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APCH231 : Chemical Analysis

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Introduction

S o far just about the only method for quantitative analysis that you have come across has involved titrations where we make use of volume measurements to determine the

concentration of an unknown. However, volumetric titrations are quite limited; especially in terms of their sensitivity – if you want to measure solutions with a concentration around about 0.01 to 1 M then a volumetric titration may be OK. On the other hand, titrations aren’t much use if you are trying to determine analytes at very low levels – for example around 10 -6 M – which we very often are called upon to do. For example you will determine trace amounts of iron in a vitamin tablet (~ 4 mg). Volumetric titrations are also limited in terms of the species that can be determined – not everything can be determined by titration. For these reasons (and others) instrumental techniques have been developed.

I n an instrumental technique, some physico-chemical property of the analyte is measured electronically – for example the absorption of light (if the analyte is coloured). Usually (hopefully!) the response of the instrument is proportional to the analyte concentration, so we get:

response concentration

or

response = constant x concentration

and we can find the analyte concentration from the instrument output if we know the value of

the proportionality constant.

Do not get confused by the various instrumental methods that we make reference to in the following examples. You will come across these in third year. The important point to realize is that each technique measures a response to a know concentration of solution. You should come across Beer's law in Physical Chemistry.

But how do we find the constant?

Calibration

W e can find the proportionality constant by calibrating the instrument. This is normally achieved by using a series of standard solutions. The instrument response is measured

for these known concentrations and a graph of response against concentration of analyte can be constructed. (What will be the equation of the line on this graph?) The instrument response for

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a sample of unknown concentration is found and the concentration of the analyte in the sample can then be determined from this calibration graph.

Calibration Graph
Instrument
Response

Concentration of Analyte

A linear response is what analytical chemists strive for in the calibration of an instrument. (You need more experimental points to fit a curve accurately.) Usually, if we have a

linear calibration, then five or six points are sufficient.

QQuueessttiioonn

1.
What is wrong (if anything) with each of the following calibration graphs?
a)
b)
Instrument
Response
Instrument
Response
Concentration of Analyte
Concentration of Analyte
c)
d)
Instrument
Instrument
Response
Response
Concentration of Analyte
Concentration of Analyte
e)
f)
Instrumen
In
strum e n t
Response
R
e sp o n se
Concentration of Analyte
Conce ntra tion of Ana lyte

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Preparation of Standards

A necessary skill for an analytical chemist is the ability to prepare a series of standard solutions with accurately known concentrations from the available chemicals and

glassware. The accuracy and precision of result of your analysis can be no better than the

accuracy and precision with which you make your standards. Sometimes the most time- consuming and laborious part of the analysis is the preparation of the standards.

Sensitivity

Sometimes one has a choice as to the exact instrumental response to measure. For example, if one is measuring the intensity of light absorbed by a solution one can change the instrument conditions to vary the wavelength of the light that is monitored. Usually, changing the conditions alters the slope of the calibration graph (ie the proportionality constant between response and concentration. The greater the slope the greater is said to be the sensitivity of the method.

QQuueessttiioonn

2. During a determination of copper using atomic absorption spectrophotometry, an analyst calibrates at two different wavelengths; 324.7 nm and 327.4 nm. The following calibration graphs are produced.

Absorption

λ = 324.7 nm
λ = 327.4 nm
Concentration of Copper

Which wavelength would you choose to use when finding the concentration of copper in some unknown solutions? Explain your answer.

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Fitting the Best Line

W hen preparing the calibration graph, the instrument response for a series of standards is plotted against the concentration of the analyte. For example, the following results

were obtained for the determination of Si by inductively coupled plasma emission spectrophotometry (a technique you will meet next year):

Si Concn

(mg/l)

Emission

Intensity

0.000

0.0250 2660

0.0500 5305

0.100 10640

0.200 21350

0.500 53300

0

It is an easy matter to fit a straight line to these points – put down a ruler and you will see that the points are almost in a perfect straight line.

