ZARA, MARIO NICO

Analytical techniques / instrumentation

INSTRUMENT
Single-Beam
Spectrophotometry

Double-beam in
space

Double-beam in
time

Flame emission
photometry

Atomic absorption
spectrophotometry
(AAS)

PRINCIPLE

IMPORTANT
COMPONENTS

APPLICATION

Measurement Of The
Amount Of Light
Transmitted By A
Solution To
Determine The
Concentration Of The
Light Absorbing
Substance In The
Solution .

1.Light Source
2.Monochromator/filt
er
3. Cuvette/sample
cell/
Analytical cell
4. Photodetector
5. entrance slit
6. read out
device/display
1. light source
2. mirror
3. entrance slit
4. monochromators
5. exit slit
6. cuvette
7. photodetector
8. Light-emitting diode
(LED)

- Calculate the concetntration

of DNA in a solution
- Determine the rate of an
enzyme-catalyzed reaction
- measure protein
concentration
- follow the growth of bacterial
cells

1. light source
2. mirror
3. sector mirror
4. half silvered mirror
5. photodetctor
6. blocking filter
7. read out device
8. cuvette
9. entrance/exit slits

- calculate the concentration

of DNA in a solution
- determine the rate of an
enzyme-catalyzed reaction
- measure protein
concentration
- follow the growth of bacterial
cells

Measurement of the
amount of light
transmitted by a
solution to determine
the concentration of
the light absorbing
substance in that
solution.
Measurement of the
amount of light
transmitted by a
solution to determine
the concentration of
the light absorbing
substance in that
solution.

- calculate the concentration

of DNA in a solution
- determine the rate of an
enzyme-catalyzed reaction
- measure protein
concentration
- follow the growth of bacterial
cells

Measurement the
1. flame
amount of light
2. internal standard
emitted by ions or
atoms excetid by heat
energy

Sodium
Lithium
Potassium

Measures
concentration by
detecting the
absorption of
electromagnetic
radiation by atoms
rather than molecules

- Calculate the concentration

of DNA in a solution
- determine the rate of an
enzyme-catalyzed reaction
- measure protein
concentration
- follow the growth of bacterial
cells

1. light source (Hallow

cathode lamp)
2. photodetector
3. read-out device
4.chopper
5. monochromator
6. sample

ZARA, MARIO NICO

Analytical techniques / instrumentation

Atomic emission
spectroscopy (AES)

Fluorometer

Chemiluminescent
technology

Nephelometer

Turbidimetry

Uses the intensity of

the light emitted from
a flame, plasma, arc,
or spark at a particular
wavelength to
determine the
quantity of an
element in a sample
Measures the
concentrations of
solutions that
containing fluorescing
molecules

Part of the chemical

energy generated
produces excited
intermediates that
decay to a ground
state with the
emission of photons.
The emitted radiation
is measured with a PM
tube, and the signal is
related to analyte
concentration.
Determine the
concentration of
particulate mtter in a
sample except that
light scattered by the
small particles is
measured at an angle
to the beam incident
on the cuvette
Determine the
concentration of
particulate matter in a
sample and the
amount of light
blocked by a
suspension of
particles depend not
only on concentration
but also on size.

1. flame
2. monochromator
3. photodetector
4. read-out device
5. cuvette

1. light source
2. attenuator
3. primary filter
4. cuvette/sample
holder
5. secondary filter
6. photodetector/
Photomultiplier
7. read-out device
1. luminometer

- calculate the concentration

of DNA in a solution
- determine the rate if an
enzyme-catalyzed reaction
- measure protein
concentration
- follow the growth of
bacterial cells
- fluoroimmunoassays

- immuneassays as antigen
labels

1. light source
2. cuvette
3. detector

- measurement of large
particles ( such as antigenantibody complexes,
prealbumin, and other
serum proteins)

1. light source
2. cuvette
3. detector

- measurement of large
particles ( such as antigenantibody complexes,
prealbumin, and other
serum proteins)

ZARA, MARIO NICO

Analytical techniques / instrumentation

pH meter

Amperometer

Measure the
concentration of
hydrogen ions

Measurement of the
current flow produced
by an oxidationreduction reaction

Gel electrophoresis Separation of

macromolecules
especially proteins
and nucleic acids

Isoelectric focusing

Capillary
electrophoresis

1. glass electrode
2. solution being tested
3. hydrogen ions formed
in the test solution
interact with the outer
surface of the glass
4. hydrogen ions formed
in the potassium
chloride solution
interact with the inside
surface of the glass.
5. meter
6. reference electrode
1. pO2 gas-sensing
Electrode

- measurement of the acidity

and basicity of a solution

1. agarose gel
2. TBE buffer
3. electrophoresis
chamber
4. power supply
5. DNA samples
6. DNA stain

- immunofixation of
immunoglobulins

- phenotyping of 1antitrypsin deficiencies

- determination of genetic
variants of enzymes and
hemoglobins
- detection of proteins in
serum and oligoclonal bands
in CSF
- isoenzyme determinations

Separates proteins on
the basis of pI

Separates
components of a
mixture by using an
electrical field

- measures numerous
analytes in biological fluids

1. power supply
2. sample
3. buffer (+) (-)
4. detector
5. capillary

- separation, quantitation,
and determination of
molecular weights of
proteins and peptides
- analysis of polymerase
chain reaction products
- analysis of inorganic ions,
organic acids,
pharmaceuticals, optical
isomers, and drugs of abuse
in serum and urine

ZARA, MARIO NICO

Analytical techniques / instrumentation

Gas
chromatography

Separate mixtures of
compounds that are
volatile or can be
made volatile

1. gas cylinder
2. NV valve
3. pressure gauge
4. PR1 PR2 regulators

- various organic molecules,

including many drugs
- detection and quantition of
drugs in body fluids

HPLC

Separates compounds
that are dissolved in a
solution

1. pumps
2. columns
3. sample injections
4. detectors
5. recorders

- separation and quantition

of various hemoglobins
associated with specific
diseases
- whole blood measurement
of glycosylated hemoglobin

Ion-exchange
chromatography

Solute mixtures are

separated by virtue of
the magnitude and
charge of ionic species

1. pumps
2. columns
3. sample injectors
4. detectors
5. recorders

- drug detection
- detection and quantition of
drugs in the body fluids

Partition
chromatography

Separation of solute is
based on relative
solubility in an organic
(nonpolar) solvent and
an aqueos (polar)
solvent

1. pumps
2. columns
3. sample injectors
4. detectors
5. recorders

- detection on quantition of
drugs in body fluids

Affinity
Chromatography

Use immobilized
biochemical ligands as
the stationary phase
to separate a few
solutes from another
unretained solutes

1. pumps
2. columns
3. sample injectors
4. detectors
5. recorders

- detection and quantition of

drugs in body fluids

Osmometer

Measurement of the
osmotic strength of a
substance

Freezing pt. osmometer

1. Refrigerant in
2. Refrigerant out
3. Thermistor
4. Sample
5. Stirring wire
6. Read-out device

- measuring the osmotic

strength of a solution,
colloid, or compound
- determination of the
concentration of osmotically
active particles that reduce
the vapor pressure of a
solution