PAKISTAN SOCIETY OF CHEMICAL PATHOLOGISTS
DISTANCE LEARNING PROGRAMME IN CHEMICAL PATHOLOGY
LESSON NO 13
ANALYTICAL TECHNIQUES AND INSTRUMENTATION
(SHORT NAME: ATI)
BY
SURG COMMODORE AAMIR IJAZ
MCPS, FCPS, FRCP (EDIN)
PROFESSOR OF PATHOLOGY /
CONSULTANT CHEMICAL PATHOLOGIST
BAHRIA UNIVERSITY MEDICAL &DENTAL COLLEGE /
PNS SHIFA KARACHI
Q:1: The analytical technique in which
optics (Photometry) is NOT used in any
form:
a. Atomic Absorption
b. HPLC
c. POCT for glucose
d. Radioimmunoassay
e. Real Time PCR
Best answer:
d. Radioimmunoassay
Optical Techniques used in Various Instruments
Atomic Absorption Photometry : Primarily a Photometric
Technique
HPLC: Photometry used in detection in many types of HPLC
Glucometers: Reflectance technique
Real Time PCR:
Fluorometry in detection
Q: 2. A Chemical Pathology autoanalyser has rejected the
blank cuvettes before the start of analysis with the
message “Cuvette Check Fail” (Dirty Cuvettes). Which of
the following phenomena is mainly prevented by this
function of autoanalyser:
a.
b.
c.
d.
e.
Absorbance of light
Emission of light
Reflectance of light
Scattering of Light
Transmission of light
Best answer:
d.Scattering of Light
Q:3: Which of the following modifications makes
a Liquid Chromatography a High Performance
Liquid Chromatography (HPLC):
a. Fixed loop injector
b. Increasing volatility by sample derivatization
c. Length of columns from 50 to 250 nm
d. Small-diameter particles as stationary phase
e. UV lamp detectors of 190 to 400 nm.
Best answer:
d. Small-diameter particles as stationary phase
Q:4: It is reported that the sensitivity of an Atomic Absorption
method is 100 times higher than Flame Emission. The reason for
this very high sensitivity is:
a. Drastic reduction in background noise
b. Most of the ground state atoms absorb light.
c. Sample derivatization before analysis
d. Use of a hollow cathode lamp made of the material to be
analysed.
e. Very high temperature is used for excitation of atoms.
Best answer:
b. Most of the ground state atoms absorb light.
Atomic Absorption Photometry
Very High Sensitivity : About 95% atoms remain in
ground state and are not excited. In AA the light is
absorbed by these 95% atoms. So the ability of this
technique to measure very small quantity of substances is
very high i.e. high analytical sensitivity.
Very High Specificity: Use of a hollow cathode lamp
made of the material to be analysed is the reason of very
high specificity of this technique.
Q:5: In an Ion Selective Electrode System glass
electrodes with sufficient selectivity are used for the
measurement of :
a. Carbon Dioxide (PCO2)
b. Chloride (Cl-)
c. Hydrogen (H+)
d. Oxygen (PO2)
e. Potassium (K+)
Best answer:
c. Hydrogen (H+)
Q:6: In Clinical Laboratories Conductometry is
used for the measurement of :
a. Calcium (Ca+2)
b. Haematocrit
c. Lithium (Li+)
d. Magnesium (Mg+2)
e. Sodium (Na+)
Best answer:
b. Haematocrit
Q:7: In Clinical Enzymology, Michaelis and
Menten Equation provides the basis of
quantitative measurement of enzymes. Which of
the following is a basic assumption of this
equation:
a. The effect of product is negligible on the concentration of EnzymeSubstrate Complex.
a.
b.
The enzyme concentration of the enzyme-catalysed reaction is fixed.
c.
The pH is neutral (7.40).
d.
The reaction is first order with respect to substrate
e.
The temperature of the reaction is 370C.
Best answer:
The effect of product is negligible on the concentration of Enzyme-Substrate
Complex.
Michaelis and Menten Equation for
quantitative measurement of enzymes
Michaelis and Menten Equation governs quantitative
estimation of enzyme activity. An important assumption
of this equation is:
‘The effect of product is negligible on the concentration
of Enzyme-Substrate Complex’.
