Chapter III

Standardisation
Methods

AAC811S
2022

1
 Chapter Content

1 Analytical Standards

2 Determining the Sensitivity (kA)

3 Standard Addition

4 Internal Standards

5 Blank Corrections

6 Isotope Dilution (ID)

2
Standardisation: the process of determining the relationship between the
signal and the amount of analyte in a sample.
(ACS Committee on Environmental Improvement “Guidelines for Data Acquisition and Data Quality Evaluation in
Environmental Chemistry,” Anal. Chem. 1980, 52, 2242–2249)

Contribution to Stotal from

Signal Stotal = kACA + Sreag sources other than sample

Method’s sensitivity for the Analyte’s concentration

analyte

(3.1)

• To standardise a method we must determine values for kA and Sreag.

• Strategies for accomplishing this are the subject of this chapter.

3
 1. Analytical Standards

•To standardise an analytical method we use substances containing known

amounts of analyte called standards.

•The accuracy of a standardisation, therefore, depends on the quality of the

reagents and glassware used to prepare these 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).

 Secondary standards: reagents that do not meet the above criteria.

Their concentration must be determined relative to a primary standard.

4
• Preparing a standard often requires additional reagents that are not
primary standards or secondary standards.
 For ex., a suitable solvent, and additional reagents may be needed to
adjust the standard’s matrix.
 These solvents and reagents are potential sources of additional analyte,
which may produce a determinate error in the standardisation.

• If available, appropriate reagent grade chemicals should be used.

 The label on the bottle of a reagent grade chemical lists either the limits
for specific impurities, or provides an assay for the impurities.
 We can improve the quality of a reagent grade chemical by purifying it, or
by conducting a more accurate assay.

• We can correct for contributions to Stotal from reagents used in an analysis

by including an appropriate blank determination in the analytical
procedure.

5
Typical packaging labels for reagent grade chemicals.
Label (a) provides the manufacturer’s assay for the reagent, NaBr.
K is flagged with (*) because its assay exceeds the limits established by ACS.
Label (b) does not provide an assay for impurities, but indicates that the reagent meets
ACS specifications. An assay for the reagent, NaHCO3 is provided.
6
Preparing Standard Solutions

It is often necessary to prepare a series of standards, each with a different

concentration of analyte.
This can be done in two ways:

• If the range of concentrations is limited to 1 or 2 orders of magnitude, then

each solution is best prepared by transferring a known mass or volume of
the pure standard to a volumetric flask and diluting to volume.

• When working with larger ranges of concentration, particularly those

extending over more than three orders of magnitude, standards are best
prepared by a serial dilution from a single stock solution.

 In a serial dilution we prepare the most concentrated standard and then

dilute a portion of it to prepare the next most concentrated standard.
 Next, we dilute a portion of the 2nd standard to prepare a 3rd standard,
continuing this process until all we have prepared all of our standards.

• Serial dilutions must be prepared with extra care because an error in

preparing one standard is passed on to all succeeding standards.

7
Calibrating the Signal (Stotal)

• The accuracy of kA and Sreag depends on how accurately we measure Stotal.

• We measure signals using equipment (e.g. glassware, balances) and
instrumentation, (e.g. spectrophotometers).
• To minimize determinate errors affecting the signal, we 1st calibrate our
equipment and instrumentation.
• We accomplish the calibration by measuring Stotal for a standard with a
known response of Sstd, adjusting Stotal until Stotal = Sstd

Examples:
 When the signal is a mass, we determine Stotal using an analytical balance.
To calibrate the balance’s signal we use a reference weight that meets
standards established by a governing agency (e.g. NIST or ASTM).
Electronic balances often includes an internal calibration weight for routine
calibrations, as well as programs for calibrating with external weights.

 We also can evaluate a spectrophotometer’s accuracy by measuring A of a

carefully prepared solution of 60.06 mg/L K2Cr2O7 in 0.0050 M H2SO4, using
0.0050 M H2SO4 as a reagent blank.
A = 0.640 ± 0.010 at λ = 350.0 nm indicates that the spectrometer’s signal is
properly calibrated. 8
 2. Determining the Sensitivity (kA)

Stot = SA + SReag To standardise an analytical method we must

determine the value of kA
= kACA + SReag
• In principle, it should be possible to derive kA by considering the chemical
and physical processes generating the signal.
• Unfortunately, such calculations are not feasible when we lack a theoretical
model of the physical processes, or are not useful because of non-ideal
chemical behavior.
• Thus, we must determine kA by analyzing one or more standard solutions,
each containing a known amount of analyte.
• For simplicity we will assume that Sreag has been accounted for by a proper
reagent blank, allowing us to write Stotal = Ssamp = SA.

