1

What are the four basic discipline in the field of analy5c chemistry?
1. Spectrometry
Includes: Spectrophotometry, Atomic absorp8on spectrometry, Mass spectrometry
2. Luminescence
Includes: Fluorescence, Chemiluminescence
3. Electroanaly5c method
Includes: Electrophoresis, Poten8ometry, Amperometry
4. Chromatography
Includes: Gas chromatography, liquid chromatography, and thin layer chromatography

Spectrophotometry

Photometric instruments: Measure light intensity without considera8on of wavelength.

Note: Most instruments today used: Filters, Prism, or gra8ngs to select a narrow range of the incident
wavelength.

What happens to the radiant energy that passes through an object?

- It will be par8ally reflected, absorbed, and transmiKed.

Electromagne5c radia5on: Photons of energy travelling in waves.

Planck’s formula: Explains the rela8onship between wavelength and energy.

Note: The frequency of the of a wave is inversely propor8onal to the wavelength, it follows that the energy
of electromagne8c radia8on is inversely propor8onal to the wavelength.

Note: that the instruments men8oned measure either absorp5on or emission of radiant energy to
determine the concentra8on of atoms or molecules.

Absorp5on and emission are two phenomena that are closely related.

o For a ray of electromagne8c radia8on to be absorbed, it must have the same frequency as a rota8onal
or vibra8onal frequency in the atom or molecule that it strikes.
o When energy is absorbed, valence electrons move to an orbital with higher energy level.
o Absorp8on or emission of energy by atoms results in a line spectrum.

Beer’s law
The concentra8on of a substance is directly propor8onal to the amount of light absorbed or inversely
propor8onal to the logarithm of the transmiKed light.

This law describes the rela8onship between absorp8on of light by a solu8on and the concentra8on of that
solu8on.

Percent transmiGance: The ra8o of the radiant energy transmiKed (T) divided by the radiant energy incident
on the sample (I).

Note: If all light is absorbed or blocked, %T is equal to zero (0). A level of 100% T is obtained if no light is
absorbed.
- Equal thickness of an absorbing material will absorb a constant frac8on of the energy incident
upon the layers.
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Absorbance (A) the amount of light absorbed.
Note: absorbance is directly propor8onal to concentra8on

Molar absorp5vity the frac8on of a specific wavelength of light absorbed by a given type of molecule.

Absorp5vity depends on the molecular structure and the way in which absorbing molecules react with
different energies.
Note:
- For any par8cular molecular type, absorp8vity changes as wavelength of radia8on changes.
- The amount of light absorbed at a par8cular wavelength depends on the molecular and ion types
present and may vary with concentra5on, pH, or temperature.
- Absorbance is directly propor8onal to concentra8on, due to the path length and molar
absorp8vity are constant for a given wavelength.

Unknown concentra5ons are determined from a calibra5on curve that plots absorbance at a specific
wavelength versus concentra8on standards of known concentra8on.

Note: not all calibra8on curves result in straight lines. Devia8ons from linearity are typically observed at high
absorbance.

Stray light (within instrument) limits the maximum absorbance that a spectrophotometer can achieve. (2.0
absorbance unit)

Spectrophotometric Instruments
Spectrophotometer is used to measure the light transmiKed by a solu8on to determine the concentra8on
of the light-absorbing substance in the solu8on.

Components of Spectrophotometer
Light Source
Incandescent tungsten or tungsten-iodide lamp – the most common source of light for work in the visible
and near-infrared regions

What is inserted between the lamp and the sample to absorb the infrared radia8on? Heat absorbing filter.

Deuterium discharge lamp and Mercury arc lamp are the lamps used for ultraviolet work.

Deuterium provides con8nuous emission down to 165nm.

Low-pressure mercury lamps emit a sharp line spectrum, with both UV and visible lines.
Medium and High-pressure mercury lamps emit a con8nuum from UV to the mid-visible region.

What are the most important factors for light source?

à Range
à Spectral distribu8on within the range
à The source of radiant produc8on
à Stability of the radiant energy
à Temperature

Monochromators
Isola8on of individual wavelength of light is an important and necessary func8on of a monochromator.
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Degree of wavelength isola5on is a func8on of the type of device used and the width of entrance and exit
slits.
Band-pass of a monochromator defines the range of wavelengths transmiKed and is calculated as width at
half the maximum transmiKance.
Devices used to obtain monochroma8c light:
Colored glass filter the least expensive, simple and inexpensive, but not precise. Usually pass a rela8vely
wide band of radiant energy and have a low transmiKance of selected wavelength.

