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Prof. of Clinical Chemistry, Mansoura University
Most methods in clinical chemistry are
based on quantitative measurement of a
coloured compound produced when a
sample containing the substance to be
measured is mixed with appropriate
reagents and subjected to certain reaction
conditions.
The radiation most often employed in
photometric analysis has the following wave
lengths .
White light can be dispersed into its constituent
wave lengths by being refracted through a glass
prism or a diffraction grating Anatural dispersion
of light occurs when a rainbow is formed with the
light from the sun being dispersed into its various
colours by the rain drops from acloud.
If a solution absorbs light completely it appears
completely black but if a solution absorbs only
part of the light energy passing through it, it will
appear coloured so a solution of haemoglobin
appears to be red because it absorbs blue green
light and transmitts the complementary colour of
red.
The ability of a substance to absorb
selectively certain wave lengths of light
while transmiting others is determined by
the molecular and atomic structure of the
substance.
The wave – length of choice is generally the
one at which the greatest absorbance
occurs.
Absorptivity constant:
This depends upon:
•
The wave length of the radiation.
•
The nature of the absorbing material.
It is reasonabe that a more concentrated
solution or longer light path should absorb
more light since in either case there are more
light absorbing molecules placed in the path of
light .
A cromophore exhibits the complementary
colour to that which it absorbs i-e-a yellow
component appears yellow because it absorbs
blue light.
Thus it must be estimated in the blue region of the spectrum.
Photometric measurements measure Light
intensity without Consideration to wavelength.
To isolate a narrow range of the incident wave length use:
Filters ( photometer )
prisim or gratings ( spectrophotometer )
Electromagnetic radiation is photons of energy packets
travelling in waves
Electromagnetic radiation includes radian
energy from short wavelength ( x rays , 6
rays )to long wavelength (radio) waves,
Visible ligh falls in between
Light: radiant energy with wavelength visible to the
human eye and with wavelength bordering on those
visible to the human eye ( 380-750nm ).
Energy (E) is
inversely proportional to the
wavelength.
UV rays with short λ has energy more than the
infrared (< 380 E > 750 nm E ).
A wave length of light is defined as the distance
(
) peaks as the light is envisioned to travel in
a wave like manner.
The distanace ( ) peaks in the UV and visible is
measeured in Angstroms (Ao), nanometers (nm)
or millimicrons (mu):
There are 1010 Ao, 109 nm , or 109 mu in 1
meter (SI unit is nm = 10 A = 1 mu)
Radient energy that passes through an object
will be parlially
Reflected
Absorbed
And transmitted
Beers law
The concentration of a substance is directly
proportional to the amount of Light absorbed or
inversely proportional to the Logarithm of the
transmitted Light.
(A) % of original incident light transmitted by equal layers of lightabsorbing solution; (B) % Tversus concentration on linear graph paper
(C) % Tversus concentration on semilog graph paper; (D) A versus
concentration on linear graph paper.
Absorbance (A) = Extinction (E)
Extinction coefficient (EC ): is the extinction
measured with a light path 1 cm long.
specific ( EC ) : is that measured with a light 1
cm long and concentration of 1%
Molecular ( EC ) : is the extinction measured with
a light path 1 cm long and a concentration of a
gram molecule per litre ( molar Absorpitivity )
Components of spectrophotometer
(1) Light source
Range of spectrum
Stability of radiant energy
Temperature
A ) Visible region :
Tungesten lamp and quartz – halogen (320- 1000
nm).
Suitable for moderatly dilute soln = colour
changes
significantly
with
change
in
concentration
Operate for 2000-5000 hr
15% visible + mostly near infrared
Quartz: withstand higher temp.
Aheat absorbed filter,between sample and the
lamp to absorb the infrared is used.
B) U.V :
Low pressure mercury – vapour lamp:
Used at certain wavelength emits a sharp.
Line spectrum with both uv and visible lines medium and high pressure mercury lamp
emits from uv to mid – visible region.
