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5 (de) vizualizări11 paginiCalibration curves

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Calibration curves

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Calibration curves

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Learning Objectives

After completing this module, the student will be able

to

find a calibration curve using the Excel function

trendline

write a macro in Excel

explain the meaning of R2

explain sources of error when estimating the

independent variable value

find a confidence interval for the independent

variable value

trendline calculation

linear regression

coefficient of determination

calibration

Prerequisites

linear equation

average and standard deviation

normal distribution

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 1

Pre-assessment

Before completing the module test whether you master the prerequisites. Linear

Equation

1.

2.

3.

4.

5.

6.

Find the equation of a horizontal line that goes through the point (2,4).

Find the equation of a vertical line that goes through the point (-1,3).

Determine the equation of the line passing through (-2,1) and (3,-1/2).

Determine the equation of the line passing through (1,-2) and (-2,4).

Determine the equation of the line with slope 3 and vertical intercept (0,2).

Determine the equation of the line passing through (-1,-1) and parallel to the line

passing through (0,1) and (3,0).

y 2x 1

7. Graph of the line given by the equation

.

3x 4y 1 0

8. Graph the line given by the equation

.

Average and Standard Deviation

9. Find the average and sample standard deviation of the following data set:

2,4,5,6,6,7,8

10.Write down the equation for calculating the average and the sample standard

x1 , x2 ,..., xn

deviation of a data set of size n:

Normal Distribution

11.Suppose X is normally distributed with mean 2 and standard deviation 1. Find (a)

the 75th percentile, (b) the 95th percentile, and (c) the 99th percentile.

12.Suppose X is normally distributed with mean 3 and variance 4. Find the

P(1 X 4)

probability that X is between 1 and 4, that is, find

.

13.Suppose X is normally distributed with mean -1 and standard deviation 4. Find an

interval centered about the mean so that with probability 0.95 X is contained in

that interval.

14.Suppose that the number of seeds a plant produces is normally distributed with

mean 142 and standard deviation 31. Find the probability that a randomly

sampled plant will produce more than 200 seeds.

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 2

Calibration

According to the NIST handbook

(http://www.itl.nist.gov/div898/handbook/pmd/section1/pmd133.htm), [t]he goal of

calibration is to quantitatively convert measurements made on one of two

measurement scales to the other measurement scale. The relationship between

two measurements is used to convert one measurement into the other

measurement. You saw one such example in your chemistry lab where you

measured absorbance to find the concentration of an unknown sample. In this case,

the relationship between absorbance and concentration was linear. You derived the

relationship by measuring absorbance of standard samples of known concentration.

The resulting line is called calibration curve. The basis for the calibration curve is

Beers Law, which states that there is a direct linear relationship between

absorbance (A) and concentration (c): When if we graph absorbance as a function of

concentration, a straight line with positive slope provides a good fit. To illustrate

this, we provide in the following table absorption measurements of standard

samples:

Concentration

[mole L-1]

0

20

40

60

80

Absorbance

0

0.2356

0.4725

0.7127

0.9507

If we graph the data points and fit a straight line through the points (Figure 1), we

find that the equation of the straight line is

A 0.0119c 0.0014

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 3

This curve is called a standard curve and is used to infer the unknown concentration

of a solution. For instance, if we find that the absorbance A of an unknown solution

is 0.6386, we find for the concentration c

0.6386 (0.0014)

53.8

0.0119

The data in our example fits Beers Law extremely well. The data was generated

using a Virtual Lab on Spectrophotometry

(http://www.chm.davidson.edu/vce/Spectrophotometry/UnknownSolution.html).

When data are obtained in actual lab experiments, measurement errors need to be

taken into account.

We assume in the following that we measure a signal y that depends linearly on a

quantity x. We call the quantity x the independent variable and the quantity y the

dependent variable. We assume that we measure x without error and that the

quantity y is measured with an error that is normally distributed with mean 0 and

standard deviation . The relationship between the two quantities is then

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 4

y a bx

To get a sense for the measurement uncertainty when inferring the quantity x from

the measurement y, we begin with simulating an experiment in which we have a set

of n standard samples and for each sample we measure the signal m times.