Interference filters produce monochroma8c light based on the principle of construc8ve interference of
waves. Can be used constructed to pass a very narrow range of wavelengths with good efficiency.

Simple glass prism A narrow beam of light focused on a prism is refracted as it enters the denser glass. Short
wavelengths are refracted more than long wavelengths, resul8ng in dispersion of white light into a
con8nuous spectrum.

Diffrac5on gra5ngs are commonly used as monochromators. It consists of many parallel grooves etched into
a polished surface.

Diffrac5on, the separa8on of light into component wavelengths, is based on the principle that wavelengths
bend as they pass a sharp corner.

Sample cell
Other term is cuveGe, typically has a flat surface.

Square cuveGes have a plane-parallel op8cal surfaces and constant light path.

Quartz cuveGes enable transmission of light and are used when substances absorb in this region (NADH at
340nm)
Note: cuveKes that has scratches in them should be discarded because its op8cal surface scaKer light.

Photodetectors
The purpose of the detector is to convert the transmiKed radiant energy into an equivalent amount of
electrical energy.

Barrier-layer cell, or photocell is the least expensive photodetector.

The photocell is composed of a film of light-sensi8ve material, frequently selenium, on a plate of iron.
Note: Photocell require no external voltage source but rely on internal electron transfer to produce a current
in external circuit.

Photocells are inexpensive and durable; however, it is temperature sensi8ve and nonlinear at very low and
very high levels of illumina8on.

Phototube is similar to a photocell in that it has a photosensi8ve material that gives of electrons when light
energy strikes. But it requires an outside (external) voltage for opera8on unlike a photocell.

Photomul5plier tube detects and amplifies radiant energy.

200 8mes more sensi8ve that the phototube.
Are used in instruments designed to be extremely sensi8ve to very low light levels and light
flashes of very short dura8on.
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Photodiode absorp8on of radiant energy by a reverse-biased PN junc8on produces a photocurrent that is
propor8onal to the incident radiant power.
Not as sensi8ve as PM tubes because of lack of intern amplifica8on, but has excellent linearity, speed, and
small size.

Spectrophotometer Quality Assurance

What are the following checks that should validate the instrument func8on?
à Wavelength accuracy- means that the wavelength indicated on the control dial is the actual
wavelength of light passed by the monochromator.
à Stray light – refers to any wavelength outside the band transmiKed by the monochromator.
Note: Reflec8on of light from scratches on op8cal surface or from dust par8cles
anywhere in the light path and higher order spectra produced by diffrac8on gra8ngs
is the most common cause of stray light.
Stray light can be detected using cutoff filters, which eliminate all radia8on at
wavelengths beyond the one of interest.
à Linearity – is demonstrated when a change in concentra8on results in a straight-line calibra8on
curve.

Atomic Absorp;on Spectrophotometry

Is used to measure concentra8on by detec8ng the absorp8on of electromagne8c radia8on by atoms rather
than by molecules.
Is sensi8ve and precise.
It is rou8nely used to measure concentra8on of trace elements that are not easily excited.
It is accurate, precise, and specific.
Disadvantage: the inability of the flame to dissociate samples into free atoms.

Light source: Hollow-cathode lamp, consists of an evacuated gas8ght chamber containing an anode, a
cylindrical cathode, and an inert gas, such helium or argon.

Note: separate lamps are required for each metal.

Electrodeless discharge lamps are rela8vely new light source for atomic absorp8on spectrophotometers.
Flame serves as the sample cell instead of a cuveKe.
Premix long-path burner the most common burner.

Photomul5plier tube is the usual light detector.

Electrothermal atomiza5on uses a an electrical furnace to break chemical bonds, which is used by flameless
atomic absorp8on.

Zeeman effect the presence of an intense sta8c magne8c field will cause the wavelength of the emiKed
radia8on to split into several components.

ICP (induc5vely coupled plasma) has been used to increase sensi8vity for atomic emission.

