Difference & derivative spectrometry

1. Presented by
Anjali.c
First year m.pharm,
Dept. Of pharmacypractice
Grace college of pharmacy

2.  Analytical methods based on measurements of UV or
visible light absorption.
 Advantage of Spectrophotometric procedures:
o Not time consuming
o Not labour-consuming
o Economical.
 The main disadvantage and limitation of the
spectrophotometry is its low selectivity.
 The measurement of absorbance is burden by
interferences derived from others components of
sample.

3.  A recorded UV-Vis spectrum is the sum of absorbance's of
analyte and matrix.
 Recorded bands are well-defined but more or less
distorted by the absorbance exhibited by reagents or
accompanied compounds in the sample.
 The problem can be omitted by measurements of sample
versus blank.
 But can be applied only in the case of :
o simple samples, which composition is stable and well known
o when highly selective reagents are used
 An isolation of an analyte from matrix is another solution
for increasing the selectivity of assay.
 But every additional operation introduced into sample
preparation procedure extents time ,costs & increases risk
of contamination of the analyte.

4.  The selectivity and accuracy of
spectrophotometric analysis of sample
containing absorbing interference may
be markedly improved by the
technique of difference
spectrophotometry.

5.  The measured value is the
difference absorbance(∆A)
between two equimolar solutions
of the analyte in different
chemical forms which exhibit
spectral characteristics.

6.  Reproducible changes may be induced in
the spectrum of the analyte by the
addition of one or more reagents.
 The absorbance of the interfering
substances is not altered by the reagents

7.  The simplest and commonly employed
technique for altering the spectral properties
is the adjustment of pH by means of aqueous
solution of acid,alkali,or buffers.
 Example:The uv-visible spectra of many
substance containing ionisible functional
groups are depend on the state of ionisation
of the functional group and pH of the
solution.

8.  . The selectivity and accuracy of
spectrophotometric analysis of samples
containing absorbing interferents may be
markedly improved by the technique of
difference spectrophotometry
 A substance whose spectrum is unaffected by
changes of pH can be determined by
difference spectrophotometric procedures.

9. another simplest method for an increasing a
selectivity is derivatisation of spectra. This
operation allows to remove spectral
interferences and as a consequence leads to
increase selectivity of assay.

10.  It involves the conversion of a normal
spectrum to it’s first,second or higher
derivative spectrum.
 The normal absorption spectrum is reffered
to as the fundamental zero order or D0
spectrum.
 The first derivative D1 spectrum is a plot of
the rate of change of absorbance with
wavelength against wavelength dA/dʎ
 The second derivative spectrum is a plot of
the curvature of the D0 spectrum against
wavelength or a plot of d2A/dʎ2 ʎ

11.  Derivative spectra may be generated by three
techniques.
 1.Modification of the optical system
 Spectrophotometers with dual
monochromators,photodetectors used
 generates a signal with an amplitude
proportional to the slope of the spectrum
over the wavelength interval.
• Disadvantage:Expensive ,Restricted to the
recording of first derivative spectra only.

12.  2.To generate derivative spectra is electronic
differentiation of the spectrophotometer
analog signal.
 Resistance capacitance (RC) modules are
highly dependent on instumental parameters,
the scan speed and the time constant.
 Standard solution of analyte is employ to
calibrate the measured value under the
instrumental condition selected

13.  3.Based upon microcomputers differentiation
 Micro computers incorporated in to or
interfaced with spectrophotometer may be
programmed
To provide
 Derivative spectra during or after scan
 To measure derivative amplitudes
between specified wavelengths
 To calculate concentrations and
associated statistics from the measured
amplitudes

14.  Multicomponent analysis
Derivative spectrophotometry (DS) has been
mainly used in pharmaceutical analysis for
assaying of a main ingredient in a presence of
others components or its degradation
product.
 Calculation of some physico-chemical
constants, e.g. reaction, complexation or
binding constants

15.  The main disadvantage of derivative
spectrophotometry is its poor reproducibility.