibration sample, using a calibration
curve, to calculate the amount in the
unknown.
 Calibration Curve

The curve is usually constructed by injecting

an aliquot of the calibration (standard)
solution of known concentration and
measuring the peak area obtained. Peak
height is sometimes used but only in
exceptional circumstances.