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SPECTROPHOTOMETRY

BASIC CONCEPTS, INSTRUMENTATION AND

APPLICATIONBy

Dr. Basil, B – MBBS (Nigeria),Department of Chemical Pathology/Metabolic Medicine,

Benue State University Teaching Hospital, Makurdi.September 2014.

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INTRODUCTION:

• Photometry is the measurement of the amount of luminous light (Luminous Intensity) falling on a surface from a source.

• Spectrophotometry is the measurement of the intensity of light at selected wavelengths.

• The method depends on the light absorbing property of either the substance or a derivative of the substance being analyzed

• Spectrophotometers use prisms and gratings for isolating wavelengths while devices that require filters for this purpose are called Filter Photometers.

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NATURE OF LIGHT:• Electromagnetic waves are characterized by their

frequency and wavelength. • Light is a spectrum of different wavelengths which the

eye recognizes as “white” but can be isolated into discrete portions and measured.

• Human eye responds to radiant energy btw 380 and 750nm, but modern instruments can measure shorter wavelengths (UV) and longer (IR) ones.

• Wavelength describes a position within a spectrum. It is the distance btw 2peaks as the light travels in a wave-like manner

• Light also is composed of discrete energy packs called photons whose energy is inversely proportional to the wavelength

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BASIC CONCEPTS:• When light passes through a solution, a certain fraction is

being absorbed. • This fraction is detected, measured and used to relate the

light absorbed or transmitted to the concentration of the substance.

• This enables both qualitative and quantitative analyses of substances.

• The spectrophotometric technique is used to measure light intensity as a function of wavelength. It does this by:– Diffracting the light beam into a spectrum of wavelengths– Direct it to an object – Receiving the light reflected or returned from the object – Detecting the intensities with a charge-coupled device– Displaying the results as a graph on the detector and then the

display device

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• The light absorption is directly related to the concentration of the compound in the sample.

• As Concentration increases, light Absorption increases linearly and light Transmission decreases, exponentially

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Transmittance and Absorbance:

• When a sample is illuminated, it absorbs some of the light and transmits the rest.

• The transmitted light (Is) is of lower intensity than the incident light (Io), and the transmitted light is defined as:

T = Is / Io

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• To ensure accuracy (by eliminating effects of reflection by surface of the cell, absorption by the cell wall and by solvent) an identical reference cell without the compound of interest is also used.

• Thus, the amount of light absorbed (A) as the incident light passes through the sample is equivalent to:

A = - log Is / IR = - log T• In practice, the Reference cell is inserted and the

instrument adjusted to an arbitrary scale corresponding to 100% transmittance, after which the percentage transmittance reading is made on the sample

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Beer’s Law:• This states that the concentration of a substance if directly

proportional to the amount of light absorbed or inversely proportional to the logarithm of transmitted light.

A = abcWhere:A = Absorbancea = proportionality constant defined as absorptivityb = light path in centimetersc = concentration in g/L of the absorbing compound

NB: - Absorbance (A) has no units, so the unit for a = reciprocals those for b and c.• When b is 1cm and c is expressed in mol/L, ἐ (epsilon) is

substituted for the constant, a.• The value of ἐ is a constant for a given compound at a given

wavelength under prescribed conditions of solvent, temperature, pH, etc., and is called the Molar absorptivity (ἐ).

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Spectrometry Nomenclature:

Application of Beer’s Law:

• In practice, a direct proportionality between absorbance and concentration must be established for a given instrument under specific conditions.

