DETECTORS

There are 3 common types of detectors which are widely used in UV Spectrophotometers. These are as
follows:-
(i) Barrier layer cell
 Simplest detectors
 This cell is also known as photovoltaic cell.
 Gives a current, which under suitable conditions, is directly proportional to the light intensity.
 The barrier cell consists of a semiconductor, such as selenium, which is deposited on a strong metal
base, such as iron or copper; this acts as one electrode. Then a very thin layer of silver or gold is
sputtered over the surface of the semiconductor to act as a second collector electrode.
 The radiation falling on the surface produces electrons at the selenium silver interface.
 A barrier exists between the selenium and iron which prevents the electrons from flowing into iron.
 The electrons are therefore accumulated on the silver surface.
 The accumulation of electrons on the silver surface produces an electrical voltage difference between
the silver surface and the base of cell. If the external circuit has a low resistance , a photocurrent will
flow which is directly proportional to the intensity of incident radiation beam.
 The sensitivity of a photovoltaic cell is only moderate and it is generally used for instruments like
photometers which allow a wide band of radiations to strike the detector.
 Photovoltaic cell is simple in design. It does not require any external power supply. However, it can be
booked directly to a micrometer or galvanometer to read its output.
 The response time on a photovoltaic cell is only fair, and thus, it cannot cause the reduction of noise.
With the pace of time, a photovoltaic cell becomes useless because of transformations of the selenium
layer.

 Consists of a metallic base plate made of iron or copper;

this acts as one electrode.
 Over this is deposited a layer of selenium or sometimes cuprous oxide.
 Over the selenium is deposited an extremely thin transparent layer of a good conducting metal like
silver, platinum or copper or gold; this acts as other electrode.
 Selenium or other oxides or sulphides have extremely small electricity conductivity. But light of suitable
frequency imparts sufficient energy to the electrons so that they leave selenium and enter transparent
metal layer.
 This produces a current proportional to the intensity of light.
 Linearity depends on resistance used in the circuit and also wavelength.
 Useful working range is 380-780 nm.

 The detector has a thin film metallic layer coated with silver or gold and act as another electrode.
 It also has a metal base plate which acts as another electrode.
 These 2 layers are separated by a semiconductor layer of selenium.
 This creates a potential difference between two electrodes and causes the flow of current.
 When it is connected to galvanometer, a flow of current observed which is proportional to the intensity
and wavelength of light falling on it.
 When light radiation falls on selenium layer, electrons become mobile and are taken up by transparent
metal layer.

Advantage
Requires no power supply.

Disadvantage
1. Lack of sensitivity
2. restricted to cheapest colorimeters and flame photometers.

(ii) Phototubes or Photocell

 Also called as Photo emissive tube or photoelectric cell.
 It consists of a high-sensitive cathode in the form of a half-cylinder of metal
which is contained in an evacuated tube.
 The anode is also present in the tube which is fixed more or less along the
axis of the tube.
 The inside surface of the photocell is coated with a light sensitive layer.
 When the light is incident upon a photocell, the surface coating emits
electrons.
 These are attracted and collected by an anode.
 The current, which is created between the cathode and anode, is regarded as
a measure of radiation falling on the detector.
 A photocell is more sensitive than photovoltaic cell because high degree of
amplification can be used.
 If quartz or fused silica windows are used, the range of the photocells can be
increased through the near-ultraviolet and the far-ultraviolet region.
 Consists of an evacuated glass tube with a photocathode and collector anode.
 The surface of photocathode is coated with a layer of elements like cesium, silver oxide or mixture of
them.
 When radiant energy falls on photosensitive cathode, electrons are attracted to anode causing current
to flow.
 More sensitive compared to barrier layer cell and therefore widely used.
 Light falls on metal surface from which electrons are easily liberated, this is enclosed in an evacuated
envelope and kept at negative voltage. Then a current which is proportional to the incident light is
produced.
 Elements of high atomic volume like potassium and cesium are used.
 And these are usually coated with composite coatings such as cesium/ cesium oxide / silver oxide to
improve sensitivity.
 This is enclosed in high vacuum and forms cathode.
 Application of sufficiently high potential between cathode and anode makes the liberated electrons
reach the anode.
 A current is obtained, which is proportional to intensity of light.
 By using different metals, the cells can be made to respond to different regions of the spectrum.
 In spectrophotometers two cells are usually used: one responsive to wavelengths upto 620 nm and
other to wavelengths form 620-1000 nm.
 Modification: Inclusion of an inert gas at low pressure to give gas-filled cells.
 Liberated electrons cause ionization of gas and consequently photocurrent increases.
 But linearity is not good and hence these cannot be used where accuracy required.

