Analytical Methods

and Instrumentation
Maria Margarette A. Dolor, RMT
Electromagnetic Energy

• Described as photons of
energy travelling in waves
characterized by their
frequency and
wavelength.
The distance between two successive peaks

Expressed in terms of nanometer (nm)

400-700nm- visible spectrum

<400nm- ultraviolet region (UV)

>700nm- infrared region (IR)

WAVELENGTH
 Frequency
• Is the number of vibrations of wave motion
per second
• The lower the wave frequency, the longer
the wavelength
• The wavelength is inversely related to
frequency and energy.
 Spectrometry
spectrophotometry, Luminescence
atomic absorption, fluorescence and
and mass chemiluminescence
FOUR BASIC spectrometry [MS]

DISCIPLINES OF
ANALYTICAL
CHEMISTRY Electroanalytic
methods
Chromatography
gas, liquid, and thin
electrophoresis,
layer
potentiometry, and
amperometry
 Kinds of Colorimetry
• Visual Colorimetry– uses the eyes in
determining end point
• Photoelectric Colorimetry
–2 Categories
✓Spectrophotometry
COLORIMETRY –(Spectrophotometric measurements)
✓Filter photometry
–(Photometric measurements:
measurement of light intensity
independent of wavelength)
SPECTROPHOTOMETRY
• is a technique used to measure solute concentration by measuring
light intensity in a narrower wavelength.
 a. Power supply e. Exit slit
COMPONENTS OF A b. Light source f. Cuvet/sample cell
SPECTROPHOTOMETER c. Entrance slit g. Photodetector
d. Monochromator h. Readout device
 BEER’S LAW

• It states that the concentration

of the unknown substance is
directly proportional to the
absorbed light and inversely
proportional to the amount of
transmitted light.
Percent Transmittance

• It is the ratio of the radiant energy transmitted

(T) divided by the radiant energy incident on
the sample (I)
𝐼𝑡
• %𝑇 = x 100
𝐼𝑜
Absorbance (A)

It is the amount of light absorbed

Proportional to the inverse logarithm of transmittance

Mathematically derived from %T.