Single-point standardisation: we measure the signal for a standard, Sstd,

containing a known concentration of analyte, Cstd.

Sstd = kA Cstd kA = Sstd / Cstd Thus, CA = Ssamp / kA

(3.2) (3.3) (3.4) 9
A single-point standardisation is the least desirable approach for
standardising a method!!!

Two reasons for this:

1st , any error in determining kA carries
over into our calculation of CA.

2nd , the experimental value of kA is for a

single CA.
Extending this to other CA requires to
assume a linear relationship between the
SA and CA, an assumption that often is not
true.

Despite these limitations, single-point standardisations find routine use

when the expected range for the analyte’s concentrations is small.
Under these conditions it is often safe to assume that kA is constant (though
it has to be verified experimentally).
This is the case, for ex., in clinical labs where many automated analysers
use only a single standard. 10
Multiple-point Standardisation (the preferred approach): Prepare a series of
standards (at least 3), each containing the analyte at a different concentration.
Standards are chosen such that they bracket the expected range for the
analyte’s concentration.

If Sstd = kA Cstd

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

• The most common method of standardisation uses one or more external

standards.

• We call them “external” because we prepare and analyse the standards

separately from the samples.

• Because this is the most common method of standardisation the resulting

relationship is called a normal calibration curve.

11
• When a calibration curve is a
straight-line, the slope of the line
gives kA.

• This is the most desirable

situation since the method’s
sensitivity remains constant
throughout CA range.

• When the calibration curve is not a straight-line, the method’s sensitivity is

a function of CA.
• In slide #10, for ex., kA is greatest when CA is small and decreases
continuously for higher CA .
• kA at any point along the calibration curve is given by the slope at that point.
• In either case, the calibration curve provides a means for relating Ssamp to
CA.
12
Example 1
A student prepared 6 solutions with a known concentration of Cr6+ and
added the necessary colouring agents. He 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)

13
 Calibration curve

0.7
Response = dependant variable = y

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

Concentration = independant variable = x

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 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

15
The student then measured his sample to have an absorbance of 0.418 and
the blank, 0.003. He, 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!!

16
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

17
The equation for a normal calibration curve for the quantitative
analysis of Cu2+ is given as follows:

Sstd = 29.59 M–1 × Cstd + 0.0015

What is [Cu2+] in a sample whose absorbance, Ssamp, is 0.114?

Compare your answer to a one-point standardization where a

standard of 3.16 × 10–3 M Cu2+ gives a signal of 0.0931.

18
 Recall: Method of Least Squares

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)

n xi yi    xi  yi  x  y   x y  x
2
m
 
i i i i i
n x    x 
c
n xi2   xi 2 i
2
i
2

19
Correlation Coefficient  used as a measure of the correlation
between two variables (x and y).

The Pearson correlation coefficient (r) is calculated as follows:

n x iyi   x i  yi
r 
n  x i
2
 2

 x i  n  y i   y i 
2 2

r = 1  An exact correlation between the 2 variables

r = 0  Complete independence of variables

In general: 0.950 < r < 0.990  fair curve

0.990 < r < 0.995  good curve
r > 0.995  excellent linearity 20
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.

21
• External standardisation allows to analyse a series of samples using a
single calibration curve.
• This is an advantage when analysing many samples.
• Thus, many of the most common quantitative analytical methods use an
external standardisation.

Limitation:

• When determining kA using equation 3.2, the analyte is present in the

external standard’s matrix (usually much simpler than samples’ matrix) and
we assume that the matrix does not affect kA.

• If this is not true, then we introduce a proportional determinate error.

• If we expect that matrix effects are important, then we try to match the
standard’s matrix to that of the sample (matrix matching).