(iii) Photomultiplier tube

 A photomultiplier tube is generally used as a detector in UV Spectrophotometers.
 A photomultiplier tube is a combination of a photodiode and an electron-multiplying amplifier.
 A photomultiplier tube consists of an evacuated tube which contains one photo-cathode and 9-16
electrodes, known as dynodes.
 The surface of each dynode is of Be–Cu, Cs–Sb or similar material.
 When radiation falls on a metal surface of the photocathode, it emits electrons.
 The electrons are attracted towards the first dynode which is kept at a positive voltage.
 When the electrons strike the first dynode, more electrons are emitted by the surface of dynode; these
emitted electrons are then attracted by a second dynode where similar type of electron emission takes
place.
 The process is separated over all the dynodes present in the photomultiplier tube until a shower of
electrons reaches the collector.
 The number of electrons reaching the collector is a
measure of the intensity of light falling on the detector.
 The dynodes are operated at an optimum voltage that
gives a steady signal.
 The photomultiplier tube is extremely sensitive as well
as extremely fast in response.
 The transit time between absorption of the photon and the arrival of the shower of electrons is
typically in the range of 10-100 μsec. For every quantum of light, approximately 106 electrons are
produced.
 Gives greater sensitivity to very weak light intensities.
 Employs multiplication of initial photoelectrons by secondary emission.
 Several anodes at a gradually increasing potential are placed in the bulb.
 Electrons from the photocathode are attracted to anode 1 where they liberate more electrons which
travel to anode 2, because anode 2 is at higher potential as compared to anode 1.
 This continues till last anode.
 The extra anodes are called as dynodes.
 Result is final photocurrent 106 – 108 times greater than primary current.
 This ‘multiplied’ current is still linear, proportional to intensity of light.

Advantages:
 Ideal for measuring weak light intensities e.g., fluorescence.
 Emission spectra is of trace elements for their determination

PHOTOMULTIPLIER TUBES
 The principle employed in this detector is that, multiplication of photoelectrons by secondary emission
of electrons.
 In a vacuum tube a primary photocathode is fixed which receives radiation from the sample.
 Some 8 to 10 dynodes are fixed each with increasing potential of 75 – 100 V higher than preceding one.
 Photomultiplier is extremely sensitive to light and is best suited where weaker or low radiation is
received.
 Near the last dynode is fixed an anode or electron collector electrode.

SILICON PHOTODIODES
 A silicon photodiode utilizes the internal photoelectric effect, the phenomenon whereby the electrical
properties of the detector itself change when light strikes it.
 As the name suggests, a silicon photodiode is a semiconductor.
 When light strikes this semiconductor, if the energy of the light is larger than the band gap, electrons in
the valence band are excited into the conduction band and holes are left in the original valence band.
 As a result, electrons accumulate and the two regions become, respectively, negatively and positively
charged.
 If this is connected to a circuit, current flows.

 Silicon photodiodes have some advantages over photomultiplier tubes:

(i) They are less expensive
(ii) There is little unevenness of sensitivity over their light-receiving surfaces
(iii) They do not require a dedicated power supply.
(iv) Even with respect to sensitivity, if the light intensity is relatively high, they can provide photometric
data that is by no means inferior to that obtained with photomultipliers.
If the light intensity is relatively low, however, because signals are amplified in the electronic circuit that
gives a current, increasing the amplification factor decreases the response speed.

DIODE
 The diode is changed to a pre-set potential by a continuously scanning electron beam.
 If radiation falls on the diode, the charge is depleted in proportion to the intensity of the radiation.
 When the electron beam scans again, it recharges the diode back to the pre-set voltage.
 This requires the flow of a current in proportion to the light intensity.
SINGLE-CHANNEL DETECTORS
 Phototubes (vacuum), photomultipliers and photodiodes are all single-channel detectors.
 They monitor the total intensity but cannot distinguish between different wavelengths.
 Spectrum may be obtained by varying the monochromatic light passing through the sample.

Photodiode array detectors:

 Consist of many.. 256, 512, 1024 diodes (array)
 Multi-channel detection
When used in combination with a dispersing system (e.g., a grating), each diode can monitor light
intensity at a different wavelength.
The array gives an almost instantaneous spectrum.
Useful in HPLC and fast reaction kinetics

Dark Current
 Seen in phototubes and photomultiplier tubes.
 In these detectors the current never falls to zero.
 A small current is produced due to spontaneous discharge at the high voltage of the dynodes or due to
thermal emission from the cathode.
 This small residual current is called as the dark current.
 Compensation has to be made by the instrument for this dark current.

RECORDING SYSTEM
 The signal from the photomultiplier tube is finally received by the recording system.
 The recording is done by recorder pen.
 The type of arrangement is only done in recording UV spectrophotometers.

POWER SUPPLY
The power supply serves a triple function:
(i) It decreases the line voltage to the instruments operating level with a transformer.
(ii) It converts A.C. to D.C. with a rectifier if direct current is required by the instrument.
(iii) It smooths out any ripple which may occur in the line voltage in order to deliver a constant voltage to
the source lamp and instruments.

SPECTROPHOTOMETERS INSTRUMENTS DESIGNS

SINGLE – BEAM SPECTROPHOTOMETERS
 A beam of radiation pass through a single cell, the reference cell is used to set the absorbance scale at
zero for the wavelength to be studied.
 It is then replaced by sample cell to determine the absorbance of the sample at that wavelength.
 This was earliest design and is still use in both teaching and industrial labs.
 It requires a stabilized voltage supply to avoid errors resulting from changes in the beam intensity.