Types of Enzyme-Substrate Reaction
Zero Order Reaction: When an enzyme activity is
to measured, the quantity of substrate is added in
excess and the amount is fixed.
First Order Reaction: is the reaction in which
quantity of substrate is to be determined. So the
quantity of enzyme is fixed.
Q:8: The Electrophoresis System with high speed
automation and miniaturization:
a. Capillary electrophoresis
b. Capillary zone electrophoresis
c. High performance gel electrophoresis
d. Isoelectric focusing
e. Microchip-based electrophoresis
Best answer:
e. Microchip-based electrophoresis
Q:9: An autoanalyser which can perform different
tests sequentially on a batch of specimen is a
called :
a. Batch analyser
b. Centrifugal analyser
c. Continuous flow analyser
d. Discrete analyser
e. Random access analyser
Best answer:
e. Random access analyser
Q:10: Point of Care Testing (POCT)
employing immunoassay is used for the
detection of:
a. Bilirubin
b. Electrolytes
c. Glucose
d. Troponins
e. Urine Albumin
Best answer:
d. Troponins
Q:11: Immunoassays have passed through an evolutionary
process of development with decreasing Limit of Detection
(Increasing Analytical Sensitivity) and ease of automation.
a. List the labelled immunoassays in the order of
ascending Analytical Sensitivity.
b. Name TWO labelled immunoassays with very high
sensitivity widely used in Hormone and Tumour
Marker auto-analysers these days. What is the
range of their lower detection limit?
c. What are the TWO technical advancements which
have resulted into such remarkable sensitivity of
these immunoassays?
Suggested Answer to Q.11a
List the labelled immunoassays in the order of ascending
Analytical Sensitivity.
(Please see next slide)
Immunoassays (In the order of Sensitivity)
• Radioimmunoassay (RIA)
• Immunoradiometric assay (IRMA)
• Enzyme linked immunosorbent (ELISA)
• Enzyme multiplied immunoassay technique (EMIT)
• Cloned enzyme donor immunoassay (CEDIA)
• Florescent immunoassay (FIA)
• Florescent polarized immunoassay (FPIA)
• Electrochemiluminescence immunoassay
• Chemiluminescence immunoassay
(Note: This is an approximate list. Some techniques may have better
sensitivity than mentioned here)
Suggested Answer to Q.11b
Name TWO labelled immunoassays with very high
sensitivity widely used in Hormone and Tumour Marker
auto-analysers these days. What is the range of their lower
detection limit?
1. Chemiluminiscence immunoassay
Range of their lower detection limit is 1 zeptomole
1. Electrochemiluminescence immunoassay
Range of their lower detection limit is 20 zeptomole
Suggested Answer to Q.11c
What are the TWO technical advancements which have
resulted into such remarkable sensitivity of these
immunoassays?
1. Enzymatic amplification of substrate to product also improve
sensitivity of immunoassay
2. Use of Luminescence material
Chemiluminescent Immunoassays
• Large number of molecules capable of
chemiluminescence
• Luminol
• Acridium esters
• Ruthenium derivatives
• Nitrophenyl oxalates
• Use sodium hydroxide as a catalyst
• Light emission ranges from quick burst or flash to light
which remains for a longer time.
• Different types of instruments are required based on
emission.
Chemiluminescent Immunoassays (Con)
• Can be used for heterogeneous or homogeneous assays.
• Can attach label to antigen or antibody.
• Heterogeneous assays use competitive and sandwich
assay.
• Competitive assays used to measure smaller analytes.
• Sandwich assays are used to measure larger analytes.
Chemiluminescent Immunoassay (cont)
• Many applications.
• Can measure antigen or antibody.
• Add chemiluminescently tagged analyte.
• Measure light which is emitted which is directly related to concentration although
competitive binding assays are available.
Chemiluminescent Immunoassays (cont)
• Best known application of chemiluminescense is luminol
• Luminol reacts with the iron in blood hemoglobin.