Hydrogen
400nm):
and
deuterium
lamps
Provide continous spectra
Deuterium :
More stable
Longer half life than hydrogen lamp
(200-
C) Laser source
To obtain an intense , narrow – wavelength
ligh source
The technique of light Amplification by
Stimulated Emission of Radiation (LASER )
is tried to be used .
Certain material has the capability of
absorbing energy →
excited state when
change to low energy level (decay) emitte
light (highly quantified light)
Different
materials
to
give
different
wavelengths (e.g argon 488 - 568 nm.)
(2) Monochromators
Isolalation of part of the spectrum (individual
wavelength of light ) depend on:
Monochromator.
Width of entrance and exit slits.
(a) Filters :
The spectral purity of a filter or other monochromat
or is described in term of its spectral bandwidth:
B.W is measured in nm at a point equal to
one half the peak transmitance of the
spectral transmittance curve
The use of high intensity light favors the use
of narrow bandpass interference filters .
(i) Transmission absorption filter :
colored- glass filters
coloured gelatin sandwiched between two glass
plates
inexpensive
simple
not precise
(ii) Interference filters:
used to obtain spectral purity and to eleminate the
harmonic wave lengths
it can be constructed to pass a very narrow range
of wavelength with good efficiency
(b) Prism:
glass prism for visibl.
quartez prism for uv.
short wave refracted more than the long.
(c) Diffraction gratings :
most common
many parrallel grooves on polished surface ( e.g
alloy of alum . copper on flat galass plate )
diffract the light = seprate it into component
wavelengths (wavelengths are bent as they pass
a sharp corner )
because the multiple spectra cause stray Light,
accessory filters are used
provide much narrower wavelength than the
filters.
the spectrum or plate is moved so that only the
specific wave –length band desired pass only
through the slit .
Fiber optics = Light pipes are a bundles
of thin transparent fibers of glass, quartz
or plastic enclosed within material of a
lower refractive index, transmit light
throughout their lengths by internal
reflection.
Adv.: better directional control and
single beam multiplexity .
Disadv: stray light and solarization (loss
of energy and decreased optical
sensitivity.
(3) Cuvets (cell) :
Glass or plastic ( 320 – 1000 nm )
Silica ( quartz ) ( below 320 nm )
May be square or round
The square is better because of :
a) flat surface to light ( no reflection or refraction)
b) easier to line up the same side
The cuvet must be:
Optically clear.
No scratching on the surface to avoid scattering
of light
Clean in soln of: conc HCL: water: ethanol (1 : 3 :
4 ), or distilled water.
Avoid hot acids or alkalies.
Optical bath 1cm – macrocuvet , microcuvet , flow
through.
(4) Photodetectors :
To convert the transmitted radiant energy into an
equivalent amount of electrical energy
a) Photocell or barrier – Layercell :
The least expensive .
Composed of:
•
film of light sensitive material on plate of iron
(selenium )
• thin transparent layer of silver over the light
sensitive material
Light excitation of the electrons on the light
sensitive material which release and flow to the
highly conductive silver where electromotive
force can be measured
used in filter photometers with
a wide band
pass producing high level of illumination so that
there is no need to amplify the signal
- temp sensitive and non linear
b) phototube :
Outside voltage is required for operation
Cathod composed of rubiduim or lithium which
emit electrons when exposed to light
c) Photomultiplier ( PM ) tube:
Detect and ampilify radiant energy
Light stricks coated cathode ,whichs absorbs
light and emit electrons
Attrating to a series of anodes (dynodes ) which
composed of material give off many secondary
electrons(multiple cascade of electrons) current
signal measured in ampers.
200 times more sensitive than the phototube
Extremly sensitive to very low light levels and
short duration light flashes .
d) photodiode :
produces current proportonal to the incident
radient power.
the cell put before the gratting
used where light is adequate
photodiode array (PDA ) detectors are available
in integrated circiuts containing 256 to 2048
photodiodes in a linear arrange ment
Each photodiode responds to a specific
wavelength and so complete uv / visible
spectrum can be obtained in one second
has excellent linearity , speed ,small size
5- Galvanometer :
1.
