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UV VISIBLE SPECTROSCOPY

What is spectroscopy?

Spectrum + Scopies

“When a beam of light is allowed to pass through a prism or grating, it will dispersed into seven

colors from red to violet and the set of colors or band produced is called spectrum” +

Examination

“Spectroscopy is the branch of the science dealt with the study of interaction of Electro

Magnetic Radiation (EMR) with matter”

So the spectroscopy means examination of spectrum. From the type of radiation, which is

absorbed, we can get idea about the nature (type) of the compound and from the amount of

the radiation, which is absorbed; we can get idea about the concentration (amount) of the

substance. So the spectroscopy is used for qualitative and quantitative analysis.

Interaction of EMR with matter

When a beam of light is passed

through a transparent cell containing

a solution of an absorbing substance,

reduction of the intensity of the light

may occur.

This is due to

1) Reflection at the inner and outer

surfaces of the cell

2) Scattered by the particles present

in the solution

3) Absorption of the light by the

molecules in the solution



Classification of Spectroscopy:

1) Absorption Spectroscopy: the type and amount of the radiation, which is absorbed, depend

upon the structure of the molecules and the numbers of molecules interacting with the

radiation. The study of these dependencies is called absorption spectroscopy. (UV, IR, NMR, X-

Ray, ESR)

2) Emission spectroscopy: if sufficient energy gets impinged upon a sample, the outer

electrons in the species will be raised from their stable ground state to higher energy level

(unstable in nature). These excited species rapidly emits a photon and return to their ground

stable energy level. The type and amount of radiation, which is emitted, is studied, this type of

spectroscopy is called emission spectroscopy. (AES, MES, Fluorimetry)

3) Scattering spectroscopy: if the incoming radiation strikes with the solid particles suspended

in the solution, the light transmitted at an angle other than 1800

from the incident light. This

spectroscopy is called scattering spectroscopy. (turbidimetry, nephelometry)

What is EMR?

• EMR is a form of energy that is transmitted through space at an enormous velocity

• It can travel in space with the same speed at that of light.

• As the name implies an EMR is an alternating electrical and associated magnetic force

field in space (It contains electrical and magnetic components)

• The two components oscillate in planes perpendicular to each other and perpendicular

to the direction of propagation of the radiation.

• EMR consist of a stream of discrete packets (particles) of pure energy, which is called

photons or quanta.

• The energy of photon is proportional to the frequency

E = hυ where E= Energy of photons, h= plank’s constant (6.624 x 10-27

erg. Sec) and

υ=frequency of radiation in cycles/second

• Wavelength (λ): it is the distance between two successive maxima on an

electromagnetic wave. (m, cm, mm, μm, nm, and A0)

• Frequency (υ): is the numbers of waves passing through a given point in unit time. (T-1

,

sec-1

, cycles/second, hertz, fresnel)

• Wave numbers (ΰ): is the numbers of waves per centimeter in vacuum. ( cm-1

)

• Velocity (V): is the product of wavelength and frequency (λ X υ =V) (cm/sec, m/sec)



Classification of EMR:

EMR is arbitrarily classified in to different regions according to wavelength.

Energy associated with the molecules:

1. The molecule as a whole may move this is called translation and the energy associate

with this movement is called transnational energy. (Etrans)

2. The part of the molecules, that is atom or groups of atoms, may move with respect to

each other. This motion is called vibration and the associated energy is called

vibrational energy. (Evib)

3. The molecule may rotate about an axis. And such rotation is characterized by the

rotational energy. (Erot)

4. Besides these modes of movements, the molecule possesses an electronic configuration

and the energy associated with this configuration is called electronic energy. (E ele)

E total = E Trans + E Vib + E Rot + E Ele



Theoretical principles

If a molecule is allowed to interact with the EMR of a proper frequency, the energy of the

molecule is raised from one level to a higher one; we say that absorption of radiation takes

place. In order for absorption to occur, the energy difference between the two energy levelmust be equal to the energy of the photon absorbed

E2 – E1 = hυ where E1 is energy of lower level and E2 is the energy of upper level

• This energy jump from one level to another is called transition

• The graph of the light absorption against the frequency is called absorption spectra.

• Visible and Ultraviolet light provides enough energy for electronic transition there for

called electronic spectra.

• On absorption of energy by a molecule in the ultraviolet region, changes are producedin the electronic energy of the molecule due to transitions of valence electrons in the

molecule.

