CHAPTER 3

SELECTING ANAYLTICAL METHODS -

ASSOC PROF DR NOR SUHAILA MOHAMAD HANAPI

CHM561: QUALITY IN ANLYTICAL MEASUREMENT 1

 STUDENT LEARNING OUTCOME:
At the end of this chapter, students should be able to:

(i) Identify the factors which have to be considered

when choosing a method.
(ii) Know where to look for suitable methods.
(iii) Decide when and how to modify standard methods
for a particular analysis.
(iv) Understand how to validate analytical methods.

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Overview of Chemical Analysis:

Analysis involves the determination of composition of

materials, such as identification and quantification the
chemical constituents.

Purpose of Analysis:

Before starting the analysis of sample, it is important to

know
why the work is being conducted,
what are the possible results and
to find out what decision will be taken (objectives must be clear)
Trace Analysis

Analysis that involve determination of a very low level of analytes e.g

ppm (mg kg-1) or ppb (ug kg-1) in a very complex matrix.

The uses of quick methods are generally less reliable (less accurate)
than those methods taken longer time and uses expensive analytical
instrumentation such as GC-MSD.

Types of methods:
Routine – 1) screening
2) surveillance (careful watch/supervision)

Regulatory – 1) confirmatory
2) reference
 Screening Method

Example:
Information about cancer screening and the types of tests used to find cancer.
Immaging test:
i) Mamograms
A fact sheet that defines screening and diagnostic mammograms and outlines
mammography screening guidelines. Discusses the benefits and some potential
limitations of screening mammograms.
ii) Computed Tomography (CT) Scans and Cancer

Laboratory Tests:
Understanding Laboratory Tests
A fact sheet that describes the role of screening and diagnostic laboratory tests.
Includes a list of the common tests used in cancer medicine and a brief discussion
of how to interpret test results.
Pap and HPV Testing
A fact sheet that describes cervical cancer screening, which includes the Pap test
and HPV testing. The fact sheet includes information about cervical cancer
screening guidelines.

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Surveillance Method

Surveillance is very useful to governments and law enforcement to

maintain social control, recognize and monitor threats, and
prevent/investigate criminal activity. With the advent of programs such as
the Total Information Awareness program and ADVISE, technologies such
as high speed surveillance computers and biometrics software, and laws such
as the Communications Assistance For Law Enforcement Act, governments
now possess an unprecedented ability to monitor the activities of their
subjects.

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Confirmatory Analysis

Inferential Statistics - Deductive Approach

Heavy reliance on probability models
Must accept untestable assumptions
Look for definite answers to specific questions
Emphasis on numerical calculations
Hypotheses determined at outset
Hypothesis tests and formal confidence interval estimation

Reference methods for domestic installations

The reference method box on the EICR is there to indicate how the particular
circuit is installed. This then allows us to verify that the cable used is capable of
carrying the current for that circuit.

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It is the laboratory's responsibility to use methods which are
appropriate for the required application.

The laboratory may use its own judgement, or select a method

in consultation with the customer, or the method that may be
specified in regulation or by the customer.

Choose the method that has the best chance of achieving the laboratory’s
service requirements

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 Analytical Methods

The following sites give free full-text access to analytical methods

(i) US Environmental Protection Agency (EPA)

publishes laboratory analytical methods used by industries and
municipalities to analyse the chemical and biological
components of wastewater, drinking water, sediment, and
other environmental samples that are required by EPA
:
regulations under the authority of the Clean Water Act and the
Safe Drinking Water Act.

(ii) Occupational Safety & Health Administration (OSHA)

.
provides an alphabetical list of chemicals that have either a
validated or partially validated OSHA method. Some chemicals
may be listed by their common synonym. The index includes the
method number, validation status, CAS number, analytical
instrument and sampling device
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(iii) National Institute for Occupational Health and Safety (NIOSH)
Manual of Analytical Methods (NMAM)

A collection of methods for sampling and analysis of contaminants in

workplace air, and in the blood and urine of workers who are
occupationally exposed. These methods have been developed or adapted
by NIOSH or its partners and have been evaluated according to
established experimental protocols and performance criteria. NMAM also
includes chapters on quality assurance, sampling, portable instrumentation.

