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Basic disciplines
1. Spectrometry
2. Luminescence
3. Electroanalytic methods
4. Chromatography

Basic disciplines Basic disciplines

3. Electroanalytic methods
1. Spectrometry
i. Electrophoresis
i. Spectrophotometry
ii. Potentiometry
ii. Atomic Emission
iii. Amperometry
iii. Atomic absorption
iv. Mass Spectrometry
2. Luminescence 4. Chromatography
i. Fluorescence i. Gas
ii. Chemiluminescence ii. Liquid
iii. Nephelometry iii.Thin Layer

1 Introduction
 Optical Instruments
 Instruments that measure light energy
 Includes:
i.

ii.
Spectrophotometers
Flame Emission
iii. Atomic Absorption

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Introduction
 Spectrophotometry takes advantage of the property of
colored solutions to absorb light of specific wavelength.
Introduction
 Light is a form of electromagnetic energy
 Transmitted via electromagnetic waves
 Waves is measured in nanometer between the peaks and
valleys (wavelength).

Introduction Introduction
 Beer’s Law
 Instruments that measure light energy
 The concentration of a substance is:
1. Spectrophotometers
 Directly proportional to the amount of light absorbed
2. Flame Emission
 Inversely proportional to the amount of transmitted light
3. Atomic Absorption

A = 2 – Log %T

I. Spectrophotometry I. Spectrophotometry
 Measures the light transmitted by a solution to  Components of a spectrophotometer
determine the concentration of the substance in the
solution

1. Light Source 4. Exit Slit

2. Entrance Slit 5. Sample cell
3. Monochromator 6. Photodetector

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I. Spectrophotometry Factors for a light source

 Components of a spectrophotometer  Range
1. Light Source
 Provide incident light for the system  Spectral distribution within the range
i. Incandescent Tungsten or Tungsten iodide lamp  Source of radiant production
 For visible and near infrared region spectrum
(320 to 700nm)  Stability of the radiant energy
Deuterium-discharge lamp and Mercury arc lamp
ii.
 temperature
 For UV spectrum (below 350nm)
iii. Silicone carbide
 For infrared spectrum

I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer  Components of a spectrophotometer
1. Light Source

1. Light Source 4. Exit Slit

2. Entrance Slit 5. Sample cell
3. Monochromator 6. Photodetector
Tungsten Lamp Deuterium Lamp

I. Spectrophotometry
 Components of a spectrophotometer
3. Monochromator
 Isolates specific wavelength from the light source
i. Interference Filter
 Based on constructive interference of waves
ii. Prism
 Separates white (visible) light into a continuous
spectrum.
iii. Diffraction gratings
 Bends light and form wave fronts. Light that are in
phase reinforce one another, if not it cancels out
 Used for UV, visible or infrared measurements

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I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer  Components of a spectrophotometer
3. Monochromator

1. Light Source 4. Exit Slit

2. Entrance Slit 5. Sample cell
Prisms Gratings 3. Monochromator 6. Photodetector

I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer  Components of a spectrophotometer
5. Sample Cell
 Also known as cuvette or analytical cell
 Holds the solution of which the absorption is to be
measured
i. Glass cuvette
 for visible range
2. Entrance Slit 4. Exit Slit
ii. Quartz or fused silica
 Exclude unwanted  Controls the
or “stray light width of the light  for UV range
beam

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I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer
 Components of a spectrophotometer
5. Photodetector
5. Sample Cell  Converts transmitted radiant energy into an equivalent amount
of electrical energy.
i. Photocell (Barrier layer cell, selenide cell)
 Generates electromotive force (no external
voltage)
 Output is not amplified ( sufficient illumination )
ii. Phototube
 Similar to photocell but requires external voltage
iii. Photomultiplier tube
Fused silica/quart Glass cuvette  Detects and amplifies radiant energy (200x
(UV region) (visible region) sensitive)

I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer  Components of a spectrophotometer
5. Photodetector 5. Photodetector
 Converts transmitted radiant energy into an equivalent amount
of electrical energy.
iv. Phototransistors and Photodiode
 Uses a photosensitive positive-negative junction
diode to produce a photocurrent.

Barrier Layer Cells

I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer  Components of a spectrophotometer
5. Photodetector 5. Photodetector

Photomultiplier tube
Phototube

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Photomultiplier tube I. Spectrophotometry

 Components of a spectrophotometer
6. Read-out Device
 A moving needle on a dial or a digital display which indicates the
amount of light passing through a sample.