2.
3.
4.
Direct reading
Ampilified reading
Digital
Microprocessor
Future instruments
1-Thermo – Lens effect
Laser → heating → refractive index change of
the soln This thermolens effect can be
extremely sensitive as an absorption detector
2- Piezelectric detector :
Absorption of energy gas volume changes or
slight temp changes sensed by membrane
current or potential generating device.
Types of spectrophotometers
1. Single beam sp .
2. Double beam sp:
All the compenents are duplicated except the light
source and the meter.
In the single beam instrument any alteration in
wavelength
of
the
beam
will
necessitate
readjustment of the output device to zero absorption
for the blank solution before an absorption on the
sample is possible.
This requirement is no longer present for the double
beam instrument which permits automatic change of
wave – length and continuous display of absorbance .
Compansate for changes in intensity of light source
And also for changes in absorbance of the reagent
blank as the
-Double beam in time spectrophotomet : if the mirror is used after exit
Source of errors in photometic
measurements
( performance characteristics )
Photometric measurements involve finding extinction at a
particular wave length and calculation of concentraton from
this measurement, many procedures involve direct
comparision with standards
In some cases the extenction of unknwn compared directly
with that pridicted for relevant pure substance when the
molecular extinction coefficiert is known as enzyme
determinations linked to NADH or to p nitr ophenol in the
standardization of thyroxine or bilirubin solutions and for the
determination of proteins .
Factors which need consideration are:
Accuray of the wavelength
Accuray of measurements of extinction
The effect of stray light
The linearity of calibration curve
1-Wave length accuracy:
Knowledge of exact wavelength becomes
critical
when
using
published
molar
absorptivities for identification of substances in
toxicological studies and in the use of
differential absorption techniques e.g Enzyme
assay using NAD – N ADH reaction are based on
a molar absorptivity constant for NADH of
6.22x10 at 340 nm.
A narrow bandwidth is needed for light
sensitivity and beers law is more likely to
be obeyed over a wider rang in
monochromatic light.
Avariety of methods is available to check
wavelength & extinction accuracy:
using spectral lamp sources : certain lamp
give eimission at certain wave length
using glass filter with earth element give
band at certain wavelength as holmium
oxide glass which is used for the narrow
spectral bandwidth instruments
holmium oxide glass may be scanned over
the range of 280 to 650 nm
W.L. accuracy
This material shows very sharp absorbance
peaks at well defined wavelengths
A solution of holmium oxide in dilute perchloric
acid may also be used .
W.L. accuracy
For broader bandpass instruments a didymium filter
may be used this filter shows a minimum percent
transmittance at 530 nm against an air blank.
W.L. accuracy
Stray light
Stray light
Stray light
Turbidimety & Nephelometry
Some analytical methods result in the formation of
an insoluble product in finely divided form, so that
the particles remain in suspension .
if abeam of ligh is passed through such a suspension
some of the light is scattered the tyndall effect, this
result in reduction of intensity of the original beam.
the variation in the intensity of the scattered light in
various directions depends on the size and shape of
the scattering particles, the wavelength of the light
and the refractive indices of the solvent and
particles.
Turbidimetry
It involves the measurement of the reduction of the
intensity of the incident beam and so similar to the
study of the absorption of light in colorimeters or
specterephotometers .
Are made with the usual types of photemeter .
The extinction & therefore the sensitivity increases with
decrease in wavelength .
However the selection of the suitable wavelength is
affected by the position of the absorption peaks of other
substances which may be present .
If small stable and reproducible particle size can be
obtained with out sedement, the extinction is
proportional to the concentration of the insoluble
material in some cases over awid range .
Nephlometry
It studies the intensity of the scattered light at
right angls to beam incident to the cuvet similar
to the measurement of the emitted light using a
fluorimeter
Advantages over specterophotometry:
Sensitivity
Wide nange of concentration measurable
Greater precisien
Specific Ag- Ab complexes and a laser saurce
have been combind to provide high spicificty and
high precision
Example for nephlomotric procedurs:
lipoperoteins
proteins by immunologicol methools
amylase (amyloclastic method).
