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Calibration of instrumental methods

Background: Calibration and standardisation processes are important in instrumental methods of analysis. These processes determine the relation between the response and concentration. In this process, chemical standards are required to obtain the correct responses. The objective of calibration is to determine the reliable, valid and accurate results. The following are the important procedures used for the calibration.

1. Comparison with standards There are two important types of comparison methods: direct comparison techniques and titration. a. Direct comparison: In some methods we compare the response of analyte with the standard substance. For this, we need to prepare the sample and standard solution in a similar way. The approach is to prepare the sample solution of similar concentration as standard giving similar responses. uch procedure is called null comparison or isomation method. For e!ample, we may prepare: Standard solution: " #$$mg made #$$ml " #ml of this diluted to #$$ml Test solution: " ! mg of substance containing about #$$mg active ingredient dissolved #$$ml " # ml of this diluted to #$$ml If absorbance reading for test is T and for standard then,

Calculation: 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. b. Titration In all titrations the analyte reacts with the standard quantitatively in a reaction of *nown stoichiometry. The amount of titrant is varied and the final end +point of chemical equivalence is determined by the use of indicator. If the ,balanced equation- of the reaction may be represented as: a. / tT 0 1roducts, where ,a- is the number of molecules of analyte ., and ,t- is the number of molecules of titrant (T). In such situation, for the calculation, we have to use the following relation. t &mol of . ' a &mol of T222(i) or, t & mmol of . ' a & ml of titrant consumed & molarity of titrant.

2. Standard addition method This is very popular method in analytical chemistry particularly in instrumental methods (spectroscopic, polarographic etc.). In many cases the sample composition may be comple! and it is impossible to prepare standard solutions of similar composition. In such a situation the method of standard addition can be employed for the determination of the quantity of specific ingredients. For e!ample, if we want to determine the lead content in the water of 3agmati 4iver by the atomic absorption method, as a general process, a calibration curve from the absorption readings of standard solutions could be obtained and the concentration of the sample could be calculated from the curve.

!bsorbance

6 6 6 6

ample signal

Conc. m ml

3ut the concentration of lead obtained (in this way) from the river water sample may not be reliable5 it may be higher or lower than the actual concentration, due to the presence of other components. This matri! effect would eliminate if the standard solutions had the same composition as the un*nown sample. In many cases this approach is not feasible. Therefore, a technique called standard addition is widely applied in spectroscopic methods in order to overcome the matri! effect. In this method, equal volumes (fi!ed volume) of the sample are ta*en (about five in number) and different amounts of *nown lead solution (standard solutions) are added to each e!cept the one. Then all solutions are diluted to the same volume. The atomic absorption signals are measured for every solution and the results are plotted on a graph paper (with the dependent variables on the y"a!is and the amount of lead added in the !"a!is. The e!trapolation of the standard curve touching at a point on the !" a!is (i.e. where y'$), provides the lead content in the river water.

Standard addition

The method of standard addition is used in instrumental analysis to determine concentration of a substance (analyte) in an un*nown sample by comparison to a set of samples of *nown concentration, similar to using a calibration curve. tandard addition can be applied to most analytical techniques and isuse d instead of a calibration curve to solve the matri! effect problem.

". #nternal standard method In most of the instrumental analysis methods, electrical response or any measured signal related to the concentration of an analyte component is proportional to the concentration. For e!ample, in flame emission, Ie (emitted light intensity) is proportional to concentration C of the metal in the sample i.e. Ie '7.C. imilarly in 89C and :19C, the pea* height or area is proportional to concentration. :owever, in using such approaches, errors may be introduced due to variations in different parameters and linearity may not be achieved for calculating the un*nown concentration. Therefore, internal standard method is employed to solve the variability problems. In flame emission, errors may be introduced due to variability in: ample si;e Change in flame performance 8as flow rate ample pre"treatment methods etc. Thus an internal standard (lithium salt) is added in the sample before injecting it into the spectrophotometer. It is assumed that internal standard is affected by any variability to the same e!tent as the analyte metal. The ratio 4 of the pea* height for analyte .s to that of internal standard .i should be constant for a certain concentration of analyte Cs in constant concentration of the internal standard. i.e. 4' .s%.i ' Cs%Ci i.e. 4 ' Cs. #%Ci ' Cs.7. (as ci is fi!ed)

The internal standard method involves the addition of a fi!ed amount of reference material (the internal standard) to the sample solution and a series of standard solutions.

