CHAPTER 5

Quality Assurance
and Calibration
Methods
1

Quality assurance
Is what we do to get the
right answer for our
purpose.
The ability to consistently
produce the same product to
meet the same
specifications time after
time
Chapter 5: Calibration methods

Importance of quality assurance

Make sure that results meet the
customers need
Analytical data and results
should be used to determine for
example whether the level of fats
in a food package is 0%.

Chapter 5: Calibration methods

Basics of quality
assurance
Important terms

Raw data: Individual

measurements e.g. volumes of a
burette in titration, weight of
precipitate,

Treated data: Concentrations

obtained from raw data.

Results: Quantities reported after
statistical treatment of data, e.g.
mean, standard deviation,
Chapter 5: Calibration methods

3 main factors of QA
1. Use objectives

States purpose for which results

will be used.(ask an unanswered Q)

2. Specifications
How good do the numbers have
to be

3. Assessment
Were specifications achieved

Chapter 5: Calibration methods

Specifications
Sampling requirements.

Quality assurance begins with sampling

How many samples, how samples are
preserved, .

How good the numbers need to be.

Accuracy and precision.

Rate of false results.
Selectivity, sensitivity.
Acceptable blank levels.
Recovery of fortification.
Calibration checks and quality control
samples.
Chapter 5: Calibration methods

False positive and false

negative
False positive:

positive= there is a problem

States that the concentration exceeds the

certified limit, BUT in fact the
concentration is below the limit.

False negative:

negative = No problem

Says that the concentration is below the

limit when it is in fact above the limit.
A "false positive" is when a good quality item gets
rejected, and a "false negative" is when a poor quality
item gets accepted

Chapter 5: Calibration methods

Examples
For drinking water; which is more
important:

To have a low rate of false negative or

false positive with respect to contaminants
levels!

Drug testing for athletes is designed to

discover cases of using steroids the
consequences of positive testing for
drugs is the ultimate prohibition of those
athletes from participation in the games

For this case which is more important in

designing those tests to have low rate of
false positive or false negative?
Chapter 5: Calibration methods

Selectivity and sensitivity

Sensitivity
Ability to respond reliably and
measurably to changes in analyte
concentration
Sensitivity = slope of calibration curve
Sensitivity = change in signal / change in
concentration

Selectivity (specificity)

Being able to distinguish analyte from

other species free from interferences.

Chapter 5: Calibration methods

Blank
Account for interference by other species
in sample and for traces of analyte in
reagents used for sample preparation,
preservation and analysis.
Method Blank

Sample containing all components except

analyte and is subjected to all steps in the
analytical method.

Reagent Blank

Same as method blank but not subjected to

all steps of analysis.

Field Blank

Similar to the method blank but it has been

exposed to the site of sampling.
Chapter 5: Calibration methods

10

Spike - Fortification
Known amount of analyte added to a
sample to test whether the response is
the same as that expected from a
calibration curve.

Matrix = everything in the sample

other than the analyte.

Spike recovery
100
Fotification is to identify interference from
matrix.
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11

Calibration check
Analyze solutions prepared to
contain known concentrations of
analyte
Perform calibration check to make
sure that our instrument continues
to work properly and our
calibration remains valid
Solutions of the calibration check
should be prepared from solutions
different from the ones used to
prepare original calibration curve.
(why?)
Chapter 5: Calibration methods
12

Performance test
samples
Also known as Quality Control
Samples or Blind Samples.
These are samples of known
composition - provided as
unknowns to analysts.
Results are compared with the
known values e.g. by a quality
assurance manager.
Used to eliminate bias introduced
by analyst who knows calibration
standards concentrations.
Chapter 5: Calibration methods

13

Assessment
A process of

Collecting data to show that the

analytical procedures are performed
correctly.

Verify that the final results meet

use objectives.
Documentation is essential for
assessment

Standard protocols - how

documentation should be done (e.g.
notebooks).
Quality control charts
Chapter 5: Calibration methods

14

Control Charts
Visual representation of confidence
intervals for a Gaussian distribution.
Used to warn from serious deviations
from a target value.
99.7% of
observations

95.5% of
observations

Chapter 5: Calibration methods

15

Control Charts
The following conditions are considered
unlikely to occur - if they occur the
process should be stopped for
troubleshooting

1 observation outside action lines

2 out of 3 consecutive measurements
between the warning and action lines
7 consecutive measurements all above or
below the center line.
6 consecutive measurements all increasing or
decreasing.
14 consecutive points alternating up and
down.
An obvious non random pattern (by common
Chapter 5: Calibration methods
16
sense)

Chapter 5: Calibration methods

17

Method validation

Is the process of proving that an

analytical method is acceptable for its
intended purpose
When should methods be validated?

1- method just developed.

2-established method adapted to a new
problem.

