APP601S 2024

CHAPTER 5: Standardizing
Analytical Methods
Standardization can be defined as: The process of
determining the relationship between the signal and the
amount of analyte in a sample.

For many analytical techniques, we need to evaluate the

response (signal) of the unknown sample against a set of
standards (known quantities).

This this is where calibration comes in!

Calibration involves the following:

1) Determine the instrumental responses for the

standards.
2) Find the response of the unknown sample.
3) Compare the response of the unknown sample to that
from the standards to determine the concentration of
the unknown.
 Analytical Standards
⚫ To standardize an analytical method we use standards.

⚫ We divide analytical standards into 2 categories: primary

standards and secondary standards.

⚫ Primary standards: reagents for which we can dispense an

accurately known amount of analyte (must have a known
stoichiometry, a known purity (or assay), and be stable
during long-term storage).
- i.e. 0.1250 g sample of K2Cr2O7 contains 4.249×10−4
moles of K2Cr2O7.

⚫ Secondary standards: Reagents that do not meet the

above criteria. Their concentration must be determined
relative to a primary standard.
 Determining the Sensitivity (kA)

Stot = SA + SReag
= kACA + SReag

To standardize an analytical method we must determine

the value of kA

Single-point standardization: we measure the signal for a

standard, Sstd, containing a known concentration of analyte,
Cstd.

If Sstd = kA Cstd then kA = Sstd / Cstd

A single-point standardization is the least desirable

method for standardizing a method!!!
Multiple-point Standardization (the preferred approach):
prepare a series of standards (at least three), each
containing the analyte at a different concentration:

If Sstd = kA Cstd

A plot of Sstd versus Cstd is known as a calibration curve.

• The most common method of standardization uses one

or more external standards.

• We call them “external” because we prepare and

analyze the standards separately from the samples.
Example 1
Rauha Primus, a research student prepares 6 solutions with a known
concentration of Co-trimoxazole and added the necessary colouring
agents to form complexes. She then used a UV-Vis spectrophotometer
and measured the absorbance for each solution at a particular
wavelength. The results are in the table below.

Concentration Absorbance Corrected

/ mg.l-1 absorbance
0 0.002 0.000
1 0.078 0.076
2 0.163 0.161
4 0.297 0.295
6 0.464 0.462
8 0.600 0.598

Corrected absorbance = (sample absorbance) – (blank absorbance)

 Calibration curve:
Response = dependant variable =

0.7
0.6
0.5
0.4
Abs

0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9
Conc / mg/l
y

Concentration = independant variable = x

 Fit best straight line:
0.7
0.6
0.5
0.4
Abs

0.3
0.2
y = 0.075x + 0.003
0.1
0
0 1 2 3 4 5 6 7 8 9
Conc / mg/l
The student then measured her sample to have an
absorbance of 0.418 and the blank, 0.003. She,
therefore, can calculate the concentration using the
obtained calibration curve.
y = 0.0750x + 0.0029

Abs = (0.0750 x Conc) + 0.0029

Conc = (Abs – 0.0029)/0.0750

For the unknown sample:

Corrected absorbance = 0.418 – 0.003 = 0.415
Conc = (0.415 – 0.0029)/0.0750
Conc = 5.49 mg.l-1

Check on your calibration curve!!

Absorbance = 0.415 Conc = 5.49 mg.l-1

0.7
0.6
0.5
0.4
Abs

0.3
0.2
y = 0.075x + 0.003
0.1
0
0 1 2 3 4 5 6 7 8 9
Conc / mg/l
 METHOD OF LEAST SQUARES
Assume:
➢ There is a linear relationship.
➢ Errors in the y-values (measured values) are greater
than the errors in the x-values.
➢ Uncertainties for all y-values are the same.

0.7
0.6 Vertical
Minimise only the 0.5 deviation
vertical deviations 0.4
= y - yi
→ assume that the
Abs

0.3
error in the y-values 0.2
are greater than 0.1
that in the x-values. 0
-0.1 0 2 4 6 8 10
Conc/ppm
Recall:
Equation of a straight line:
y = mx + c
where m = slope and c = y-intercept

We thus need to calculate m and c for a set of points.