6* Anti-bonding

π* Anti-bonding n Non-bonding

π Bonding

6 Bonding

Types of transitions:

1. 6 to 6*: A transitions of electrons from a bonding sigma orbital to the higher energy

antibonding orbitals. ( eg. Alkane). Sigma bonds are, in general, very strong, there for

they require high energy for the transitions and this transitions requires very short

wavelength (near about 150 nm)

2. n to 6*: This transition involves saturated compounds with one hetero atom with

unshared pair of electrons (n electrons). Corresponding band appears at 180-200 nm.



3. π to π*: This transition is available in compounds with un-saturation (eg. Alkene).

Corresponding band appears at 170-190 nm.

4. n to π*: This type of transitions are shown by the unsaturated molecules containing one

or more hetero atoms. (O, N, S)

5.

Conjugated system: In conjugated dines, the π orbital of the separate alkenes groupcombine to give new orbital i.e. the two new bonding orbital which are designated π1

and π2 and new two anti-bonding orbital designated as π3* and π4*. So for the π2

π3* transition very low energy is requires corresponding to the higher wavelength.

Some important terms:

1. Chromophore: It is a group of molecules, which is responsible for the absorption of light

by molecules. It is conjugated dienes. It is minimum structural requirements for the

absorption of radiation in UV range.

2. Auxochrome: It is a saturated group containing unshared electrons which when

attached to a Chromophore changes both intensity as well as the wavelength of the

absorption maxima. e.g. OH, NH2, Cl etc.

3. λ-max: It is a wavelength at which there is a maximum absorption or absorption

intensity. It is a physical constant and characteristic of structure and so useful for

identification of compounds. It is independent of concentration.

4. Bathochromic shift: The shifting of absorption to a longer wavelength due to

substitution or solvent is called as bathochromic shift. It is also called as Red shift. e.g.,

λmax of Ascorbic acid=243nm, λmax of Ascorbic acid in alkali medium=299nm.

5. Hypsochromic shift (Blue shift): Shifting of λmax to lower value or left hand side due to

substitution, solvent, pH etc is called as Hypsochromic shift. e.g. λmax of Phenol in basic

media=297nm, λmax of Phenol in acidic media=277nm.

6. Hyperchromism: Increase in absorption intensity (e) due to solvent, pH or some other

factors called hyperchromic effect.

7. Hypochromism: Decrease in absorption intensity due to substituent, solvent, pH etc.

called hypochromic effect.

8. A1%

1cm (A one percent one centimeter): Is the absorbance of the solution having

concentration 1 gm per 100 ml of the solution.

9. Molar absorptivity (ε): Is the absorbance of the solution having concentration

gm.mol.weight/1000 ml of the solution. [ε = (A1%

1cm X Mol. Wt.)/10]

10. Transmittance (T): is the ratio of IT /I0 and % transmittance (%T) is given by %T=100 IT /I0

11. Absorbance (A): Is the degree of absorption of light by a medium through which the

energy passes. It is expressed as the logarithm of the ratio of light transmitted through a



pure solvent to the intensity of light transmitted through the medium. It is the area

under the curve.

A= log I 0 /I T A= log I 0 - log I T A=2- log % T

Absorption Spectra

The graph of the light absorption against the frequency is called absorption spectra. It is

characterized by 1) λ-max: - Position of spectra 2) Intensity of absorbance:- the amount of the

radiation absorbed by the molecule.

1) Factors affecting the position of the spectrum (λ-max)

A) Structural factors

• Substitution: Placing a substituent on a Chromophore may produce change in λ-max by

two mechanisms: introduction of an entirely new transition and/or shifting the

wavelength of existing transitions. E.g. each alkyl substituent produces 5 nm

bathochromic shifts.

• Solvent: the solvent effect arises because solvation is frequently different for the

ground and excited states. If the ground state is solvated more strongly than the excited

state, the energy difference between the levels is increased. The increase in energy

difference is reflected in a shift of the absorbance to shorter wavelengths.

• Geometry: e.g. stilbene. Trans-stilbene absorbs at a longer wavelength than cis-stilbene

due to steric effects. Co-planarity is needed for the most effective overlap of the π-

orbitals. The cis-isomer is forced in to a non planar conformation due to steric effects.

The cis isomers are twisted slightly out of plane by steric interactions so that the degree

of conjugation in the π system is slightly less than the Trans isomers, resulting in greater

energy for the transitions.

B) Non Structural factors

• PH: e.g. Phenolphthalein: in alkaline medium it is pink and in the acidic medium it is

colorless

• Temperature: Temperature provides more energy to ground state. As a result energy

required for excitation will be less, so there is bathochromic shift.



2) Factors affecting the intensity of absorption of radiation.