22
Calibration curves for an analyte in the standard’s matrix and in the sample’s
matrix. If the matrix affects kA, as is the case here, then we introduce a
determinate error into our analysis if we use a normal calibration curve.

• If we are unsure of the sample’s matrix, then we must show that matrix
effects are negligible, or use an alternative method of standardisation.

• Both approaches are discussed in the following section. 23

 3. 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.

24
We can avoid the complication of matching the matrix of the
standards to the matrix of the sample by conducting the
standardization in the sample.

STANDARD ADDITION!

Add a small volume of concentrated standard solution

(“spike”) 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.

25
 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.
26
 • 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 27

Example 1:

A spectrophotometric method for the quantitative

analysis of Pb2+ in blood yields an Ssamp of 0.193
when a 1.00 mL sample of blood is diluted to 5.00
mL.

A 2nd 1.00 mL sample of blood is spiked with 1.00 µL

of a 1560 ppb Pb2+ external standard and diluted to
5.00 mL, yielding an Sspike of 0.419.

What is the concentration of Pb2+ in the original

sample of blood?

28
Solution
Begin by making appropriate substitutions into the previous eqn
and solving for CA.
Note that all volumes must be in the same units; thus, first covert
Vstd from 1.00 µL to 1.00 × 10–3 mL.

29
• 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

30
Example 2:
A spectrophotometric method for the quantitative analysis of Pb2+
in blood yields an Ssamp of 0.712 for a 5.00 mL sample of blood.

After spiking the blood sample with 5.00 µL of a 1560-ppb Pb2+

external standard, an Sspike of 1.546 is measured.

What is the concentration of Pb2+ in the original sample of blood.

Solution
To determine the concentration of Pb2+ in the original sample of
blood, we make appropriate substitutions into the corresponding
eqn and solve for CA.

31
32
 Multiple 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 (or 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)

33
• At the top is a set of 6 standard
additions for the determination
of Mn2+.

• The flask on the left is a 25.0

mL sample diluted to 50.0 mL.

• The remaining flasks contain

25.0 mL of sample and, from
left to right, 1.0, 2.0, 3.0, 4.0,
and 5.0 mL of an external
standard of 100.6 mg/L Mn2+.

• Below are 2 ways to plot the

standard additions calibration
curve.

• The absorbance for each

standard addition, Sspike, is
shown by the filled circles.

34
How is this best done in practise?

The solutions in all the flasks have

all 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.

35
The result:

standard

5 mL
sample

36
Example 3
Gold was determined in a waste stream using
voltammetry. The peak height of the current
signal is proportional to concentration.
A standard addition analysis was done by adding specific volumes of
10 ppm Au solution to the sample as shown in the table below.
All solutions were made up to a final volume of 20 ml. The peak
currents obtained from the analyses are also tabulated below.
Calculate the concentration of Au in the original sample.

Volume of Volume of std Peak current

sample / ml / ml /A
10 0 8
10 2 16
10 5 25
10 10 41 37
1 - Calculate the concentration of added Au to each sample.
CiVi = CfVf or Cspike = Cstd X (Vstd/Vf) (Vf = 20 ml)
Volume of std Conc. of added std Peak current
/ ml / ppm /A
0 0 8
2 1 16
5 2.5 25
10 5 41
Peak current /  A

50
40
30
20
10
0
0 1 2 3 4 5 6
Conc added std / ppm 38
 2 - Find the best straight line
n xi yi    xi  yi  x  y   x y  x
2

n x    x 
m i i i i i
n x    x 
2 2 c 2 2
i i i i

Conc. Added Peak hgt

xi yi xi yi xi2
0 8 0 0
1 16 16 1
2.5 25 62.5 6.25
5 41 205 25
 8.5  90  283.5  32.25

(4)(283.5)  (8.5)(90) (32.25)(90)  (283.5)(8.5)

m 2
 6.50 c  8.68
(4)(32.25) - (8.5) (4)(32.25) - (8.5) 2

y = 6.50x + 8.68 39
 3 - Extrapolate to the x-axis (y = 0)

y = 6.50x + 8.68 50

Peak current /  A
40
30
Y=0
20
Thus, X = CA y = 6.50x + 8.68
= -8.68/6.50 10
R2 = 1.00
= 1.33 ppm 0
(Absolute value) -2 -10 0 2 4 6

Conc added std / ppm

4 - Take dilutions into account

CA (Diluted) x 20 ml = CA (Original) X 10 ml
1.33 ppm x 20 ml = CA (Original) X 10 ml
CA (Original) = 1.33 ppm X (20/10)

Conc of original sample = 2.66 ppm 40

• Since we construct a standard additions calibration curve in the sample,
we can not use the calibration equation for other samples.