Points = (xi, yi) for i = 1 to n
(n= total number of points)

m=
n (xi yi ) −  xi  yi  (
xi2 ) yi −  (xi yi ) xi
( )
n xi2 − ( xi )2
c=
n (xi2 )− ( xi )2
Example 1

xi yi xiyi xi2
0 0.000 0 0
1 0.076 0.076 1
2 0.161 0.322 4
4 0.295 1.180 16
6 0.462 2.772 36
8 0.598 4.784 64
xi = 21 yi = 1.592 xiyi = 9.134 (xi2) = 121

Slope: y-intercept:

m=
n (xi yi ) −  xi  yi  (
x i2 ) yi −  (x i yi ) x i
( )
n xi2 − ( xi )2
c=
n (xi2 )− ( x i )2
Recall:

0.7
0.6
0.5
0.4
Abs

0.3
0.2
y = 0.075x + 0.003
0.1
0
0 1 2 3 4 5 6 7 8 9
Conc / mg/l
CORRELATION COEFFIECIENT → used as a measure
of the correlation between two variables (x and y).

r = 1  An exact correlation between the 2 variables

r = 0  Complete independence of variables

In general: 0.90 < r < 0.95  fair curve

0.95 < r < 0.99  good curve
r > 0.99  excellent linearity
Example 1
xi yi xiyi xi 2 yi2
0 0.000 0 0 0.000
1 0.076 0.076 1 0.00578
2 0.161 0.322 4 0.0259
4 0.295 1.180 16 0.0870
6 0.462 2.772 36 0.213
8 0.598 4.784 64 0.358
xi = yi = xiyi = (xi2) = (yi2) =
21 1.594 9.134 121 0.690
Correlation coefficient:
n x i yi −  x i  yi
r=
n  x i
2
− 
( x i )2 n  y i 2 − ( y i )2 
(6)(9.134) − (21)(1.594)
r=
(6)(121) − (21)2 (6)(0.690) − (1.594)2 
r = 0.999
Note:
➢A linear calibration is preferred, although a non-linear curve
can be used.
➢ It is not reliable to extrapolate any calibration curve.
➢With any measurement there is a degree of uncertainty. This
uncertainty is propagated as this data is used to calculate
further results.
 STANDARD ADDITION

In a sample, the analyte is generally not isolated from

other components in the sample.

The MATRIX:

Some times certain components interfere in the analysis

by either enhancing or depressing the analytical signal
→ matrix effect.

BUT, the extent to which the signal is affected is

difficult to measure.
How do we circumvent the problem of matrix effects?

STANDARD ADDITION!

Add a small volume of concentrated standard solution

to a known volume of the unknown.

Assumption:
The matrix will have the same effect on the
analyte in the standard as it would on the
original analyte in the sample.
 Single Standard Addition
⚫ The simplest version of a standard addition.

⚫ First, add a portion of the sample, Vo, to a volumetric flask,

dilute it to volume, Vf, and measure its signal, Ssamp.

⚫ Next, add a 2nd identical portion of sample to an equivalent

volumetric flask along with a spike, Vstd, of an external standard
whose concentration is Cstd.

⚫ After diluting the spiked sample to the same final volume, measure
its signal, Sspike.

⚫ The following 2 eqns relate Ssamp and Sspike to the

concentration of analyte, CA, in the original sample.
 As long as Vstd< Vo, the effect
of the standard’s matrix on the
sample’s matrix is insignificant.

Under these conditions the value

of kA is the same for Ssample and
Sspike. Solving both equations for
kA and equating gives

Vf/V0: Dilution Factor

 Example 1:
A 10.0 g sample that contains Isoniazid is transferred to a 250 ml
volumetric flask and diluted to volume. When a 10.0 ml aliquot of the
resulting solution is diluted to 25.0 ml, it gives an absorbance of 0.235. A
second 10 ml portion of the same solution is spiked with 10.0 ml of a 1.0
ppm standard solution of isoniazid and diluted to 25.0 ml. The signal of
the spiked solution is 0.502.

a) Calculate the concentration of Isoniazid in the sample.

b) Calculate the weight percent of Isoniazid in the sample.

Solution
Begin by making appropriate substitutions into the previous eqn and
solving for CA by making it the subject of the formula.

Vo= 10.0 ml
Vf= 25.0 ml
Cstd= 1.00 ppm
Vstd= 10.0 ml
Ssamp= 0.235
Sspike= 0.502
EXAMPLE 2

A spectrophotometric method for the quantitative analysis of Pb2+ in

blood yields a Ssamp of 0.193 when a 1.00 mL sample of blood is diluted to
5.00 mL. A 2nd 1.00 mL portion of blood sample is spiked with 1.00 µL of
a 1.560 mg/ml standard solution of Pb2+ and diluted to 5.00 mL, yielding
an signal, Sspike = 0.419.
What is the concentration of Pb2+ in the original sample of blood?
It also is possible to make a standard addition directly to the
sample, measuring the signal both before and after the spike.

In this case the final volume after the standard addition is

Vo + Vstd and the previous equations become
Example 2:
A spectrophotometric method for the quantitative analysis of Aspirin in
blood yields a signal, Ssamp = 0.712 for a 5.00 mL sample of blood. After
spiking the blood sample with 5.0 µL of a 1.560 mg/ml Aspirin standard, a
signal, Sspike = 1.546 is obtained.
What is the concentration of Aspirin in the original sample of blood.

Solution
To determine the concentration of Aspirin in the original sample of
blood, we make appropriate substitutions into the corresponding eqn
and solve for CA.

