6 ANALYTICAL METHODS

CHAPTER

LEARNING OUTCOMES

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

1. discuss the principle and significance of each analytical method;

2. contrast the components and distinct feature of the analytical methods;

3. apply Beer's law to determine the concentration of the unknown analyte; and
4. relate the clinical applications of the emerging techniques.

Measurement of colors is the historical basis of quantifying the concentrations of the unknown analytes in the
clinical chemistry laboratory. Portable spectrophotometers are utilized to determine the transmittance which is then
converted to absorbance following Beer's Law.
Currently, fully automated biochemistry analyzers have replaced manual photometric readings to meet the desired
turnaround time of each assay. The principles and distinguishing characteristics of each analytical method are
presented in this chapter.

Light Energy, Wavelength, and Radiant Energy

Energy is transmitted via electromagnetic waves that are characterized by their frequency and wavelength.
Wavelength is the distance between two successive peaks and it is expressed in nanometer (nm).
Nominal wavelength represents the wavelength in nanometer at peak transmittance.
A slight error in wavelength adjustments can introduce a significant error in absorbance readings.
The relationship between wavelength and energy (E) is described by
Planck's formula.

motion per second.

Frequency is the number of vibrations of wave
The lower the wave frequency, the longer the wavelength.
inversely related to frequency and energy; the shorter the wavelength, the higher the
The wavelength is

frequency and energy, and vice versa.

Wavelength Regions Planck's formula: E = hv

Visible spectrum: 400-700nm

where:
Ultraviolet region (UV): E - is the energy of a photon in Joules or eV
Infrared region (IR): > 700nm
h - constant (6.626 x 10-34 erg sec)
v - frequency

MAJOR ANALYTICAL METHODS IN THE CLINICAL CHEMISTRY LABORATORY

I, COLORIMETRY
a. Spectrophotometric measurement - is the measurement of light intensity in a narrower wavelength.
b. Photometric measurement - is the measurement of light intensity using a specific wavelength.
 40 REVIEW HANDBOOK IN CLINICAL CHEMIS-

The primary analytical utility of spectrophotometry or filter photometry is the isolation of discreet portin
of the spectrum for purposes of measurement.

A. Spectrophotometry
It involves measurement of the light transmitted by a solution to determine the concentration of the
ligh
absorbing substances in the solution.
A spectrometer is a device that measures the wavelengths of light or the intensity of radiation.
Single-beam Spectrophotometer
It is the simplest type of an absorption spectrophotometer.
It is designed to make one measurement at a time at one specified wavelength.
The maximum absorption of the analyte must be known in advance when a single-beam instrument is used.
Double-beam Spectrophotometer
It is an instrument that splits the monochromatic light into two components: one beam passes through

sample, and the other through a reference solution or blank.

The additional beam corrects for variation in light source intensity.
In this type of spectrophotometer, the absorbance of the sample can be recorded directly as the electrical outpu
of the sample beam.
Types of Double-beam Spectrophotometer
a. Double-beam in space with 2 photodetectors, for the sample beam and reference beam
b. Double-beam in time - with 1 photodetector; alternately passes the monochromatic light through the sample
cuvette and then through the reference cuvette using a chopper (breaks out radiation beam) or rotating secto
mirror

Six Basic Components of Single- or tmobong h onet

Entrance tha Exit
Double-beam Spectrophotometer slit slit

Light
Sample PM tube
(1) Stable source of radiant energy, (2) filter that source Monochromator
isolates a specific region of the electromagnetic spectrum, A/D Display
(3) sample holder, (4) radiation detector, (5) signal
processor, and (6) readout device

Parts of the Spectrophotometer FIGURE 3. Single-beam Spectrophotometer.

1. Light/Radiant Source Source: Bishop et al., 2010

Itprovides polychromatic light and must

generate sufficient radiant energy or power to
measure the analyte of interest. Grating
An intense beam of light is directed through the
monochromator and the sample.
To give accurate absorbance measurements
throughout its absorbance range, its response to
change in light intensity must be linear.
Cell
Types of Light Source

a. Continuum source - emits radiation that changes Slit

in intensity; widely used in the laboratory Lamp

Examples: tungsten, deuterium, and xenon
lamps
Photodiode
Tungsten light bulb is the commonly used array
light source in the visible and near infrared
region.
Deuterium lamp is routinely used to provide
UV radiation in analytic spectrometers. FIGURE 4.
Double-beam Spectrophotometer.
Source: Bishop et al., 2010
ANALYTICAL METHODS 41

Xenon discharge lamp produces a continuous source of

radiation, which covers both the UV and the
visible range.
b.
Line source - emits limited radiation and wavelength
Examples: Mercury and sodium vapor lamps in spectrophotometers (UV and visible regions), and the
hollow cathode lamp (AAS)
Line sources that emit a few discrete
lines find wide use in atomic absorption, molecular, and
fluorescent spectroscopy.
Light Amplification by Stimulated Emission of Radiation (LASER) is also used as light sources for
spectrophotometry.
Factors for choosing a light source: Range, spectral distribution within the range, the source of radiant
production, stability of the radiant energy, and temperature
Alternatives for Tungsten Light Bulb
Mercury arc (visible and UV) / Xenon lamp - UV
Deuterium lamp (165nm) - UV Merst glower - IR
Hydrogen lamp - UV Lobar (silicone carbide) - IR
2. Entrance Slit

It minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator
system.
Stray light refers to any wavelengths outside the band transmitted by the monochromator; it does not
originate from the polychromatic light source; it causes absorbance error.
maximum absorbance that a spectrophotometer can achieve.
Stray light limits the
Straylight is the most common cause of loss of linearity at high-analyte concentration.0p 2 7291901
3. Monochromator
absorbence
It isolates specific or individual wavelength of light.

Kinds of Monochromators
Prisms

These are wedge-shaped pieces of glass, quartz, or sodium chloride.

These can be rotated, allowing only the desired wavelength to pass through an exit slit.
A narrow light focused on a prism is refracted as it enters the more dense glass.
Diffraction gratings
These are the most commonly used; better resolution than prism. untersm
These are made by cutting grooves (parallel grooves) or slits into an aluminized surface of a flat piece of

crown glass; wavelengths are bent as they pass sharp corner.

Holographic gratings are special types of diffraction gratings.
Filters

These are simple, least expensive, not precise, but useful monochromators.
These are made by placing semi-transparent silver films on both sides of a dielectric such as magnesium
fluoride.
produce monochromatic light based on the principle of constructive interference of waves-light
Filters
the second surface.
waves enter one side of the filter and are reflected at
have a low transmittance of the selected wavelength.
Filters usually pass : wide band of radiant energy and

4, Exit Slit
of the spectrum to reach
It controls the width of the light beam (bandpass); allows only a narrow fraction
the sample cuvette.
Bandpass is the total range of wavelengths transmitted.
1/5 the natural bandpass of the
Accurate absorbance measurement requires a bandpass less than
spectrophotometer.
42 REVIEW HANDBOOK IN CLINICAL CHEMISTRY

The degree of wavelength isolation is a function of the type of device used and the width of entrance and exit
slits.
narrower the bandpass
the bandpass, that is, the
Spectral purity of the spectrophotometer is reflected by
the greater the resolution.

