WEEK 4 Flashcards
two primary considerations in colorimetric analysis
- Quality of the color
- Intensity of the color
4 BASIC DISCIPLINES: ANALYTICAL
CHEMISTRY
- spectrometry
- luminescence
- electroanalytic methods
- chromatography
fluorescence, chemiluminescence, and nephelometry
luminescence
gas, liquid, and thin- layer
chromatography
spectrophotometry, atomic
absorption, and mass spectrometry
spectrometry
electrophoresis, potentiometry, and amperometry
electroanalytic methods
The primary analytical utility of spectrophotometry or filter
photometry is the isolation of discreet portions
of the spectrum for purposes of measurement.
Photoelectric Colorimetry
two types of photoelectric colorimetry
- Spectrophotometric measurement
- Photometric measurement
it is measurement of light intensity in a narrower wavelength.
Spectrophotometric measurement-
is measurement of light intensity at multiple wavelengths
Photometric measurement
MEASUREMENTS OF RADIANT ENERGY
+ Emitted
+ Transmitted
+ Absorbed
+ Scattered
+ Reflected
transmitted via electromagnetic waves that
are characterized by their frequency and Wavelength
Energy
visible spectrum
400-700m -
Ultraviolet region(UV)
<400nm -
Infrared region (IR)
> 700nm
it is the section of the electromagnetic radiation spectrum that is visible to the human eye
The visible light spectrum
the relationship between
wavelength and energy is being described by
Planck’s formula
Planck’s formula is
E = hv
TYPES OF RADIANT ENERGY
+ Cosmic rays
+ Gamma rays
+ X-rays
+ Visible
+ Ultra-violet (UV)
+ Infrared (IR)
+ Radio, TV, microwave, etc
is the number of vibrations of
wave motion per second
Frequency
represents the wavelength in nanometers at peak transmittance.
Nominal wavelength
A slight error in wavelength adjustments can introduce
significant error in absorbance readings.
the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator.
Wavelength accuracy
used to check wavelength accuracy
(wavelength calibration).
Didymium or holmium oxide filter -
It involves measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substances in the solution
Spectrophotometry
ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample.
TRANSMITTANCE
Formula of transmittance
%T = It/Iox100
The amount of light absorbed
Proportional to the inverse log of transmittance
Mathematically derived from %T
ABSORBANCE
States that the concentration of a substance is directly
proportional to the amount of the light absorbed or
inversely proportional to the logarithm of transmitted
light
BEER’S LAW/ BEER-LAMBERT’S-LAW
A=abc
A = absorbance
a = molar absorptivity
b = length of light through the solution
c = concentration of absorbing molecules/solution
It is designed to make one measurement at a time at one specified wavelength.
Single beam spectrophotometer
Is an Instrument that splits the monochromatic light into two components - one beam passes through the sample, and the other through a reference solution or blank
Double-beam spectrophotometer
7 basic components of single or double-
beam configuration spectrophotometer:
- Stable source of radiant energy
- Filter that isolates a specific region of the electromagnetic spectrum
- Cuvets
- Sample holder
- Radiation detector
- Signal processor and
- Readout device
2 Types of Double-beam
Spectrophotometer
- double-beam in space
- double-beam in time
with 2 photodetectors, for the sample
beam and reference beam
double-beam in space
with one photodetector and alternately
passes the monochromatic light through the sample cuvet and
the reference cuvet using a chopper or rotating sector mirror
double-beam in time
emits radiation that changes in
intensity; widely used in the laboratory
Continuum source
Parts of the Spectrophotometer:
- Light/Radiant source
- Entrance Slit
- Monochromator
- Exit Slit
- Cuvet
6, Photodetector - Meter or read-out device
it provides polychromatic light and must generate sufficient
radiant energy or power to measure the analyte of interest.
Light/Radiant source
example of Continuum source
tungsten, deuterium and xenon lamps
the commonly used light source in the
visible and near infrared region
Tungsten light bulbs
produces a continuous source of
radiation, which covers both the UV and the visible range
Xenon discharge lamp
routinely used to provide UV radiation in
analytic spectrometers.