Calibration Graph for the Determiantion of

Si
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50000
40000
30000
20000
10000
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Intensity

Si Concn (mg/l)

Calibration Graph for the Determiantion of

Si
60000
50000
40000
30000
20000
10000
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Intensity

Si Concn (mg/l)

However, it is not always so easy. What do we do in a case like this? (The results are from the determination of Sr by flame emission spectrophotometry):

Sr concn

(mg/l)

Emission

Intensity

0.00

1.00 16.6

2.00 37.8

3.00 43.2

4.00 68.7

0.0

5.00 95.2

Calibration Graph for the Determiantion of

Sr
100
80
60
40
20
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Intensity

Sr Concn (mg/l)

In this case it is not quite so easy to see where the straight line should go – it certainly does not join up all the points… In other words, the experimental points do not exactly obey the calibration equation (in this case, Intensity = const x Sr Concn). Why not? Because of experimental errors in the measurement of the intensity values. Back to our old friend uncertainty!

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S o how do we find the best line for “imperfect” points? We could try putting down a ruler and moving it until we get what we would (subjectively) call the best line. The points will

not all fall on the same straight line – whatever line we draw will leave us with differences between the experimental intensities and the intensities we should get if the calibration equation

was being followed exactly. These differences (on the y- axis) are known as residuals and the best line is the one that minimizes the sum of the squares of all the residuals – this is the method of least squares and is an example of linear regression. Linear regression is a statistical technique that allows us to find the equation of the best straight line through a set of points. From the equation we can draw the line on the calibration graph. The technique also gives us ways of deciding whether a straight line is the best line to draw through the points and also an estimate in the uncertainties involved. How exactly does the least squares method work? Look in a textbook or ask a statistician! In this module, we will not consider the derivation of the method but only how we can use the results. Fortunately for us, “doing least squares” is very easy – many calculators have least squares functions as do spreadsheets (such as MS Excel) and other specialized graph drawing software.

Results of Linear Regression

S o what do we get when we do a least squares analysis? We get a value for the slope (m) of the best straight line and also a value for the intercept (c) on the y-axis (ie at x = 0). Of

course, we can use these values for slope and intercept to find the concentration of an unknown solution; making use of the equation

= We can also obtain estimates of the standard error for the slope (se m ) and intercept (se c ) – these tell us about the uncertainty in the calculated values. We can calculate confidence intervals for the slope and intercept using:

y

mx

+ c

m ± t. se m and:

c ± t. se c

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In this case, we use n – 2 degrees of freedom when finding the t value, where n is the number of points on the graph. (we use n – 2 because we know both the slope and the intercept; losing one degree of freedom for each.)

QQuueessttiioonnss

3. A linear regression analysis gave values of 777 and 110234 ppm -1 for the intercept and slope of a calibration graph for the determination of P by ICP emission spectrophotometry. If a sample gave an emission intensity of 27630 what is the P concentration (ppm) in the sample?

4. A calibration graph for the determination of the fungicide carbendazim by absorption at 281.5 nm in a 1.00 cm cell gave the following parameters:

 Parameter Value Standard Error Slope 0.02293 (mg/l) -1 0.00008 (mg/l) -1 Intercept 0.0085 0.0016 R 2 0.99996 n 5

a) A sample of leather (0.5886 g) was treated with 0.1 M HCl to extract the carbendazim. The extract was made up to 100.0 ml and then diluted by taking 10.00 ml of the extract solution and making it up to 25.00 ml. The absorbance of the diluted extract solution in a 1.00 cm cell at 281.5 nm was found to be 0.3683. Find the % by mass of carbendazim in the leather sample.

b) Calculate the 95% confidence interval for the intercept.

c) Comment on how well Beer’s Law is followed.

The correlation coefficient (R) tells us how well the x and y values are correlated – if R = 1.000 then the points lie just about on a perfect straight line, whereas R = 0.000 means that there is absolutely no relationship whatsoever between the x and y values (not very useful for a calibration graph!!!) Often, the computer will give R 2 values rather than R. In practice, if you get R 2 values less than about 0.99 you should not be very happy about your calibration. Even

with R 2 values above 0.99 you should be careful

In the graphs below the same points are

shown fitted to a straight line and to a curve (also shown are the equations of the best lines and the R 2 values). Clearly the correct choice is to fit a curve even though the straight line gives R 2 = 0.99.