• Thickness of the medium: Lambert’s law: “when a beam of monochromatic light is

allowed to pass through a transparent medium, the rate of decrease of intensity with

the thickness of medium is directly proportional to the intensity of incident radiation”. It

gives relationship between absorbance and the thickness of the medium.• Concentration of absorbing solute: Beer’s law: “when a beam of monochromatic light is

allowed to pass through a transparent medium, the rate of decrease of intensity with

the concentration of absorbing solute is directly proportional to the intensity of incident

radiation”. It gives relationship between absorbance and the concentration of the

medium.

A = a b c (Fundamental equations of spectroscopy)

Deviation from the Beer’s curve (Errors in spectrophotometric measurement)

Errors may arise from instrumental of from chemical factors. Instrumental errors can arise from

several sources. Noise, fluctuation in light source.

The ideal absorbance range for most measurement is in the range of 0.2 to 0.8. The calibration

curve is relatively linear in this range. Other factor includes Spectral Slit Width (SSW).

As slit width is increased, the fine structure of the absorption band is lost as the incident light is

no more monochromatic. Generally, fast scan rates tend to distort spectra, altering the

positions of both maxima and minima as well as diminishing peak intensities.

This introduces both qualitative and quantitative errors in the measurement.

A numbers of chemical factors may also produce errors in the analysis.

Solute-solute interaction, e.g. aggregation, precipitation, dimerization etc. Ionization or even

complexation of the analyte in solution can also lead to apparent deviation from the Beer’s

curve.

Fluorescence from absorbing species in solution may also contribute to interference.



INSTRUMENTATION

1. Light Source: (source of electromagnetic radiation):

• The tungsten filament lamp is a satisfactory light source for the region 350 to 2000 nm.

It consists of a tungsten filament contained in a glass envelope. The most convenient

light source for UV radiation is discharge lamp. Generally deuterium discharge lamp is

used. It is consisting of deuterium-filled silica envelope. It gives radiation from 185 to

380 nm.2. Slit: (Radiation intensity controlling device):

• Enough light must pass through the sample to elicit a measurable response from the

detector.

3. Monochromator: (wavelength selecting device).

• It converts polychromatic light in monochromatic light (light having one wavelength).

3.1 Filters:

o Glass filters are pieces of colored glass, which transmit limited wavelength

ranges of the spectrum. The color is produced by incorporating oxides of suchmetals

3.2 Prisms:

o When a beam of light passes through a prism, it is bent or refracted. The amount

of deviation is dependent on the wavelength.

o The prism is made up of quartz for use in the UV light, since glass absorbs

wavelengths shorter than about 330 nm.

o Glass prism is preferable for the visible region of the spectrum, as the dispersion

is much greater than that obtained with quartz.

3.3 Grating: o Most modern UV spectrophotometer uses diffraction grating as a

Monochromator. It consisting of a very large number of equispaced lines (200-

2000 per mm) ruled on a glass plate.

o They can be used either as transmission grating or when aluminized, as

reflection grating.



4. Sample Holder:

The sample holder is known as cuvettes. Cuvettes must be transparent to the light, so

the glass cells are used in the visible region and quartz or silica cells are used in the UV

region. The cells used in the UV spectrophotometers are usually 1 cm in path length but

cells are available from 0.1 cm to 10 cm or more.

5. Detectors (Radiation measuring device):

It is also known as photocell. They convert radiation energy in electrical energy. For the

determination of substances by spectrophotometric techniques, precise determinations

of the light intensities are necessary. Photoelectric detectors are most frequently used

for this purpose. They must be employed in such a way that they give a response

linearly proportional to the light input and they must not suffer from drift or fatigue.

5.1 Barrier-layer photocell:

o It one of the simplest detectors, which has the advantage that it requires no

power supply but gives a current, which, is directly proportional to the light



intensity. It is consists of a metallic plate, usually copper or iron, upon which

is deposited a layer of selenium.

o An extremely thin transparent layer of a good conducting metal, e.g. silver,

platinum or copper, is formed over the selenium to act as one electrode, the

metallic plate acting as the other. Light passes through the semitransparentsilver layer causes release of an electron, which migrates, to the collector.

o The electron accumulating on the collector resulting in a potential difference

between the base and collector, which can be measured by a low resistance

galvanometer circuit.

o The useful working range of selenium photocell is 380-780 nm. Their lack of

sensitivity compared to phototube and photo multiplier tube, restricts their

use to the cheapest colorimeters and flame photometers.