In-class Activity 1

In the spreadsheet CalibrationWorkbook under the tab Simulation, you will find

x 10,20,40,60,80

the simulation of standard samples with values

a 0

b 1

the intercept

and the slope

. Each signal is measured 3 times. The

simulated data are in the gray-colored box. The input parameters for the slope, the

intercept, and the standard deviation s.d. for the error are in the yellow-colored box.

The trendline is calculated using the Excel function LINEST. (This function is difficult

to use and you will not need to learn how at this point.)

To investigate how the estimated value of the independent variable x depends on

the error , we proceed as follows. We assume that the (unknown) value of the

y ax b

independent variable x is equal to 50 (Cell F11). Using the equation

a1

with

b 0

and

with s.d. 1, we can calculate the measured value of the quantity y

(Cell F12). We can then use the estimated trendline to find the estimate for x (Cell

F13) . The graph displays the simulated data from the calibration experiment, the

trendline, and the data point corresponding to the unknown sample.

When you press F9, you will see that Excel runs another simulation. By repeatedly

pressing F9, you can get a sense for the variability of the estimated value of x in our

simulation experiment. It is tedious to record manually the values of repeated

simulations. Excel has a feature, called Macro, that records repeated key strokes.

Lets write a macro to record the outcome of repeated simulations for the estimate

of x.

(a) To write a macro to simulate values of x, proceed as follows:

1. Open the Developer tab and click on Record Macro in the Code group.

Citation: Neuhauser, C. Calibration

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 5

2.

3.

4.

5.

6.

Give the macro a name and select a key, for instance, Ctrl-a works.

Select the Home tab.

Copy the value of x from Cell F13.

Paste the value of x into Cell Q3 as Paste Value.

Click on Insert in the Cells group and click on Shift cells down in the

Insert window.

7. Go to the Developer tab and click on Stop Recording in the Code group.

If you press Ctrl-a, the simulated values will be copied into your spreadsheet in

Column Q. Repeat the simulation 100 times. (The numbers in Column P help you

keep track of the simulations.)Sort the simulated values from Smallest to Largest.

Find the middle 90%.

x 15

x 50

spread out compared to when

measurement error affects the uncertainty in the calculation of x.

Figure 2: Screenshot of the simulation. The input parameters are listed in the yellow box; the

simulated data are listed in the gray box; the estimated values of the slope and vertical

intercept are listed in the green box together with the calculation of the unknown quantity x

Citation: Neuhauser, C. Calibration

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 6

based on the measurement of the unknown sample y. The graph displays the simulated data

(blue symbols), the trendline (black line), and the unknown measurement (red data point).

Linear Regression

When two quantities are linearly related, such as absorbance and concentration, a

straight line provides a good fit. In Excel, a straight line can be fitted using the

Trendline option. The Trendline option is under the Layout in the Chart Tools.

When clicking on the blue triangle under Trendline and choosing More Trendline

Options, a window opens that offers additional options, such as Display Equation

on chart and Display R-squared value on chart. We already know the meaning

of the Equation. We will now look at the meaning of R-squared.

y a bx

deviation . We obtained data points

,

, and used the Trendline

option to fit a straight line. This results in estimates for the slope and the intercept.

a

We denote the estimated value of the intercept by and the estimated value of the

b

slope by

The method that Excel uses to estimate the slope and the intercept is called method

n

j 1

and

)

yj (a bx

j

2

is as small as possible. We say that the sum of the squared deviations is minimized.

Expressions for the estimated intercept and slope can be given. It is not important

to memorize the expressions.

Citation: Neuhauser, C. Calibration

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 7

y a bx

with

b

(xj x)(yj y)

j 1

(xj x)2

j 1

y bx

a

To measure how good the fit is we calculate a quantity called the coefficient of

(xj ,yj )

2

, we can

bx

j a

y

j

define

yj y

mean,

j ) (y

j y)

yj y (yj y

A somewhat lengthy calculation shows that the total sum of squared deviations

(yj y)2

j 1

(Explained) and a part that reflects the stochastic errors (Unexplained)

n

j y)2 (yj y

j )2

(yj y)2 (y

1j 14 2 4 3 1j 14 2 4 3 1j 14 2 4 3

Total

Explained

Unexplained

The ratio between the explained variation and the total variation is the coefficient of

determination

Citation: Neuhauser, C. Calibration

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 8

Explained

R2

Total

the model.