• A linear relationship exists up to a certain concentration or absorbance beyond which the solution is said to no longer obey Beer’s Law

• Within this limitation, a calibration constant (K) may be derived and used to calculate the concentration of unknown solutions by comparison

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Recall, a = A/bcThus, A1 / b1c1 = A2 / b2c2

Where 1 and 2 refers to the calibrating (c) and unknown (u) solutions respectively

• But because the Light path b remains constant, b1 = b2

• Then, A1/c1 = A2 /c2 or Ac/cc = Au/cu

• Hence, concentration of the unknown: cu = Au/Ac x cc

And, cu = Au x cc / Ac = Au x K

Where, K = cc / Ac

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• The constant cannot be used once Beer’s Law have been violated

• A non-linear calibration curve can be used if a sufficient number if calibrators of varying concentration is included to cover the entire range encountered for reading on the unknowns

• Published constants should be used only if the method is followed in detail and readings are made on a spectrophotometer capable of providing light of high spectral purity at a verified wavelength.

• Use of broader band light leads to some decrease in absorbance

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• Beer’s Law is followed only if the following conditions are met:– Incident radiation on the substance of interest is

monochromatic– Solvent absorption is insignificant compared to the

solute absorbance– Solute concentration is within given limits– Optical interferant is not present– Chemical reaction does not occur between the

molecule of interest and another solute or solvent molecule.

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INSTRUMENTATION: The Spectrophotometer

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• The basic components of a spectrophotometer include: a light source, a means to isolate light of desired wavelength, fiber optics, cuvets, a photodetector, a readout device, a recorder and a computer.

• Three different types of the device available are:

The Single Beam Spectrophotometer:

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Double-beam-in-space Spectrophotometer:

Double-beam-in-time Spectrophotometer:

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Light Sources:• This provides a sufficient amount of light which is

suitable for making a measurement. • The light source typically yields a high output of

polychromatic light over a wide range of the spectrum.

Electromagnetic spectrum:

• Types of light sources used in spectrophotometers include: Incandescent lamps and lasers.

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Incandescent Lamps:• Tungsten Filament Lamp: The most common source of visible and near

infrared radiation ( at wavelength 320 to 2500 nm)• Deuterium lamp: Continuous spectrum in the ultraviolet region is produced

by electrical excitation of deuterium at low pressure. (160nm- 375nm)

• Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from 200 to 800 nm): high-pressure mecury and xenon arc lamps are commonly used in UV absorption measurements as well as visible light.

• Globar (silicon carbide rod): Infra-Red Radiation at wavelengths: 1200 -40000 nm

• NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm): for IR Region

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Laser Sources:• These devices transform light of various frequencies

into an extremely intense, focused, and nearly non-divergent beam of monochromatic light

• Through selection of different materials, different wavelengths of light emitted by the laser are obtained.

• Used when high intensity line source is required• Unique properties of laser sources include:– Spatial coherence: a property that allows beam diameters in

the range of several microns– Production of monochromatic light– Have pulse widths that vary from microseconds to (flash

lamp-pulsed lasers) to nanoseconds (nitrogen lasers), to picoseconds or less (mode-locked lasers)

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Spectral Isolation:• A system for isolating radiant energy of a desired

wavelength and excluding that of other wavelength is called a Monochromator.

• Monochromator consists of these parts:– Entrance slit– Collimating lens or mirror– Dispersion element: A special plate with hundreds of parallel

grooved lines. The grooved lines act to separate the white light into the visible light spectrum

– .

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• Focusing lens or mirror: Combinations of lenses, slits, and mirrors which relays and focuses light through the instrument

• Exit slit

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• Devices used for spectral isolation include: Filters, Prisms, and Diffraction gratings.

• Variable slits are also used to permit adjustments in total radiant energy reaching the photocell

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• The spectral purity of a monochromator is usually described as its spectral bandwidth – width, in nm, of the spectral transmittance curve at a point equal to half the peak transmittance

• Filters:– Simplest type is a thin layer of coloured glass which is not a true

monochromator because it transmits light over a relatively wide range of wavelength

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– Commonly used glass filters have spectral bandwidth of about 50nm and are refered to as wide-bandpass filters

– a cut-off filter shows a sharp rise in transmittance over a narrow portion of the spectrum and is used to eliminate light below a given wavelength

– Narrow-bandpass filter is constructed by combining two filters like shown above, however, the availability of high intensity light sources now favours the use of narrow bandpass interference filters.