Vo= 5.0 ml
Vo+Vf = 5.005 ml
Cstd= 1.560 mg/ml
Vstd= 5.0 µl
Ssamp= 0.712 AU
Sspike= 1.546 AU
 Multiple Point Standard Additions

⚫ We can adapt the single-point standard addition into a

multiple-point standard addition by preparing a series of
samples containing increasing amounts of the external
standard.

⚫ We plot a standard addition calibration curve based on

the previous equation for Sspike.

⚫ We plot Sspike against the volume (or concentration) of the

spikes, Vstd (Cstd). If kA is constant, then the calibration
curve is a straight-line.

⚫ It is easy to show that the x-intercept is equivalent to

-CAVo/Cstd (or –CAVo/Vf).
How is this best done in practise?

The solutions in all the flasks all

have the same concentration of
the matrix.

Add a quantity of standard

solution such that the signal is
increased by about 1.5 to 3
times that for the original
sample.

Analyse all solutions.

The result:
Example 3
A fifth spectrophotometric method for the quantitative analysis of Aspirin in
blood uses a multiple-point standard addition based on the previous graph.
The original blood sample has a volume of 1.00 mL and the standard used for
spiking the sample has a concentration of 1.560 mg/ml aspirin. All samples
were diluted to 5.00 mL before measuring the signal. A calibration curve of
Sspike versus Vstd has the following equation:
Sspike=0.266 + 312 ml-1 x Vstd

What is the concentration of aspirin in the original sample of blood.

Solution
• Find the value for the x-intercept by setting Sspike = 0
−𝐶 𝑉
• x-intercept = 𝐶 𝐴 𝑜
𝑠𝑡𝑑

• Setting Sspike to zero gives: 0 = 0.266 + 312 ml-1 x Vstd

 INTERNAL STANDARDS
An internal standard is a known concentration of a
compound, different from the analyte, that is added to
the unknown.

Why add an internal standard?

The signal from the analyte is compared to the signal

from the internal standard when determining the
concentration of analyte present.

If the instrument response varies slightly from run to

run, the internal standard can be used as an indication of
the extent of the variation.
Assumption:
If the internal standard signal increases 10% for the
same solution from one run to the other, it is most
likely that the signal from the sample also increases by
10%.

Note: If there are 2 different components in solution

with the same concentration, they DO NOT need to have
the same signal intensity. The detector will generally give
a different response for each component.
Say analyte (A) and internal standard (IS) have the
same concentration in solution.
The signal height for A may be 1.5 times greater than
that for IS, so we calculate the sensitivity (K)

4
3 A
IS • For the sample, we use then
2
value of K to determine the
1 concentration
0
0 1 2 3 4 5 6

The sensitivity for this analysis is 1.5 times greater

for A than for IS.
Example 1:
A standard sample contains 10.0 mg/L of analyte and 15.0 mg/L
internal standard. Analysis of the sample gives signals of the analyte
and the internal standard as 0.155 and 0.233 AU respectively.
Sufficient internal standard is added to an unknown sample to make
its concentration 15.0 mg/L. Analysis of the unknown yields signals
for the analyte and the internal standard of 0.274 and 0.198 AU
respectively. What is the concentration of the analyte in the
unknown?

Solution:

Calculate sensitivity for the standard:

𝐶𝐼𝑆 𝑆𝐴
𝐾= 𝑠𝑡𝑑 ×
𝐶𝐴 𝑆𝐼𝑆
15.0 0.155
𝐾= ×
10.0 0.233
𝐾 = 0.998
Use ‘K’ to calculate the concentration using the formula in the
previous slide.
Example 2:
A sixth spectrophotometric method for the quantitative analysis of Pb2+
in blood uses Cu2+ as an internal standard. A standard containing 1.75
ppb
Pb2+ and 2.25 ppb Cu2+ yields a ratio of (SA/SIS)std of 2.37. A sample of
blood is spiked with the same concentration of Cu2+, giving a signal ratio,
(SA/SIS)samp, of 1.80. Determine the concentration of Pb2+ in the sample
of blood.
 Summary
➢ Standardization is a process of determining the relationship between
the analyte signal and its concentration by using solutions of known
concentration called standards.

➢ Analyte concentration is obtained by the determining the sensitivity (kA)

through a single or multiple point calibration.

➢ External standards (the most widely used) are those prepared/analyzed

separately from the sample.

➢ Standard addition is when a known quantity of analyte is added to an

unknown to increase the concentration of analyte. It is especially useful
when matrix effects are considered.

➢ Matrix effect: change in the analytical signal caused by anything in the

sample other than analyte.

➢ An internal standard is a known amount of a compound, different from

analyte, that is added to the unknown.

➢ Signal from analyte is compared with signal from the internal standard
to find out how much analyte is present