Calibration Curves

Statistics - Part 3

Statistical Tools in Quantitative Analysis

The Method of Least Squares
Calibration Curves
Using a Spreadsheet for Least Squares

Analytical Response

Lecture #5 - Overview

Measure of Unknown

Amount/Concentration
of Unknown

Concentration of Standard

= Known Amount/Concentration of Standard

Construction of Calibration Curves

Standard Solutions = Solutions containing known
concentrations of analyte(s)

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
Blank Solutions = Solutions containing all the reagents
and solvents used in the analysis, but no deliberately
added analyte

e.g.

1x

1/5x

1/25x

1/125x 1/625x

Blank

Measure response with analytical procedure

Construction of Calibration Curves

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 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 2: Subtract the average response of the blank samples

from each measured standard to obtain the corrected value.

Corrected

Measured - Blank

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.

Analytical Response

Calibration Curves

Measure of Unknown

Amount/Concentration
of Unknown

Concentration of Standard

= Known Amount/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

Slope (m) = y
x

y-intercept (b)

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=

Intercept:

b=

D=

yi

(xi2)

(xiyi)

xi

(xi2)

xi

xi

yi

Determinants

AD - BC

Method of Least Squares

m

n(xiyi) - xiyi
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

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

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

6 data points

x iy i

0
0.43
1.83
3.69
6.52
9.60
22.07

x i2

0
25
100
225
400
625
1375

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.

n(xiyi) - xiyi
n

(xi2)

xi

(xi)2

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

0
5.0
10.0
15.0
20.0
25.0
75

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

x iy i

0
0.43
1.83
3.69
6.52
9.60
22.07

x i2

0
25
100
225
400
625
1375

b=

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

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

n=6

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 =

sy =

sy =

(d1 - d)2
(degrees of freedom)
(d1)2
(degrees of freedom)
(d1)2
n-2

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

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

x iy i

0
0.43
1.83
3.69
6.52
9.60
22.07

x i2

0
25
100
225
400
625
1375

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

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.

(d1)2
n-2

sy =

d i2

0.00012321
0.00000540
0.00030442
0.00001036
0.00003589
0.00017525
0.00065442

=
=

(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

sb2 =

sy2(xi2)
D

xi

0
5.0
10.0
15.0
20.0
25.0
75.0

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

xi yi

0
0.43
1.83
3.69
6.52
9.60
22.07

xi 2

0
25
100
225
400
625
1375

d i2

0.00012321
0.00000540
0.00030442
0.00001036
0.00003589
0.00017525
0.00065442

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.

xi

0
5.0
10.0
15.0
20.0
25.0
75.0

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

xi yi

0
0.43
1.83
3.69
6.52
9.60
22.07

xi 2

0
25
100
225
400
625
1375

n=6
sy = 0.012790808, D=2625

d i2

0.00012321
0.00000540
0.00030442
0.00001036
0.00003589
0.00017525
0.00065442

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

(xi2)

D=

xi

xi

1375

75

75

D=
= (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.

xi

0
5.0
10.0
15.0
20.0
25.0
75.0

yi

0
0.086
0.183
0.246
0.326
0.384
1.225

xi yi

0
0.43
1.83
3.69
6.52
9.60
22.07

xi 2

0
25
100
225
400
625
1375

n=6
sy = 0.012790808, D=2625

d i2

0.00012321
0.00000540
0.00030442
0.00001036
0.00003589
0.00017525
0.00065442

sb2 =

sy2 (xi2)
D

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

Uncertainty and Least Squares

Linearity

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.

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)