Fluorometry

Energy is lost by: Collision, heat loss, transfer to other molecules, and emission of radiant energy.

Stokes shi[ the difference between the maximum wavelengths, excita8on, and emiKed fluorescence.
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Fluorometry Instrumenta;on
Filter fluorometers measure the concentra8ons of solu8ons that contain fluorescing molecules.

In spectrofluorometers, the filters are replaced by a gra8ng monochromator.

Gas discharge lamps are the most frequently used sources of excita8on radiant energy.
Mercury vapor lamps are commonly used in filter fluorometers.
High-pressure xenon lamp what most spectrofluorometers used. This lamp produces a nearly con8nuous
spectrum of wavelengths.

PM tubes are the exclusively used light detector, because of their higher sensi8vity to low light intensi8es.

Note: Fluorescence is directly propor8onal to concentra8on.

Fluorescence Polariza;on
Radiant energy is polarized in a single plane.

Advantages and Disadvantages of Fluorometry

What are the two advantages of fluorometry over conven8onal spectrophotometry?
Specificity and Sensi5vity

Is 1000 8mes more sensi8ve than most spectrophotometric methods.

What are its disadvantages?

Biggest disadvantage is that fluorescence is very sensi8ve to environmental changes.

Quenching is any decrease in fluorescence resul8ng from change in pH, temperature change, Contamina8ng
chemicals or change in solvents.

Chemiluminescence
In chemiluminescence reac8ons, part of the chemical energy generated produces excited intermediates that
decay to a ground state with the emission of photons.

PM tube is used to measure the emiKed radia8on.

No excita8on radia8on is required, and no monochromators are needed is what makes it different from
fluorescence.

Advantages: subpicomolar detec8on limits, speed, ease of use, and simple instrumenta8on.
Disadvantages: Impuri8es can cause a background signal that degrades the sensi8vity and specificity.

Turbidimetry
Turbidimetric measurements are made with a spectrophotometer to determine the concentra8on of
par8culate maKer in a sample.

Nephelometry
Similar to turbidometry, except that light scaKered by the small par8cles is measured at an angle to the beam
incident on the cuveKe, instead of at 180°.
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Laser Applica;ons
(read the book page 102)

Electrochemistry
Is the basis for many types of analyses used in the clinical laboratory.
Includes: Poten5ometry, amperometry, coulometry, and polarography.

Galvanic cell and electroly5c cells are the basic type of electrochemical cells involved in these analyses.
Galvanic and Electroly;c cells
Electrochemical cells consist of two half-cells and a salt bridge, which can be a liquid, or a piece of filter
paper saturated with electrolytes.
Each half-cell contains one electrode, either anode or cathode.

In galvanic cell, as the electrodes are connected, there is spontaneous flow of electrons from the electrode
with the lower electron affinity.

Electroly5c cell, current may be forced to flow through the dead cell only by applying an external
electromo8ve force E.

Note: galvanic cell can be built from an electroly8c cell.

Half-Cells
Note: It is impossible to measure the electrochemical ac8vity of one half-cell; two reac8ons must be coupled
and one reac8on compared with the other.

The electrode defined as 0.00 V is the standard hydrogen electrode: H2 gas at 1 atm.

Hydrogen electrode is used to determine the accuracy of reference.

Indicator electrodes the stability of standard solu8ons, and the poten8al of liquid junc8ons.

Ion-Selec;ve Electrode (ISE)

Are designed to be sensi8ve toward individual ions.

pH Electrodes
Universally used ISE in clinical laboratory.

Components of pH electrodes:
à Indicator electrodes- Silver wire coated with AgCl.
à Reference electrode- Calomel electrode (commonly used reference electrode)
à Liquid Junc5ons –
à Readout meter
à Nernst equa5on describes the electromo8ve force generated because of H+ at the glass
à Calibra5on is a step necessary to standardize a pH
à pH Combina5on Electrode

Gas-sensing electrodes
Similar to pH glass electrode but are designed to detect specific gases in solu8on and are usually separated
from the solu8on by a thin, gas-permeable hydrophobic membrane.
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Enzyme Electrodes
Immobilized enzyme that can catalyze a specific chemical reac8on.

Coulometric Titra;on
A constant current is applied, and the poten8al of a working electrode is monitored.
Is used clinically for sweat chloride determina8on.

