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Lecture #5 - Overview

Analytical Response Statistics - Part 3

Calibration Curves

Statistical Tools in Quantitative Analysis The Method of Least Squares Calibration Curves Using a Spreadsheet for Least Squares

Measure of Unknown

Amount/Concentration of Unknown

= Known Amount/Concentration of Standard

Concentration of Standard

Construction of Calibration Curves


Standard Solutions = Solutions containing known concentrations of analyte(s) Blank Solutions = Solutions containing all the reagents and solvents used in the analysis, but no deliberately added analyte

Construction of Calibration Curves


Step 1: Prepare known samples of analyte covering a range of concentrations expected for unknowns. Measure the response of the analytical procedure for these standards.

Serial Dilution e.g.

1x

1/5x

1/25x

1/125x 1/625x

Blank

Measure response with analytical procedure

Construction of Calibration Curves


Step 1: Prepare known samples of analyte covering a range of concentrations expected for unknowns. Measure the response of the analytical procedure for these standards. Step 2: Subtract the (average) response of the blank samples from each measured standard to obtain the corrected value.

Construction of Calibration Curves


Step 1: Prepare known samples of analyte covering a range of concentrations expected for unknowns. Measure the response of the analytical procedure for these standards. Step 2: Subtract the average response of the blank samples from each measured standard to obtain the corrected value. Step 3: Make a graph of corrected versus concentration of standard, and use the method of least squares procedure to find the best straight line through the linear portion of the data. Step 4: To determine the concentration of an unknown, analyze the unknown sample along with a blank, subtract the blank to obtain the corrected value and use the corrected value to determine the concentration based on your calibration curve.

Corrected

Measured - Blank

Calibration Curves
Analytical Response

Measure of Unknown

Amount/Concentration of Unknown

= Known Amount/Concentration of Standard

Concentration of Standard

Method of Least Squares


to draw the best straight line through experimental data points that have some scatter and do not lie perfectly on a straight line

Method of Least Squares


Vertical Deviation = di = yi - y = yi - (mxi + b) di2 = (yi - y)2 = (yi - mxi - b)2

y = mx + b y !y !x y-intercept (b) x Slope (m) = !y !x

We wish to minimize to minimize the magnitude of the deviations (regardless of sign) so we square the terms. This is where Method of least Squares takes its name.

Method of Least Squares


"(xiyi) "xi Slope: m= "yi "(xi2) Intercept: b= "xi "xi n n "(xiyi) "yi D

Determinants
A B D AD - BC

"(xi2) D= "xi

Method of Least Squares


m = n"(xiyi) - "xi"yi n" (xi2) - ("xi)2

"(xi2)"yi - ("xiyi)"xi n" (xi2) - ("xi)2

Method of Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Amount Protein (mg) 0 5.0 10.0 15.0 20.0 25.0

Absorbance 0.099 0.185 0.282 0.345 0.425 0.483

Corrected* 0.000 0.086 0.183 0.246 0.326 0.384

* Absorbance - Average Blank (=0.0993)

Method of Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line.

Method of Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line.

m=

xi

" n=6

0 5.0 10.0 15.0 20.0 25.0 75

0 0.086 0.183 0.246 0.326 0.384 1.225

yi

0 0.43 1.83 3.69 6.52 9.60 22.07

x iy i

0 25 100 225 400 625 1375

x i2

n"(xiyi) - "xi"yi n" (xi2) ("xi)2

xi

= (6)(22.07) - (75)(1.225) (6)(1375) - (75)2 m = 0.015445714 " n=6

0 5.0 10.0 15.0 20.0 25.0 75

0 0.086 0.183 0.246 0.326 0.384 1.225

yi

0 0.43 1.83 3.69 6.52 9.60 22.07

x iy i

0 25 100 225 400 625 1375

x i2

b=

"(xi2)"yi - ("xiyi)"xi n" (xi2) - ("xi)2

= (1375)(1.225) - (22.07)(75) (6)(1375) - (75)2 b = 0.01109524

6 data points

Method of Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line.