R2

j y)2

(y

j 1

n

(yj y)2

j 1

In-class Activity 2

Return to the spreadsheet CalibrationWorkbook. Under the tab Simulation, you

have already worked on the simulation of standard samples with values

x 10,20,40,60,80

a 0

b 1

and the slope

. Each signal

is measured 3 times. The simulated data are in the gray-colored box. The graph has

a small textbox where the equation of the trendline and the coefficient of

determination is listed. You will see that when you increase the standard deviation,

the coefficient of determination decreases. Give a verbal explanation as to why you

would expect this.

In your Calibration Lab, you were asked to prepare a calibration curve. The

spreadsheet CalibrationLab.xlsx will help you do the analysis. Open the

spreadsheet. The Calibration Lab Analysis sheet is set up so that you can enter your

data into the yellow cells. To calculate the calibration curve, enter the data from the

absorbance measurements of the standard samples into C4:C21 (Step 2). The

spreadsheet will calculate the slope and intercept in the cells I19 and I20,

respectively, (see blue cells and Step 3). In Step 4, the spreadsheet calculates the

coefficient of determination. Compare the values in the cell to the textbox in the

figure that has the same information.

(a) To include the uncertainty of the calibration curve in your lab report, record the

coefficient of determination together with the equation of the trendline. Explain in

words the meaning of the coefficient of determination.

Citation: Neuhauser, C. Calibration

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 9

(b) In the chemistry lab, you then determined the concentration of an unknown

sample based on the calibration curve. Enter the three measurements into cells

B25-B27 (Step 5). The spreadsheet is set up so that it calculates the estimated

concentration. Use paper and pencil to verify the result in Cell B 31 (estimated

concentration) the spreadsheet.

(c) While the theory is beyond this course, the spreadsheet is set up to calculate a

x*

confidence interval for the estimated concentration

. In Cell K25, you can set the

confidence level, for instance 95%. The lower and upper limits of the confidence

interval are listed in Cells K27 and K28, respectively. Record the confidence interval.

The Cell K26 contains the value of half the length of the confidence interval, which

x* Cx

Cx

we denote by

If you want to read more about Linear Calibration, consult the statistics and data

analysis paper by Burke, S. Regression and Calibration. LC GC Europe Online

Supplement.

Homework

1. Find a linear regression line through the given points and compute the coefficient

of determination

x

y

-3.0

-6.3

-2.0

-5.6

-1.0

-3.3

0.0

0.1

1.0

1.7

2.0

2.1

temperature, the following data were obtained by Pierce, 1949 (The Songs of

Insects. Cambridge, Mass. Harvard University Press):

Temperature

(F)

Chirps/sec

69

70

72

75

81

82

83

84

89

93

15

15

16

16

17

17

16

18

20

29

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 10

used. The calibration curve is obtained from the following data

Added glucose,

[glucose] (mM)

Absorbance

0.00

0

0.23

1

0.05

0

0.27

9

0.10

0

0.31

4

0.20

0

0.42

3

0.30

0

0.54

0

0.40

0

0.66

5

(a) Find the equation of the calibration curve and the coefficient of

determination.

(b) Suppose the absorbance of an unknown sample is measured as 0.356. Use

the calibration curve to estimate the glucose level.

Created: October 18, 2009 Revisions:

Copyright: 2009 Neuhauser. This is an open-access article distributed under the terms of the

Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted use,

distribution, and reproduction in any medium, and allows others to translate, make remixes, and

produce new stories based on this work, provided the original author and source are credited and the

new work will carry the same license.

Funding: This work was partially supported by a HHMI Professors grant from the Howard Hughes

Medical Institute.

Page 11

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