5.2Photo emissive tube:



o It consists of an anode and a cathode sealed in an evacuated glass tube,

which may have a quartz or silica window for UV measurement.

o The cathode is coated with a layer of light sensitive material that emits

electrons upon absorption of photons.

o A power supply maintains the anode positive with respect to the cathode sothat the photoelectrons are collected at the anode.

o This current is directly proportional to the light intensity. Phototubes are

available for use over the entire UV/visible region of the spectrum, but no

single tube covers the entire range satisfactorily.

o Therefore many instruments with phototube detectors employ

interchangeable blue and red sensitive phototube in order to provide

sufficient sensitivity over the entire spectrum.

5.3 Photo multiplier tube:

o It is very sensitive detectors with very short response times. It contains a

photo cathode and a series of dynodes, which are also photosensitive.

o A higher successive potential is maintained between each dynodes.

o A photoelectrons released from the photo cathode is accelerated toward the

first dynode by their voltage difference, where it strikes to release several

electrons.



o The secondary electrons are then accelerated toward the second dynode

where the process repeats. In this way multiplication of the electrons can be

achieved.

o The current from phototubes and photo multiplier tubes never falls to zero. A

small residual current called dark current is produced, due to long exposureof the light.

6. Display (Read out meter):

• The signal from the detector is normally proportional to the intensity of light, and after

amplification may be displayed as % T or after passing through a logarithmic conversion

circuit as absorbance (Log 1/T)

TYPES OF INSTRUMENTS:

Instruments for measuring the absorption of light may be of the single beam or double beam

type. In a single beam instrument, light from the sources passes through a filter and then

through the sample and in to the detector. The signal from the detector is proportional to the

intensity of the light beam striking it.

To make a measurement of absorbance using a manually controlled single-beam instrument,

the Monochromator is adjusted to the required wavelength and the appropriate lamp and

photocell are selected by means of switches.

The first step is to close a shutter in the path (or adjust dark filter) and adjust 0 %T.

The second step is to open the shutter and place the cell containing only the solvent in the light

beam and adjust the scale on 100 % T (equivalent to 0 absorbance).

The third step is to place the sample cell in the light path and measure the intensity IT or its

equivalent absorbance.

In double beam spectrophotometer, the monochromatic light is split by the beam splitter in to

two equal intensity light beam, which are directed alternatively in rapid succession through acell containing the sample and one containing the solvent only. This instrument measures the

ratio of the intensity of the beam coming through the sample and through the solvent.

Changes in the intensity of the source affect both beams proportionately so the ratio of their

intensities is not altered.



Therefore, a high degree of stability in the light source is not required in these instruments.

Difference in the lamp output, optical system throughput, and detector sensitivity with

wavelength also affect both beams in the same way.

APPLICATIONS:

1. Qualitative Analysis:

• The UV spectra of most compounds are of limited value for qualitative analysis as

compared to IR and Mass spectra. Qualitative analytical use of UV spectra has largely

involved λ-max and absorptivities, occasionally includes absorption minima. In

pharmacopoeias, absorption ratios have found use in identity tests, and are referred toas Q-values in USP.

2. Quantitative Analysis:

• UV spectroscopy is perhaps the most widely used spectroscopic techniques for the

quantitative analysis of chemical substances as pure materials and as components of

dosage forms.

2.1 Single component Analysis:

• Direct Analysis: Essentially all compounds containing conjugated double bond or

aromatic rings, and many inorganic species absorb light in the UV-visible regions. In

these techniques the substance to be determined is dissolved in suitable solvent and

diluted to the required concentration by appropriate dilutions and absorbance is

measured.

• Indirect Analysis: (Analysis after addition of some reagent) indirect methods are based

on the conversion of the analyte by a chemical reagent that has different spectral

properties. Chemical derivatization may be adopted for any of the several reasons.

o If the analyte absorbs weakly in the UV region.

o The interference form irrelevant absorption may be avoided by converting the

analyte to a derivative, which absorbs in the visible region, where irrelevant

absorption is negligible.

o This technique can be used to improve the selectivity of the assay in presence of

other UV radiation absorbing substance.

o Cost.



Methods of calculating concentration in single component analysis

• By using the relationship: A = a b c

• By using the formula: Cu = (Au/As) X Cs

• By using the equations: Y = mX + C

• By using the Beer’s curve

A) Multi component Analysis:

a) Simultaneous Equations method:

• If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λ -max

of the other (λ1 and λ2), it may be possible to determine both the drugs by the

simultaneous equations method.