 The light travels in a single continuous optical path between the light source and the detector.
 In manually controlled instruments, the monochromator is adjusted to required wavelength and the

lamp and photocell selected by means of levers or switches.

 In the single-beam system, UV radiation is given off by the source.

 A convex lens gathers the beam of radiation and focuses it on the inlet slit.
 The inlet slit permits light from the source to pass, but blocks out stray radiation.
 The light then reaches the monochromator, which splits it up according to wavelength.
 The exit slit is positioned to allow light of the required wavelength to pass through.
 Radiation at all other wavelengths is blocked out.
 The selected radiation passes through the sample cell to the detector, which measures the intensity of
the radiation reaching it.
 By comparing the intensity of radiation before end after it passes through the sample, it is possible to
measure how much radiation is absorbed by the sample at the particular wavelength used. The output
of the detector is usually recorded on graph paper.
 One problem with the single-beam system is that it measures the total amount of light reaching the
detector, rather than the percentage absorbed.
 Light may be lost at reflecting surfaces or may be absorbed by the solvent used to dissolve the sample.
 Furthermore, the source intensity may vary with changes in line voltage.
 For example, when the line voltage decreases, the intensity of the light coming from the source may
decrease unless special precautions are taken.
 Consequently, the intensity of radiation may be constantly changing.
 Another problem is that the response of the detector varies significantly with the wavelength of the
radiation falling on it.
 Even if the light intensity is constant at all wavelengths, if the wavelength is steadily increased from 200
to 750 nm, the signal from the detector starts at a low value, increases to a value that is steady over a
wide range, and then decreases once more.
 This relationship between the signal from the detector and the wavelength of radiation is called the
response curve.
 It indicates the wide variation in signal that than be expected from the detector even though the light
intensity falling on it is constant.
 Different detectors respond differently at different wavelengths.
 The detector selected must operate over the desired range of the experiment. The problem of
instrument variation can be largely overcome by using the double-beam system.

PROCEDURE
1. Close the shutter,, adjust detector to ‘zero’. This set the scale to 0% T or infinite absorbance.
2. Open the shutter and place cuvette containing only the solvent
Set the scale to 100% T, or zero absorbance
Switch labelled ‘zero absorbance’
3. Place cuvette containing sample, and measure intensity IT, or absorbance, on the scale.

ADVANTAGES:
 Relatively inexpensive
 Useful when absorbance of many samples is being measured at same wavelength.

DISADVANTAGE
 Have to reset the 100% T at each wavelength since intensity of light from lamp varies with wavelength.

DOUBLE-BEAM SPECTROPHOTOMETERS

 The description
of a double-
beam ultraviolet
spectrophotometer is as follows:
(i) The radiation from the source is allowed to pass via a mirror system to the monochromator unit.
The function of the monochromator is to allow a narrow range of wavelengths to pass through an exit
slit.
(ii) The radiation coming out of the monochromator through the exit slit is received by the rotating
sector which divides the beam into two beams, one passing through the reference and the other
through the sample cell.
(iii) After passing through the sample and reference cells, the light beams are focused onto the
detector.
(iv) The output of the detector is connected to a phase sensitive amplifier which responds to any
change in transmission through sample and reference.
 (v) The phase sensitive amplifier transmits the signal to the recorder which is followed by the
movement of the pen on chart.
The chart drive is coupled to the rotation of the prism and thus the absorbance or transmittance of the
sample is recorded as a function of wavelength.

Advantages of Double Beam Instruments

Although the double beam instruments are more complicated and expensive, they do offer the
following advantages:
(i) It is not necessary to continually replace the blank with the sample or to zero adjust at each
wavelength as in the single beam units.
(ii) The ratio of the powers of the sample and reference beams is constantly obtained and used.
Any error due to variation in the intensity of the source and fluctuation in the detector is minimized.
(iii) Because of the previous two factors, the double beam system lends itself to rapid scanning over a
wide wavelength region and to the use of a recorder or digital read out.

 The monochromatic light is split into two beams by a rapidly rotating beam chopper/ beam splitter.
 Two cells – one containing sample and other containing only the solvent
 The beams are directed alternately in rapid succession through each cell.
 The beams enter the detector after passing through the cells.
 The detector produces current voltages proportional to light intensities of the beams.
 I0 is the light intensity of beam coming from reference cell and IT is the light intensity of beam coming
from sample cell.
 The ratio of voltages is recorded as %T
IT / I0 × 100
 Or as absorbance after logarithmic conversion
log I0 / IT
 Automatically compensate for variation of I0 with wavelength
 Equipped with wavelength scanning device which allows rapid automatic scanning.

