Spectrophotometry

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Spectrophotometry and photometry
Kefaya EL- Sayed Mohamed
Prof. of Clinical Chemistry, Mansoura University
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• 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 .

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• 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.

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• 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.

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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.
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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

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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 ? has energy more than the
  infrared (lt 380 E gt 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.

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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.

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(A) of original incident light transmitted by
equal layers of light-absorbing solution (B)
Tversus concentration on linear graph paper (C)
Tversus concentration on semilog graph paper
(D) A versus concentration on linear graph paper.
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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 )

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Components of spectrophotometer
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(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.

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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 and deuterium lamps (200-400nm)

• Provide continous spectra
• Deuterium

More stable Longer half life than hydrogen lamp
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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.)

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(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 .
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(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

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(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 .

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• 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.

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(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

1. flat surface to light ( no reflection or
   refraction)
2. 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.

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(4) Photodetectors

• To convert the transmitted radiant energy into an
  equivalent amount of electrical energy

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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

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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 .

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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

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5- Galvanometer

1. Direct reading
2. Ampilified reading
3. Digital
4. 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.

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Types of spectrophotometers

1. Single beam sp .
2. Double beam sp

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• 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

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• Double beam in time spectrophotomet if the
  mirror is used after exit

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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 .

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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.

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• 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

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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 .

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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.

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W.L. accuracy
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Stray light
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Stray light
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Stray light
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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.

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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
  .

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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

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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).

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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

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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 )

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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.

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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.

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Flame photometry
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• 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.

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Fluorometry
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• 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.).

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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.

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(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.

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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.

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Checking performance

• A set of fluorescence glass standards.
• Known concentrations of quinine sulphate.

Uses

• Fluoroimmunoassay and immunofluorimetric assay.

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Chemiluminescence or bioluminescence
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• The excitation results from chemical reaction.
• This chemical reaction is oxidation process
  require O2 or H2O2. e.g. Luminol reaction,
  acridinium esters.

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• 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.

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• Adv. Subpicomolar detection lemit.
• Disadv. impurities can cause background signal
  that degrade sensitivity and specificity.

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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.

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Mass spectrometry
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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).

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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.

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Thank you