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450

400

350

300

250

200

150

100

50

0

y = 75x + 18.667
R 2 = 0.9906

0123456

400

350

300

250

200

150

100

50

0

y = -5x 2 + 100x + 2
R 2 = 1.000
0123456

QQuueessttiioonn

5. Two different analysts who were determining the phosphate concentration in a lake water sample obtained the calibration graphs shown below. The method involves taking a 10.00 ml sample of the water (sample or standard), adding reagents (which cause a blue colour to develop) making the solution to a final volume of 25.00 ml and measuring the absorption of the resulting solution.

 Calibration Graph Calibration Graph Analyst: Lucky Analyst: Hopewell
1
y = 0.8524x + 0.001
0.8
R 2 = 0.9988
0.6
0.4
0.2
0
0
0.5
1
Absorbance

PO 4 3- Concn (mg/l)

1.2
y = 0.9316x + 0.001
1
0.8
R 2 = 0.9505
0.6
0.4
0.2
0
0
0.5
1
Absorbance

PO 4 3- Concn (mg/l)

a) Comment on the calibration graphs.

b) Samples from each of two lakes were treated in the same way as the standards and the following results were obtained:

Lake

Absorbance

 Baringo 0.564 Bogoria 2.152

Calculate the phosphate concentration in each lake.

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1.

a) The line does not pass through the origin – this sometimes happens with calibration graphs. Also, there is no “blank” plotted – a blank is a standard with a concentration of zero.

b) The calibration line is horizontal – what does that tell us about how the instrument responds to the analyte? It tells us that the response is independent of the concentration of the analyte – is that any good?

c) At first sight this line looks fine – a straight line passing through the origin. (Why is that good…?) But look more closely and you will see that the line has been extrapolated past the point with the highest concentration? That’s dangerous – how do you know how the instrument will respond at high concentrations? Perhaps the line will begin to curve…

d) How many points should you plot?

e) This calibration line begins to curve at higher concentrations and when we have a curve we need a lot more points before we can fit it accurately. (Now you can see why extrapolating is dangerous – what would have happened if you hadn’t measured the last point but had extrapolated the straight line part?)

f) There is a straight line passing through the origin – but does it fit the experimental points well?

2. Which line gives the greater sensitivity? For a given concentration, which line will give the greater absorbance? For solutions of similar concentration, which line will give the bigger difference in absorbance? Which line will make it easier to measure low concentrations?

3. The calibration equation is:

Intensity = slope x concn + intercept So, for our sample we have:

27630 = 110234 ppm -1 x concn + 777 and so the concentration is 0.244 ppm. (How do we know the units of concentration? Look at the units of the slope… What are the units of the intensity and the intercept? They don’t have units – they are relative values.)

4.

a)

Concentration in the diluted extract =

0.3683

0.0085 =

0.02293

15.69

So concn in original extract solution =

25.00 x

10.00

So mass carbendazim extracted =

39.23x

100

1000

15.69

=

= 3.923

39.23

mg

mg/l

mg/l

So carbendazim in leather =

3.923

mg

x

1

g

0.5886 g

1000

mg

x

100

=

0.666

%(m/m)

b) 95% confidence interval = 0.0085 ± 3.18 x 0.0016 = 0.0034 – 0.0136

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Law, should be zero. On the other hand, the high value for R 2 and the small standard error for the slope indicate that the instrument response is linear – which it should be according to Beer’s Law.