– Alternatively, narrow bandpass filter can be constructed by use of dielectric material of controlled thickness sandwiched btw two silvered pieces glass

• Prisms and Gratings:– Prisms seperates white light into a continous spectrum by

refraction with shorter wavelengths being bent or refracted more than longer ones

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– Diffraction gratings are prepared by depositing a thin layer of aluminuin-copper alloy on the surface of a flat glass plate, then ruling many small parallel grooves into the metal coating

Selection of a Monochromator:– Narrow spectral bandwidth – for resolving and

identifying sharp absorption peaks that are closely adjacent.

– Lack of agreement with beer’s law will occur when a part of the spectral energy transmitted by the monochromator is not absorbed by the analyte as commonly observed with wide-bandpass instruments

– Increase in absorbance and improved linearity with concentration is usually observed with instruments that operate at narrower bandwidths of light

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Cuvets:

• This is a small vessel used to hold a liquid sample to be analyzed in the light path of a spectrophotometer.– May be round, square or rectangular and are constructed

from glass, silica (optical grade quartz) or plastic.– It should be without impunities that may affect

spectrophotometric readings– Reference solution must be transparent to the radiation

which will pass through them. – Quartz or fused crystalline silica cuvettes for UV

spectroscopy. – Glass cuvettes for Visible Spectrophotometer.– NaCl and KBr Crystals for IR wavelengths.

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Photodetectors:• These are devices that convert light into an

electric signal that is proportional to the number of photons striking its photosensitive surface.

• The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking them

• The Photomultiplier tube (PMT) is a commonly used photodetector for measuring light intensity in the UV and Visible region of the spectrum. They are extremely rapid, very sensitive and slow to fatigue.

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• The PMT consists of:– A photoemissive cathode (a cathode which emits

electrons when struck by photons)– Several dynodes (which emit several electrons for

each electron striking them) – An anode – Produces an electric signal proportional

to the radiation intensity– Signal is amplified and made available for direct

display– A sensitivity control amplifies the signal– Examples: Phototube (UV); Photomultiplier tube

(UV-Vis); Thermocouple (IR); Thermister (IR)

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• Other photodetectors include: Barrier layer cells (photovoltaic cells), Photodiodes,

• Photodiodes are made of photosensitive semi-conductor materials like silicon, gallium, arsenide etc which absorb light over a characteristic wavelength range e.g 250nm to 1100nm for silicon. They are capable of measuring light at a multitude of wavelengths.

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Display or Readout Devices:

• Electrical energy from the detector is displayed on a meter or readout system such as an analog meter (obsolete), a light beam reflected on a scale, or a digital display, or LCD

• Digital readout devices operate on the principle of selective illumination of portions of a blank of light emitting diodes (LEDs), controlled by the voltage signal generated.

• Compared to meters, digital read out devices have faster response and are easier to read

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Computers:• Using the computer; – output from calibrator is digitally stored– digital signals from blanks are subtracted from

calibrators and unknowns, and– the concentration of unknowns is automatically

calculated• Data from multiple calibrators often are used to:– store a complete calibration curve– display or print out the curve for visible inspection– calculate result of unknowns based on the curves or

some mathematical transformation of the data• Computers are also used to convert kinetic data

into concentration or enzyme activity.

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Recorders:• Spectrophotometers may be equipped with recorders

in addition to or instead of a digital display• They are synchronized to provide line traces of

transmittance or absorbance as a function of either time or wavelength.

• When a continuous tracing of absorbance versus wavelength is recorded, the resultant figure is called an absorption spectrum. If a substance absorbs light, distinct peaks of absorbance will be observed.

• Measuring the absorption spectra of an unknown sample and comparing them with spectra from known compounds is very useful for qualitative purposes.