A = abc = 2-log%T
Light Source
• Provides polychromatic light and must generate sufficient
radiant energy or power to measure the analyte of interest.
• Light is divided into visible and invisible light. Visible light
falls in between, with the color violet at 400 nm and red at
700 nm wavelengths being the approximate limits of the
visible spectrum.
• The most common source of light for work in the visible and
near-infrared regions is the incandescent tungsten or
tungsten-iodide lamp.
• The lamps most commonly used for ultraviolet (UV) work
are the deuterium discharge lamp and the mercury arc
lamp.
 Two Types of Light Source
a. Continuum source- emits radiation that changes in intensity. It is
widely used in the laboratory.
oTungsten light bulb- commonly used light source in the visible and
near infrared region. Does not supply sufficient radiant energy for
measurements below 320nm.
oDeuterium lamp- routinely used to provide UV radiation in
analytic spectrometers. More stable and has a longer life than a
hydrogen lamp.
oXenon Discharge lamp- produces a continuous source of
radiation. It cover both the UV and visible range
 Two Types of Light Source
b. Line source- emits limited radiation and wavelength
oMercury vapor lamps and Sodium vapor lamps- exits narrow bands
of energy at well defined places in the spectrum (UV and visible)
oLight Amplification by Stimulated Emission of Radiation (LASER)-
transform light of various frequencies into an intense, focused, and
nearly non-divergent beam of monochromatic light.
ENTRANCE SLIT
• Minimizes unwanted or stray light and prevents the entrance of
scattered light into the monochromator system
MONOCHROMATOR
• It isolates specific or individual or desired wavelength of light.
• Monochromatic light - light radiation of a single wavelength
Types of Monochromators
• Colored Glass Filter
• least expensive, pass a relatively wide band of radiant energy and have a low
transmittance of the selected wavelength. Although not precise, they are
simple, inexpensive, and useful.
Types of Monochromators
Interference Filters
• produce monochromatic light based on the principle of constructive
interference of waves
• Two pieces of glass, each mirrored on one side, are separated by a
transparent spacer that is precisely one-half the desired wavelength.
 Prism
• Wedge-shaped pieces of
glass, quartz or sodium
chloride
• A narrow beam of light
focused on a prism is
refracted as it enters the
more dense glass.
• The prism can be rotated,
allowing only the desired
wavelength to pass through
an exit slit.
Types of Monochromators
 Diffraction Gratings
• most commonly used,
consists of many parallel
grooves (15,000 or 30,000
per inch) etched onto a
polished surface.
• based on the principle that
wavelengths bend as they
pass a sharp corner
• better resolution than
prism
Types of Monochromators
EXIT SLIT
• Controls the width (bandpass) of light beam- allows only a narrow
fraction of the spectrum to reach the cuvette
SAMPLE CELL
• absorption cell/ analytical cell/ cuvette, may be round or square
• Holds the solution whose concentration is to be measured
• Path length is 1 cm, however, to increase sensitivity, some cuvettes
are designed to have path length of 10 cm, increasing the absorbance
for a given solution by a factor of 10
 Different Types of Cuvette
• Alumina silica glass
• Most commonly used (350 to 2000nm)
• Quartz/ Plastic
• Used for the measurement of solution requiring visible and ultraviolet
spectra
• Borosilicate glass
• Soft Glass
PHOTODETECTOR
• convert the transmitted radiant energy into an equivalent amount of
electrical energy
 Types of Photodetector
Barrier Layer Cells / Photocell / Photovoltaic Cell
• least expensive, composed of a film of light-sensitive material,
frequently selenium, on a plate of iron
• require no external voltage source but rely on internal electron
transfer
• used mainly in filter photometers with a wide bandpass
• inexpensive and durable; however, it is temperature sensitive and
nonlinear at very low and very high levels of illumination.
Types of Photodetector
Phototube
• contain a negatively charged cathode and a positively charged
anode enclosed in a glass case
• Has a photosensitive material that gives off electron when light
energy strikes it
• Requires external voltage
Types of Photodetector
Photomultiplier Tube
• Most commonly used detector- measures visible and UV regions, 200
times more sensitive than the phototube
• Has excellent sensitivity and has rapid response- detects very low
level of light
• Should never be used to room light because it will burn out
Types of Photodetector
Photodiode
• Not as sensitive as PMT but with excellent linearity, speed and small
size
• Measures light at a multitude of wavelength- detects less amount of
light
• Most useful as a simultaneous multichannel detector
METER / READ- OUT DEVICE
• It displays output of the detection system
• E.g. Galvanometer, ammeter, LED
 2 Types of
Spectrophotometer
A. SINGLE BEAM SPECTROPHOTOMETER
• Simplest type of spectrophotometer
• Designed to make one measurement at a time at one specified
wavelength
B. DOUBLE BEAM SPECTROPHOTOMETER
• Instrument that splits the monochromatic light into two components:
• One beam passes throughout the sample
• Another beam passes through a reference solution or reagent blank
• The additional beam corrects for variation in light source intensity
Two Types of
A. DOUBLE-BEAM IN SPACE- uses
Double-Beam two(2) photodetectors
Spectrophotometer
 b. Double-beam in time- uses one
Two Types of photodetector and alternately passes the
Double-Beam monochromatic light throughout the sample
Spectrophotometer cuvet and the reference cuvet using a chopper
or rotating sector mirror
 SPECTROPHOTOMETER QUALITY ASSURANCE
• Performing at least the following checks should validate instrument
function: wavelength accuracy, stray light, and linearity.