COMPARISON
SINGLE BEAM INSTRUMENT DOUBLE BEAM INSTRUMENT
 Calibration should be done with blank every  Calibration is done only in the beginning.
time, before measuring the absorbance or
transmittance of sample.
 Radiant energy intensity changes with  It permits a large degree of inherent
fluctuation of voltage. compensation for fluctuations in the intensity
of the radiant energy.
 It measures the total amount of transmitted  It measures the percentage of light absorbed
light reaching the detector. by the sample.
 In single beam it’s not possible to compare  In double beam it’s possible to do direct one
blank and together. step comparison of sample in one path with a
standard in the other path.
 In single beam radiant energy wavelength has  In this scanning can be done over a wide
to eb adjusted every time. wavelength region.
 Working on single beam is tedious and time  Working on double beam is fast and non-
consuming. tedious.
APPLICATIONS OF UV / VISIBLE SPECTROSCOPY
 Qualitative & Quantitative Analysis:
- It is used for characterizing aromatic compounds and conjugated olefins.
- It can be used to find out molar concentration of the solute under study.
 Detection of impurities:
- It is one of the important methods to detect impurities in organic solvents.
 Detection of isomers are possible.
 Determination of molecular weight using Beer’s law.

Quantitative Spectrophotometric Assay:

 Done for any absorbing substance
 Just prepare solution in transparent solvent and measure its absorbance
 Measurements done at λ max
 Concentration is then calculated by using various procedures.

 Single component Analysis

 Multi component Analysis
 Spectrophotometric Titrations

I] SINGLE COMPONENT ANALYSIS

1. Use of Standard Absorptivity value
2. Use of a Calibration Graph
3. Single Point Standardization
4. Double Point Standardization

1.) Use of Standard Absorptivity Value:

 Adopted by official compendia
 Used for stable substances which have broad absorption bands and which are unaffected by variation of
instrumental parameters like slit width, scan speed.
 Use of A(1%, 1 cm) or ϵ values
 No need to prepare standard solution of reference
 So useful where reference substance is difficult or expensive to obtain
2.) Use of a Calibration Graph:
 The absorbances of a number of standard solutions of reference (pure) substance at concentrations
encompassing the sample concentrations are measured.
 A calibration graph is constructed
 The concentration of sample is read from the graph (concentration corresponding to the absorbance of
the graph)
 Some methods are based on conversion of colorless substances to colored derivatives by chemical
reaction which may be accompanied by heating, change in pH; color produced.
 Absorbance is directly proportional to concentration of analyte.

3.) Single Point Standardization:

 Involves the measurement of the absorbance of a sample and a standard solution of the reference
substance.
 Both are prepared in similar manner.
 Concentration of standard should be close to that of sample.
 Concentration of sample is calculated from proportional relationship between absorbance and
concentration
Ctest / Atest = CStd / AStd

Ctest = Cstd × Atest

Astd

4.) Double Point Standardization:

 When a linear but non-proportional relationship between concentration and absorbance occurs.
 This is indicated by a significant positive or negative intercept in the Beer’s Law plot.
 Standard solutions chosen such that concentration of one standard is greater while the other is lower
than the sample concentration.
 ( A test − A Std 1 )( Cs t d 1−C s t d 2 ) +C std1 ( A Std 1− A Std2 )
C test =
( A ¿ ¿ Std 1− A Std2 )¿

CHEMICAL DERIVATISATION
 Indirect spectrophotometric assays.
 Based on conversion of analyte to a derivative
 Derivative has different spectral properties
 Derivative formed by addition of chemical reagent
 If conversion is complete (when excess reagent is used) absorbance of derivative is usually, but not
always proportional to concentration of analyte.
 Procedures usually involve the conversion of analyte to a derivative that has longer λ max and/or higher
absorptivity.

Reasons for Chemical Derivatization

1) To obtain a more sensitive method of assay:
– When analyte absorbs weakly in the UV region
– By converting to a derivative with a more intensely absorbing chromophore
– For e.g., sugars do not absorb significantly above 220 nm; they are heated with anthrone in
concentrated sulphuric acid and colored derivative measured at 625 nm.

2)
To

avoid interference from irrelevant absorption:

– By converting analyte to a derivative which absorbs in the visible region where irrelevant absorption
is negligible
– For e.g.,
- Coextraction of absorbing components from tablet matrix in Methyltestosterone tablets.
- Condensation of ketosteroids with hydrazide reagents.
OR
- Oxidation of α -ketol group by tetrazolium salts produces derivatives which absorbs in the
visible region free of interferences from irrelevant absorption.

3) To improve selectivity of the assay (Indirect Spectrophotometric procedure):

– When sample contains other UV absorbing substances besides the analyte
– For e.g.,
- Low concentration of adrenaline in Procaine and Adrenaline injection; both procaine and the
bactericide interfere with measurement of absorbance at λ max of adrenaline.
- Only adrenaline forms the purple derivative in the presence of iron (II), and measured
colorimetrically, free of interference.

4) To reduce cost:
– Single-beam manually adjusted visible spectrophotometers (may be called colorimeters) are much
cheaper than uv-visible spectrophotometers.