Deuterium lamp
emits limited radiation and wavelength.
Line source
Example of line source
mercury and sodium vapor lamps in
- spectrophotometers
- hollow cathode lamp
also used as light sources for spectrophotometry
Light Amplification by Stimulated Emission of Radiation (LASER)
It minimizes unwanted or stray light and prevents the
entrance of scattered light into the monochromator system
Entrance Slit
refers to any wavelengths outside the band
transmitted by the monochromator; it does not originate from
the polychromatic light source; it causes absorbance error.
Stray light
most common cause of loss of linearity at
high-analyte concentration.
Stray light
Factors for choosing a light source
- Range
- Spectral distribution within the range
- The source of radiant production
- Stability of the radiant energy and
- Temperature
it isolates specific or individual
wavelength of light
Monochromator
Alternatives for tungsten bulb
+ Mercury arc (visible and UV)
+ Deuterium lamp (165nm) - UV
+ Hydrogen lamp - UV
+ Xenon lamp - UV
+ Merst glower -IR
+ Globar (silicone carbide) - IR
Kinds of Monochromators:
a. Prisms
b. Diffraction gratings
c. Filters
These are wedge-shaped pieces of glass, quartz or sodium chloride. Allowing only the desired wavelength
to pass through an exit slit
prism
most commonly used; better resolution than prism. these are made by cutting grooves (parallel grooves) or slits
into an aluminized surface of a flat piece of crown glass
Diffraction gratings
It controls the width of light beam (bandpass) - allows only a
narrow fraction of the spectrum to reach the sample cuvette.
exit slit
the total range of wavelengths transmitted
Bandpass
Accurate absorbance measurement requires a bandpass less than _______ the natural bandpass of the spectrophotometer.
1/2
reflected by the
bandpass, that is, the narrower the bandpass, the greater the
resolution
Spectral purity of the spectrophotometer
These are simple, least expensive, not precise but useful.
filters
It holds the solution whose concentration is to be
measured.
cuvet
Kinds of Cuvets:
- Alumina silica glass
- Quartz/plastic
- Borosilicate glass
- soft glass
most commonly used cuvet (can be used
in 350-2000m)
Alumina silica glass
used for measurement of solution requiring visible and ultraviolet spectra
Quartz/plastic
solutions that do not etch
Borosilicate glass
Silica cuvettes transmit light effectively at wavelengths ______.
220mm
The path length of cuvets is _____
1 cm
To increase sensitivity, some cuvets are designed to have path lengths of
______, increasing the absorbance for a given solution by a factor of 10.
10 cm
It detects and converts transmitted light into
photoelectric energy.
Photodetector
It is the most commonly used detector - measures visible and UV regions. has excellent sensitivity and has a rapid response - detects very low levels of light. It should never be exposed to room light because it will burn out.
Photomultiplier tube (PMT)
contains cathode and anode enclosed in a glass case. It has a photosensitive material that gives off electron
when light energy strikes it. It requires an external voltage for operation
Phototube
not as sensitive as PMT but with excellent linearity. It measures light at a multitude of wavelengths - detects less amount of light. It is most useful as a simultaneous multichannel detector.
Photodiode
It is the simplest detector; least expensive; temperature-sensitive.
Barrier layer cell/Photocell/Photovoltaic cell
Excitation of electrons from lower to higher energy state.
principle of Flame Emission Photometry (FEP)
It displays output of the detection system.
Meter or read-out device
method of Flame Emission Photometry
Indirect internal Standard Method
breaks up the solution into
finer droplets so that the atom will absorb heat energy from the flame and get excited.
ATOMIZER OR BURNER
means the blank contains serum but without the reagent to complete the assay.
Blanking technique
corrects absorbance caused by the color of the reagents - the
absorbance of reagents is automatically subtracted from each of unknown reading.
Reagent 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.