5.

a) There doesn’t seem to be much wrong with Lucky’s graph – a good straight line passing very close to the origin and very close to all the points. Hopewell’s graph leaves a lot to be desired… looks like the experimental errors are quite high.

b) Which calibration graph would you use to answer this part? I’d go for Lucky’s. We’ve got the equation so it’s an easy matter to find the concentration of the unknowns:

0.564

0.001

For Lake Baringo:

(How do I know the units of concentration? Look at the x axis label of the graph.)

concn =

0.8524

= 0.660 mg/l

For Lake Bogoria: watch out!!! The absorbance of the sample is not within the range of the standards… So what? Well, that means that you will have to extrapolate beyond the region known to be linear – and that is dangerous (see the answer to Q 1 c). Does that mean that we cannot find the concentration of phosphate in the Lake Bogoria sample? Why not dilute the sample (so that it is within the range of the standards used) and try again…

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Using Microsoft Excel

You can use Excel to plot and display calibration curves. You must always use the "scatter plot" to produce your calibration curves. You can select whether you want the data points only or lines connecting the dots. This is not a "join the dots" scenario; you are trying to establish a relationship (constant) between a response from an instrument to a set of standards of known concentration. Therefore, you should normally just plot the data points and then add on the trendline afterwards. Remember to label the axis appropriately and give the graph a title, do not simply state that it is "x versus y".

Calibration graph for silicon by ICP-OES
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[Si] mg/L
Calibration graph for silicon by ICP-OES
60000
y = 106632x - 7.9377
50000
R 2 = 1
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30000
20000
10000
0
0
0.1
0.2
0.3
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0.5
0.6
-10000
[Si] mg/L
Emission Intensity
Emission Intensity

How do you add the trendline? Once you have plotted your data, select a data point and right click the mouse. There is a list of options that you can choose. Choose Add Trendline, a screen comes up with two tabs, select the tab that has Options. Choose what you want displayed on your graph, the equation of the line is useful and possibly the R 2 value. You can then use the equation of the line to determine the concentration from the response of the instrument to your samples. You can control the number of significant figures in the equation by double clicking the equation and choose the Number tab and then choosing the appropriate type e.g "number" or "scientific" and choosing the appropriate number of figures after the decimal.

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There are other ways to deal with data without having to plot a graph. You can use the Excel functions SLOPE and INTERCEPT. You can manually type them in or you can insert function and use the statistical category.

1

2

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13

A

B

CD

 Si Concn Emission (mg/l) Intensity 00 0.025 2660 0.05 5305 0.1 10640 0.2 21350 0.5 53300 slope 106632 intecept -7.9377 equations for slope SLOPE(C2:C7,B2:B7) for intercept INTERCEPT(C2:C7,B2:B7)

LINEST Another useful function is to use the LINEST function. Here we start by highlighting a 3-row × 2-colum.This is where the output will go. You then can either type in the function LINEST(C2:C7,B2:B7,TRUE,TRUE) followed by the key stroke CTRL+SHIFT+ENTER. Or once you have the cells highlighted you can use the INSERT menu and insert the statistical function LINEST. You then fill in the appropriate boxes tying TRUE for the last two entries and before accepting these data points you use the key stroke CTRL+SHIFT+ENTER. You should then get an output that looks like the following.

 Si Concn Emission (mg/l) Intensity Output from LINEST 0 0 slope Intercept 0.025 2660 parameter 106631.57 -7.937685 0.05 5305 std dev 49.328325 11.087448 0.1 10640 R 2 0.9999991 20.666204 0.2 21350 0.5 53300

Highlight cells E3:F5 Type "=LINEST(C2:C7,B2:B7,TRUE,TRUE)" followed by the key stroke CTRL+SHIFT+ENTER or you could insert the statsitical function LINEST (select True and True for the last entries.) You then need to press CTRL+SHIFT+ENTER to make the function work.

I have typed in and highlighted what the cells actually are. Reading down the column labelled slope you have the slope the standard deviation of the slope and the correlation coefficient. In the column labelled Intercept you have the intercept and the standard deviation of the intercept below it. In the bottom right hand side there is the standard deviation around the regression. It approximately corresponds to the average deviation of each y-data point (residual) from calculated line. You do not need to use this at this point in time. As you can see from the above data, the data fit the straight line extremely well and there is a linear relationship between the response of the instrument to the different concentrations of standards used. The relative error of the slope is 0.05%, this is good. The only poor part about using LINEST is that you need to know what the output is; there is nothing to tell you what the values are and the HELP is of no help!

As stated previously these can all be calculated manually and you can see how this is done in the textbook. At this point in time we are simply making you aware of Excel's functions and getting you to use the graphing capabilities of Excel.