Reactions Used For Chemical Derivatization

a) Diazotization and Coupling of Primary Aromatic amines
The amine is first diazotized with an aqueous solution of nitrous acid, (generated in situ by the reaction of
hydrochloric acid and sodium nitrite) at 0-5°C

The colourless diazonium salt is very reactive and when treated with suitable coupling reagent (Ar’-H):

E.g., a phenol or aromatic amine, produces an azo derivative.

 The azo derivatives are coloured and have absorption maximum in the visible region.
 The λ max and ϵ max depend on Ar and Ar’ groups.
 The most widely used coupling reagents are 1-naphthol, 2-naphthol and N-(1-naphthyl)-ethane-1,2-
diammonium dichloride (the Bratton-Marshall reagent) which gives high absorptivities.
 The azo derivative of diazotized sulphadiazine coupled with the Bratton Marshall reagent is:

 Which absorbs intensely around 545 nm owing to its excessive conjugation.

b) Condensation Reactions
 It is based on rapid reaction that occurs under suitable conditions between amines and carbonyl
compounds.
 Substances containing a carbonyl group react with variety of reagents containing amino group.

 Reaction involves nucleophilic attack by the amine on carbonyl cation with elimination of water.
 Many hydrazine and hydrazide (isoniazid) reagents are used for ketosteroids.
 INH reacts with 4-en-3-one and 1,4-dien-3-one steroids (ketosteroids) to give yellow derivatives in
acidic solution at λ max of 400 nm.

c) Reduction of Tetrazolium Salts

 In presence of a steroid with an α -ketol (21-hydroxy-20-keto) side chain group, tetrazolium salts are
reduced to their coloured formazan derivatives
 Many corticosteroids are assayed using triphenyl tetrazolium chloride.
 Reaction is carried out in alkaline medium (Tetramethylammonium hydroxide) at 30-35°C for 1-2 hrs.
 Absorbance of red product is measured at around 485 nm.
 The oxidation of the α -ketol and the reduction of triphenyl tetrazolium chloride to triphenyl formazan
are shown below:

d) The acid-dye method:

 The addition of an amine in its ionized form to an ionized acidic dye, e.g., methyl orange or bromocresol
purple, yields a salt (ion-pair) that may be extracted into an organic solvent such as chloroform or
dichloromethane.
 The indicator dye is added in excess, pH adjusted if needed to value where both the amine & dye are in
ionized forms.
 The ion pair is separated from the excess indicator by extraction into organic solvent.
 Absorbance is measured at λ max of the indicator in the solvent.
 Used in the assay of formulations containing quaternary ammonium salts or amines e.g., clonidine HCl
injection & tablets, biperidine lactate injection.

e) Oxidation Methods:
 Oxidation of the side chain of weakly absorbing compounds containing a simple phenyl group produces
a carbonyl derivative that has a much higher absorptivity than parent compound.
 Commonly used oxidation reagents are alkaline potassium permanganate solution, acidified potassium
dichromate solution, or perchlorate solution.
 The product formed from simple monophenyl compounds like ephedrine or propanol amine, is the
corresponding benzaldehyde derivative which exhibits intense absorption at around 240 nm.
 The assay of Ephedrine Hydrochloride Elixir involves the extraction of the benzaldehyde into
cyclohexane, and measurement of the absorbance at its λ max 241 nm.
f) Metal-ligand Complexation:
 Many organic reagents (called ligands) form complexes with metal atoms by the formation of
coordinate bonds (in which both the electrons are donated by the ligand) and covalent bonds.
 Ligands with 2 or more donating groups are called multidentate and coordinate with a single metal
atom.
 Such chelates are often coloured and may be determined by visible spectrophotometry.
 Metals (addition of excess chelating agent) as well as organic substances (adding excess of suitable
complexing metal) can be assayed.
 E.g., adrenaline with buffered solution of iron (II) sulphate, giving purple complex, pH 8-8.5 for
maximum intensity ( λ max 540 nm).

SPECTROPHOTOMETRIC TITRATIONS
 In classical visual titrimetry  equivalence point in a reaction is detected by observing a change in
colour.
 Under favorable conditions, precision is easily attainable by operators with normal vision.
 Good results are difficult to obtain, if colour change is gradual or if colours of two forms do not contrast
sharply.
 Such difficulties can be overcome by carrying out titration in cuvette in spectrophotometer or filter
photometer.
 An optimum wavelength or filter is selected and zero adjustments made in advance.
 Photometric reading is taken following each incremental addition from burette.
 Conventional spectrophotometers require some structural modifications to permit insertion of titration
vessel of convenient size, as well as burette tip & stirrer.
 Numerous automatic and semiautomatic titrators are available.
 Here end point is evacuated from data on absorbance of solution.
 Usual photometric titration curve is plot of absorbance against volume of added reagent.
 Since spectrophotometric titrations are carried out in a vessel for which light path is constant,
absorbance is proportional to concentration.
 If absorbing substances (titrant, substance titrated or both) follow Beer’s Law, then titration curve,
corrected for dilution will consist of 2 straight lines intersecting at equivalence point.
 Intersection is likely to show some degree of curvatures as a result of incompleteness of reaction at
equivalence point.
 Shape of photometric titration curve will depend on optical properties of reactant, titrant and products
of reaction at wavelength used.

