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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.

1x

1/5x

1/25x

1/125x 1/625x

Blank

## 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

## 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

## 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

yi

x iy i

x i2

xi

yi

x iy i

x i2

b=

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.

## 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)

## 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

yi

x iy i

x i2

d i2

sy =

= =

= 0.012790808

## 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 =

xi

yi

xi yi

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

## 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

yi

xi yi

xi 2

d i2

xi

yi

xi yi

xi 2

d i2

sb2 =

sy2 "(xi2) D

## 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

## 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)