UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application in Pharmaceutical Industry Dr. A. Amsavel. UV &Visible Spectroscopy-Absorption Theory Electronic Transitions Beer- Lambert Law Chromophores & Auxochrome Factors Influence the Absorption UV-Vis Spectrophotometer-Instrumentation Operation of the Spectrophotometer Qualification & Calibration Application

1. Principle, Theory, Instrumentation and
UV- VISIBLE
SPECTROPHOTOMETRY
Principle, Theory, Instrumentation and
Application
in Pharmaceutical Industry
Dr. A. Amsavel, M.Sc., B.Ed., Ph.D.

2. An Overview
 Introduction
 UV &Visible Spectroscopy-Absorption Theory
 Electronic Transitions
 Beer- Lambert Law
Beer- Lambert Law
 Chromophores & Auxochrome
 Factors Influence the Absorption
 UV-Vis Spectrophotometer-Instrumentation
 Operation of the Spectrophotometer
 Qualification & Calibration
 Application

3. UV- Vis Spectroscopy
UV-Visible spectrometry is one of the most widely used analytical
technique in Industries as well Academic Research. It is very
powerful and popular method for identification and estimation of
elements & organic molecules.
Advantage:
 Readily available
 Simple & Easy to operate
 Relatively less expensive
 Does not require complex sample preparation
 Require small amount of sample & non-destructive method
 To test wide verity of organic & Inorganic chemicals
 Qualitative and quantitative analysis

4. UV-Vis Spectroscopy
 Spectroscopy is the branch of science dealing the study of interaction of
electromagnetic radiation with matter. Absorption of molecule due to
electromagnetic radiation is based on the energy levels.
 Ultraviolet-visible (UV-Vis) spectroscopy , electronic transition happens
due to interaction electromagnetic radiation and electrons. One or more
of the outer or the bonding electrons promote from a ground state into a
of the outer or the bonding electrons promote from a ground state into a
higher-energy state.
Energy (E) = hν = (hc/λ) × 109
Where h = Planck's constant (6.63 × 10-34J · s)
ν = frequency (Hz), related to the energy change ΔE, induced when
electromagnetic radiation is absorbed (ΔE = hν per photon)
c = velocity of light (2.998 × 108 ms-1)
λ = wavelength (nm)

5. Electromagnetic Radiation
Wavelength

6. UV & Visible Light Absorption Theory
 Ultraviolet and visible (UV-Vis) Spectrophotometry is based on the
ability of atoms, molecules and ions to absorb light at wavelengths
in the ultraviolet (180-400 nm) and visible (400-800 nm) range.
 This absorption is associated with changes in electronic energy in
the form of temporary transitions of electrons to an excited state at
a higher energy orbital. As each energy level of a molecule or
molecular ion also has associated vibrational and rotational sub-
levels.
 Absorption band is characteristic of the functional groups and
bonds in a molecule.

7. Electronic Transitions
 Most of the Organic compounds observed transitions of electrons
in σ or  or n non-bonding electron & orbitals of atoms such as
H, C, N, O etc.
 UV- Vis range only n → ∗ &  → ∗ transition

8. UV-Vis Spectrophotometry
 In UV-Vis spectrophotometry , transmittance T is a measure of the
attenuation of a beam of monochromatic light based upon the
comparison between the intensities of the transmitted light (I) and the
incident light (Io)
Transmittance (T)= I / Io
Transmittance (T)= I / Io
Absorption (A) = log 1/T = log (Io/I) or = - log T
Specific Absorption coefficient Es =A/ Cl or I= Io X10 –EsCl
Molar Absorption coefficient ε =A/Cl
 Quantitative analysis: Laws of molecular absorption is used in all spectroscopic
quantitative Analysis

9. Beer- Lambert Law
 French mathematician Lambert and German physicist Beer proposed a
hypothesis about absorption of molecules by electromagnetic radiation.
 Lamberts Law: Absorbance (A) is proportional to the Path length (l) of the
absorbing medium.
 Beers law: Absorbance (A) is proportional to the Concentration (C) of the
 Beers law: Absorbance (A) is proportional to the Concentration (C) of the
sample.
 Beer- Lambert Law - Absorbance is (A) proportional to Concentration
and Path length of the sample.
 A  Cl ; A = εCl
C-Concentration (Moles /litre) ; l- Path length (cm) & ε - Molar absorption coefficient
(molar absorptivity)
Absorption for a mixture of two compounds A = l x (ε1C1 + ε2C2) = A1 +A2

10. Beer- Lambert Law
“When monochromatic light passes to the transparent,
homogeneous medium, absorption of light is
proportional to the Concentration and Path length of
the sample”.
the sample”.
Beer- Lambert Law will obey:
 the light used must be monochromatic
 the concentrations must be low
 the solution must not be fluorescent or heterogeneous
 the solute must not undergo to photochemical transformations
 the solute must not undertake variable associations with the
solvent.

