2) Determination of the concentration by UV Spectrophotometric method Molecular absorption spectroscopy is based on the measurement of absorbance (A) or transmittance (T)

of the solution contained in transparent cells. Thus, when using a spectrophotometer, we get the absorbance (A) of solution of the test substance and solution of its reference standard. In some cases reference standard may not be available. After getting the absorbance value, there are different possibilities for the determination of the concentration of a sample solution. a) As the instrument directly provides the absorbance (A) reading, we can use the formula, A= $nown and c&A 'r, c & !(T )ence by preparing a series of solutions of the standard in different concentrations, we can measure absorbance of each of the solutions. *y plotting A or !(A, against the concentration we can get a straight line that may pass through the origin (c=+, A= +(T=!++). This calibration line may then be used to determine un$nown concentration of solution of the same material after measurement of absorbance. The linearity of the curve is established by statistical treatment of data facilitated by a computer or calculator. The line of best,fit can be established and the value of -c. is determined by the relation A= bc / a, where 0b1 is the slope of the line and 0a1 is the intercept on the y,a%is. The 0a1 and 0b1 parameters can also be obtained by the following relation2 a = 3(4y)( 4%5) 6 (4%)( 4%y)7( 384%5 6(4%)57
!
!" ! cm

b# c, for the calculation of concentration, where b is

!" ! cm

is available from the reference boo$s.

b) If we e%amine the relation A = abc,

b= 384%y 6 (4%)( 4y)7( 384%5 6(4%)57, where % is related to 0c1 and y is related to 0A1 c) 9e prepare the sample solution and the standard solution of appro%imately similar concentrations and determine their absorbance. :or e%ample, dissolve !++mg of standard in !++ml of solvent. Then dilute !ml to this solution to !++ml solvent (standard solution). 9eigh a ;uantity e;uivalent to !++mg of the sample and prepare the solution as above(test solution) . Measure the absorbance of both the solutions. 9e have, <tandard solution2 !++mg ===.!++ml !ml ===.. !++ml Test solution2 % mg of substance containing about !++mg active ingredient======.!++ml ! ml ===...!++ml If absorbance reading for test is T and for standard < then, >alculation2 T(< # standard dilution( test dilution = T(s # (!++mg(!++ml#!ml(!++ml) # (!++ml(!++mg#!++ml(!ml) mg of active ingredient in !mg of the test substance. This information can be used to calculate the percentage purity or the active ingredient content per tablet. %amples2 !. >alculate the concentration of methyltestosterone in an ethanolic solution of which the absormance in a i,cm cell at its ? ma% 5@!nm, was found to be +.AB+. The A!"!cm of methyltestosterone is given in *C as D@+ at5@!nm.
5

5. >alculate the concentration in Eg(ml of a solution of tryptophan (molecular weight 5+@.5) in +.!M hydrochloric acid, giving an absorbance at its ?ma% 5FFnm, of +.G!H in a @cm cell. The molar absorption at 5FFnm is D@H5. H. The absorbance values at 5D+nm of D standard solutions, a blan$ solution and a sample solution of a drug are given in the following Table. >alculate the concentration of the sample. >oncentration (Eg(ml) +.+ !+ 5+ H+ @+ D+ <ample A (at 5D+nm) +.++5 +.!GA +.H5B +.D+A +.GG+ +.A@G +.G!!

@. In an assay of a sample of progesterone, a solution was prepared by dissolving B.GF@mg of sample in alcohol to ma$e !++ml, and then ma$ing a ! in !+ dilution of the first solutionI the e%tinction of this dilution in a !cm cell at ?ma% 5@+mE was found to be +.D!I for the pure progesterone in alcohol,
!" ! cm

at

?ma% 5@+mE is D@+. >alculate the percentage of progesterone in the sample. 3) Application of Beers Law to Mi t!res
*eer1s law also applies to a medium containing more than one $ind of absorbing substance. Crovided that the species do not interact, the total absorbance for a multi,component system is given by A (total) = A! / A5 / =/ An = J!bc / J5bc / JHbc / =./ Jnbc, where the subscripts refer to absorbing components !,5,H1=.,n. @. The spectrophotometric readings of the standard solutions of a colored cpd at @G+nm were as shown in the table. >alculate the ;uantity of the substance present in syrup. The sample included Dml of the syrup diluted to !++ml with B+" ethanol. (use a scientific calculator) H

>onc. (mg(ml) !.+++ 5.+++ H.+++ @.+++ D.+++ <ample thanol (BD")

Absorbance +.!@A +.5D5 +.HD! +.@@G +.D@F +.H!5 +.+++