Appendix 3: Instructions for Using MS Excel

A. Calibration Curve for ELISA

A calibration curve of absorbance vs. concentration for the ELISA analysis is non-linear and can
be fit to a log function as shown below.

Absorbance vs. Concentration

1.2

0.8
Absrobance

0.6

0.4

0.2

0
0 1 2 3 4 5 6
Concentration (ppb)

However, in creating a calibration curve for this experiment a calibration curve of absorbance vs.
log concentration will be used. The following formulas can be used to simplify calculations in a
spreadsheet. The raw data is shown in columns A and B and the calculations are performed in
columns D-G.

To calculate the average absorbance of the 0.5 ppb std. in cell e2 the formula “=(b2+b3)/2” was
applied.
To calculate the log of concentration (0.5 ppb) in cell f2 the formula “=log(d2)” was applied.
To find the range in absorbance measurement of the 0.5 ppb standard in cell g2, the formula
(=e2-b3) was applied. This range can be used to place error bars on absorbance measurement.

Example:
A B C D E F G
Ave. Log Range in
Conc. Abs. Conc. Abs. conc. Abs.
0.5 0.9876 0.5 0.98205 -0.30103 0.00555
0.5 0.9765 1 0.73875 0 0.00125
1 0.74 5 0.3811 0.69897 0.0012
1 0.7375
5 0.3823
5 0.3799

1
The resulting calibration curve of Abs. vs. log concentration in ppb can then be fit to the best
straight line using linear regression. Y-error bars showing the range in absorbance values of the
two standards should be displayed.

Absorbance vs. Log conc. y = -0.5852x

2
+ 0.7783
R = 0.9865

1.2

0.8
Absorbance

0.6

0.4

0.2

0
-0.4 -0.2 0 0.2 0.4 0.6 0.8
Log Conc. (ppb)

To create the calibration curve. Go the insert menu and choose chart (x-y scatter)
Select the “series” tab and choose “add.”
For x values, highlight the column in the spreadsheet containing log concentration values.
(f2..f4)
For y values, highlight the column in the spreadsheet containing ave. absorbance values. (e2..e4)
To add a trendline using the method of least squares, click on a data point on the chart to select
the data series. Right click and select “add trendline.” Under options choose “display equation
and r2 value.”
To add y error bars. click on a data point on the chart to select the data series. Right click and
select “format data series.” Choose “y error bars,” “both,” “custom.” In both the + and – range
select the column in the spreadsheet containing the range in abs. measurements (g2..g4).

Now use the calibration curve and the absorbance of your unknown to calculate the
concentration in the unknown.

2
B. Uncertainty in unknown based on error in the calibration curve. (GC-MS data)

Sections 5-1, 5-2 and 3-5 in Quantitative Analysis 6th Ed. by

Daniel Harris, W.H Freeman has a detailed discussion of finding the uncertainty from a
calibration curve and propagation of uncertainty in mathematical operations. A copy of this text
has been placed on reserve in the chemistry library.

Calibration Curves and Propagation of Uncertainty

The following data is used as an example of generating a calibration curve and finding the
uncertainty.

Protein amount (x) Absorbance (y)

5 0.0887
10 0.1727
15 0.2477
20 0.3307

The method of least squares finds the “best” straight line through experimental data by
minimizing the vertical deviations between the points and line.

Calibration Curve y = 0.016x + 0.0097

R2 = 0.9995
0.35

0.3

0.25
Absorbance

0.2

0.15

0.1

0.05

0
0 5 10 15 20 25
Protein amount (ug)

What is the amount of protein in the unknown if the absorbance is 0.2730? What is the
uncertainty in the answer?

3
Finding the amount of protein in the unknown:

The equation for the straight line and the absorbance of the unknown was used to find the
unknown concentration (x).

0.2730 = 0.016x + 0.0097

x = 16.45

Uncertainty in x can be found from the following formula:

x=
Sy 1 x 2 n ∑ xi
+ +
( ) − 2 x∑ x
2
i

m k D D D

k is the number of measurements of the unknown

∑ (d )
2

sy =
i

n−2

d i = y i − mxi − b

( )
D = n∑ xi − (∑ xi )
2 2

Spreadsheet calculation:
xi yi xi2 b di di2
5 0.0887 25 0.0097 -0.001 0.000001
10 0.1727 100 0.0097 0.003 9E-06
15 0.2477 225 0.0097 -0.002 4E-06
20 0.3307 400 0.0097 0.001 1E-06
Sum 50 750 1.5E-05

sy =
(1.5 x10 ) = 0.0027386
−5

4−2

D = (4)(750)-(50)2 = 500

Solving for the uncertainty:

x=
Sy 1 x 2 n ∑ xi
+ +
( ) − 2 x∑ x
2
i

m k D D D

4
 0.0027386 16.45 2 (4) (750) 2(16.45)(50)
x= 1+ + − = 0.38
0.016 500 500 500

Answer: 16.45 ± 0.38