• Each sample, therefore, requires its own standard additions calibration

curve.

• This is a serious drawback if you have many samples.

• For example, suppose you need to analyze 10 samples using a 3-point

calibration curve.

• For a normal calibration curve you need to analyse 13 solutions (3

standards + 10 samples).

• If you use standard additions, however, you must analyse 30 solutions

(each of the 10 samples must be analyzed 3X, once before spiking and
after each of 2 spikes).

41
Using a Standard Addition to Identify Matrix Effects

We can use standard additions to validate an external standardisation when

matrix matching is not feasible.

How?

 Prepare a normal calibration curve of Sstd vs Cstd and determine kA from

its slope.

 Prepare a standard additions calibration curve.

The slope of this standard additions calibration curve provides an
independent determination of kA.

 If there is no significant difference between the 2 values of kA, then we

can ignore the matrix effect.

 When the values of kA are significantly different, then using a normal

calibration curve introduces a proportional determinate error.

42
 4. 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.

Internal standards are useful when:

 the quantity of sample analyzed is not reproducible,
 the instrument response varies from run to run (signal drift),
 sample losses occur in sample preparation.

43
Because the analyte and the internal standard in any sample or standard
receive the same treatment, the ratio of their signals is unaffected by any
lack of reproducibility in the procedure.

Assumption:
If the internal standard signal increases by 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.

44
Say analyte (X) and internal standard (S) have the same
concentration in solution.
The signal height for X may be 1.5 times greater than that for S.

IX  IS 
F 
4 [X]  [S] 
3
X
S
2
1 [X] and [S]: concentrations of
analyte and standard after they
0 have been mixed together.
0 1 2 3 4 5 6

The response factor (F) is 1.5 times greater for X than for S.
45
Example: In a preliminary experiment, a solution containing 0.083 7 M X and
0.066 6 M S gave peak areas of Ix = 423 and Is = 347 (areas are measured in
arbitrary units). To analyse the unknown, 10.0 mL of 0.146 M S were added
to 10.0 mL of unknown, and the mixture was diluted to 25.0 mL in a
volumetric flask. This mixture gave a chromatogram, for which Ix = 553 and
Is = 582. Find the concentration of X in the unknown.

IX  IS 
Solution: 1st use the standard mixture to find
the response factor
F 
[X]  [S] 

In the mixture of unknown + standard,

the concentration of S is

46
Using the known
IX  IS 
response factor, we find F 
the concentration of [X]  [S] 
unknown in the mixture:

Because X was diluted from 10.0 to 25.0 mL when the mixture with S
was prepared, the original concentration of X in the unknown was

= 0.143 M

47
 Every sample should be analyzed with an internal standard (IS)!!!

• Recall: what is an IS?

– Element that is added to EVERY sample/ blank/calibration standard/QA
sample/etc., that is not expected to be in the sample in appreciable
quantities and is not an element of interest.
– Use IS to monitor machine drift (both short and long term) and matrix
effects.

• Choice of IS depends upon which elements you are quantifying.

• The IS should have similar properties as element(s) of interest.
• ICP-MS: similar in mass/ionization potential.

• Example (For ICP-MS analysis):

attempting to quantify U (m/z: 238) - use Th (m/z: 232)
attempting to quantify most transition metals - use As
115In and 103Rh are common IS for general use
alternatively, you can add several IS to each sample

•In GC analysis with non-MS detector: if target is, for ex. tri-chlorophenol,
use tri-bromophenol or di-chlorophenol.
48
• From previous slide, we assume that samples have little or no Th or As.

• It’s important to have an idea of what’s in your sample prior quantitative

analysis.

• Solid samples can use a naturally occurring element as IS, provided that
you know the concentration in each sample.