SPECTROPHOTOMETRIC TITRATION: TYPICAL PLOTS

(a) is typical of titration where titrant alone absorbs

(b) corresponds to systems where substance titrated and titrant are colourless and product alone absorbs.
(c) is characteristic of systems where substance titrated is converted into non-absorbing product.
(e) is obtained when a colourless reactant is converted into a colourless product by a coloured titrant.
(d) and (f) curves represent successive addition of ligands to form 2 successive complexes of different
absorptivity.

APPPARATUS

Spectrophotometric titration: Perspex cell with quartz

windows
 Special titration cell is necessary which completely fills cell compartment of spectrophotometer.
 Cell is made from 5 mm Perspex sheet, cemented together with special Perspex cement and with
dimensions suitable for instrument to be used.
 Since Perspex is opaque to UV light, two opening are made in cell to accommodate circular quartz
windows  23 mm in diameter and 1.5 mm thick.
 Windows are inserted in such a way that beam of monochromatic light passes through their centers to
photoelectric cells.
 Perspex cover of cell has two small openings for tip of 5 ml micro burette and
micro stirrer, held by means of rubber bungs, stirrer is sleeved.
 Whole of cell, exception of quartz windows, is covered with black paper.
 Further precaution  top of cell is covered with black cloth as it is important
to exclude all extraneous light.
 Sometimes, probe type photometer may be used, based on fibre optics.

 The titration in which the titrant, analyte or reaction product absorbs.

 The plot of absorbance vs volume of titrant added will consist of the reaction
is complete, two straight lines intersecting at the end point.
 Thus, determination of changes in absorption to follow the changes in the
concentration of light absorbing constituents in the solution during titration is
the basis of spectrophotometric titration.
 The absorbance of a solution can be monitored during a titration.
 In this example, stirring will cause the solution to circulate in the cuvette and
the response can be measured.
 You could also use a pump or simply transfer a sample at known intervals.

TECHNIQUE:
 Experimental technique is simple.
 Cell containing solution to be titrated is placed in light path of spectrophotometer
 A wavelength appropriate to particular titration is selected and absorption is adjusted to a convenient
value by means of sensitivity and slit width controls.
 Measured volume of titrant is added to stirred solution and absorbance is read.
 This is repeated at several points before end point and several more points after end point.
 End point is found graphically.
 Optimum concentration of solution to be analyzed depends on molar absorption coefficient of
absorbing species involved and is of order 10-4 to 10-5 M.
 Effect of dilution can be made negligible by using sufficiently concentrated titrant.
 If relatively large volume of titrant are added, effect of dilution may be corrected by multiplying
observed absorbance by factor (V + v)/V, where V is initial volume & v is volume added.
 If dilution is of order of only few % lines in titration plots appear straight.
 Operating wavelength is selected to avoid interference by other absorbing substances and obtain an
absorption coefficient so the change in absorbance falls in convenient range.
 Range is particularly important, as serious photometric error is possible in high absorbance regions.
 Light leakage must be avoided.
ADVANTAGES
1. Presence of other substances absorbing at same wavelength does not cause interference, since only
change in absorbance is significant.
2. Precision of locating the titration line by pooling the information derived from several points is greater
than precision of any single point.
3. Method is useful for reactions which tend to be appreciably incomplete near the equivalence point.
4. Precision of 0.5% is often attainable.
5. Linear response of absorbance to concentration often produces appreciable break in a
spectrophotometric titration even though changes in concentration are insufficient to give a clearly defined
inflexion point in photometric titration.

APPLICATIONS
 Many Organic compounds may be determined by neutralization reactions using spectrophotometric
titrations.
 Complexation titrations can also be performed.
 NOTE: There is no difficulty in using non-aqueous solvents & it is useful feature of Spectrophotometric
titrations.

Optimum Conditions for Spectrophotometric Measurements

Accuracy and precision of the method depends on the selection of most appropriate sample and
instrumental conditions.
(1) Sample Conditions: Solvent, concentration and pathlength
(2) Instrumental Parameters: wavelength, slit width, scan speed, stray light

SAMPLE CONDITIONS
(1) Solvent
 Choice of solvent governed by the solubility of the absorbing substance and by absorption of solvent at
analytical wavelength.
 Water is ideal solvent as it is transparent at all wavelengths in the UV and visible regions, above 180
nm; also, cheap and readily purified
 Organic solvents are restricted to measurements at wavelengths where solvents are reasonably
transparent.
 Weak absorption by solvent is compensated by using reference cell containing only solvent

(2) Concentration and Pathlength

 Every absorbance value has a small random error associated with it (due to small random fluctuations
in light transmitted, light source intensity, detector and amplifier noise)
 The % relative error is given by:
100 ∆ c
% Relative Error=
c
 Relative error is minimum, and accuracy & precision is optimum when absorbance is around 0.9
 Practically combination of concentration and pathlength is adjusted to give ideal absorbances (0.2 to
1.0)