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SOURCES OF METHODS
Analytical methods: a) Qualitative b) Quantitative; The methods used
must be fit for the purpose of study:
i) In-house method (developed by one laboratory for their own special
needs

ii) Method published in the open scientific literature, eg. Journal of

Chromatography, Analytical Chemistry, Water Research, etc. Saim et al
(1998) An Experimental approach... Anal.Chem,70,420-424.pdf

iii) Method supplied by trade organisations (Application Note)

Application note 336.pdf

iv) Methods in books published by professional organisations eg RSC (The

Royal Society of Chemistry.

v) Methods from standards organisations, e.g. USA (ASTM); Europe

(CEN); UK (BSI); International (ISO).

vi) Methods from statutory publications, e.g. The Fertilisers (Sampling

and Analysis) Regulations 1991 13
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Group 1: Determination of MTBE in Water

Group 2: Determination of MTBE in Soil

Group 3: Determination of organophosporus pesticides in Water

Group 4: Determination of organophosporus pesticides in

Vegetables

Group 5: Determination of organophosporus pesticides in

Sediment

MTBE = Methyl Tert-Butyl Ether

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Factors to be considered:

1. suitable for the purpose intended

2. adequately validated and documented

3. provide results that are traceable to stated references at

an appropriate level of uncertainty.

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Factors to be considered (continued):
1. Sample type
2. Sample size
3. Sample preparation required
4. Concentration and range
5. Selectivity needed (interferences)
6. Accuracy/precision needed
7. Tools/instruments available
8. Expertise/experience
9. Cost
10. Speed
11. Can it be automated?
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Sample Preparation
Sample preparation is the most crucial step in the analytical procedure
designed for implementation in any analytical application (food analysis,
bionalysis, forensics, toxicology, environmental monitoring etc).

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SAMPLE PREPARATION

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SOXHLET EXTRACTION

The sample (solid), which has

been homogenized in sodium
sulphate, is covered with
glass wool and contained in a
porous cellulose thimble. The
thimble is placed in
the extraction tube, which
itself sits on a flask containing
an organic solvent (like
hexane).

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List of extraction technique

 Liquid-Liquid Extraction (LLE)

 Solid Phase Extraction (SPE)
 Magnetic solid phase extraction (MSPE)
 Micro- Solid Phase Extraction (μSPE)
 dispersive solid phase extraction (dSPE)
 Molecularily imprinted Solid Phase Extraction
 Solid phase Microextraction (SPME)
 Headspace-SPME
 In-tube SPME
 Microextraction in Packed Syringe (MEPS)
 Liquid phase microextraction (LPME)

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 Two phase and three phase LPME
 Single drop microextraction (SDME)
 dispersive liquid liquid microextraction (DLLME)
 Hollow fiber supported Liquid phase microextraction (HFLPME)
 Supercritical fluid extraction (SFE)
 Accelerated solvent extraction (ASE), or Pressurized liquid extraction (PLE)
 Stir bar sorptive extraction (SBSE)
 Quick, Easy, Cheap, Effective, Rugged and Safe extraction (QuEChERS)
 Membrane based extraction
 Electromembrane extraction
 Cloud point extraction
 Soxhlet extraction
 Microwave assisted solvent extraction (MASE)
 Vortex assisted extraction
 Ultrasound assisted extraction
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PRESSURISED LIQUID EXTRACTION (PLE)

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High-Pressure Liquid Chromatography (HPLC)
Sample is introduced in a high pressure solvent (mobile phase) through a column
packed with sorbent (stationary phase)

(1) Column
a. Column Parameters
Material: glass, stainless steel, plastic
Dimensions: length, inner diameter
Frit size
Filter type
Precolumn and/or guard column type, if used

b. Packing Material
• Particle type: size, shape, pore diameter
• Surface modification (e.g., bonded surface type, surface coverage, percent
carbon, additional silylation)
• Recommended pH range for column use
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(2) System Suitability Testing
Each analytical procedure submitted should include an appropriate number of
system suitability tests defining the critical characteristics of that system. Criteria
for all system suitability testing should be provided.
• Tailing factor
• Relative retention
• Resolution
• Relative standard deviation (RSD)
• Capacity factor
• Number of theoretical plates

(3) Operating Parameters

The sequence of injection of blanks, standards, and samples should be defined.
Flow rates, temperatures, and gradients should be described.
Complete details should be provided for the preparation of the mobile phase,
including the order of addition of the reagents and the methods of degassing and
filtration.

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Gas Chromatography (GC)
The following parameters should be included in the description of a GC procedure:
(1) Column
Column dimensions: length, internal diameter, external diameter
Stationary phase depend on polarity (e.g., HP5-MS)
Column material (e.g., silica, glass, stainless steel)
Column conditioning procedure (temperature setting)

(2) Operating Parameters

Gases: purity (eg 99%), flow rate (eg 1.5mL/sec), pressure (psi)
Temperatures: column, injector, detector (including temperature program, if used)
Injection (e.g., split, splitless, on-column)
Detector type (FID/ECD/MSD)
Typical retention time and total run time

(3) System Suitability Testing

Appropriate system suitability criteria should be defined and included in all
analytical procedures.
If an internal standard is used, the minimum acceptable resolution between the
internal standard and one or more analyte should be specified.
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DEVELOPING THE ANALYTICAL CHEMISTRY METHOD

Example: Bioanalytical Chemistry Method

The first step is to select the analytical technique.