m = 0.015445714 b = 0.01109524 y = (0.015445714)x + (0.01109524)

Method of Least Squares


to draw the best straight line through experimental data points that have some scatter and do not lie perfectly on a straight line

y = mx + b #y y

(xi,yi)

Vertical Deviation (di) = yi - y

di = yi - y = yi - (mxi + b) (di)2 = (yi - mxi - b)2 x

Uncertainty and Least Squares


#y ! sy = "(d1 - d)2 (degrees of freedom) "(d1)2 (degrees of freedom) "(d1)2 n-2

sy =

sy =

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

xi

" n=6

0 5.0 10.0 15.0 20.0 25.0 75.0

0 0.086 0.183 0.246 0.326 0.384 1.225

yi

0 0.43 1.83 3.69 6.52 9.60 22.07

x iy i

0 25 100 225 400 625 1375

x i2

di (=yi - mx - b) -0.0111 -0.0022 0.0174 0.0032 0.0060 -0.0132

0.00012321 0.00000540 0.00030442 0.00001036 0.00003589 0.00017525 0.00065442

d i2

sy =

"(d1)2 n-2 (0.00065442)/(6-2) 0.0001636

= =

= 0.012790808

Uncertainty and Least Squares

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

sm 2 =

sy2n D sy2"(xi2) D

sb2 =

0 5.0 10.0 15.0 20.0 25.0 " 75.0

xi

yi

0 0.086 0.183 0.246 0.326 0.384 1.225

0 0.43 1.83 3.69 6.52 9.60 22.07

xi yi

0 25 100 225 400 625 1375

xi 2

0.00012321 0.00000540 0.00030442 0.00001036 0.00003589 0.00017525 0.00065442

d i2

"(xi2) "xi

"xi n 75 6

D=

1375 D= 75

n=6 sy = 0.012790808

= (1375 x 6) - (75 x 75) = 2625

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

0 5.0 10.0 15.0 20.0 25.0 " 75.0

xi

yi

0 0.086 0.183 0.246 0.326 0.384 1.225

0 0.43 1.83 3.69 6.52 9.60 22.07

xi yi

0 25 100 225 400 625 1375

xi 2

0.00012321 0.00000540 0.00030442 0.00001036 0.00003589 0.00017525 0.00065442

d i2

sm2 = sy2n D = (0.012790808)2 (6) (2625) = 0.000000373954 sm = 0.000611518

0 5.0 10.0 15.0 20.0 25.0 " 75.0

xi

yi

0 0.086 0.183 0.246 0.326 0.384 1.225

0 0.43 1.83 3.69 6.52 9.60 22.07

xi yi

0 25 100 225 400 625 1375

xi 2

0.00012321 0.00000540 0.00030442 0.00001036 0.00003589 0.00017525 0.00065442

d i2

sb2 =

sy2 "(xi2) D

= (0.012790808)2 (1375) (2625) = 0.0000856977 sb = 0.009257307

n=6 sy = 0.012790808, D=2625

n=6 sy = 0.012790808, D=2625

Uncertainty and Least Squares


Example: To analyze protein levels, you use a spectrophotometer to measure a colored product which results from chemical reaction with protein. To construct a calibration curve, you make the following measurements of absorbance (of the colored product) for several known amounts of protein. Use the method of least squares to determine the best fit line. Calculate the uncertainty associated with this line.

Linearity

m = 0.015445714 0.000611518 = 0.0154 0.0006 b = 0.01109524 0.009257307 = 0.011 0.009

Linear Range vs. Dynamic Range


Dynamic Range

Linear Range

Determining Linearity
Square of Correlation Coefficient R2 = ["(xi - x)(yi - y)]2 "(xi - x)2 "(yi - y)2 R2 close to 1 (e.g. " 0.99, 0.98, 0.95) R2 High (>0.95) R2 Low (<<0.95)