Instrumental Parameters
(1) Slit width
 An increase in slit width increases the spectral bandwidth and reduces the monochromaticity of light.
 Deviations from Beer-Lambert Law will be observed.
 If slit width is too narrow, there will be reduction in incident energy and hence it lowers signal-to-noise
ratio and decreases the precision.
 Optimum slit width is the widest setting that provides proper spectral bandwidth.
(2) Scanning speed
 If scan speed is too fast, electronic and mechanical damping of the spectrophotometer’s signal to the
recorder may prevent the recorder pen from responding quickly enough to the rapid changes of
absorbance.
 In progressively faster replicate scans, the apparent λ max is displaced in the direction of the scan, Amax
are decreased and Amin are increased; also, resolution between adjacent bands is reduced.
 Optimum speed is the fastest speed at which the recorder pen operates properly.

(3) Stray Light

 Stray light is any radiation reaching the detector other than the narrow range of wavelengths normally
transmitted by the monochromator.
 It arises from scattering and refraction inside the monochromator, mainly due to imperfections on the
optical surfaces.
 May result in spurious maxima and deviations from Beer-Lambert law.
 Stray light ideally should be kept at a minimum.

MULTI COMPONENT ANALYSIS

 Used when single component is rare.
 UV spectrophotometric techniques are mainly used for multicomponent analysis, thus minimising the
cumbersome task of separating interferents and allowing the determination of an increasing number of
analytes, consequently reducing analysis time and cost.
 Multicomponent UV spectrophotometric methods are based on recording and mathematically
processing absorption spectra. The multicomponent analysis methods offer the following advantages:
1. Avoiding prior separation techniques, e.g., extraction, concentration of constituents, and clean-up
steps that might be required.
2. Spectral data are readily acquired with ease.
3. The process is fast, accurate, and simple.
4. Wide applicability to both organic and inorganic systems.
−4 −5
5. Typical detection limits of 10 to 10 M and moderate to high selectivity.

 Usually, concentration of one or more substances is required to be measured in presence of other

absorbing substances which may interfere in the assay.
 Modifications may be done to remove interferences… Many procedures available
 The basis of all the spectrophotometric technique for multicomponent analysis is the property that at
all wavelengths:
 Absorbance of the solution is sum of absorbances of individual components
 Measured absorbance is difference between total absorbance of solution in sample cell and
absorbance of solution in reference (blank) cell.

Fixed Dose Combinations

Fixed-dose combinations (FDCs) are also known as combination products are combinations of two or more
active drugs produced in a single dosage form.

List of some fixed dose combinations available in Indian market

1) Clonidine and Hydrochlorothiazide Antihypertensive
2) Clonidine and Chlorthalidone Antihypertensive
3) Tizanidine, Nimesulide and Paracetamol NSAID
4) Tizanidine and Mefenamic acid NSAID
5) INH and Ethambutol and Rifampicin Anti Tuberculosis
6) Zidovudine and Lamivudine and Nevirapine Anti-viral
7) Stavudine and Lamivudine and Nevirapine Anti-viral
8) Metformin and Glibenclamide Anti-diabetic
9) Metformin and Glimepiride Anti-diabetic

Advantages of Fixed Dose Combination Products

1) Increase in the therapeutic ratio
2) Reduced administration costs stem from simplified packaging, fewer prescriptions and fewer dispensing
fees.
3) Reducing the number of pills diminishes the complexity of the regimen, so that improved patient
adherence is expected with combination products.
4) Combination products make particular sense in the treatment of infectious disease, where partial
adherence can lead to the development of drug-resistant strains and a threat to public.

Disadvantages of Fixed Dose Combination Products

1) These fixed dose combinations can lead to polypharmacy
2) Dose of one ingredient cannot be altered
3) Different pharmacokinetic properties can pose difficulty in frequency of administration and in case of
development of an Adverse Drug Reaction (ADR).
4) It is difficult to withdraw the suspected drug alone.
5) A combination makes it more difficult to pinpoint the offending agent responsible for the adverse
reaction.
6) Some FDCs when combined lead to increased toxicity. For instance, the anti-TB drugs, Streptomycin,
Kanamycin and Capreomycin cannot be combined, as they have the same side effects (oto and nephro-
toxicity)

Need of Multicomponent Analysis

 The spectrophotometric assay of drugs rarely involves the measurement of absorbance of sample
containing only one absorbing component.
 The pharmaceutical analyst frequently encounters the situation where concentration of one or more
substances is required to be determined in presence of other absorbing substances which potentially
interfere in the assay.
 Example: Brimonidine Tartrate + Timolol Maleate
Olmesartan Medoxomil + Hydrochlorothiazide
Ezetimibe + Simvastatin

 If the recipe of sample formulation is available to the analyst. The analyst can identify concentration of
substance interfering and its extent of interference.
 If the unknown interference arises due to manufacturing impurities, decomposition products and
formulation excipients, if not removed imparts a systematic error to the analysis of drug in sample.

 A number of modifications to the simple spectrophotometric procedure used for determination single
component are available, which may eliminate certain sources of interference and permit the accurate
determination of one or all of the absorbing components.

