This document discusses the technique of difference spectrophotometry and derivative spectrophotometry. It explains that these methods can improve the selectivity and accuracy of spectrophotometric analysis for samples containing absorbing interferents. Difference spectrophotometry works by measuring the difference in absorbance between two equimolar solutions with different chemical forms, while derivative spectrophotometry converts a normal absorption spectrum into its derivative to remove spectral interferences. Both techniques allow determination of a substance's spectrum that is unaffected by pH or other changes.

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.