DIFFERENCE & DERIVATIVE SPECTROMETRY

Presented by

Anjali.c

First year m.pharm,

Dept. Of pharmacypractice

Grace college of pharmacy

Analytical methods based on measurements of UV or visible light absorption.

Advantage of Spectrophotometric procedures: o Not time consuming o Not labour-consumingoEconomical.

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.

INTRODUCTION

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.

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

DIFFERENCE SPECTROPHOTOMETRY

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

FEATURE OF DIFFERENCE SPECTROPHOTOMETY

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

CRITERIA FOR APPLYING DIFFERENCE SPECTROPHOTOMETRY TO THE ASSAY OF A SUBSTANCES IN THE PRESENCE OF OTHER ABSORBING SUBSTANCE

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.

. 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.

ADVANTAGES

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.

DERIVATIVE SPECTROMETRY

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 vs ʎ

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.

INSTRUMENTATION

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

3.Based upon microcomputers differentiation

Micro computers incorporated in to or interfaced with spectrophotometer may be programmedTo provide

Derivative spectra during or after scan To measure derivative amplitudes

between specified wavelengths To calculate concentrations and

associated statistics from the measured amplitudes

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

Analytical application of derivative spectrophotometry

The main disadvantage of derivative spectrophotometry is its poor reproducibility.

DISADVANTAGE