It is also called absorption cell/analytical cell/sample cell.

It holds the solution whose concentration is to be measured.

Kinds of Cuvettes
Alumina silica glass - most commonly used (available in 350 nm to 2000 nm)
Quartz/plastic = used for measurement of solution requiring visible and ultraviolet spectra
Borosilicate glass
Soft glass

Notes to Remember

Cuvettes with scratches on their optical surface scatter light and should be discarded.
Silica cuvettes transmit light effectively at wavelengths 2220nm.
alkali slowly dissolves glass
Alkaline solutions should not be left standing in cuvettes for prolonged periods because
producing etching.
The path length of cuvettes is 1 cm, although much smaller path lengths are used in automated systems.
10 cm, increasing the absorbance
To increase sensitivity, some cuvettes are designed to have path lengths equivalent to
for a given solution by factor of 10.

6. Photodetector
It detects and converts transmitted light into photoelectric energy.
It detects the amount of light that passes through the sample cuvette.

Kinds of Photodetectors
Barrier Layer Cell/Photocell/Photovoltaic Cell
It is the simplest detector; least expensive; temperature-sensitive.
It is used in filter photometers with a wide bandpass.
It is the basic photodetector for detecting and measuring radiation in the visible region.
It iscomposed of selenium on a plate of iron covered with transparent layer of silver.
It requires external voltage source but utilizes internal electron transfer for current production low

internal resistance.

This cell typically has a maximum sensitivity at about 550 nm and the response falls off to about 10% of the
maximum at 350 and 750 nm.
Phototube

It contains cathode and anode enclosed in a glass case.

/ It has a photosensitive material that gives off electrons when light energy strikes it.

It requires an external voltage for operation.

Photomultiplier Tube (PMT)
/ It is the most sensitive photodetector,
The response of the PMT begins when incoming photons strike a photocathode.
the
/ It is limited to measuring low power radiation because intense light causes irreversible damage to
photoelectric surface.
It should never be exposed to room light because it will be damaged.
Photodiode
/ It is not as sensitive as PMT but with excellent linearity.
It measures light at a multitude of wavelengths : detects less amount of light.
ANALYTICAL METHODS 43

It has a lower dynamic range and

It is most useful as
higher noise compared to PMT.
a simultaneous multichannel detector.
7. Meter or Readout Device

It displays the
output of the detection system.
Example: Galvanometer/ammeter/light-emitting diode (LED) display
Quality Assurance Monitoring of Spectrophotometers
It includes monitoring the
wavelength/photometric accuracy, absorbance check, linearity, and detects straylight.
Wavelength accuracy is the actual wavelength of light that passes through the monochromator.
Didymium or holmium oxide filter is used to check
wavelength accuracy (wavelength quality assurance
calibration).
Didymium glass has an absorption peak around 600 nm.
Holmium oxide has multiple absorption with a sharp peak at 360 nm.
Neutral density filters and dichromate solution verify linearity.
An absorbance check is performed using glass filters or solutions that have known absorbance values for a
specific wavelength - the operator simply measures the absorbance of each solution at a specified wavelength
and compares the results with the stated values.
Absorption spectroscopy is preferred for solutions with absorbance values of less than 2.0.
Absorbance values higher than 2.0 may yield unreliable results, and thereby cause a deviation from Beer's law,
resulting in bending of the linear plot
Beer's Law
It states that the concentration of the unknown substance is directly proportional to the absorbed
light (absorbance or optical density) and inversely proportional to the amount of transmitted light
(% Transmittance).
It mathematically establishes the relationship between concentration and absorbance.

Absorbance (A)
It is the amount of light absorbed.
It is proportional to the inverse log of transmittance.
It is mathematically derived from % T.

A = abc = 2

where:

A= Absorbance
a = Molar absorptivity; absorptivity of the compound under standard conditions
b = Length of light through the solution
c = Concentration of absorbing molecules/solution

Au
Unknown solution: =
AS

Percent Transmittance (%T)

/ It is the ratio of the radiant energy transmitted
(T) divided by the radiant energy incident on the sample (I).

where:

I, = Transmitted light thru the sample

= Intensity of light striking the sample
 44 REVIEW HANDBOOK IN CLINICAL CHEMISTRY

the ratio of the sample transmitted beam divided

The %T measured by commercial spectrophotometers is
100).
by the blank transmitted beam (sample beam signal/blank beam signal *
In actual practice, the light transmitted by blank is substituted for •
TABLE 16. Colors and Complimentary Colors of the Visible Spectrum

Wavelength Color Absorbed Complementary Color

Yellow-blue
350-430 Violet
Yellow
431-475 Blue
Orange
476-495 Green-blue
Red
496-505 Blue-green
Green
Purple
506-555
Violet
556-575 Yellow-green
Blue
576-600 Yellow

Orange Green-blue
601-650

651-700 Red Blue-green

NOTE: If a solution absorbs light of a certain color (2nd column), the observed color of the solution is the complementary color.

Notes to Remember

The amount of light absorbed at a particular wavelength depends on molecular and ion types present and may vary
with concentration, ph, or temperature.
The light path must be kept constant to have absorbance proportional to concentration.
Deviations from Beer's law may be caused by changes in instrument functions or chemical reactions. astile
Instrument deviation is commonly a result of the finite band pass of the filter or monochromator. chordal ddail
Changes in solution pH, ionic strength, and temperature can alter Beer's law.
Turbidity readings on a spectrophotometer are greater in the blue region than in the red region of the spectrum.

Blanking Technique A

It means the blank contains serum but without the reagent to complete the assay.
Reagent blank corrects absorbance caused by the color of the
reagents the absorbance of reagents is

automatically subtracted from each of unknown reading.

/
Sample blank measures absorbance of the sample and reagent in the absence of the end
product, and corrects the
measurement for optical interference (like hemoglobin) absorbing the wavelength of measurement.
A blanking process may not be effective in some
cases of turbidity, and ultracentrifugation may be necessary to
clear the serum or plasma of chylomicrons.
Lipids interfere mainly by increasing light scatter (turbidity).
To correct for artifactual absorbance readings, "blanking" procedures or dual-wavelength methods may be used.
B. Flame Emission
Photometry (FEP)
Principle: Excitation of electrons from lower to higher energy state.
Light source: Flame (also serves as the cuvette)
Method: Indirect Internal Standard Method
Internal standard: Lithium/Cesium corrects variations in flame and
It measures the light emitted by atomizer characteristics
single atom burned in a flame.
It is used in the
measurement of excited ions(sodium and potassium).
Flickering light indicates changes in the fuel reading of the instrumen
Atomic Absorption Spectrophotometry (AAS)
C.