< #. >. ?. A. @. C.

tandard%sample olution Concentration #$ ppm (std #) >$ ppm (std >) @$ ppm (std ?) B@ ppm (std A) #$$ ppm (std @) " (spl) =olume #$ ml #$ ml #$ ml #$ ml #$ ml #$ml

Internal standard volume #$ ml #$ ml #$ ml #$ ml #$ ml #$ ml

Total volume (final sol.) >$ ml >$ ml >$ ml >$ ml >$ ml >$ ml >$ ml

From the above solutions, the following data can be obtained from the spectrophotometric readings. $inal solution #. ( >. ( ?. ( A. ( @. ( C. ( td #) td >) td ?) td A) td @) ample) 1ea* of height standards%sample from chromatogram in cm(1s) 1s # 1s > 1s ? 1s A 1s @ ample of 1ea* height of %atio &'s '#( the I TD in cm (1i) 1i 1i 1i 1i 1i 1i 1s#%1i 1s>%1i 1s?%1i 1sA%1i 1s@%1i 1 %1i

Thus the ratio of the standard solutions and the pea* height of the internal standard is plotted with the concentration of the standard solutions. This gives a calibration curve. The concentration of the sample is read from the graph for the ratio of pea* height of sample to the internal standard.

%atio of 'eak height

4' 1s%1i

Conc. of spl

Conc. of standard solutions

The plot of the ratio of the pea* heights against concentration should be linear and pass through the origin or close to origin. The linearity of the calibration may be tested by calculating the correlation coefficient which should be close to #. The line of best fit can be calculated by using a computer%calculator or relevant formula. ). *+ternal standard calibration In this method an e!ternal standard solution is separately prepared from the standard substance related to the component of interest (analyte). From this solution, a series of solutions of the standard in different concentrations can be prepared. Ee can measure the response (e.g absorbance) of each of the solutions. 3y plotting the response (.) or #%., against the concentration we can get a straight line that may pass through the origin (C'$, .' $(T'#$$). This calibration line may then be used to determine the concentration of the sample, i.e. the absorbance of the sample can be used to read the concentration of the sample directly from the graph. 3ut if the calibration curve does not pass through the origin or if some of

the points are not in the straight"line Fthe least square method- is used to find the linearity or the line of best fit. Determination of the line of best fit or linearit, of the calibration cur-e: .fter plotting the points of the responses and the concentration of standard solutions, in a graph, if all the points do not fall in a straight line we will have problem to decide the standard curve. 3esides, even if all points may fall in a straight"line, the line may not pass through the origin. In such cases, we cannot directly read the concentration of the sample from the calibration graph. In this situation, we need to use a ,least square method- or ,linear regression- method. This method provides the means for getting the best line by specifying certain uncertainties. It is based on the mathematical model y' 3! / ., where ,3- is the slope of the line and ,.- is the intercept cut by the line in the y"a!is. This equation (with *nown value of 3 and) . is called the line of best fit. .s we have the value of y (response of the sample), we get the value of ! (conc. of sample) from the equation. The linearity of the curve is also established by statistical treatment of data facilitated by a computer or calculator. The statistical treatment should be followed for all techniques which use a calibration graph or linear regression approach for the determination of the analyte concentration. For a series of standard solution, the value of . and 3 can be calculated by the following relations. . ' G(Hy)( H!>) I (H!)( H!y)J% G<H!> I(H!)>J 3' G<H!y I (H!)( Hy)J% G<H!> I(H!)>J Ehere K is the instrumental signal (response) value at L concentration and < is the number of pairs of values.

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