Anodic Stripping Voltammetry

The analyte is first concentrated onto the surface of an electrode at a constant poten8al and then goes back
into solu8on as the voltage is changed.
Is used for the analysis of lead in point-of-care and laboratory sehngs.

Electrophoresis
Is the migra8on of charge solutes or par8cles in an electrical field.

Iontophoresis refers to the migra8on of small ions.

Zone electrophoresis is the migra8on of charged macromolecules in porous support medium such as paper,
cellulose acetate, or agarose gel film.

Electrophoretogram is the result of zone electrophoresis and consists of sharply separated zones of
macromolecules.

Macromolecules of interest in clinical laboratory are:

à Proteins in serum
à Urine
à Cerebrospinal fluid
à Other biologic body fluids
à Erythrocytes
à Tissues

Five components of Electrophoresis:

à Driving force (electrical power)
à The support medium
à Buffer
à Sample
à Detec5ng system

Power supply (read book page 8)

Buffer
Two buffer proper8es that affect the charge of ampholytes are pH and ionic strength.

Monovalent ions is what made up for most widely used buffers.

Support materials.
à Cellulose Acetate
à Agarose Gel
à Polyacrylamide Gel
à Starch Gel
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Treatment and applica5on of sample (read book page 9)

Detec;on and Quan;fica;on

Visualiza5on under UV light is the simplest way to accomplish detec8on.

Densitometry is the most common and reliable way to quan8tate the protein bands.

Electroendosmosis
The movement of buffer ions and solvent rela8ve to the fixed support.

Isoelectric Focusing
Is a modifica8on of electrophoresis.
Charged proteins migrate through a support medium that has a con8nuous pH gradient.

Immunofixa;on Electrophoresis
Used in the clinical laboratory to characterize monoclonal proteins in serum, urine, or cerebro spinal fluid.

Capillary Electrophoresis
Separa8on is performed in a narrow-tube, fused silica capillaries.

Has been used for separa8on, quan8ta8on, and determina8on of molecular weights of proteins and
pep8des; for the analysis of polymerase chain reac8on products, and for the analysis of inorganic ions,
organic acids, pharmaceu8cals, op8cal isomers, and drugs of abuse in serum and urine.

Electroosmo5c flow a fundamental CE concept.

EOF is the bulk flow of liquid toward the cathode upon applica8on of an electric field, is superimposed on
electrophore8c migra8on.
It controls the amount of 8me the solutes remain in the capillary.

Two-Dimensional Electrophoresis
This electrophoresis assay combines two different electrophoresis dimensions to separate proteins from
complex matrices such as serum or 8ssue.

Osmometry
Is the principle of measuring the concentra8on of solute par8cles in a solu8on using one of the four
colliga8ve proper8es: Osmo5c pressure, Vapor pressure, Boiling point, and Freezing point.

Osmometer used to perform this measurement.

Measures osmolality indirectly by measuring on of these colliga8ve proper8es, which change
propor8onally with osmo8c pressure.
Expressed in milliosmolal pre kilogram (mOsm/kg) units.

Freezing point osmometry commonly used in the clinical laboratory.

Super cooled solu5on when water cool to -40°C and s8ll have liquid water, provided no crystals or par8culate
maKer is present.

Thermistor is a material that has less resistance when temperature increase.

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Newer Op;cal Techniques
Surface plasmon resonance (SPR) and Biolayer interferometry (bli)

SPR enables the study of binding of ligands to surface receptors such as membrane proteins in real 8me.
Can be used to measure interac8ons between immobilized an8bodies and freely circula8ng
analytes.
Biolayer interferometry is a similar label-free technique that used sensor 8ps instead of a flat metal surface.

Chromatography
Refers to the group of techniques used to separate complex mixtures on the basic of different physical
interac8ons between the individual compounds and the sta8onary phase of the system.

Basic components in chromatographic techniques are:

Mobile phase (gas or liquid), which carries the complex mixture (sample)
Sta5onary phase (solid or liquid), through which the mobile phase flows

Modes of Separa;ons
Adsorp7on
Adsorp5on chromatography also known as liquid-solid chromatography, is based on the compe88on
between the sample and the mobile phase for adsorp8on sites on the solid sta8onary phase.