Principle: Element is not excited but

merely dissociated
unexcited, ground state. from its chemical bond and place in an unionized,
Light source: Hollow-cathode lamp
ANALYTICAL METHODS 45

Interferences: Chemical, matrix (differences in viscosity), and ionization

It measures the light absorbed by atoms dissociated by heat.
It is used for
measurement of unexcited trace metals (calcium and magnesium).
It is more sensitive than FEP: it is
accurate, precise, and very specific.
Internal standard is not needed changes in aspiration have litte effect on the number of ground state atoms.
An atomizer (nebulizer/graphite furnace) is used to convert ions to atoms; a chopper is used to modulate the
light source.
Lanthanum or strontium chloride is added to samples to form stable complexes with phosphate.
Sample PM tube
Chopper (atoms)

Light
source Readout
Monochromator
Burner
head

Oxidant

Mixing baffles

Aspirating air

Sample

FIGURE 5. Single-beamn atomic absorption spectrophotometer.

Source: Bishop et al., 2010

Principle: The unknown sample is made to react with a known solution in the presence of an indicator.
Examples: Schales and Schales method (Chloride test)
EDTA Titration method (Calcium test)

III, TURBIDIMETRY
Principle: It determines the amount of light blocked (reduction of light)
by a particulate matter in a turbid
solution.

It depends on specimen concentration and particle size.

formation.
The measurement of the reduction of light is due to particle
For measuring abundant large particles (proteins) and bacterial suspensions.
using visible photometers or visible
Solutions requiring quantitation by turbidimetry
are measured

spectrophotometers.
to detect bacterial growth in broth cultures, antimicrobial test
Uses: Protein measurements (CSF and urine);

(broth method); and to detect clot formation

IV. NEPHELOMETRY

Principle: It determines the amount of scattered light by a particulate matter suspended

in a turbid solution.

Photodetector: Photomultiplier tube

Use: For measuring the amount of antigen-antibody complexes (proteins)
Light scattering depends on wavelength and particle size.
measured at an angle, typically 15 to 90 degrees to the beam incident on the
Light scattered by particles is

of 250 nm to 1500 nm, and the wavelengths used are

Most antigen-antibody complexes have a diameter
320 nm to 550 nm, thus light is scattered forward (Rayleigh-Debye type).
 46 REVIEW HANDBOOK IN CLINICAL CHEMISTRY

Basic Components: Light source (mercury-arc lamp, a tungsten-filament lamp, light emitting diode, and a laser),
collimator, monochromator, sample cuvette, stray light trap, and photodetector

etector, spectrophotometer
FIGURE 6. Nephelometer
turbidimetry
versus Spectrophotometer.
Light
source Source: Bishop et al., 2010

Detector, nephelometer
forward light scatter
Detector, nephelometer
90° light scatter

V. FLUOROMETRY
Primary filter
Principle: It determines the amount of light emitted by a Source
Sample
molecule after excitation by electromagnetic radiation. holder
It measures the amount of light intensity present over a zero
background.
It uses two monochromators (either filters, prisms, or Secondary
filter
gratings) - wavelength that is best absorbed by the
the
solution to be measured is selected by the primary filter or
excitation monochromator; the incident light is prevented Detector

from striking the photodetector by the secondary filter or (photomultiplier)

Readout
emission monochromator.

To avoid potential interference from the excitation signal, FIGURE 7. Filter Fluorometer.
fluorescence measurements detect the emitted light at a Source: Bishop et al., 2010

right angle to the incident light.

It is 1000 x more sensitive than spectrophotometer - emitted radiation is measured directly.
It is affected by quenching pH and temperature changes, chemical contaminants, and UV
light changes.
It is a subset of molecular luminescence spectrometry (MLS); fluorescence and phosphorescence are types of

Fluorescence is the light emission from a single excited state, while phosphorescence is the light emission from
an excited triplet state.
Use: For measurement of porphyrins, magnesium, calcium, and catecholamines
Basic components: Light source (mercury arc or xenon lamp), primary monochromator, secondary
monochromator, cuvette, and photodetector (photomultiplier tube or phototube)
VI. CHEMILUMINESCENCE

Principle: The chemical reaction yields an electronically excited compound that emits light as it returns to its
ground state or transfers its energy to another compound which then produces an emission.
In this method, no excitation radiation is required and no
monochromators are needed because the
chemiluminescence arises from one sample.
It quantifies sample analytes in a wide range of wavelengths not observed in the visible spectrum.
The measurement of light signal is against a dark background since no excitation light is required.
The emission of light is produced from a chemical or electrochemical reaction
and not from absorption of
electromagnetic energy-the light that is emitted is detected at a right angle to the incident light to eliminate
any excitation interference signal.
It involves the oxidation of anorganic compound (dioxetane) by an oxidant (hydrogen peroxide), and the
reactions may occur in the presence of catalysts, such as enzymes, metal ions, and hemin-it
produces
chemiluminescence reaction as it returns to the singlet state.
A typical signal from a chemiluminescent compound rises rapidly with time and reaches a maximum limit when
reagent and analyte are completely mixed.
This method is best when
differentiating two compounds having excitation reaction at the same wavelength but
emit at different wavelengths.
ANALYTICAL METHODS 47

It is widely used nowadays due to its high sensitivity while even more sensitive than fluorometry.
Methods: Chemiluminescence Immunoassay (CLIA)
Uses: Autoantibody testing; measurement of hormones, drugs, vitamins, tumor markers; forensic analysis;
microbial and infectious disease marker studies; and toxicology
Photodetector: Photomultiplier tube (luminometer)
Notes to Remember

In chemiluminescence, the wavelength of the emittedlight is longer than that of the exciting light because some energy
is lost before emission from the excited state due to collisions with the solvent or other molecules.
Thesignal-to-noise ratio in chemiluminescence is very high because light is measured against a nearly dark
background.
The light emission from a single excited state is called fluorescence. INTEM

VII. OSMOMETRY

Principle: Measurement of changes in the colligative properties of solutions that occur owing to variations in

particle concentration.
It is the measurement of the osmolality of an aqueous solution such as serum, plasma, or urine.
When active osmotic particles are added to a solution, osmolality increases and four other properties of the
solution are also affected.

Osmotic particles: Glucose, urea nitrogen, and sodium

Colligative properties of the solution: Osmotic pressure, boiling point, freezing point, and vapor pressure
As the osmolality of a solution increases, the following reactions occur: osmotic pressure increases; boiling point
is elevated; freezing point is depressed; and vapor pressure is also depressed

Freezing-point Depression Osmometry

It is the most commonly used method for measuring the changes in colligative properties of a solution.
It is based on the principle that addition of solute molecules lowers the temperature
at which a solution freezes.

A 1.0 mOsm/kg solution has a freezing point depression of

0.00186° C when compared with pure solvent (usually water).
285 mOsm/kg has
Blood plasma, with an osmolality of about Readout
a freezing point of about -0:53° C.
freezing
During measurement of the sample osmolality, the Thermistor

point of the solution remains stable for several minutes once Stirring
wire
equilibrium temperature is reached. Refrigerant
out
sample is rapidly
Procedure: In a refrigeration chamber, the
several degrees below its freezing point
supercooled to Sample Refrigerant in
stirrer to start the freezing process,
then mixed with a

Crystallization of the sample produces heat which then raises

the temperature of the specimen. FIGURE 8. Freezing-point osmometer.
chamber (with ethylene
Basic components: Refrigeration Source: Bishop et al., 2010

glycol), stirrer, thermistor (temperature-sensing

readout device
device,
detects the freezing point), and
VIII. ELECTROPHORESIS
Principle: Migration of charged particles in an electric field.
the basis of their electric charge densities.
It separates proteins on

the net charge on protein, hence its electrophoretic mobility.