• Criteria for obtaining maximum precision, below mentioned ratio should lie out side the

range 0.1-2.0

(A2/A1) / (aX2/aX1) and (aY2/aY1) / (A2/A1)

The information required is

• The absorptivities of X at λ1 and λ2, aX1 and aX2

• The absorptivities of Y at λ1 and λ2, aY1 and aY2

• The absorbances of the diluted sample at λ1 and λ2, A1 and A2

• Let Cx and Cy be the concentration of X and Y respectively in the sample

The absorbance of the mixture is the sum of the individual absorbances of X and Y

At λ1 A1 = aX1* Cx + aY1* Cy (1)

At λ2 A2 = aX2* Cx + aY2* Cy (2)

Multiply the equation (1) with aX2 and (2) with aX1



A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 (3)

A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 (4)

A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1

A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)

Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5)

Same way we can derive

Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6)

Equations 5 and 6 are known as simultaneous equations and by solving these simultaneous

equations we can determine the concentration of X and Y in the sample.

b) Q-Absorbance ratio method

• The absorbance ratio method is a modification of the simultaneous equations

procedure. It depends on the property that, for a substance, which obeys Beer’s law at

all wavelength, the ratio of absorbances at any two wavelengths is a constant value

independent of concentration or path length.

• In the quantitative assay of two components in admixture by the absorbance ratio

method, absorbances are measured at two wavelengths, one being the λ-max of one of

the components (λ2) and other being a wavelength of equal absorptivity of two

components (λ1), i.e. an iso-absorptive point.

At λ1 A1 = aX1* Cx + aY1* Cy (1)

At λ2 A2 = aX2* Cx + aY2* Cy (2)

Now divide (2) with (1)

A2/A1= (aX2* Cx + aY2* Cy)

(aX1* Cx + aY1* Cy)

Divide each term with (Cx + Cy)



A2/A1 =

(Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy)

(aX2* Cx + aY2* Cy) / (Cx + Cy)

Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)

A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy]

Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx

Therefore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)]

= [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]

At iso-absorptive point aX1 = aY1 and Cx = Cy

Therefore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1

= (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1)

Let Qx = aX2/aX1, Qy = aY2/aY1 and absorption ratio Qm = A2/A1

Qm = Fx Qx + Qy - Fx Qy

= Fx (Qx-Qy) + Qy

Fx = (Qm – Qy) / (Qx – Qy) (3)

From the equations (1)

A1 = aX1 (Cx + Cy) therefore Cx + Cy = A1 / aX1

Therefore Cx = (A1/aX1) – Cy (4)

From the equation (3)

Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy)

There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy)

There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) (5)



a. Derivative spectroscopy

• Derivative spectroscopy involves the conversion of a normal spectra to its first, second

or higher derivative spectra. The normal spectrum is known as fundamental, zero order

or D0

spectra. The first derivative spectrum (D1) is a plot of the rate of change of

absorbance with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ.

• The second derivative spectrum is a plot of Δ2A/ Δλ

2vs. λ. For the quantitative

estimation of binary mixtures by the derivative spectroscopy, first of all we have to find

out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP

for A and B so that at that particular ZCP other component shows remarkable

absorbance. Now prepare calibration curve of A at the ZCP of B and of B at the ZCP of A.• Find out the unknown concentration using calibration curves.

1) Determination of Dissociation constant of an indicators



Indicators give different color at different pH. Methyl red is red in color in acidic

medium and is yellow in alkaline medium because in acidic medium it remains as HMR

(Unionized form) and in alkaline medium as MR- (Ionized form).

HMR=MR-+ H

+

Ka = [(MR

-

) (H

+

)] / (HMR) Therefore Pka = pH – Log [(MR

-) (H

+)]

2) Determination of composition of Complex.

M + L = Complex

There are two methods for the determination of composition of complex first is Mole

ratio method. In this technique concentration of on of the components of the complex

is kept constant and other is increased and the absorbance of the resulting solution is

measured. Now from the plot of absorbance Vs concentration. Another method is

Job’s curve method (Continuous variation method).

5) As a detector in HPLC

References:

1) Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4th

Edn., PartOne, CBS Publishers, New Delhi, 2000.

2) Schirmer, R.E., In; Modern Methods of Pharmaceutical Analysis, 2nd

Edn., Volume-I,

CRC Press, Florida, 2000.

3) Christian, G.D., In; Analytical Chemistry, 6th

Edn., John Wiley and Sons, Inc., Singapore,

2004.

4) Ohannesian, L. and Streeter, A.J., In; Handbook of Pharmaceutical Analysis, Marcel

Dekker, Inc., New York, 2002.

5) Connors, K.A., In; A Text Book of Pharmaceutical Analysis, 3rd

Edn., A Wiley-

Interscience Publications, New York, 1982.6) Kalsi, P.S., In; Spectroscopy of Organic Compounds, 6

thEdn., New Age International

Publishers, new Delhi, 2004.

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