For a drug with a molecular weight of <2000, instrumental methods such as HPLC or GC,
with a variety of possible detectors such as UV, florescence, FID, ECD, NPD, MS, may be
employed.

Selection of columns, eluents, instrumental conditions that can resolve the drug from
potential interfering substances is an important consideration to be addressed.

The detection system should have sufficient sensitivity to be able to quantify the drug at
the low concentrations expected in physiological fluid specimens.

Also important during method development is the definition and evaluation of sample
preparation procedures for isolating the drug from matrix components.

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 Analytical Methods Requirements

(i) Method Detection Limit (MDL)

is a measure of the minimum concentration of a substance
that can be measured and reported with 99-percent confidence
that the analyte concentration is greater than
Zero.
(ii) Practical Quantitation Limit (PQL)
is the lowest level that can be reliably achieved within specified
limits of precision and accuracy during routine laboratory
operations.

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TERMS
Sensitivity
Terms used to describe the
Analytical Sensitivity, smallest concentration of a
Functional Sensitivity, measurand that can be
reliably measured by an
Lower Limit of Detection, analytical procedure.
(LoD, and LoQ)

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DETECTION LIMIT EXPERIMENT

Blank sample
-has a zero concentration of analyte
of interest.
Spike sample
-has a low concentration of analyte
of interest (the known amount of
analyte added to the blank solution).

Both the blank and spike samples

are measured repeatedly (~10
times).
Calculate the means and standard
deviations (SDs) from the values
observed for the samples.

Z value is usually 2 or 3 times the

SD
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LLD: Lower Limit of Detection

is estimated as the mean of the blank sample plus 2 or 3 times the SD

obtained on the blank sample
(i.e., LLD= meanblk + ZSblk (Z=2 or 3) ; S=SD

BLD: Biologic Limit of Detection

is estimated as the LLD plus 2 or 3 times the SD obtained from the

spike sample
(i.e., BLD= LLD + ZSspk (Z=2 or 3) ; S=SD

FS: Functional Sensitivity

is estimated as the mean concentration of a spiked sample having

coefficient of variance 20%.
(i.e., FS = meanspk @ Sspk = 20%)
To estimate FS, several spiked concentration must be studied to
determine the pricision profile at the low concentration range.
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Quantity to be estimated:

There are at least three different

concepts (and terms) that are
commonly used, as illustrated in
the accompanying figure (LLD, BLD
and FS)

The determination of these

different quantities involves
different calculations with the
data from the blank and spiked
samples.

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Functional sensitivity (FS)

A manufacturer's claim is 10 ug/L for the functional sensitivity of a method based on 10 replicate measurements.

Calculate the SD in concentration units for the 10 ug/L standard (e.g., 200 units time the calibration factor of 1
ug/L per 100 units gives an SD of 2.0 ug/L)

Calculate the CV for the 10 ug/L standard (e.g., CV is 2.0 ug/L times 100 divided by 10 ug/L, or 20%).
Coeficient of Variance (CV%) = Standard deviation x 100
Mean

FS is 10 ug/L by definition, i.e., 10 ug/L is the concentration at which the CV of the method is 20%.

FS = meanspk @ Sspk = 20%

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 When comparing various instruments:

• The measurement of interest is the analyte

concentration
• Not only are LOD and sensitivity of the method
important, but also the linear range.
• The linear range is the concentration range over
which a single slope applies.
• instrumental methods which have large linear
ranges coupled to low limits of detection
• illustrates a typical instrumental calibration
curve with the linear range denoted.

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Surrogates?

Surrogates are measured amounts of certain compounds added

before preparation or extraction of a sample. These compounds
are similar to the chemical of interest, but differ by molecular
weight or chemical configuration (e.g., ortho vs. meta).

The recovery of a surrogate is measured to determine

systematic extraction losses of analytes.

Six surrogate standards will be added prior to extraction of the

sample to monitor the extraction efficiency of the method.

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Matrix Spike?
A Matrix Spike is an aliquot of sample to which known
amounts of analyte have been added. It is subjected to the
sample preparation or extraction procedures and analyzed
as samples.

The stock solutions used for spiking are prepared

independently of calibration standards.

The spike recovery (%R) measures the effects of

interferences in the sample matrix and reflects the
accuracy or the determination

%R = Amount detected x 100

Amount spike
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