The acidic and basic amino acids determine
a

During electrophoresis, proteins are negatively charged (anions) and they move towards the anode.
Electrical power, Support medium, Buffer, Sample, and
Detector
Basic Components:
Buffer: Barbital (pH 8.6)
 48 REVIEW HANDBOOK IN CLINICAL CHEMISTRY

Factors Affecting Rate of Migration

Net electric charge of the molecule Nature of the supporting medium
Size and shape of the molecule / Temperature of operation
Electric field strength
Supporting Media
1. Cellulose acetate - separates by molecular size
2. Agarose gel - separates by electrical charge; does not bind protein
3. Polyacrylamide Gel - separates on the basis of charge and molecular size; separates proteins into 20 fractions;
used to study isoenzymes

Cathode(-) Marker compound

Anode(+)
loaded into well

Sample loaded into well FIGURE 9. Gel Electrophoresis.

Source: ScienceDirect Topics. (n.d.). Agarose
Gel Gel Gel Gel Gel
Gel Electrophoresis - An Overview. https://
www.sciencedirect.com/topics/agricultural-and-
Buffer Buffer
biological-sciences/agarose-gel-electrophoresis
Electric field and direction of
migration of molecules
Plastic gel box Plastic gel box

Ionic Strength of the Buffer

The ionic strength of the buffer determines the amount of current and the movement of the proteins for a fixed
voltage. If ionic strength is low, more current is carried by the charge proteins which move a longer distance (fast
mobility). If ionic strength is high, less current is carried by the proteins which move shorter distance (slow
mobility).
A high ionic strength prevents effective separation of electrophoretic bands.
Increasing the strength of the field (increased current) also increases migration.
The more the ph of the buffer differs from the isoelectric point (pl), the greater is the magnitude of the net
charge of that protein and the faster it will move in the electric field.

Electrophoretic Mobility
Electrophoretic mobility is directly proportional to net charge and inversely proportional to molecular size and
viscosity of the supporting medium.
The larger the protein molecule, the more it interacts with the environment, creating a "solvent drag" effect
which slows down the movement of the protein.
A particle without a net charge will not migrate, and it remains in the point of application.
The ions carry the applied electric current and allow the buffer to maintain a constant pH during electrophoresis.
Precautions

Improper alignment of the electrodes may cause the current on one side of the gel to be denser than on the other.
This imbalance drives the migration ofproteins to the side with more current.
The proteins may migrate off the gel and into the buffer if the electrophoresis lasts too long.
If the electrical circuit is interrupted and no current flows, the proteins will not
migrate from the point of
application.
Gels frequently display the "smile artifact,"
in which migration of samples at the center of the gel is farther than
that of the samples at the margins.

Stains for Visualization of Fractions/Bands

Amido black

Nitrotetrazolium Blue O i l Red 0

Sudan Black Fat Red 7B
ANALYTICAL METHODS 49

/ Coomassie Blue for

CSF Protein Electrophoresis
/ Gold/Silver stain - very
sensitive even to nanogram quantities of proteins
Albumin

FIGURE 10. Typical normal

pattern for serum protein
Alpha, Alpha, electrophoresis.
Beta Source: O'Connell et al., 2005
Gamma

Densitometry
It measures the absorbance of stain - concentration of the dye and protein fraction.
It scans and quantitates electrophoretic pattern.
Basic components: Light source, monochromator, scanner, optical system, and photodetector
Notes to Remember

At pH 8.6, the gamma globulins move toward the cathode despite the fact that they are negatively charged a condition
known as endosmosis.

The actual distance traveled by a particular protein migrating in an electrical field is determined by the combined

magnitudes of the electromotive force (a feature of the protein itself and the pH) and the electro-osmotic force
(a function primarily of the support medium).
at the
After electrophoresis, the gel is treated with a mild fixative, such as acetic acid, that precipitates the proteins
positions to which they have migrated. They are then stained, and the gel is dried and cleared of excess stain.
Because electrophoresis separations sometimes necessitate high voltages (50-200 V DC), the power source should
maintain a consistent DC voltage at these depths.
Isoelectric Focusing (IEF)
It separates molecules by migration through a pH gradient.
It is ideal for separating proteins of identical sizes but with different net charges.
by adding acid to the anodic area of the electrolyte cell and adding base to the cathode
ph gradient is created
area.

Proteins move in the electric field until they reach a pH equal to their isoelectric point.
cellulose acetate. t r S STRINI POMSINT9
Supporting media: agarose gel, polyacrylamide gel, and

Advantages of IEF
1. The ability to resolve mixture of proteins
To detect isoenzymes of ACP, CK, and ALP in
serum
2.
as alpha-1-antitrypsin
3. To identify genetic variants of proteins such
4. To detect CSF oligoclonal banding

Capillary Electrophoresis
In this method sample molecules are separated by electro-osmotic flow (EOF),
It utilizes nanoliter quantities of specimens.
in the specimen emerge
early at the capillary outlet because the EOF and the ion
Positively charged ions
movement are in
the same direction.
the specimen
move towards the capillary outlet but at a slower rate.
Negatively charged ions in
ves Servration, quantitation, and determination of molecular weights of proteins and peptides: analysis
of PCR

pros lees arniysis of organic and inorganic substances and drues

electrolyte buffer reservoirs, a high-voltage power supply, and a
Basic components:
Fused silica capillary,
detector volume, short turnaround time (10 minutes), and higher
Advantage over routine
electrophoresis: Low sample
resolving power
 CLINICAL CHEMISTRY
50 REVIEW HANDBOOK IN

Terminologies
on pH conditions
1. Amphoteric - has a net charge that can be either positive or negative depending
the fixed support
Electroendosmosis/Endosmosis - is the movement of buffer ions and solvent relative
to
2.

3. lontophoresis - is the migration of small charged ions

4. Zone electrophoresis - is the migration of charged macromolecules

IX. MASS SPECTROMETRY (MS)

Principle: It determines the components of : mple where the x-axis represents the mass-to-charge (m/z) ratio
and the y-axis is their relative abundance within the sample.
It is based onthe fragmentation and ionization of molecules using a suitable source of energy.
It separates diverse molecules from one another in the gas phase (gas-liquid chromatography) utilizing the so-
called mass spectroscopy-mass spectroscopy (MS-MS) methodology.
It is vital in structural identification and determination of molecular weight.
The relative abundance of each ion produces a distinct mass spectrum of the original molecule.
Uses: Detection and quantification of analytes in human fluids and tissues; utilized in proteomics, metabolomics,
and toxicology; differentiates normal tissues from malignant tissues; quanti-assay of both therapeutic and illicit
drugs; and drug development
Requirement: Before a compound can be detected and quantified by MS, it must be separated by GC.
Advantage: Qualitative and quantitative analysis of complicated samples; in biomedical research

Basic components: Sample inlet, ion source, mass analyzer, and ion detector
Samples: Blood (serum and plasma), urine, tissues, and other body fluids
Sample Preparation

Samples should be volatilized then ionized prior to measurement, to generate charged molecular ions and
fragments.
During Molecular Fragmentation
The original/native molecule ions undergo fragmentation to lower molecular mass forms.
Molecular ions are transported via an inert gas through a tube comprising a section that is in an alternating
electric field.