A. Wavelength Accuracy
• means that the wavelength indicated on the control dial is the actual
wavelength of light passed by the monochromator
• Most commonly checked using standard absorbing solutions or filters
(Didymium or holmium oxide in glass). Some instruments with narrow
bandpass use a mercury vapor lamp to verify wavelength accuracy.
 SPECTROPHOTOMETER QUALITY ASSURANCE
B. Stray Light
• any wavelengths outside the band transmitted by the monochromator
• most common causes of stray light are reflection of light from scratches on optical
surfaces or from dust particles
• major effect is absorbance error
• detected by using cutoff filters or liquids, such as NiSO4, NaNO2, and acetone,
absorb strongly at short wave- lengths and can be used in the same way to detect
stray light in the UV range.
C. Linearity
• demonstrated when a change in concentration results in a straight line calibration
curve
• Colored solutions may be carefully diluted and used to check linearity
 ATOMIC ABSORPTION SPECTROPHOTOMETRY
• used to measure concentration by detecting the absorption of electromagnetic
radiation by atoms rather than by molecules, measures the light absorbed by atoms
dissociated by heat
• Principle
• Element is not excited but merely dissociated from its chemical bond and place in an unionized,
unexcited, ground state
• Light source:
• Hollow Cathode Tube (a separate lamp is required for each metal, example, a copper hollow-
cathode lamp is used to measure Cu)
• Electrodeless discharge lamps are a relatively new light source for atomic absorption
spectrophotometers.
• Interferences: chemical, matrix and ionization
 ATOMIC ABSORPTION SPECTROPHOTOMETRY
• DISADVANTAGES
• Inability of the flame to dissociate samples into free atoms (phosphate may interfere
with calcium analysis by formation of calcium phosphate)
• SOLUTION: Lanthanum or strontium is added to samples to form stable complexes with phosphate
• ionization of atoms following dissociation by the flame
• SOLUTION: reduce the flame temperature
• Matrix interference, due to the enhancement of light absorption by atoms in organic
solvents or formation of solid droplets as the solvent evaporates in the flame
• SOLUTION: pretreatment of the sample by extraction
 COMPONENTS OF THE AAS
• BURNER – uses flame to dissociate the chemical bonds and form free,
unexcited atoms.
TYPES:
a. Total consumption burner – flame is more concentrated and can be made
hotter, thus lessening chemical interferences.
• produces large droplets in the flame and produces a high acoustical noise.
b. Premix burner –gases are mixed and the sample is atomized before entering
the flame and the large droplets go to waste and not in the flame.
• It has less noisy signals with longer path length and greater absorption and
sensitivity.
• flame is less hot and therefore cannot dissociate metal complexes.
 • MONOCHROMATOR – selects the desired
COMPONENTS wavelength from a spectrum of wavelength which
could either be a prism or diffraction gratings.
OF THE AAS • ATOMIZER(NEBULIZER/GRAPHITE FURNACE) is
used to convert ions to atoms
• CHOPPER- is used to modulate the light source
• DETECTOR – uses photomultiplier tubes to
measure the intensity of the light signal.
• READ OUT DEVICE
FLAME EMISSION PHOTOMETRY
• measures light emitted by excited atoms, was widely used to
determine concentration of excited atoms such as Na+, K+, or Li+.
• Principle: Excitation of electrons from lower to higher state energy
• Light Source: Flame
• Method: Indirect Internal Standard Method
• Internal Standard: Lithium/Cesium (corrects variations in flame and
atomizer characteristics)
COMPONENTS OF THE FLAME PHOTOMETER
GASES – using a mixture of hydrogen and oxygen gas,
(acetylene, propane or natural gas)
ATOMIZER OR BURNER – breaks up the solution into finer
droplets so that the atom will absorb heat energy from the
flame and get excited.
COMPONENTS OF THE FLAME PHOTOMETER
INTERFERENCE FILTERS AS MONOCHROMATOR
• Na filter – transmit yellow light (589 nm)
• K filter – transmit violet light (767 nm)
• Lithium – transmit red light (761 nm)

DETECTOR – uses photocell as detector.

