INSTRUMENTAL CHEMICAL ANALYSIS

LECTURE TWO
CHM 2203
BY

Godfrey Muhwezi

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 Outline of lecture Two

Figures of merit

Calibrations

Standardizations

Blank correction
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 Figures of merit
• A figure of merit is a numerical expression
representing the efficiency of a given instrument
or procedure. Figures of merit are a way for
analytical chemists to characterize a method. The
six figures of merit we use are;
Precision-A measure of the
reproducibility(Relative SD, Absolute SD)
Accuracy-How close a measured value is to the
true value
 Sensitivity-A measure of the methods ability to
distinguish between two samples reported as
change in signal per unit change in the amount of
analyte
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 Figures of merit cont’d

Linear dynamic range-Concentration range-(range

over which measurements can be made)
 Detection limit -smallest amount of analyte that
can be determined with confidence or the smallest
amount of analyte that has a signal larger than the
signal from the reagent blank
Selectivity-A measure of a methods freedom from
interferences

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 Calibrations

• Calibration is defined as the act of making sure that a

scientific process or instrument will produce results

which are accurate. In more complex terms, calibration

is the act which determines the functional relationship

between measured values and analytical quantities.

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 Calibration cont’d

• The goal of calibration is to minimize any

measurement uncertainty by ensuring the

accuracy of test equipment. Calibration

quantifies and controls errors or uncertainties

within measurement processes to an acceptable

level.
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 When do you calibrate instruments?

After an event e.g. if there is a knock,

bump anything that can impact accuracy

When measurements don’t seem right

When instructed by manufacturer

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 Methods of calibration

•Calibration curve method

•Standard addition method
•Internal standard method

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 Calibration curve method

• In the calibration method, also known as

working curve method, a set of
standards must be prepared. They will each
contain a known amount of the analyte being
measured. These standards are then measured
using the instrument in question, and
a calibration curve will be plotted. This curve
will show the relationship between the
response of the instrument and the
concentration of the analyte. An example of a
calibration curve can be found below.
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 Calibration curve

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 Calibration cont’d

• When using the working curve method, it is important that each standard is

prepared individually and not all from the same stock solution. Any errors

in the stock solution will carry through the entire calibration process, and

thus the instrument will not be calibrated correctly.

• Also, the calibration curve should be checked for any outliers. An outlier is

a measurement which is significantly different from the other

measurements. Put simply, these results will shift the regression line (line of

best fit) and give inaccurate results, and should, therefore, be removed.

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 Steps to follow during Calibration
• The calibration standards should cover the range
of interest – this is so, during your actual
experiment, you are sure to get the most accurate
results from your curve
• A ‘blank’ should be included in your calibration –
this is a standard which contains no analyte
• Don’t automatically set your regression line
intercept to zero! Only do this if you have enough
evidence to show that the intercept point is not
actually statistically different from zero.

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 Example: Pb in Blood by GFAAS
9(Students plot)
[Pb] Signal
(ppb) (mAbs)

0.50 3.76
1.50 9.16
2.50 15.03
3.50 20.42
4.50 25.33
5.50 31.87

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 35

30
y = 5.56x + 0.93
25

20
mAbs

15

10

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

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 .EXAMPLE
A sample containing an unknown amount of
Pb gives a signal of 27.5 mAbs. Calculate
the Pb concentration

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Standard addition calibration
The standard-addition method of calibration helps to remove bias that may arise
from a number of factors, including the temperature and composition of the actual
matrix.

For the standard-addition method of calibration, two requirements must be met:

1.The calibration curve has to be linear

2.The regression line must pass through zero

• In this method, the signal intensity of the sample solution is measured. Then, the
analyte is added to this solution at known concentrations – the signal intensity is
measured after each addition of the analyte. This, therefore, gives a calibration curve
which is linear and shows signal intensity vs. added concentration.
The concentration of the analyte is determined from the point where
the regression line crosses the axis at zero.

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 Standard addition calibration

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 Standard addition cont’d

• In this method, the matrix itself remains

completely unchanged – it is for this
reason that this method is useful in cases
where the matrix is either very
complicated or hard to reproduce.