The higher m/z fragments migrate onto the tube at closer positions to the electron beam, whereas the smaller
cationic fragments migrate farther away from the beam the smaller cationic fragments migrate farther along
the tube than the larger cationic fragments.
A change in voltage happens everytime a fragment "hits" the tube, representing a specific m/z ratio.
The size of thevoltage change indicates the amount of a specific fragment.
Generation of Mass Spectrum
The mass spectrum is achieved whenever the plot of the
voltage changes is compared to the m/z ratio.
Each compound or macromolecule has a unique mass spectrum, which appears in a "fingerprint" pattern.
Given the electron beam intensities, temperature, and other parameters, fragments, as well as their relative
abundances, define the parent molecule, allowing the original molecule present in a patient sample to be
identified.
The ions or analytes are separated by m/z in a mass spectrometer, which is shown in the mass
x-axis of a
spectrum againsttheir relative abundance (y-axis).
The sample is measured by a detector which provides the ion current (charge) for each analyte.
To connect signal intensity to concentration, absolute quantification by mass spectrometry requires an internal
standard, a chemical that is
similar to but not identical to the analyte of interest.
Mass Spectrometry Imaging
Rather than assessing absolute concentrations, this technique focuses on monitoring various biomolecules such
as lipids and peptides.
ANALYTICAL METHODS 51

This is tool in chemical to track

a
profiling where healthy biomolecule ratios are determined and compared
disease-related biomolecule ratios.
Time-of-flight (TOF)
It distinguishes ions based on their velocity, which is accelerated by an electric field, and each ion receives the
same amount of kinetic energy.
Tandem Mass Spectrometry (MS/MS or MS2)
It involves doing mass analysis twice while performing a dissociation process or a chemical reaction to change
an ion's mass or charge.
Determines two (2) physical properties of the analytes: Precursor (parent source) and Product ion mass.
It enhances specificity beyond molecular weight separation, gives clarification on complex structures, and
assists in the identificationof basic composition of the sample.
In newborn screening test, it enables the GC-MS to detect several inborn errors of metabolism from a single dried
blood sample through analysis of amino acids.
When MS/MS is coupled with liquid chromatography (LC), the retention time adds another property to correctly
identify the analyte, resulting in enhanced specificity (Jannetto, 2015).

Sample Vaporizer
Magnet
Sample (Filament)
Injection
Port 1606001 FIGURE 11. Mass Spectrometry.
Magnetic
Field Source: Priyam Study Centre. (n.d.).
Mass Spectrometry. https://www.
priyamstudycentre.com/2022/02/mass-
spectrometry.html
Sample Accelerated
Molecule Ion Beam
Electron
Beam

Detector

Direct Sampling Ionization Techniques

Paper Spray Ionization

It is procedure utilized in mass spectrometry (MS) for direct detection of the analytes in biofluid samples such
as blood.

It is both applied in therapeutic drug monitoring and testing of illicit drugs.

It is also applied in cancer study and analysis of immunosuppresants.
the procedure)
Sample volume: 0.4 uL (may vary depending on

Substrate used: Chromatographic paper

Solvent: Methanol (water solution may be added)
substrate, it is then treated with the solvent while the
Procedure: After allowing the sample to dry on the the wetted substrate to
solvent. An electrical current (approx. 3 kV) is applied to
analytes are eluted into the will be measured through MS.
create an electrospray that produces ions that
utilizing "dried blood spot" sample.
It can be used in POCT especially in the analysis of drugs of abuse by
groups, PSI-MS
control identified a
Afteranalyzing 239 fingerprint samples from habitual drugdetection
users and
of cocaine (Costa et al., 2017, as cited in
99% true-positive rate and a 2.5% false-positive rate for the
McPherson and Pincus, 2022).
cross-validating a PSI
Tacrolimus, an immunosuppressant drug that has high clinical value, was evaluated
by

method with FDA-approved immunoassays and LC-MS/MS methods. PSI showed a significant correlation and
McPherson and Pincus, 2022).
adequate capabilities (Shiet al,, 2015, as cited in
Desorption Electrospray Ionization (DESI)
It has similarity with MALDI, however, it utilizes a charged solvent stream instead of a
laser.

biofluids even in dried blood spots.

It is a rapid analysis of drugs in
 52 REVIEW HANDBOOK IN CLINICAL CHEMISTRY

It is also used in cancer biology specifically in the study of brain cancer markers.
the charged solvent
The charged sample ions that is formed between the sample (blood or urine) and
are

determined by MS.
under ambient environments
It provides a great advantage in biomolecule profiling and imaging capabilities
(McPherson and Pincus, 2022).
X. PROTEOMICS
Principle: It is basically the study of proteins in aid of disease diagnosis.
biomarkers that will assist in the detection of diseases.
It identifies potential
at given time
It involves the characterization of complete set of proteins present in a cell, organ, or organism
(Chandramouli and Qian, 2009).
It determines the actual, real-time condition of the cells.
High-resolution MS are often used to improve the specificity of highly charged peptides and proteins, and can
separate isotopic peptides that differ in their composition (Mann and Kelleher, 2008, as cited in McPherson and
Pincus, 2022).
When coupled with chromatography and mass ratio, proteomics is a reliable method for amino acid sequencing.
Uses: For proteome profiling, comparative expression analysis of two or more protein samples, localization and

identification of posttranslational modifications, and study of protein-protein interactions (Chandramouli and

Qian, 2009).
Types of Proteomics: Bottom-up, top-down, and shotgun
Adjunct methodologies: Two-dimensional electrophoresis and LC-MS/MS
Samples: Blood, urine, tissue extract, and other body fluids
XI. METABOLOMICS
Principle: Determines the concentrations of metabolites with very small sizes in biological samples utilizing
separation and mass-to-charge ratio techniques.
It involves the comprehensive study of metabolites and their chemical properties in biofluids.
It aims to conduct precise analyses of hundreds to thousands of metabolites and to identify a significant subset of

metabolites that can differentiate disease states.

Uses: Quantification of smaller compounds, generally less than 2 kDa/1kDa in size (amino acids, nucleotides,
antioxidants, and vitamins); study of inborn errors of metabolism; and drug development
Protein expression depends not only on transcription but also on mRNA stability and rates of protein synthesis
and degradation, so the presence or absence of mRNA may not accurately reflect levels of the corresponding

protein (Mann et al., 2019).

Because proteins (proteomics) and metabolites (metabolomics) are downstream of genetic variation
and transcriptional changes, they can provide instantaneous "snapshots" of the state of a cell or organism (Mann
et al., 2019).