49
 Advantages:
• Fluctuations are monitored in each sample/calibration / blank.

Disadvantages:
• Assume that behavior of IS is the same as the analyte.

We can correct for instrument drift with:

 Internal standardisation (common procedure).
 Drift corrector solutions (DCS) also can be used.

Drift Corrector Solutions (DCS):

• Measure the same solution intermittently throughout the course of the
analytical session.
• Change in ion signal is assumed to be linear between each DCS
measurement.
• The DCS should contain all elements of interest and can be matrix
matched to samples.
– Example: use standard reference materials (SRMs) for DCS.
50
Advantages of DCS correction:

• All analytes are monitored for drift

• Nothing added to sample solutions

Disadvantages of DCS correction:

• Assume change is linear

• Cannot easily monitor matrix effects

51
 5. Blank Corrections
• So far, we have assumed the use of a suitable reagent blank to correct for
signals arising from sources other than the analyte.

• But, what constitutes an appropriate reagent blank?

• Surprisingly, the answer is not immediately obvious.

Case Study
About 200 analytical chemists were asked to evaluate a data set
consisting of a normal calibration curve, a separate analyte-free blank,
and 3 samples of different size but drawn from the same source.

The normal calibration curve for the data is

Sstd = 0.0750 × Wstd + 0.1250

The y-intercept (= 0.1250) is the calibration blank.

A separate reagent blank gives the signal for an analyte-free sample.

52
The analytical chemists used 4 different approaches for correcting signals:
(a) ignoring both the calibration blank, CB, and the reagent blank, RB;
(b) using the calibration blank only;
(c) using the reagent blank only; and
(d) using both the calibration blank and the reagent blank.

53
• That all 4 methods give a different result for CA shows the importance of
choosing a proper blank, but does not tell us which blank is correct.
• Because they all fail to predict the same CA for each sample, none of these
blank corrections properly accounts for an underlying constant source of
determinate error.
54
• To correct for a constant method error, a blank must account for signals from
any reagents and solvents used in the analysis, as well as any bias resulting
from interactions between the analyte and the sample’s matrix.
• Both the calibration blank and the reagent blank compensate for signals from
reagents and solvents.
• Any difference in their values is due to indeterminate errors in preparing and
analyzing the standards.

• Unfortunately, neither a calibration blank nor a reagent blank can correct for
a bias resulting from an interaction between the analyte and the sample’s
matrix.
• To be effective, the blank must include both the sample’s matrix and the
analyte and, consequently, must be determined using the sample itself.

• One approach is to plot of Ssamp vs the amount of sample.

• The resulting y-intercept gives the signal in the absence of sample, and is
known as the Total Youden Blank., which is the true blank correction.

The regression line for the 3 samples is: Ssamp = 0.009844 × Wsamp + 0.185
giving a true blank correction of 0.185 with 3 identical values for CA.
55
• Total Youden Blank is not common in analytical work.

• Most chemists rely on a calibration blank when using a calibration curve,

and a reagent blank when using a single-point standardisation.

• As long we can ignore any constant bias due to interactions between the
analyte and the sample’s matrix, which is often the case, the accuracy of
an analytical method will not suffer.

• It is a good idea, however, to check for constant sources of error before

relying on either a calibration blank or a reagent blank.

56
• Standard blank
Blank used to monitor polyatomic ion interferences, gas peaks, and
contamination from reagents; used for background subtraction.

• Procedural blank
Blank used to monitor contamination acquired during all stages of
sample preparation; grinding, digestion, acidification, powdering, etc.

Use of blanks during an analytical session

• ALWAYS begin an analytical session with at least one standard blank.

• Analyse standard blanks periodically throughout the course of the
session in particular to monitor memory effects.
• Process and analyse at least one procedural blank at some point during
your research study; it’s preferable to measure it early in order to avoid
any potential memory effects.
• The more standard blanks that are run during an analytical session, the
more information you will have with regards to monitoring change(s) in
background levels throughout the entire session.
57
How to determine “the background”:

1. Just use the first standard blank.

2. Average all standard blanks.

3. Take median of all standard blanks.

4. Apply statistical analysis to standard blanks and select some of them

(outlier tests).

Outlier tests:

Option 1. “I know the truth”: should be avoided – unscientific and invalid.