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 Standard Addition Method

1. Most convenient when a small number of

samples are to be analyzed.

2. Useful when the analyte is present in a

complicated matrix and no ideal blank is
available.

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 Standard Addition Procedure

1. Add one or more increments of a standard

solution to sample aliquots of the same size. Each
mixture is then diluted to the same volume.

2. Prepare a plot of Analytical Signal versus:

a) volume of standard solution added, or
b) concentration of analyte added.

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 Standard Addition Procedure

3. The x-intercept of the standard addition plot

corresponds to the amount of analyte that must
have been present in the sample (after accounting
for dilution).

4. The standard addition method assumes:

a) the curve is linear over the concentration range
b) the y-intercept of a calibration curve would be 0

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 Example: Fe in Drinking Water

Sample Standard
Volume Volume The concentration of
(mL) (mL) Signal (V) the Fe standard
solution is 11.1 ppm
10 0 0.215
All solutions are
10 5 0.424 diluted to a final
10 10 0.685 volume of 50 mL
10 15 0.826
10 20 0.967

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 1.2

0.8
Signal (V)

0.6

-6.08 mL 0.4

0.2

0
-10 -5 0 5 10 15 20 25

-0.2
Volume of standard added (mL)

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 [Fe] = ?

x-intercept = -6.08 mL

Therefore, 10 mL of sample diluted to 50 mL would give a

signal equivalent to 6.08 mL of standard diluted to 50 mL.

Vsam x [Fe]sam = Vstd x [Fe]std

10.0 mL x [Fe] = 6.08 mL x 11.1 ppm

[Fe] = 6.75 ppm

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 Internal Standard Method

1. Most convenient when variations in

analytical sample size, position, or matrix
limit the precision of a technique.

2. May correct for certain types of noise.

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 Internal Standard Procedure

1. Prepare a set of standard solutions for

analyte (A) as with the calibration curve
method, but add a constant amount of a
second species (B) to each solution.

2. Prepare a plot of SA/SB versus [A].

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 Notes

1. The resulting measurement will be

independent of sample size and position.

2. Species A & B must not produce signals that

interfere with each other. Usually they are
separated by wavelength or time.

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 Example: Pb by ICP Emission
Each Pb solution contains 100
ppm Cu.

Signal

[Pb]
(ppm) Pb Cu Pb/Cu

20 112 1347 0.083

40 243 1527 0.159
60 326 1383 0.236
80 355 1135 0.313
100 558 1440 0.388
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 Standardization

• Standardization is the process of determining the

exact concentration of a solution. It is the process
of experimentally determining the relationship
between the signal and the amount of analyte

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 Blank correction

• In blank determination all steps of the analysis are

performed in the absence of a sample. A blank solution
contains the solvent and all the reagents in an analysis
but none of the sample. The results from the blank are
then applied as a correction to the sample
measurements. Blank determinations reveal errors due
to interfering contaminants from the reagents and
vessels employed in analysis.

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Questions that follow are based on this
Excel sheet

Microsoft Excel
Worksheet

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 Calculations
1. Using the data on the Precision and Accuracy tab, calculate the
average, standard deviation, percent relative standard
deviation, and 95% confidence interval.
a. What is the precision of the data?
b. The known value for the data is 0.1500. Is there evidence of
bias? Support your answer.
2. A calibration curve is presented in the Calibration Curve tab, where
the response of a method is plotted versus the analyte
concentration.
a. Estimate the linear region of the data. One way to approach this
problem is to selectively remove data points from the plot until
you maximize the R2 value.
b. What is the sensitivity in the linear region?

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 Questions continued

3. Data are presented in the Limit of Detection tab for 10

replicate measurements of a 4.00 mM sample ([A] =
4.00 mM) and 10 replicate measurements of a blank
([A] = 0 mM). Perform the following calculations in the
spreadsheet. Use the calibration curve from part (2)
to determine the slope.
a. Calculate the signal detection limit.
b. Calculate the limit of detection.
c. Calculate the signal quantitation limit.
d. Calculate the limit of quantitation.

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