XII. CHROMATOGRAPHY
Principle: It is the components in a solution by specific differences in physical-chemical
separation of soluble
characteristics of the different constituents, with a mobile phase and a stationary phase.
Bases of Separation
1. Rate of Diffusion

2. Solubility of the solute

3. Nature of the solvent

4. Sample volatility/solubility
5. Distribution between two liquid phases
6. Molecular Size (molecular sieving)
7. Hydrophobicity of the molecule
8. Ionic attractions
ANALYTICAL METHODS 53

9. Differential distribution
between two immiscible liquids
10. Selective separation of substances
11. Differences in adsorption and desorption of solutes

Sample Preparation for Column-type Chromatography

Samples (urine or blood) are introduced into the column using a hypodermic syringe or an automated sampler
(loop injector).
The use of loop injectors has better sampling result and higher reproducibility.
Purpose of the Chromatogram in Column Chromatography
To detect the sample-analytes through the retention time and to measure their concentration by the area under
the peak

The retention time is utilized to determine analytes versus the standard retention time run in the same
conditions.
Peak is created by the sample analytes and it is proportional to the concentration of the sample.
FORMS OF CHROMATOGRAPHY hon

A. Planar

Paper Chromatography
It is used for fractionation of sugar and amino acid.
Sorbent (stationary phase): Whatman paper
Thin Layer Chromatography (TLC)
It is a semiquantitative drug screening test.
Sample components are identified by comparison with standards on the same plate.
Extraction of the drug is pH-dependent the ph must be adjusted to reduce the solubility of the drug in the
aqueous phase.
When all drug spots including the standards have migrated with the solvent front, it is caused by incorrect
aqueous to nonaqueous solvent mixture.
Biological samples such as blood, urine, and gastric fluid can be used for the test.
Each drug has characteristic Re value
and it must match the R value of the drug standard.

Sorbent: Thin plastic plates impregnated with a layer of silica gel or alumina.
the relative distance of migration from the point application
Retention factor (R) value - is

Distance leading edge of component moves

Re =
Total distance solvent front moves

B. Column

Gas Chromatography (GC)

It is used for separation of steroids, barbiturates, blood, alcohol, and lipids.
can be easily converted into a volatile form.
It is useful for compounds that are naturally volatile
or

direct injection, it is necessary to derivatize or volatilize the

If the molecule of interest is not volatile enough for
sample.
Flame ionization is used as a detector for gas liquid chromatography (GLC).
Elution order of volatiles is based on their boiling point.
the retention time of the solute in GC or HPLC
hydrogen, and argon
Mobile phase (inert carrier gases): Helium (most commonly used), nitrogen,
Stationary phase: Liquid solution
be injected in gaseous form or the temperature of the injection port should be
Sample preparation: Sample should
of the analytes.
higher than the boiling points
 REVIEW HANDBOOK IN CLINICAL CHEMISTR
54

The flow of the sample must be Carefully

Procedure: The specimens are vaporized and swept onto the column.
and test results repeatability.
managed to ensure maximum column efficiency
Advantage: Short turnaround time, sensitivity, accuracy, and high resolution
conductivity or
Basic components: Gas cylinder (mobile phase), sample injector, detector (thermal flame

ionization), column, and a computer for data

Types

Gas Solid Chromatography (GSC)

surfaces.
Separation occurs based on differences in absorption at the solid phase
Gas Liquid Chromatography (GLC)
mobile phase and the liquid
Separation occurs by differences in solute partitioning between the gaseous
g stationary phase.

Gas Chromatography-Mass Spectroscopy (GC-MS)

It is the gold standard in drug testing for identification and quantitation of drugs in body fluids.
It involves the separation and identification of compounds in the gas phase from their mass/charge ratios and
fragmentation patterns.
It determines xenobiotics, anabolic steroids, and pesticides.
In this method, quantitative measurement of drug can be performed by selective ion monitoring.
It uses an electron drug emerging from the column into its component ions - drugs are detected
beam to split the
by means of the presence of decomposition fragments which arise after degradation of the analytes.
The position of the parent molecule ion and degradation products give rise to fingerprint patterns which will

provide the final identity of the analyte of interest.

Every drug or analyte has its own fingerprint pattern which is compared to a computer library of known
fragmentation patterns.
The sample-analytes are "broken" into fragments following their characteristic molecular structure.
Requirement: Volatile and thermally stable samples
Preferred ionization technique: Electron ionization/EI (the sample-molecules are exposed to high energy
electrons resulting in the formation of charged ions and fragments)

Pressure Regulator

Injection Port
Computer
Controller
and Data
FIGURE 12. Gas Chromatography.
Acquisition Inlet Gas Filters
Oven Source: ScienceDirect Topics. (2017). Gas
Chromatography - An Overview. https://www.
Flow sciencedirect.com/topics/agricultural-and-
Controller biological-sciences/gas-chromatography
Gas Chromatograph

Carrier Gas
Detector
Supply
Transfer Line

Liquid Chromatography

the distribution of solutes between a liquid mobile phase and a stationary phase.
It is based on

It is the method of choice for thermolabile

compounds to achieve better separation of analytes.
It confirms positive results from screening of illicit drugs - a complementary method to GC-MS.
HPLC is the most widely used liquid chromatography.
LC-MS/MS has higher specificity for the analyte because the
quadrupole detects both the target molecule in the
sample and its fragments.
ANALYTICAL METHODS 55

The sensitivity of LC-MS/MS may allow lower limits of detection for some analytes, such as steroids, compared
to
immunoassays and other methods while giving clinical laboratories the ability to multiplex, identifying and
quantifying several analytes of interest simultaneously (Jannetto, 2015).
Basic components: Liquid
mobile phase, sample injector (manual or automatic), mechanical pump, column,
detector, and a data recorder
Advantage: Uses lower temperatures for separation of analytes, thereby achieving better separation of
thermolabile compounds

High Performance Liquid Chromatography (HPLC)

It uses pressure
for fast separations, controlled temperature, in-line detectors, and gradient elution technique.
It is ideal for separation of tricyclic antidepressants and their metabolites.
It is also used for
detection of cocaine and heroin in urine samples.
In reverse phase HPLC, the mobile phase is more polar than stationary phase; a buffer is also included in the
assay system.
Stationary phase: Column silica gel
Detector system: Photodiode array, amperometric or electrochemical detector, and fluorescence detector

Stationary phase for Reversed HPLC: Nonpolar octadecyl C-18 hydrocarbon

Mobile phase for Reversed HPLC: Acetonitrile, methanol, water, or combination of solvents
Use: Fractionation of drugs, hormones, lipids, carbohydrates, and proteins; separation and quantitation of

various hemoglobins associated with specific diseases (e.g., thalassemia); rapid HbA1c test (within 5 minutes)
Sensitivity level: Nanomolar to micromolar range .291431

Requirement: An internal standard to compensate for variation in extraction and injection

TABLE 17. Separation Techniques Used in Liquid Chromatography

Separation Technique Description

Gel/Gel Permeation/Gel Filtration/ It separates molecules based on differences in their size and shape. As solutes travel through
Size Exclusion/Molecular Sieve the gel, large molecules remain in the mobile phase and are eluted rapidly from the column.