I. Spectrophotometry I. Spectrophotometry
 Components of a spectrophotometer

Single- beam spectrophotometer

1. Light Source 4. Exit Slit

2. Entrance Slit 5. Sample cell
3. Monochromator 6. Photodetector

Double-Beam Spectrophotometer

Quality Assurance of Spectrophotometer

Optical Instruments
 Wavelength Accuracy  Instruments that measure light energy
 Didymium or Holmium oxide 1. Spectrophotometers
 Mercury-vapor lamp 2. Flame (Atomic) Emission
 Stray light 3. Atomic Absorption
 Cut-off filters
 NiSO4, NaNO2, Acetone
 Linearity
 Neutral density filters

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II. Flame Emission Spectrophotometry II. Flame Emission Spectrophotometry

 Measures light emitted by excited atoms
 used to measure sodium, potassium and lithium
because they are easy to excite
 Principle:
A. Flame using propane is used to excite the atoms
(higher energy state)
B. Excited atoms return to the ground state by
emitting light energy, characteristic for that atom.

II. Flame Emission Spectrophotometry Optical Instruments

 Components of FES
 Instruments that measure light energy
1. Nebulizer (atomizer) 1. Spectrophotometers
 Deliver a fine spray of sample containing the 2. Flame Emission
metallic ion to the burner.
3. Atomic Absorption
2. Burner
 A fuel gas (propane) with an oxidizing agent
(compressed air) burned to produce the flame.
3. Monochromator system
 Allow only emitted line spectrum of specific
element to strike the PMT.
4. Photosensitive detector (photomultiplier tube)

III. Atomic Absorption Spectrophotometry III. Atomic Absorption Spectrophotometry

 Measures light absorbed by ground state atoms  Components of an AAS
 Used to measure concentration of calcium atom Beam Chopper
Flame
(not easily excited)
 100 times more sensitive than FES

Nebulizer

1. Light Source 4. Burner

2. Beam Chopper 5. Monochromator
3. Nebulizer 6. Photodetector

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III. Atomic Absorption Spectrophotometry

Hollow cathode lamp
 Components of an AAS
1. Light Source
 Provide incident light for the system
i. Hallow cathode lamp
 Consists of an evacuated gas tight chamber
filled with inert gas (helium or argon)
ii. Electrodeless discharge lamp
 Consists of bulb filled with argon and the
element to be tested.
 Radiofrequency generator excites the element

III. Atomic Absorption Spectrophotometry

Single beam AAS
 Components of an AAS
2. Beam Chopper
 Modulates the hollow cathode light beam.
 Produces an alternating signal to be electronically
eliminated.
Beam
Flame
Chopper

Nebulizer

III. Atomic Absorption Spectrophotometry Atomic Emission and Atomic Absorption

 Components of an AAS
3. Nebulizer
 Deliver a fine spray of sample containing the metallic ion
4. Burner
 Long narrow slit (permits absorption of incident radiation)
 A fuel gas (acetylene) with an oxidizing agent (compressed air)
is burned to produce the flame.
5. Monochromator (Gratings)
6. Photodetector (Photomultiplier tube) Measures the intensity Measures the absorption
7. Read-out Device of emitted light when of light of a by atoms in
an activated atom the ground state
returns to the ground
state

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LIGHT EMISSION AND SCATTERING

2 TECHNIQUES
 Instruments that measure light energy
1. Fluorometry
2. Chemiluminescence
3. Turbidity and Nephelometry
4. Laser

I. Flourometry
I. Fluorometry
 Principle:  Components of a Filter Flourometer
 Energy emission that
occurs when certain
compounds absorb
electromagnetic
radiation, becomes
excited gives off light.
 Fluorometer
 measure the
concentrations of solution
that contain fluorescing 1. Light Source 4. Sample Holder
molecules 2. Attenuator 5. Secondary Filter
3. Primary filter 6. Readout device

I. Flourometry I. Flourometry
 Components of Filter Flourometers  Components of Filter Flourometers
1. Light Source (Gas discharge lamps) 4. Sample holder
 Emits short-wavelength high-energy excitation light  The fluorescing sample emits fluorescent light in all
i. Mercury-vapor lamps (filter fluorometers) directions

ii. Xenon-arc lamp (spectrofluorometers) 5. Secondary filter

 Passes longer wavelengths of fluorescent light, preventing
2. Attenuator
incident light from striking the photodetector
 Controls the light intensity
6. Photodetector
3. Primary Filter
 Selects the wavelength that is best absorbed by the sample

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LIGHT EMISSION AND SCATTERING

