Difference & derivative spectrometry
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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.
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.