Option 2. “Looks different”: better but only if the measurement is repeated.

Option 3. Statistical “proof”: the best approach, but needs to be carried out
carefully in order to avoid false negatives and positives.

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 DETECTION LIMITS
All instrumental methods have a degree of noise associated with the
measurement.  limits the amount of analyte that can be detected.

Detection limit (DL or LOD): the lowest concentration level that can be
determined to be statistically different from the analyte blank.
LOD = 3*STDEVblank
Limit of Quantification (QL or LOQ): the smallest (minimum) concentration of
a measurand that can be reliably (i.e. with a high degree of confidence)
measured by an analytical procedure: LOQ = 10*STDEVblank
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A ‘good’ analytical method will:
1. provide the means to calculate an accurate background level
2. allow for correction of instrument drift
3. use Internal standardisation to monitor matrix effects
4. provide some method for monitoring/ correcting interferences
5. Use a proper calibration strategy

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 6. Isotope Dilution (ID)

• Most accurate and precise calibration method available

• Requires analyte with two stable isotopes

• Monoisotopic elements cannot be determined via ID

• Spike natural sample with enriched isotope spike of analyte

111Cd
111Cd

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 ID Scheme

‘‘ideal ’’ Internal
Standard

Abce Isotope 1 Abce Isotope 2 Abce

(%) (%) (%)
Isotope 1
Isotope 2 Isotope 1 Isotope 2

Natural Enriched Isotope Mixture

Sample standard
62
The «ratio» concept
Sample: Enriched Standard 199Hg « spiked» sample
Natural Abundance Hg
35 100 91,95 R 202Hg/199Hg
29,86
30 modified
abundance (%)

23,1 80
25
20 16,87 60
13,18
15 9,97 40
10 6,87
20 4,92 0,66 0,73 0,11
5 0,15 0 1,63
0 0
196 198 199 200 201 202 204 196 198 199 200 201 202 204 198 199 200 201 202 204
isotopes isotopes isotopes

A known amount of the tracer, called ’’spike’’, with an abnormal isotopic composition
of the element to be analysed is added to the sample, preferably before any
chemical treatment.
The isotopic ratio between the tracer and the element is known; we measure the
isotopic ratio in the spiked sample with a mass spectrometry method. The element
concentration can then be deduced from the isotopic ratio and the amount of the
added spike.
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64
• The amount of spike is selected so that the resulting ratio between
spiked isotope and unspiked isotope is near unity – maximizes precision
• Typically use the most abundant isotope as the reference -- maximizes
sensitivity

• Check isotope ratio in unspiked sample to determine if the “natural

ratio” in the sample matches with the predicted ratio
• If not -- interference is acting on one or both of the isotopes
• Always attempt to use interference free isotopes

• Prepare the spike to desired concentration

• Add spike as early as possible – after equilibration of spike and sample
you don’t have to have complete sample recovery
• During any stage of the process complete equilibration is absolutely
necessary

• Analyse the solution on the MS using many repetitive scans (to

maximize precision)
• Need to measure isotopic ratios on standards of a known ratio in order
to correct for machine mass discrimination
• Use previous equation to calculate concentrations!
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• Advantages:

 Most accurate and precise method for quantitative elemental

concentrations
 Partial loss of analyte during preparation is compensated for since
physical and chemical interferences are not an issue -- will cancel out
as they will affect each isotope identically
 Ideal form of internal standardisation since another isotope of the
same element is used in this capacity
 Quantification is a direct mathematical calculation from determined
isotopic ratios and known constants and does not depend on a
calibration curve or sample recovery.

• Disadvantages:

– Generally only applicable to multiple-isotopic elements

– Need an enriched isotope spike for the analyte of interest - not always
available or sometimes at very high cost
– Need two interference free isotopes
– VERY time consuming

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 Hg Enriched Isotope Standards

119 (Butyltin mix, 0.1/0.7/0.1ppm) 280€/ml

67
 Summary

• Standardisation is a process of determining the relationship between the

analyte signal and its concentration by using solutions of known
concentration called standards.

• External standards (the most widely used) are those prepared/analysed

separately from the sample.
• Analyte concentration is obtained by the determining the sensitivity (kA)
through a single or multiple point calibration.

• A 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 important.
• 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.
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