Standardization and calibration -dr.mallik

It will be helpfull for those who wants to be part of pharma companies and want to be analytical chemists

1. Standardization and Calibration Prepared by- Dr . Mallikarjunaswamy C Assistant Professor Postgraduate Department of Chemistry JSS College of Arts, Commerce and Science Ooty Road, Mysuru-570025

2. The Language of Analytical Chemistry Analysis - A process that provides chemical or physical information about the constituents in the sample or the sample itself. Analyte - The component of interest in the sample Matrix - All other constituents in a sample except for the analytes . Determination - An analysis of a sample to find the identity, concentration, or properties of the analyte. Measurement - An experimental determination of an analyte’s chemical or physical properties. Technique - A chemical or physical principle that can be used to analyze a sample. Method - A means for analyzing a sample for a specific analyte in a specific matrix. Procedure - Written directions outlining how to analyze a sample . Protocol- A set of written guidelines for analyzing a sample specified by an agency. The Analytical Perspective

3. What is calibration? Calibration determines the relationship between the analytical response and the analyte concentration . Usually this is accomplished by the use of chemical standards .

4. Why is calibration important? Calibration is an act of evaluating and adjusting accuracy and precision of measuring instrument Instrument calibration intended to eliminate or reduce bias in an instrument’s readings. A well calibrated instrument provides confidence to users as well as it ensures the acceptance in international instrument.

5. STANDARDIZATION Standardization is the technique used to find the exact concentration of a solution Primary standard A primary standard is a compound of sufficient purity from which a standard solution can be prepared Properties of primary standard: 1 . It must be easy to obtain, to purify, to dry (preferably at 110-120 °C) and to preserve in a pure state. 2. The substance should be unaltered in air during weighing 3. The total amount of impurities should not exceed 0.01-0.02 per cent . 4. It should have a high relative molecular mass 5. The substance should be readily soluble under the conditions in which it is employed 6. The reaction with the standard solution should be stoichiometric and practically instantaneous

6. Comparison with Standards Two types of comparison methods are described Direct comparison technique and Example: Determination of creatinine by Jaffe’s picrate method 2. Titration procedure. HCI + NaOH  NaCI + H 2 O Example: titration of the strong acid HCI with the strong base NaOH

7. External Standard Calibration An external standard is prepared separately from the sample. Example- Determination of Na/K in drinking water by Flame Photometric method

8. External Standards   Single-point standardization - Any standardization using a single standard containing a known amount of analyte . A single standard containing a known concentration of analyte, C S , is prepared and its signal, S stand , is measured. The value of k (sensitivity) is calculated as = (1) Multiple-point standardization - Any standardization using two or more standards containing known amounts of analyte. Once standardized, the concentration of analyte, C A , is given as C A = (2 ) Multiple-point standardization is accomplished by constructing calibration curve , this is most frequently used method of standardization.  

9. Example : A spectrophotometric method for the quantitative determination of Pb 2+ levels in blood yields an S stand of 0.474 for a standard whose concentration of lead is 1.75 ppb. How many parts per billion of Pb 2+ occur in a sample of blood if S samp is 0.361? Solution: Equation (1) calculate the value of k for this method using the data for the standard = = = 0.2709 Once k is known, the concentration of Pb 2+ in the sample of blood can be calculated using equation (2) C A = = C A = = 1.33 ppb  

10. Method of least squares (linear least square)-how to plot a right straight line. In applying the method of least squares , the following two assumptions are fundamentally made : We assume that a linear relationship actually exists between the measured response ‘ y’ and the standard analyte concentration ‘ x ’ . The mathematical relationship describing this assumption is called the regression model , which may be represented as y = mx + b Where, y dependent variable x independent variable m slope of the curve b intercept on the ordinate (y-axis) y is usually measured variable, plotted as a function of changing x We also assume that any deviation of the individual points from the straight line arises from error in the measurement . whenever there is significant uncertainty in the x data, basic linear least-squares analysis may not give the best straight line. For example: in a spectrophotometric calibration curve y would represent the measured absorbance and x would be the concentration of standards. Then, our problem is to establish the values of ‘ m’ and ‘ b’ It can be statistically that the best straight line through a series of experimental points is that line for which the sum of the squares of the deviations of the points from the line is minimum . This is known as the ‘ method of least squares ’.

11. The square of the sum of the differences, S , is then

12. Correlation Coefficient and Coefficient of Determination The correlation coefficient is used as a measure of the correlation between two variables.(X and Y) When variables x and y are correlated rather than being functionally related (i.e ., are not directly dependent upon one another), we do not speak of the “best” y value corresponding to a given x value, but only of the most “probable” value. The Pearson correlation coefficient is one of the most convenient to calculate. This is given by

13. standard is added directly to the aliquots of analyzed sample This method is used where sample matrix also contributes to the analytical signal – MATRIX EFFECT Standard addition is frequently used in chemical instrumental analysis such as AAS and GC. STANDARD ADDITIONS METHOD Procedure- A typical procedure involves preparing several solutions containing the same amount of unknown, but different amounts of standard. For example, five 25 mL volumetric flasks are each filled with 10 mL of the unknown. Then the standard is added in differing amounts, such as 0, 1, 2, 3, and 4 mL. The flasks are then diluted to the mark and mixed well . The total concentration of the analyte is the sum of the unknown and the standard , and that the total concentration varies linearly . If the signal response is linear in this concentration range, then a plot similar.

14. ( Bear in mind that the chemical species X and S are the same) Signal is directly proportional to analyte concentration, so initial concentration of analyte [X] i gives a signal intensity I X Then a known concentration of standard, S, is added to an aliquot of the sample and a signal I SX Let’s call the diluted concentration of analyte [X] f , where “f” stands for “final.” We designate the concentration of standard in the final solution as [S] f . Standard addition equation: For an initial volume V o of unknown and added volume V S of standard with concentration [S] i , the total volume is V V o V S and the concentrations in Equation

15. If all standard additions are made to a constant final volume, plot the signal I SX versus the concentration of diluted standard, [S] f . In this case, the x -intercept is the final concentration of unknown, [X] f , after dilution to the final sample volume. The initial concentration of unknown, [X] i , is calculated from the dilution that was applied to make the final sample

16. References- Fundamentals of Analytical Chemistry - Douglas A. Skoog , F. James Holler, Stanley R. Crouch Analytical Chemistry 7e by Gary D. Christian Vogel’s Textbook of Quantitative Chemical Analysis by Arthur Israel Vogel Principles of Instrumental Analysis 7th edition Skoog by Douglas A. Skoog , F. James Holler, Stanley R. Crouch

17. Thank you