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PERFORMANCE PARAMETERS:

Parameters tested to verify that the spectrophotometer is performing satisfactorily include:• Wavelength Accuracy:– Holmium oxide in dilute perchloric acid is used for

calibration of Narrow-spectral-bandwidth instruments– sharp absorbance peaks are obtained at defined

wavelengths which can be compared with established values.

– Didymuin filter is used in broader-bandpass instruments• Spectral Bandwidth: • Width of and emission band at half-peak height– Maybe calculated from the manufacturer’s specifications– Interference filters maybe used to check the instrument

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• Stray Light: • radiation of wavelengths outside the narrow band nominally

transmitted by the monochromator that hits the detector– Defined as a ratio or percent of the total detected light– Causes an absorbance error and can be minimized by an inbuilt

stray light filter– Cutoff filters are used for the detection of stray light which they are

capable of absorbing with 0% transmission. In the UV range, liquid cutoff filters are very effective

– Emanates from scattering and diffraction inside the monochromator, light leaks, and fluorescence of the sample

• Photometric Accuracy:• Neutral density filters are used to check an instrument’s

photometric accuracy.– Solutions of potassium dichromate may be used for overall checks

on photometric accuracy which is compared with literature values.

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APPLICATIONS:1. Measurement of Concentration:– Prepare samples– Make series of standard solutions of known concentrations – Set spectrophotometer to the λ of maximum light

absorption– Measure the absorption of the unknown, and from the

standard plot, read the related concentration

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2. Detection of impurities:– UV absorption spectroscopy is one of the

best methods for determination of impurities in organic molecules

– Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material

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3. Elucidation of the structure of Organic Compounds:• From the location of peaks and combination of

peaks UV spectroscopy elucidate structure of organic molecules:– the presence or absence of unsaturation, – the presence of hetero atoms

4. Chemical Kinetics:– Kinetics of reaction can also be studied using

UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed

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5. Detection of Functional Groups:– Absence of a band at particular wavelength regarded as an

evidence for absence of particular group

6. Molecular weight determination:– Molecular weights of compounds can be measured

spectrophotometrically by preparing the suitable derivatives of these compounds.

– For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate

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REFLECTANCE PHOTOMETRY• In Reflectance photometry, diffused light illuminates a reaction

mixture in a carrier and the reflected light is measured. – Alternatively, the carrier is illuminated and the reaction

mixture generates a diffuse reflected light which is measured.• The intensity of the reflected light from the reagent carrier is

compared with the intensity of light reflected from a reference surface.

• The intensity of reflected light is non-linear in relation to the concentration of analyte, – The data is first converted to linear format using the Kubelka-

Munk equation or the Clapper-Williams transformation.• Reflectance photometry is used as the measurement method with

dry-film chemistry systems.• The Electro-optical components used in reflectance photometry

are essentially the same as that used in absorbance photometry.

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FLAME EMISSION SPECTROPHOTOMETRY• This is based on the characteristic emission of light by atoms of

many metallic elements when given sufficient energy, such as that supplied by a hot flame

• The wavelength to be used in the measurement of an element depends on the selection of a line of sufficient intensity to provide adequate sensitivity and freedom from other interfering lines at or near the selected wavelength (e.g lithium – red, sodium – yellow, potassium – violet, rubidium – red, magnesium – blue)

• These colours are characteristic of the metal of the metal atoms present as cations in solution.

• Under constant and controlled conditions, the intensity of the characteristic wavelength produced by each of the atoms is directly proportional to the number of atoms that are emitting energy, which in turn is directly proportional to the concentration of the substance of interest in the sample.

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REFERENCES:• Tietz Textbook of Clinical Chemistry• The principles of use of a spectrophotometer

and its application in the measurement of dental shades[2003]

• Fundamentals of UV-visible spectroscopy, Tony Owen, 1996

• Spectrophotometry FUNDAMENTALS (Chapters 17, 19, 20), Dr. G. Van Biesen, Win2011

• http://www.bio.davidson.edu

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