Precautions for turbidimetry and nephilometry:
•
•
Particle size of the standard must be the same as
the substance measured
Avoid setting of particle by adust time for reading
and use gelatin or arabic gum to provide a viscous
meduim retard settling
Atomic absorption spectrophotometry
light source.
hollow –cathod lamp (inert gas ,anode and cylindrical cathod)
when voltage is applied the gas is ionized
ions attracted to the cathode collide with the metal excited
metal ions emite light (this metal the same as that will be
measured (alloye may be used).
burner ( O2, H2, air ):
The sample is aspirated atomized and excited →
to the ground state and emitte light
The emitted light from the light source excite the sample
atoms in the ground state which excited , then emited light
which passed to monochromator to isolate the specific
wavelength measured by PM tube.
To differentiate between light beem
emitted by the hollow-cathod lamp and
that emitted by excited atoms in the flam
use:
A mechanical rotating chopper between light
and the flam or;
by pulsing the electric supply to the lamp.
Used to measure light emitted by excited
atoms e.g., Na+, K+.
The flame is used to break up the chemical
bands to produce atoms.
Then the atoms absorb energy and become
excited.
Return to the ground state and emote light
energy → monochromator → detector.
Fluorescence is a physical energy process
that occurs when certain compounds absorb
electromagnetic radiation, become excited
and then return to their original energy
level. Since the energy given off is less than
or equal to that absorbed, the waver length
of the light being given off will longer (will
be emitted).
The excited state persists for less than 10
nanoseconds (decay time 10-8 sec.).
Components of the fluoromiter:
(1) Energy source:
Mercury are lamps or xenon lamp
Will produce enough energy.
Absorption transion to high energy level
within the molecular.
(2) Inlet and exit slits:
Perpendicular
Prevent the incident high energy from
reaching the detector.
(3) Monochromators:
The first to isolate wavelengths before
excitation of the substance in the cell.
The second will selectively remove
unwanted wavelengths before they fall
upon the detector.
Types:
Filter fluorimeter
Spectrofluorimeter
Precautions (Interference):
PH and temp. control.
Avoid contamination to avoid extrafluorescence or
diminished fluorescence by other substances
(quenching).
The use of high purity solvents (fluorescence grade).
Avoid turbidity and air bubbles, to avoid light
scattering.
Nitric acid is a good cleaning agent for glassware.
Checking performance:
A set of fluorescence glass standards.
Known concentrations of quinine sulphate.
Uses:
Fluoroimmunoassay and immunofluorimetric assay.
Chemiluminescence
or bioluminescence
The excitation results from chemical
reaction.
This chemical reaction is oxidation
process require O2 or H2O2. e.g.;
Luminol reaction, acridinium esters.
In the luminol reaction → dicarboxylate is
excited → emits photoms when return to it's
ground state.
No monochromators are required because
chemiluminescence arise from one species.
Adv.: Subpicomolar detection lemit.
Disadv.:
impurities
can
cause
background
signal
that
degrade
sensitivity and specificity.
Enhanced chemiluminescence technique:
Increase
efficiency
by
enhancer
system in the reaction with an
enzyme.
The time course of the high intensity is
much longer (60 min.) vs 30 sec. in the
conventional.
Mass
spectrometry
It is a technique that can provide information
concerning the elemental composition and
structure of organic compounds:
The arrangement of the functional groups.
The molecular weigh (upto 10.000 a.m.u).
Submicrogm quantities in biological matrix
(compounds, drugs, metabolites).
Principle:
Ionization of the compound.
Filtration through electrostatic or magnetic
field.
Identification according to mass (m) and to
charge (z) ratio (m/z).
Sufficient excess energy can be imported to
the molecular ion to generate many
fragment ions that can be separated,
measured and recorded from the smallest
fragment to the intact ion to produce a mass
spectrum.