There are various spectrophotometric methods available which can be used for the analysis of combination
samples.
Following methods can be used:-
1) Simultaneous equations method (Vierodt’s method)
2) Absorbance ratio method (Q-Absorbance method)
3) Derivative spectrophotometric method
4) Geometric correction method
5) Orthogonal polynomial method
6) Difference spectrophotometric method
7) Chemical derivatisation method

Many methods available:-

1) Assay as single component:
Provided other substances have sufficiently small absorbance at wavelength of measurement.
2) Assay using absorbance corrected for interference:
If identity, concentration and absorptivity of absorbing interferences are known, it is possible to calculate
their contribution to total Absorbance of mixture
3) Assay after solvent extraction of sample:
If interference is large or contribution to absorbance cannot be calculated, separate the analyte from
absorbing interferants by solvent extraction procedures
 Second derivative spectrophotometry discriminates in favour of the narrow band of the fine structure
of the drug and eliminates the broad band absorption of the excipients.

Limitations / Disadvantages:
 The enhanced resolution and bandwidth discrimination increases with increasing derivative order
 However, it is also found that there is an increase in electronic noise along with the increase in the
derivative order, which place serious practical limitations on the higher order spectra.
 For quantitative purposes, second and fourth derivative spectra are the most frequently employed
derivative orders employed

GEOMETRIC CORRECTION METHOD:

 Number of mathematical correction procedures which reduce or eliminate background irrelevant
absorption that may be present in samples of biological origin.
 Simplest procedure ----→ 3 point geometric procedure....that is applied if irrelevant absorption is linear
at 3 wavelengths selected.
 3 point correction procedures are simply algebraic calculations of what baseline technique in infrared
spectrophotometry does graphically.

ORTHOGONAL POLYNOMIAL METHOD:

 This technique was introduced by Glenn in the year 1963.
 Mathematical correction procedure which involves more complex calculations than 3 point correction
procedure.
 Accuracy of orthogonal functions procedure depends on correct choice of polynomial order and set of
wavelengths.
 Usually, quadratic or cubic polynomials are selected depending on shape of absorption spectra of drug
and irrelevant absorption.
 Set of wavelength is defined by number of wavelengths, intervals & mean wavelength of the set.

DIFFERENCE SPECTROPHOTOMETRY
 Difference spectrophotometry improves selectivity and accuracy of the analysis of compound of
interest in presence of absorbing interferents.
 It measures the value , which is difference of absorbance (∆A) between two equimolar solutions of the
analyte in different chemical forms which exhibit different spectral characteristics.
 Following criteria should be followed for difference spectrophotometry:
a) Reproducible changes can be produced in the spectra of analyte by the addition of one or more
solvents.
b) The spectra and the absorbance of the interfering substance should not get affected by the reagent.
 The simple and most commonly employed technique for altering the spectral properties of the analyte
is the adjustment of the pH by means of aqueous solution of acid, alkali or buffers.
 The ultraviolet-visible absorption spectra of many substance containing ionizable functional groups,
E.g.: phenol, aromatic carboxylic acids and amines, are dependent on the state of ionization of the
functional groups and consequently on the pH of solution.
 The difference absorption spectrum is a plot of difference in absorbance between the solution at

 pH =13 and that pH = 1 against wavelength.

 It may be generated automatically using double beam recording spectrophotometer with a solution at
pH = 13 in sample cell and the solution at pH=1 in reference cell (blank) cell.

 At 257 and 278 nm both solutions have identical absorbance.

 Such wavelength of equal absorptivity of the two analytes are called ISOBESTIC OR ISOABSORPTIVE
POINTS.

 The measured value in quantitative difference spectrophotometric assay is the ∆A at any suitable
wavelength measured to the baseline, e.g. ∆A1 at 1 or amplitude between adjacent maximum and
minimum, e.g. ∆A2 at 2

 At 1 ∆A1 = Aalk – Aacid

Where,
Aalk and Aacid are the individual absorbances at 1 in 0.1 M sodium hydroxide and 0.1M hydrochloric
acid solution respectively.

 If individual absorbances, Aalk and Aacid, are proportional to the concentration of analyte and
pathlength, the, ∆A also obeys the Beer- Lambert Law and modified equation may be derived as:

∆A = ∆abc

Where, ∆a is the difference absorptivity of the substance at the wavelength of measurement

 If one or more other absorbing substances is present in the sample which at analytical wavelength has
identical absorbance (Ax) in the alkaline and acidic solutions, its interference in the spectrophotometric
measurement is eliminated.

∆A = (Aalk + Ax) – (Aacid + Ax)

= Aalk – Aacid

 A substance whose spectrum is unaffected by changes of pH may be determined by difference

spectrophotometric procedure if it can be quantitatively converted by means of suitable reagent to a
chemical species that has different spectral properties to its unreacted parent substance.

 The ∆A between equimolar solutions of the unreacted substance and its derivative is free of
interference if the irrelevant absorption is unaffected by the reagent.