Chromatography Hydrophilic gel (Gel Filtration)

For separation of enzymes, antibodies, and proteins
Example: Dextran and agarose
Hydrophobic gel (Gel Permeation)
For separation of triglyceride and fatty acid
Example: Sephadex
It involves the exchange of sample ions and mobile-phase ions with the charged group of the
Ion Exchange Chromatography
stationary phase.
It is for separation of amino acids, proteins, and nucleic acids.
on the sign and ionic charge
Separation of nucleic acids and proteins depends primarily
density.
the partition between a liquid mobile phase and a
Partition Separation of compounds is based on
Chromatography
stationary phase coated on a solid support.
liquid
(Liquid-Liquid Chromatography)
It is for detection of therapeutic drugs and their metabolites.
tuses immobilized biochemical ligands as the stationary phase to separate a few solutes
Affinity Chromatography
from other unretained solutes.
This type of separation uses the so-called lock-and-key binding that is widely present in
biologic systems.
It is for separation of lipoproteins, carbohydrates, glycated hemoglobins, and antibodies.
based on the
differences (competition) between the adsorption and desorption
Adsorption Chromatography Separation is
of solutes at the surface of a solid particle.
(Liquid-Solid Chromatography)
are adsorbed to a solid support such as silica or alumina.
The compounds
 56 REVIEW HANDBOOK IN CLINICAL
CHEMISTRY

Liquid Chromatography-Mass Spectrometry (LC-MS)

It is utilized for detecting nonvolatile substances in body fluids; used as a screening and confirmatory method
It is utilized to confirm positive results from screening of illicit drugs; it is a complementary method to GC-MS

It requires interface methods to convert nonvolatile to volatile compounds.

LC-MS/MS is utilized in both quantitative and qualitative analyses of drugs and hormones.
Ionization/Interface techniques: Atmospheric Pressure Chemical Ionization (APCI, preferred) and Electrospray
(ES)
Use: Micronutrient study, hormone assay, TDM, and drug development
Major interference: [on suppression
XIII. MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT-MASS
SPECTROMETRY (MALDI-TOF MS)
Principle: Ionized samples are separated based on their mass-to-charge ratio and measured by determining the
time it takes for the ions to travel to a detector at the end of a time-of-flight tube.
It is a strong analytic counterpart that uses a TOF analyzer to perform high-resolution mass spectrometry.
To produce intact gas-phase ions, samples are covered in a chemical matrix and ionized by a laser.
It is mostly utilized in clinical microbiology in the rapid identification of bacteria and fungi generating specific
results.

Uses: HbA1c assay, drug detection, and lipid analysis

MALDI-TOF imaging is useful in histological studies, tumor detection, characterization of protein expression in
tissues, and observation of cellular distribution of organic molecules such as lipids

XIV. ELECTROCHEMISTRY TECHNIQUES

The measurement of current or voltage generated by the activity of a specific ion.
It measures blood pH and gases, electrolytes in blood and sweat samples, toxins, glucose, and urea.

1. Potentiometry
It is the measurement of electrical potential due to the activity of free ions - change in voltage indicates
activity of each analyte.
It measures the differences in voltage (potential) at a constant current.

It follows the Nernst equation - an ideal reference electrode should conform to this equation.
Concentration of ions in a solution can calculated from the measured potential difference between the
be

two electrodes (reference electrode and measuring electrode).

Reference electrodes: Standard hydrogen, calomel, and silver-silver chloride (electrodes with a constant
voltage)
Use: Blood PH and pCO2 measurements
Reference Electrodes

1.
Saturated calomel electrode - consists of mercury in contact with a solution that is saturated with mercurous
chloride (calomel) and known concentration of potassium chloride
2. Silver-silver chloride - consists of a silver electrode immersed in solution that has
potassium chloride
been saturated
with silver chloride; ideal for temperatures higher than 60° C and react with more sample
components compared to calomel

ph Electrode (glass pH Electrode)

It measures the potential difference between a measuring electrode which contains the sample in contac
with a special glass membrane permeable only to H* ions, and a reference electrode which has a knowl
stable pH (Trulock III, 1990).
It is made up of a small bulb of
hydrated and nonhydrated glass, which contains a chloride ion buffer
solution.

From the voltage across these electrodes, the sample ph is calculated.

ANALYTICAL METHODS 57

pCO2 Electrode (Severinghaus Electrode)

It employs an adaptation of the pH
measurement.
It is
pH electrode contained within a plastic jacket- the plastic jacket is filled with a sodium bicarbonate
buffer and has a gas-permeable membrane.
Carbon dioxide from the blood sample equilibrates across gas-permeable membrane with a bicarbonate
solution in a reaction that generates
Ht ions (Trulock III, 1990).
pCO2 level in the sample is
determined indirectly by sensing the pH change in this solution (Trulock III,
1990).
Glass Electrode
It is the
first and still the most commonly used electrodes for measuring hydrogen ion activity or pH.

Ion-Selective Electrode (ISE)

It is an electrochemical transducer capable of responding to one given ion.
Its ionic selectivity depends on the membrane/barrier composition used.
It is very sensitive and measures the activity of one ion (selective).
It is a sensitive methodbut nonspecific it does not discriminate between ions in causing voltage
differences between the measuring electrode and the standard electrode.
ISE analyzers measure the electrolyte dissolved in the fluid phase of the sample in mmol/L of plasma water.
The ISE method uses a semipermeable membrane to create a potential from having different ion
concentrations on either side of the membrane, with two electrodes (reference and measuring electrodes) to
complete the assay.
Electrodes are used (one electrode with a constant potential acts as the reference electrode) to determine
the "concentration" of the analyte or ion in the sample.
Examples of ISE: Glass electrodes, liquid membrane electrodes, precipitate-impregnated membrane
electrodes, solid-state electrodes, gas electrodes, and enzyme electrodes
TABLE 18. Types of Ion-Selective Electrode
Direct ISE Indirect ISE

With sample dilution

Without sample dilution
In this method, the patient's serum sample is brought into
It involves diluting the patient's serum with a buffer in the

range of 1:16 to 1:34 before the sample comes in contact with

direct contact with the electrode surface and the activity of the
the (SE membranes.
relevant ion is measured in the water portion of the serum.
It calculates the electrolyte concentration in the diluted sample
Advantage over Indirect ISE: Not affected by hyperproteinemia
by assuming that the original sample had a plasma water
and hyperlipidemia
concentration of 93% (dissolved solids, mostly proteins and
Use: POCT electrolyte test and blood gas analysis lipids, typically make up the remaining 7% of a patient's plasma
volume).
Use: Routine electrolyte test

Source: Datta, 2018

Examples of ISE Membranes

(potassium), organic liquid membrane ion exchangers
Glass aluminum silicate (sodium), valinomycin gel

(calcium and lithium), gas, and enzyme electrodes

Interferences
to excess protein, an

Protein coating
the ISE membrane would cause a response error--if interference is due
ISE in undiluted specimen can be employed to yield correct electrolyte activity
alternative mode of analysis such
(i.e., equivalent of osmolality).
Causes of Malfunctions of ISE
of counter voltages from liquid junction potentials at the salt bridge, and
Defective ISE membrane, buildup
buildup of proteins at the electrodes
 58 REVIEW HANDBOOK IN CLINICAL CHEMIST