Q:12
Chromatography is an important component of any Analytical
Chemical Pathology Laboratory. A Chemical Pathologist should have a
broad knowledge base upon which detailed technical knowhow can be
built. So please answer following questions regarding Chromatography:
a. Give basic characteristics of Chromatography. Give ONE
feature of Electrophoresis which differentiates it from
Chromatography.
b. Name two basic forms of chromatography with sub-classification
of each form.
c. What are the various separation mechanisms used in
chromatography? Please write one line for each of these
mechanisms.
d. Name components of a column chromatograph.
Suggested Answer to Q.12a
Give basic characteristics of Chromatography. Give ONE feature
of Electrophoresis which differentiates it from Chromatography.
• In electrophoresis migration of charged solutes or
particles in a liquid medium under the influence of an
electric field
• Chromatography is a physical process where the
component of a sample mixture are separated as a
result of their differential distribution between stationary
and mobile phase.
Suggested Answer to Q.12b
Name two basic forms of chromatography with sub-classification of
each form.
Planer
•
Paper
•
Thin layer chromatography (TLC)
•
High performance thin layer chromatography(HPTLC
Column
•
Gas (GC)
•
Liquid (LC)
•
HPLC
Suggested Answer to Q.12c
What are the various separation mechanisms used in
chromatography? Please write one line for each of these
mechanisms.
(Plz see next slide)
Separation Mechanisms Used in Chromatography
1.Ion Exchange Chromatography :
Ion exchange chromatography is based on an exchange of ions
between a charged stationary surface and ions of the opposite
charge in a mobile phase.
2.Partition Chromatography
The differential distribution of solutes between two immiscible
liquids is the basis for sepration by partition chromatography.
3.Adsorption Chrpmatography
The basis of sepration by adsorption chromatography is the
difference between adsorption and desorption of solutes at the
surface of a solid particles.
Separation Mechanisms Used in Chromatography
(Contd)
4.Size- Exclusion Chromatography
In this technique, solutes are separated on the basis of
their molecular size, shape and hydration.
5.Affinity Chromatography
In affinity chromatography, the unique and specific
biological interaction of the analyte and ligand is used for
separation.
Suggested Answer to Q.12d
Name components of a column chromatograph.
•
Mobile phase supply
•
Flow controller (GC) or pump (LC)
•
Injector
•
Computer/controller
•
Integrator/ recorder
•
Column (s)
•
Detector (s)
•
Waste
Q:13 Mass Spectrometry (MS) can be regarded as one of the most
important technical advances in the field of Analytical Chemistry in
recent times. It has enhanced identification and quantification of small
quantities to a greatest degree. Please answer following questions
regarding Mass Spectrometry:
a. What is a Mass Spectrometer and on which principle Mass
Analysis is carried by these instruments?
b. What is the technical innovation in MS which has led to the
development of definite methods of various routine analytes?
c. Name various instruments in which MS is used as a primary
technique or used as a coupled component to greatly
enhance the specificity and sensitivity.
Suggested Answer to Q.13 a
What is a Mass Spectrometer and on which principle Mass
Analysis is carried by these instruments?
Mass spectrometer (MS) is an instrument in which ionized molecules
are separated and measured according to their mass-to-charge ratio.
Principle of Mass analysis:
Mass analysis is the process by which a mixture of ionic species is
identified according to the mass-to-charge (m/z) ratios (ions).
Mass spectrometer (universal detector) is a powerful qualitative and
quantitative analytical instrument that first ionizes a target molecules
and then separates and measures the mass of a molecule or its
fragments.
Suggested Answer to Q.13 b
What is the technical innovation in MS which has led to the
development of definite methods of various routine analytes?
Isotope dilution mass spectrometry (IDMS) Ability of MS
to quantify a compound relative to an isotope species of
known or fixed concentration is called IDMS. The IDMS
technique has been used to develop definitive methods
for various routine analytes
Suggested Answer to Q.13 c
Name various instruments in which MS is used as a primary
technique or used as a coupled component to greatly enhance
the specificity and sensitivity.
(Please see next slide)
.
Instruments in Which MS is Used
• Primary Technique
•
•
•
•
•
MS/MS (Tandem MS)
Time of Flight (TOF) MS
MALDI Mass spectrometry
SELDI Mass spectrometry
ICP Mass Spectrometry
• Coupled Technique
• Liquid Chromatography - Mass Spectrometry
• Gas Chromatography Mass Spectrometry
Thank You and Best Of Luck