Sample blank
Na filter
transmit yellow light (589 nm)
K filter
transmit violet light (767 nm)
Lithium
transmit red light (761 nm)
referred internal standard; also acts as a radiation buffer
Lithium
For measuring abundant large particles (proteins) and bacterial
suspensions
TURBIDIMETRY
It determines the amount of light blocked (reduction of light) by a particulate matter in a turbid solution.
principle of turbidimetry
Element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited, ground state
principle of Atomic Absorption Spectrophotometry (AAS)
Light source of Atomic Absorption Spectrophotometry
Hollow-cathode lamp
for measuring the amount of antigen-antibody complexes (proteins)
NEPHELOMETRY
it determines the amount of scattered light by a particulate matter suspended in a turbid solution
principle of nephelometry
The unknown sample is made to react with a
known solution in the presence of an indicator
principle of volumetric
Is the migration of charged particles in an electric field. it
separates proteins on the basis of their electric charge and densities.
ELECTROPHORESIS
has a net charge that can be either
positive or negative depending on pH conditions
Amphoteric
Is the movement of
buffer ions and solvent relative to the fixed support
Electroendosmosis/Endosmosis
is the migration of small charged ions
iontophoresis
is the migration of charged
macromolecules
Zone electrophoresis
very sensitive even to nanogram
quantities of proteins
Gold/Silver stain
Factors Affecting Rate of Migration:
- Net electric charge of the molecule
- Size and charge of the molecules
- Electric field strength
- Nature of the supporting medium
- Temperature of operation
it measures the absorbance of stain - concentration of the dye and protein fraction.
Densitometry
Supporting Media:
a. Cellulose acetate
b. Agarose gel
c. Polyacrylamide Gel
separates by electrical charge; it does
not bind protein
Agarose gel
separates by molecular size
Cellulose acetate
separates on the basis of charge and molecular size; separates proteins into 20 fractions; used to study isoenzymes
Polyacrylamide Gel
it is ideal for separating proteins of identical sizes but with
different net charges
Isoelectric Focusing
In this method, sample molecules are separated by electro-osmotic
flow (EOF).
It utilizes nanoliter quantities of specimens.
Capillary Electrophoresis
It involves separation of soluble components in a solution by specific differences in physical-chemical characteristics of the different constituents.
CHROMATOGRAPHY
It is used for fractionation of sugar and amino acid.
Sorbent (stationary phase) - Whatman pap
Paper chromatography
It is a semiquantitative drug screening test.
Thin Layer Chromatography (
it is based on the fragmentation and ionization of molecules using a suitable source of energy.
Mass Spectroscopy (MS)
it is used for separation of steroids, barbiturates, blood, alcohol and
lipids
Gas Chromatography (
Separation occurs based on differences in absorption at
the solid phase surfaces.
Gas Solid Chromatography (
Separation occurs by differences in solute partitioning
between the gaseous mobile phase and the liquid stationary
phase
Gas Liquid Chromatography (
can detect 20 inborn errors of metabolism from a single blood spot.
Tandem mass spectroscopy
it is based on the distribution of solutes between a
liquid mobile phase and a stationary phase.
Liquid Chromatography
The mechanism in this type of chromatography is the
exchange of sample ions and mobile-phase ions with the
charged group of the stationary phase.
Ion Exchange Chromatography
It uses immobilized biochemical ligands as the
stationary phase to separate a few solutes from other unretained solutes.
Affinity Chromatography
it is based on measuring changes in the colligative properties of solutions
that occur owing to variations in particle concentration
principle of OSMOMETRY
The measurement of current or voltage
generated by the activity of a specific ion.
ELECTROCHEMISTRY TECHNIQUES
it is the measurement of the amount of
electricity at a fixed potential.
Coulometry
The measurement of current after which a
potential is applied to an electrochemical
cell.
Voltammetry
It is the measurement of the current flow
produced by an oxidation-reaction
Amperometry
two types of colorimetry method
- visual colorimetry
- photoelectric colorimetry
two types of photoelectric colorimetry
- spectrophotometric measurement
- photometric measurement
absorbance formula
A=2-log%T
A = -log%T
= 1/log%T
principle of diffraction gratings
wavelengths are bent as they pass a sharp corner
cuvet also called as
absorption cell/analytical cell/sample cell
indicates changes in the fuel reading of the instrument
Flickering light