3-when established method is used in

different laboratories, with different
analyst or with different equipment.
Chapter 5: Calibration methods

18

Method validation
Specificity

The ability of an analytical method to

distinguish analyte from everything else.

Linearity

Measures how well a calibration curve

follows a straight line.
Measured by Square of Correlation
Coefficient

R2 can be obtained by LINSET

R2 close to
1 5:indicates
curve
Chapter
Calibration methodsgood linear
19

Method validation
Accuracy
Nearness to the truth
Determined by

Analyze certified reference material

Compare results from two different
methods
Analyze blank spiked with known
addition of analyte
Use standard addition method.

Example of analysis of spiked

sample accuracy is expressed as
100.50.8%
Chapter 5: Calibration methods

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Method validation

Precision

Show how replicate measurements agree

with one another (usually expressed by s)

Instrument precision

Reproducibility observed
when the same quantity of
one sample is repeatedly
injected in an instrument.

Intra-precision

Evaluated by analyzing
aliquots of a material several
times by one person on one
day using the same
instrument.
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21

Method validation
Intermediate precision

Is the variation observed when an

assay is performed by different people
on different instruments on different
days in the same lab.

Interlaboratory precision

Reproducibility observed when aliquots

of the same sample are analyzed by
different people in different
laboratories.

Chapter 5: Calibration methods

22

Method validation
Range

Concentration interval over which linearity,

accuracy, and precision are all acceptable

Example: Spike recovery 10010

Limit of detection

Smallest quantity of the analyte that is

significantly different from the blank.

Limit of quantitation

The smallest amount that can be

determined with reasonable accuracy

Chapter 5: Calibration methods

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Determination of detection limit

(DL)

Using an estimate of DL prpare a sample

with concentration 1-5 times DL
Measure the signal (ysample) from n
replicates of sample (n>=7)
Obtain s
Measure signal of n blank (yblank)
Determine minimum detectable signal
(ydl)

Chapter 5: Calibration methods

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Example :
From

previous
measurements
of
a
low
concentration of analyte, the signal detection
limit was estimated to be in the low nanoampare
range. Signals from seven replicate samples with
a concentration about three times the detection
limit were 5.0, 5.0, 5.2, 4.2, 4.6, 6.0 and 4.9 nA.
reagent blanks gave values of 1.4, 2.2, 1.7, 0.9,
0.4, 1.5 and 0.7 nA. the slope of the calibration
curve is m=0.229nA/uM.

a) find the signal detection limit and the minimum

detectable concentration
b) what is the concenyration of analyte in a
sample that gave a signal of 7.0nA
Chapter 5: Calibration methods

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Another way for DL

From the least square equation of a

calibration curve.

ydl = b + 3Sy
LD = 3Sy/m
Instrument detection limit

is
obtained by replicate measurements of
one sample.

Chapter 5: Calibration methods

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Reporting limit
The concentration below which
regulation say that a given analyte is
reported not detected
This does not mean that it is not
observed.

Reporting limit for trans fat is

0.5 g/serving

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Robustness
Is the ability of an analytical
method to be unaffected by small
deliberate changes in operating
conditions.
Example the method pH is 7.5 to
check for robustness we change it
to 7 and to 8 and evaluate the error
in the obtained concentrations.

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Standard addition

Known quantities of analyte are added to

the unknown
From the increase in signal, we deduce
how much analyte was in the original
unknown.
Standard addition must be used
whenever the matrix of a sample changes
the analytical sensitivity of the method

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Standard addition
Matrix effect

Matrix = everything in

the unknown other than

analyte
Matrix effect =The
change in analytical
signal caused by
anything in sample other
than analyte

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5.3 Standard addition

Known amounts of analyte are added to the unknown solution

Ix = k[X]i (Response for unknown sample)

Is+x = k([X]f+ [s]f (Response for unknown sample + standard)

X i
S f X f
Xf
s f

Ix
I s x

Vo
X i
VT
Vs
s i
VT

Example: A 25.0 mL sample of

Ni2+ gave a current of 2.36 A in

an electrochemical analysis.
Upon addition of 0.500 mL of
solution containing 28.7 mM Ni2+
the current increased to 3.79 A.
Find the concentration of nickel
in the sample.

Chapter 5: Calibration methods

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Graphical procedure for standard

addition

Unknown without any

added standard

Chapter 5: Calibration methods

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5.4 Internal standard

A known amount of a compound different from the analyte
is added to the unknown sample.

Analyte signal
Standard signal

concentration of analyte
concentration of standard

Ax
As
F

X S
Example: A solution containing 3.47 mM X
and 1.72 mM S gave peak areas of 3473 and
10222, respectively. 10.00 mL of 0.146 mM S
when added to 10.00 ml of unknown X and
diluted to 25.0 mL gave peak areas of 5428
and 4431 for X and S respectively. Find the
concentration of X in the original unknown.
Chapter 5: Calibration methods

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