 FLUOROMETRY / MOLECULAR LUMINESCENCE
SPECTROPHOTOMETRY
• measure the concentrations of solutions that contain fluorescing
molecules
• measures the amount of light intensity present over a zero background
• Principle: It determines the amount of light emitted by a molecule after
excitation by electromagnetic radiation
• Light Source: Gas discharge lamps (Mercury Arc or Xenon lamp 365 to 366
nm)
• Light Detectors: Photomultiplier tube or phototube
FLUOROMETRY / MOLECULAR LUMINESCENCE
SPECTROPHOTOMETRY
• It uses 2 monochromators (filters, prisms or gratings)
• Primary filter: select the wavelength that is best absorbed by the solution to
be measured
• Secondary Filter: prevents the incident light form striking the photodetector
• About 1000x more sensitive than spectrophotometer- emitted
radiation is measured directly.
• It is affected by quenching- pH and temperature changes, chemical
contaminants, UV light changes
• Uses: measurement of porphyrins, magnesium, calcium and
catecholamines
 CHEMILUMINESCENCE
• Differs from fluorescence and phosphorescence in that the emission of light is
created from a chemical or electrochemical reaction and not from the
absorption of electrochemical energy
• No excitation radiation is required, and no monochromators are needed
• More sensitive than fluorescence
• Principle:
• The chemical reaction yields an electronically excited compound that emits light as it
returns it to its ground state, or that transfers its energy to another compound, which
then produces emission
• Involves the oxidation of an organic compound (dioxetane, luminol,
acridinium ester) by an oxidant (H2O2, hypochlorite or O2). These oxidation
reactions may occur in the presence of a catalyst, such as enzymes, metal ions
and hemin
CHEMILUMINESCENCE
• Use: Immunoassays
• Photodetector: Photomultiplier Tube (Luminator)
• Advantages
• subpicomolar detection limits, speed (with flash-type reactions, light is only
measured for 10 seconds), ease of use (most assays are one-step procedures),
and simple instrumentation
• Disadvantage
• impurities can cause a background signal that degrades the sensitivity and
specificity.
TURBIDIMETRY
• determines concentration of solute by measuring the amount of light
blocked by the analytes
• Light blocked depends on concentration and size of solute
• Uses: for measuring abundant large particles such as protein (CSF and
urine) to detect bacterial growth in broth cultures; measuring
antibiotic sensitivities, detecting clot formation
NEPHELOMETRY
• measures the amount of light scattered by a small particle
• light scattering depends on wavelength and particle size
• detectors placed at various forward angles, as well as at 90° to the
incident light
• for macromolecules with a size close to or larger than the wavelength
of incident light, sensitivity is increased by measuring the forward
light scatter
• more sensitive for protein measurement
• Used for measuring the amount of antigen-antibody complexes
Electrochemistry
• used for the measurement of blood gas, blood pH, electrolytes, glucose,
urea, ionized calcium, lead and chloride
• Principle: It involves the measurement of current or voltage generated by
the activity of specific ions
METHODS
• Potentiomentry
o Ion Selective Electrode
• Coulometry
• Amperometry
o Polarography
• Voltammetry
POTENTIOMETRY
• Measurement of electrical potential due to the activity of free ions
• Measurement of differences in voltage (potential) at a constant
current
• Follows the Nernst Equation
• Reference Electrodes: Saturated Calomel and Silver-Silver Chloride
• Uses: for pH and pCO2 measurements
Ion Selective Electrode (mmol/L)
• Very sensitive and selective for the ion it measures- measures the activity of one ion
much more than the other ions present in the sample
• Interference: excess protein
• Two types of ISE
• Direct ISE: without sample dilution
• Indirect ISE: with sample dilution
• Ion selectivity depends on the membrane/ barrier composition used
• ISE Membrane:
• Glass aluminum silicate or Glass Ion Exchange Membrane (Na)
• Valinomycin gel (K)
• Ion Exchange membrane (Cl)
• Organic liquid membrane ion exchanger (Ca and Li)
• Gas and enzyme electrode
COULOMETRY
• Measurement of the amount of electricity (in coulomb) at a fixed
potential
• An electrochemical titration in which the titrant is electrochemically
generated, and the endpoint is detected by amperometry
• Follows the Faraday’s Law
• Use: Chloride Test (CSF, Serum and sweat)
• Interference: Bromide, cyanide and cysteine
AMPEROMETRY
• The measurement of the current flow produced by oxidation-reaction
• Used: pO2, glucose, chloride and peroxidase determination
• Polarography
• Measurement of differences in current at a constant voltage
• Follows the Ilkovic equation
VOLTAMMETRY
• Measurement of the current after which a potential is applied to an
electrochemical cell
• Allows sample to be preconcentrated, thus utilizing minimal analyte
• Anodic Stripping Voltammetry: Lead and Iron Testing
 ELECTROPHORESIS
• The migration of charged solutes or particles in an electrical field.
• Iontophoresis refers to the migration of small ions, whereas zone
electrophoresis is the migration of charged macromolecules
• Separates proteins on the basis of their electric charge densities
• The acidic and basic amino acids determine the net charge on a protein
• During electrophoresis, proteins are negatively charged (anions) and they
move towards the anode
ELECTROPHORESIS: Factors affecting Rate of
Migration
1. Net electrical charge
2. Size and Shape of the molecule
3. Electric Field Strength
4. Nature of the supporting medium
5. Temperature of operation
 COMPONENTS OF AN ELECTROPHORETIC
SYSTEM
1. Support media
–Paper
–Starch Gel – separates by surface charge and molecular size
–Cellulose acetate – Separates by molecular size
–Agarose gel - neutral; separates by electrical charge; does not bind protein
(lipoprotein)
–Polyacrylamide gel – neutral; separates on the basis of charge and
molecular size; separates proteins into 20 fractions; used to study
isoenzymes
2.Electrophoretic chamber
3.Power supply
 Stains for Visualization of Fraction
A. For serum electrophoresis
• Coomasie Brilliant Blue
• Ponceau S
• Amido Black
B. For lipoproteins
• Oil Red O
• Sudan Black
• Fat Red 7B
C. For glycoproteins
• Periodic Acid Schiff
D. others also use
• Coomassie Blue – CSF proteins
• Gold/Silver Stain – very sensitive
Densitometer
• Detector of electrophoretic machine
• Measures the absorbance of the stain
• Scan and quantify electrophoretic pattern
• Reads gel and cellulose acetate membrane
 THINGS TO CONSIDER DURING
ELECTROPHORESIS
• Electrophoretic Mobility is directly proportional to net charge and inversely
proportional to molecular size and viscosity
• The ionic strength of the buffer determines the amount of current and the movement
of proteins for a fixed voltage
• At pH 8.6, the gamma globulins move toward the cathode, despite the fact that they
are negatively charged (endosmosis)
• If the electrodes are not properly aligned, the current may be denser on one side of
the gel than the other
• If electrophoresis proceeds too long, the proteins may migrate off the gel into the
buffer
• If there is a break in the electric circuit and no current passes, the proteins will not
move
• Frequently, gel shows “smile artifact” in which samples at the center of the gel migrate
further than those at the edges
 MODIFICATONS OF ELECTROPHORESIS
A. ISOELECTRIC FOCUSING
• Separating molecules migrate through pH gradient; uses a constant gradient
• It is ideal for separating proteins of identical sizes but with different net charges
• Proteins move in the electric field until they reach a pH equal to their isoelectric point
• Supporting Media: Agarose gel, Polyacrylamide Gel, Cellulose Acetate
• ADVANTAGES
• The ability to resolve mixtures of proteins
• Detects isoenzymes
• Identify genetic variants
• Detects CSF oligoclonal band
 MODIFICATONS OF ELECTROPHORESIS
B. CAPILLARY ELECTROPHORESIS
• Sample molecules are separated by electro-osmotic flow
• (+) charged ions move faster
• (-) charged ions move slower
• It uses nanoliter quantities of specimen
• Uses:
• Separation, quantitation and determination of MW of proteins
• Analysis of PCR
• Analysis of organic and inorganic substances and drugs
CHROMATOGRAPHY
• refers to the group of techniques used to separate complex mixtures
on the basis of different physical interactions between the individual
compounds and the stationary phase of the system
• The amount of the mixture are separated by continuous
redistribution between 2 phases:
 MODES OF SEPARATION
1. ADSORPTION CHROMATOGRAPHY
• known as liquid–solid chromatography, is based on the competition
between the sample and the mobile phase for adsorptive sites on the solid
stationary phase.
2. PARTITION CHROMATOGRAPHY
• also referred to as liquid– liquid chromatography. Separation of solute is
based on relative solubility in an organic (nonpolar) solvent and an aqueous
(polar) solvent.
 MODES OF SEPARATION
3. STERIC EXCLUSION CHROMATOGRAPHY
• a variation of liquid–solid chromatography, is used to separate solute
molecules on the basis of size and shape.

