Quantitative analysis (UV SPECTROSCOPY)

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Quantitative analysis: 

Quantitative analysis ( SINGLE COMPNENT ) MINU.K.P M.Pharm (Pharmacology)


TYPES Single Component Analysis Sample contain only one analyte Multi Component Analysis Sample contain more than one analyte

Single component analysis Different methods used: 

Single component analysis Different methods used I. Use of standard absorptivity value. II. Use of a Calibration graph. III. Method of least square. IV. Single Point Standardisation . V. Double Point Standardisation

Use of standard absorptivity value. : 

Use of standard absorptivity value. Used for stable compounds such as methyltestosterone , have broad absorption bands and practically unaffected by instrumental parameters. Use of Standard value avoid needs to prepare standard drug solution of the reference subtance . Advantage in situations where it is difficult or expensive to obtain a sample of the reference substance

Examples : 

Examples Paracetamol Standard absorptivity value at 257nm is 715 methyltestosterone Standard absorptivity value at 241nm is 540

Use of a Calibration graph. : 

Use of a Calibration graph. In this procedure the absorbances of a number (typically 4-6) of standard solutions of the reference substances at concentrations encompassing the sample concentration are measured and a calibration graph isconstructed .

PowerPoint Presentation: 

Concetration of analyte in the sample is read from the graph as the concentration corresponding to the absorbance of the solution. Calibration data are essential if the absorbance has a non-linear relationship with concentration , absorbance or linearity is dependent on the assay condition. pH , temperature and time of heating may gives a significant variations of absorbance, and experimentally derived calibration data is reqired for each set of samples.

Method of least square. : 

Method of least square. If the absorbance values and concentrations bear a linear relation ship the regression line y= α + β x can be estimated, where x is concentration and y is absorbance. (∑y)(∑x 2 ) - (∑x) (∑x y ) α = ---------------------------- N∑x 2 - (∑x) 2 N ∑x y - (∑x) (∑y ) β = -------------------------- N∑x 2 - (∑x) 2 N :- Number of pairs of values x :- Concentration and y:- absorbance

Single Point Standardisation. : 

Single Point Standardisation . Used when the relationship between concentration and absorbance is linear and proportional Involves the measurement of the absorbance of a standard solution and sample substance which are prepared in a similar manner in identical conditions. Concentration of the standard solution should be close to that of sample solution.

PowerPoint Presentation: 

Concentration of the substance in the sample is calculated from the proportional relationship exist between absorbance and concentration. A test x C Std C test = ------------------ A Std

Double Point Standardisation : 

Double Point Standardisation Used when the relationship between concentration and absorbance is linear and non proportional. Two standard solutions are used, one having greater concentration than sample solution and one having lesser concentration than sample solution. (A test - A std1 )(C std1 - C std2 ) + C std1 (A std1 - A std2 ) C test = --------------------------------------------------------- A std1 - A std2 Std1- more concentrated std Std2- less concentrated std


REFERENCE Practical pharmaceutical chemistry - Fourth edition-part two by A.H Beckett , J.B Stenlake . page:-278-281