Not widely used in clinical laboratory because of the technical problem with prepara8on of sta8onary phase
that has homogenous distribu8on of adsorp8on sites.

Molecules that are most soluble in the mobile phase move fastest, the least soluble move slowest. Thus, a
mixture is typically separated into classes according to polar func8onal groups.

Mobile phase can be a single solvent or a mixture of two or more solvents, depending on the analytes to be
desorbed.
Sta5onary phase can be acidic polar, basic polar, or nonpolar.

Par77on
Par55on chromatography also referred to as liquid-liquid chromatography.
Separa8on of solute is based on rela8ve solubility in an organic (nonpolar) solvent and an aqueous (polar)
solvent.

Par55on coefficient the ra8o of the concentra8on of the solute in the two liquids.

Steric Exclusion
A varia8on of liquid-solid chromatography, is used to separate solute molecules on the basis of size and
shape.

Gel filtra5on the use of hydrophilic beads of cross linked dextran, polyacrylamide, or agarose, which formed
a gel when soaked in water.

Gel permea5on chromatography a similar separa8on process as using hydrophobic gel beads of polystyrene
with nonaqueous mobile base.

Ion-Exchange Chromatography
Solute mixtures are separated by virtue of the magnitude and charge of ionic species.
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Is used to remove interfering substances from a solu8on, to concentrate dilute ions solu8ons, and to separate
mixtures of charged molecules, such as amino acids.

Sta5onary phase: Resin, consis8ng of large polymers of subs8tuted benzene, silicates, or cellulose deriva8ve,
with charged func8onal groups.
The resin is insoluble in water, and the func8onal groups are immobilized as side chains on resin beads that
are used to fill the chromatographic column.

Anion-exchange resins are made with exchangeable hydroxyl ions such as the diethylamine func8onal group.
They are used like ca8on-exchange resins, except that hydroxyl ions are exchanged for anions.

Note: Anion and Ca5on resins mixed together are used to deionize water.

Chromatographic Procedures
Thin-layer Chromatography
Is a variant of column chromatography.
Is commonly used as a semiquan8ta8ve screening test.

The solvent migrates up the thin layer by capillary ac5on, dissolving and carrying sample molecules.

Reten5on factor is the distance a component migrates, compared with the distance the solvent front moves.

High-Performance Liquid Chromatography

Uses pressure for fast separa8ons, controlled temperature, inline detectors, and gradient elu8on techniques.

Pumps
Forces the mobile phase through the column at a much greater velocity than that accomplished by gravity
flow columns and includes pneuma8c, syringe, reciproca8ng, or hydraulic amplifier pumps.
Mechanical reciproca5ng pump most widely used pump today, which is used as a mul8head pump with two
or more reciproca8ng pistons.
Pneuma5c pumps are used for preopera8ve purposes.
Hydraulic amplifier pumps are no longer commonly used.

Columns
Silica gel most common material used for column

Reverse-phase HPLC is now popular; the sta8onary phase is nonpolar molecules bonded to silica gel
par8cles.
Mobile phase: Acetonitrile, methanol, water, or any combina8on of solvents.
Reverse-phase column can be used to separate ionic, nonionic, and ionizable samples.

Sample Injectors
Small syringe can be used to introduce the sample into the path of the mobile phase that carries it into the
column.
Loop injector the best and most widely used method. The sample is introduced into a fixed-volume loop.
High reproducibility and are used at high pressures.

Detectors
Modern HPLC detectors monitor the eluate as it leaves the column and, ideally, produce an electronic signal
propor8onal to the concentra8on of each separated component.
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Recorders
Produces a graph, called chromatogram, that shows detector response versus the 8me it takes for the mobile
phase to pass through the instrument, star8ng from the 8me of sample injec8on.

Peak area is propor8onal to the concentra8on of the compounds that produced the peaks.

Isocra5c elu5on is when strength of the mobile phase is constant throughout the separa8on.

Gas Chromatography
Is used to separate mixture of compounds that are vola8le or can be made vola8le.

GC may be gas-solid chromatography, with a solid sta8onary phase, or gas-liquid chromatography, with a
nonvola8le sta8onary phase.

Similar set-up to HPLC but the mobile phase is a gas, and samples are par88oned between a gaseous mobile
phase and a liquid sta8onary phase.