I. Flourometry TECHNIQUES
 Advantage
 Instruments that measure light energy
 1,000x more sensitive
1. Fluorometry
than most
2. Chemiluminescence
spectrophotometry.
3. Turbidity and Nephelometry
 Disadvantage
4. Laser
 Quenching interference
 reduces intensity of
fluorescence

LIGHT EMISSION AND SCATTERING

II. Chemiluminescence TECHNIQUES
 Principle
 Instruments that measure light energy
 Process of exciting molecules by chemical means and
1. Fluorometry
measuring the light emitted as the molecules return to
2. Chemiluminescence
their stable unexcited state.
3. Turbidity and Nephelometry
 Requires no excitation radiation and Monochromator
 Oxidation reactions of luminol 4. Laser
 Acridinium esters
 dioxetanes
 Advantage
 Sub-Picomolar detection limits
 Speed (10 seconds)
 Simple instrumentation

III. Turbidimetry and Nephelometry III. Turbidimetry and Nephelometry

 Turbidimetry  Nephelometry
 Principle:  Principle:
 Measures the amount of light blocked (absorbance) by  Light scattered by the small particles is measured at an
suspension of particles angle (forward or 90 degrees) to the incident light.
 Dependent on particle size and concentration  Dependent on wavelength and particle size.

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LIGHT EMISSION AND SCATTERING IV. Laser

TECHNIQUES
 Light amplification by stimulated emission of radiation
 Instruments that measure light energy  Based on the interaction of radiant energy and suitably excited
1. Fluorometry atoms or molecules
 Polarized and coherent and has narrow spectral width and
2. Chemiluminescence
small cross-sectional area with low divergence
3. Turbidity and Nephelometry  Coulter counter
4. Laser  Differential of WBC

3 ELECTROPHORESIS
 Definition:
 The process of separating the charged constituents of a
sample by means of an electrical current.
i.

Iontophoresis
Migration of small ions
ii. Zone electrophoresis
 Migration of charged macromolecules in a porous support
(paper, cellulose acetate or agarose gel)

ELECTROPHORESIS ELECTROPHORESIS
 Electrophoretogram
 Components of Electrophoresis
 Result of electrophoresis consisting of separated zones
i. Driving force (electrical power)
of a macromolecule
ii. Support medium
iii. Buffer
iv. Sample
v. Detecting System

Agarose Gel Electrophoresis.

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ELECTROPHORESIS ELECTROPHORESIS
 Components of Electrophoresis  Components of Electrophoresis

ELECTROPHORESIS ELECTROPHORESIS
 Components of Electrophoresis  Components of Electrophoresis

Detecting System (UV transillumination)

ELECTROPHORESIS ELECTROPHORESIS
 Components of Electrophoresis  Components of Electrophoresis

Detecting System (Densitometer) Detecting System

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ELECTROPHORESIS ELECTROPHORESIS
 Electrophoretogram
 Components of Electrophoresis
 Result of electrophoresis consisting of separated strands
i. Driving force (electrical power)
of a macromolecule
ii. Support medium
iii. Buffer
iv. Sample
v. Detecting System

Agarose Gel Electrophoresis.

ELECTROPHORESIS ELECTROPHORESIS
 Charged particles migrate toward the opposite  Power Supply
 Constant current or voltage
charged electrode
 Velocity of migration is controlled by:  Buffers
 If a protein is placed in a
i. net charge of the particle
solution that has a pH
ii. Size and shape of the particle
higher than the pI, the
iii. Strength of the electric fields protein will bear a negative
iv. Chemical and physical properties o the supporting charge
medium  Whereas at a pH less than
v. Electrophoresis temperature the pI, the protein will be
positively charged.

ELECTROPHORESIS ELECTROPHORESIS
 Support materials  Electroendosmosis
i. Cellulose acetate  Movement of buffer and solvent relative to their fixed support.
 Cellulose acetylated with acetic  Isoelectric focusing
anhydride  Charged proteins migrate through a support medium that has
 Separates serum proteins into 5 a continuous pH gradient .
bands
 Capillary electrophoresis
ii. Agarose Gel
 Separation is performed in narrow-bore fuse silica capillaries
 Purified fraction of agar
 10 -15 bands
iii. Polyacrylamide gel
 Separates proteins with more
fraction than cellulose
acetate/agarose (>20 bands)

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Basic disciplines End of Part I

1. Spectrometry
2. Light Emission and Scattering Techniques
3. Electrophoresis
4. Chromatography
5. Electrochemistry
6. Others (Mass spectrometry, Proteomics and
Osmometry)

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