Indicator
Reference
Electrode
Electrode

FIGURE 13.
Ion-Selective Electrode.
Source: Lindner and Pendley,
2012
Bridge Electrolyte-1
Inner filling
Solution
Bridge Electrolyte-2
Ion-selective
Liquid Junctions Membrane

Sample Solution

2. Coulometry
It is the measurement of the amount of electricity (in coulombs) at a fixed potential.
It is an electrochemical titration in which the titrant is electrochemically generated and the endpointi
detected by amperometry.
It follows Faraday's law-the number of silver ions generated after ionization can be calculated fron
Faraday's law, and the product of the calculation is exactly equal to the chloride ions in the sample.
Use: Chloride test (CSF, serum, and sweat)
Procedure: A constant current is applied across the two silver electrodes, which liberates silver ions into
specimen at a constant rate. Chloride ions in the sample combine with silver ions to produce insoluble silve
chloride. A pair of indicator and reference electrodes senses the excess silver ions and stops the titration
(McPherson and Pincus, 2022).
Interferences: Bromide, cyanide, and cysteine

3. Amperometry
It is the measurement of the current flow produced by an oxidation reaction.
Several immobilized enzyme electrodes use this amperometry principle.
Use: pO2, glucose, chloride, and peroxidase determinations
Polarography
It is the measurement of differences in irrent at a constant voltage.
It follows the Ilkovic equation.

pO2 measurement
It is determined amperometrically.
In this assay, the oxygen from the blood sample diffuses across a semipermeable membrane and is reduce
at the cathode of a polarographic electrode, thereby producing a measurable current that is directl
proportional to the sample p02 (Trulock 1II, 1990).
It uses Clark electrode.

4. Voltammetry
It is the
measurement of current after which a potential is applied to an electrochemical cell.
It allows the sample to be preconcentrated, thus utilizing minimal analyte concentration.
Advantage: High sensitivity and the capability for multielement measurements (analytes can be detecte
in the parts-per-billion range)-several analytes can be measured simultaneously in a single voltammel"
assay

Anodic stripping voltammetry: For lead and iron testing

ANALYTICAL METHODS 59

(POCT)
It is also known as the
decentralized test, bedside test, or near-patient test.
It is an analytical testing performed outside the laboratory usually by nonlaboratorian personnel.
It is used both
in chemical analysis and microbiological tests.
It is
currently routine in all hospitals and has become the standard for patient care in a variety of other health
care settings (Nichols et al., 2020).
A separate certification
is usually not necessary when it is performed under the supervision of the laboratory
director responsible for laboratory services in a health care facility.
Examples: bloodglucose meter, electrolytes, blood gases, enzymes, cardiac markers, and therapeutic drug
monitoring (TDM)
Types of POCT: Wet chemistry, cartridge-based, and strip-based

FIGURE 15. The Abbott i-STAT

System offers test results for the most
FIGURE 14.
commonly ordered tests in surgical and
Point of care (POC)
specialty care areas, including: blood
diagnostic devices
gases, chemistries, lactate, electrolytes,
for glucose.
hemoglobin and hematocrit, and
coagulation (including PT/INR).
Source: http://sandor.co.in/wp-content/
uploads/2015/10/istat.jpg?fbclid=

Disclaimer: The author is not in any way connected with the manufacturer/
distributor of nor promoting this product. The descriptions and photo are
used only for lecture/discussion purposes.

IMMUNOASSAY TECHNOLOGY IN CLINICAL CHEMISTRY

It is used to detect the presence of a molecule (analyte) by measuring its interaction with an antibody or antigen.
Because the interaction between antigen and antibody is an immune response, this analysis is referred to as an
"immunoassay."
In most instances, the analyte is a protein or an antigen present in the test sample that reacts with an anti-

analyte antibody (the antibody that specifically targets the molecule being measured). lodds 19th
This kind of assay is mostly used in TDM, hormone studies, and toxicology.
Chemistry Laboratory b sdal3edW 0f
Examples of Immunoassays/Immunochemical assays used in the Clinical
Enzyme-Linked Immunosorbent Assay (ELISA)
Chemiluminescence Immunoassay (CLIA)
Immunoradiometric Assay (IRMA)
Immunochemiluminometric Assay (ICMA)
Fluorescence Polarization Immunoassay (FPIA)
Enzyme-Amplified Sensitivity Immunoassay (EASIA)
NOTE: For the principles and discussions of the immunoassays, refer to Chapters 19 and 21.
 60 ANALYTICAL METHOD~

ASSESSMENT QUIZ
1. Which type of filter is best for measuring stray light?
didymium
b. sharp cut-off d. gratings
2. Which part of the spectrophotometer allows only a narrow light to reach the sample cell?
entrance slit c. exit slit

b. prism d. photomultiplier tube

This electrochemistry technique measures chloride in sweat.
a. potentiometry C. chloridometry

b. coulometry d. polarography
4. It involves dissociation of analytes by heat energy into atoms and measures minimal concentrations.
a. electrophoresis c. nephelometry
b. fluorometry d. atomic absorption spectrophotometry
5. Ion selective electrode is a "selective" method due to which factor?

a. membrane composition c. quality of reference electrodes

b. use of stable voltage d. specificity of indicator electrode
6. What is the most sensitive photodetector that measures light in the visible and UV regions?
a. photocell C. photodiode
b. photomultiplier tube d. holographic gratings
7. This photoanalytical method does not require an excitation radiation and monochromators to determine the
concentration of the unknown substances.
a. turbidimetry c. chemiluminescence
b. flame emission d. voltametry
8. Which type of molecular luminescence spectrometry utilizes two monochromators?
a. proteomics C. nephelometry
b. mass spectrometry d. fluorometry
9, It has a great advantage over other analyticals in its capacity to measure substances with small molecular
sizes.

C.
tandem mass spectroscopy
b. d. MALDI-TOF
10. What is the
ideal concentration of the buffer in serum protein electrophoresis?
a. pH 5.2 C. pH 7.4
b. pH 6.5 d. pH 8.6
11. In electrophoresis, if
the electrodes are not fixed and properly aligned, the proteins will migrate
on the side with more current.

a. closer
C. very near
b. farther
d. faster
12. Which separation technique is a requirement in mass
spectrometry before the unknown analytes can be
detected and quantified?
a. gas chromatography C. time of flight
b. electrophoresis
13. In electrochemistry, it is a type of pH electrode with
measurement of carbon dioxide.
a sodium bicarbonate buffer utilized in the

a. Clark
C. Calomel
b. Valinomycin d. Severinghaus
ANALYTICAL METHODS 61

14. It
measures proteins with large molecular complexes using scattered light and sensitive enough
quantitate concentrations less than
to even

grams per deciliter.

a.
spectrophotometry
C. proteomics
b. nephelometry
d. tandem mass spectrophotometry
15.
What is the preferred method in measuring ions with minimal serum levels such as magnesium?
a. AAS
C. fluorometry
b.
mass spectrometry d. Desorption Electrospray Ionization