Carrier gas: Nitrogen, Helium, or Argon.

Note: the selec8on of carrier gas is determined by the detector used in the instrument.

The sample. Which is injected through a septum, must be injected as a gas or the temperature of the injec8on
port must be above the boiling point of the component so that they vaporize upon injec8on.

Columns
GLC columns are generally made of glass or stainless steel and are available in a variety of coil configura8ons
and sizes.

Detectors
Most stable detectors in GLC: Thermal conduc5vity and Flame ioniza5on detectors.

Thermal conduc5vity detectors contain wires that change electrical resistance with change in temperature.

Flame ioniza5on detectors are widely used in clinical laboratory. They are more sensi8ve than TC detectors.

Mass Spectrometry
When mass spectrophotometer is used as a detector the defini8ve iden8fica8on of samples elu8ng from GC
and HPLC columns is possible.

Coupled techniques that have powerful analy8c capabili8es with widespread clinical applica8ons.
GC/MS and LC/MS

Four basic components that are standard in all MSs:

à Sample inlet.
à Ioniza5on source
à Mass analyzer
à Ion detector

Note: molecule iden8fica8on is based on the forma8on of characteris8c fragments.

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Sample Introduc;on and Ioniza;on
Direct infusion is commonly used to interface a GC or LC with an MS

Electron Ioniza5on
The most common form of ioniza8on used in GC/MS
It requires a source of electron in the form of a filament to which an electric poten8al is applied, typically at
70 eV.

Atmospheric Pressure Ioniza5on

Note: most LC/MS ioniza8on techniques are conducted at atmospheric pressure.

Three types of ioniza8on for LC/MS discussed here:

à Electrospray ioniza8on
à Atmospheric pressure chemical ioniza8on
à Matrix-assisted lase desorp8on ioniza8on.

Electrospray Ioniza5on
Has wide mass range and high sensi8vity, and can be applied to a wide range of biological macromolecules
in addi8on to small molecules and has become the most common ioniza8on source for LC/MS.

Atmospheric Pressure Chemical Ioniza5on

Similar to ESI in that the liquid from LC is introduced directly into the ioniza8on source.

Matrix-Assisted Laser Desorp5on Ioniza5on

Is used for the analysis of biomolecules such as pep8des and proteins.

Mass Spectrometer Analyzer

Mass analyzers generate electric fields that can manipulate the charged molecules to sort them according
to their m/z.

Quadrupole
The most common mass analyzer in use today. The electric field on the two sets of diagonally opposed rods
allows only ions of s single selected m/z value to pass through the analyzer to the detector.

This technique will generate a full-scan mass spectrum.

Selected ion monitoring is a technique that select a specific mass to monitor a few target analytes. Allows
for longer dwell 8me and therefore higher sensi8vity.
More suitable for target compound analysis.
Full scan provides more informa8on than SIM since ions not specifically selected in SIM are not detected.
Preferable for general unknown screening.

Ion Trap
Can be thought of as a modified quadrupole.

Unique feature: trap and store ions generated over 8me, effec8vely concentra8ng the ions of interest and
yielding a greater sensi8vity.

Tandem Mass Spectrometry

Use for greater selec8vity and lower detec8on limits.
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Collision induced dissocia5on, the ions are accelerated to high kine8c energy and allowed to collide with
neutral gas molecules to fragment the ions.

The single ion that passed through the first analyzer is called the precursor (parent)
The ion formed during fragmenta8on of the precursor ions are called product (daughter) ions.

High-Resolu5on MS
(read the book page 122-124)

Detector
(read the book page 124)

Applica;ons of MS in the Clinical Laboratory

Small Molecule Analysis

(Read the book page 125)

Mass Spectrometry in Proteomics and Pathogen Iden;fica;on

Genomics uses the known sequences of the en8re human genome for determining the role of gene8cs in
certain human diseases.
Proteomics is the inves8ga8on of the protein products encoded by these genes.

Note: A “shotgun” approach is onen used in the discovery of new biochemical markers.

Pathogen Iden5fica5on
MALDI-TOF MS is increasingly being used for the iden8fica8on of pathogens in modern microbiology
laboratories.

Mass Spectrometry at the Point of Care