4. ION EXCHANGE CHROMATOGRAPHY

• solute mixtures are separated by virtue of the magnitude and charge of ionic
species.
5. GEL/ GEL PERMEATION/ GEL FILTRATION/ SIZE EXCLUSION/ MOLECULAR
SIEVE CHROMATOGRAPHY
• Separates molecules based on differences in their size and shape
• As solute travel through the gel, large molecules remain in the mobile phase are
eluted rapidly in the column
• Hydrophilic Gel (Gel Filtration)
• For separation of enzymes, antibodies and proteins
• Hydrophobic Gel (Gel Permeation)
• For separation of triglycerides and fatty acids
 MODES OF SEPARATION
6. AFFINITY CHROMATOGRAPHY
• Uses immobilized biochemical ligands as the stationary phase to separate a
few solutes from other unretained solutes
• Uses the so-called lock and key binding
GAS CHROMATOGRAPHY
• Used for the separation of steroids, barbiturates, blood, alcohol and lipids
• Useful for compounds that are naturally volatile or can be easily converted into a
volatile form
• Specimens are vaporized and swept onto the columns
• Flame ionization is used as detector
• Elution order of volatile is based on their boiling point
• Mobile phase: Nitrogen, Helium. Hydrogen and Argon
Mass Spectrometry (MS)
• Based on fragmentation and ionization of molecules using suitable source of energy
• Substance first be first separated by Gas Chromatography
• Can also detect structural information and determination of MW
• Gas Chromatography with Mass Spectrometry (GC-MS)
• Gold standard for drug testing
• Uses an electron beam to split the drug
• Used for xenobiotics, anabolic steroids, pesticides

• Tandem Mass Spectroscopy (MS/MS)

• Can detect 20 inborn errors of metabolism from a single blood spot
• Liquid Chromatography
• is based on the distribution of solutes between a liquid mobile phase and a
stationary phase
• HPLC is the most widely used liquid chromatography

High Performance Liquid Chromatography

• Uses pressure for fast separations, controlled temperature, in-line
detectors and gradient elution technique
• Uses: Fraction of drugs, hormones, lipids, carbohydrates, and proteins;
separation and quantitation of various hemoglobins associated with
specific diseases, rapid HbA1c