11. Chromophores
What are the atoms or groups in the molecules shows
responses to UV- Vis spectrum?
 Group /part of a molecule responsible for its Colour is called
Chromophores.
 The functional groups containing multiple bonds capable of
absorbing radia ons above 200 nm due to n → π* & π → π*
transitions.
 Chromophore group:
Eg -C=C-, -C=O, -C=N, -C≡N, -C=S, -NO2, -N=O, etc

12. Auxochrome
Auxochrome:
The functional groups attached to a Chromosphores which
modifies the ability of the Chromosphores s to absorb light ,
altering the wavelength or intensity of absorption.
Auxochrome eg. –OH, -NH2, -OCH3 , -halogens etc
Benzene absorption - λmax = 255 nm
- OH group in Benzene -Phenol λ max = 270 nm
- NH2 group in Benzene Aniline λmax = 280 nm

13. Chromophores in Organic Compounds
Group/
Name
Chromophore λ max (nm) ε max
Amine -NH2 195 2800
Oxime = NOH 190 5000
Nitro -NO 210 3000
Nitro -NO2 210 3000
Nitrite -ONO 230 1500
Nitrate -ONO2 270 12
Nitroso -N = O 300 100
Nitrite -ONO 220-230 1000-2000
Ethylene -C=C- 190 & 195 8,000, 1000

14. Chromophore of Organic Compounds
Group/
Name
Chromophore λ max (nm) ε max
Ketone -C=O 270–285 18–30
Aldehyde -CHO 210, 280–300 Strong, 11–18
Azo -N=N- 285–400 3–25
Azo -N=N- 285–400 3–25
Benzene
184 46,700
202 6,900
255 170
Naphthalene 220 112,000
275 5,600
312 175

15. Factors Influence the Absorption
Bathochromic Shift (Red Shift):
 Absorption maxima (λmax) of a compound shifts to longer
wavelength.
 It is due to presence of an auxochrome or by the change of solvent.
p-Nitrophenol- λ =255nm, but in alkaline medium λ = 265 nm
 p-Nitrophenol- λmax =255nm, but in alkaline medium λmax = 265 nm
Hypsochromic Shift (Blue Shift):
 Absorption maxima (λmax) of a compound shifts to shorter
wavelength.
 It is due to presence of an group causes removal of conjugation or by
the change of solvent.
 Aniline (λmax 280nm) & shows blue shift in acidic medium λmax
265nm. It loses conjugation.

16. Factors Influence the Absorption
Hyperchromic Effect:
 An increase in molar absorptivity (ε) of a compound.
 If Auxochrome introduces in the molecule , the intensity of
absorption increases
absorption increases
 Eg.Pyridine λ max = 257nm & 2-Methyl pyridine-λ max = 260 nm
Hypochromic Effect:
 An increase in molar absorptivity (ε) of a compound.
 Eg. Naphthalene ε = 19000 & 2-Methyl naphthalene ε = 10250

17. Wavelength Shift
RED SHIFT
BLUE SHIFT
HYPERCHROMIC SHIFT
Absorbance
(A)
BLUE SHIFT
HYPOCHROMIC SHIFT
Absorbance
(A)
Wavelength ( λ)
λmax

18. UV & Visible Spectrum Range
Wavelength Range
Absorbed (WL-nm)*
Colour
Absorbed
Colour
Seen By Eye
380 - 450 Violet Yellow - Green
450 - 495 Blue Yellow
450 - 495 Blue Yellow
495 - 570 Green Violet
570 - 590 Yellow Blue
590 - 620 Orange Green - Blue
620 - 760 Red Blue - Green

19. UV-Vis Spectrophotometer
UV-Vis Spectrophotometer consists:
 Light source
 Monochromator or Polychromator
 Sampling area
 Sampling area
 Detector
 Computer with processing
UV-Vis Spectrophotometers are available as single beam or double beam

20. UV-Vis Spectrophotometer
 Light source : Deuterium lamp for the UV region and , a tungsten-
halogen lamp for the visible region or a xenon lamp to cover the
entire UV-Vis range.
 Filters or Monochromators: Wave Selectors
Note: Basic models will have Filters. Gelatin coloured with organic dyes that
are sealed between Glass plates. Monochromators are commonly used.
 Sample Holder Area: To hold the Cuvette (s ) for blank /sample on
the path of monochromatic light.
Cuvettes : Normally 1 cm Rectanle cell made in high-purity quartz
or sapphire transparent to UV-Vis radiation.
Note: Normal Glass absorbs will absorb uv radiation

21. UV-Vis Spectrophotometer
 Slit –entry of polychromatic light from the
source.
 Collimating device – lens or mirror which
helps in reflecting the polychromatic light
Monochromators or Polychromators:
Concave
lens
helps in reflecting the polychromatic light
to the dispersion device.
 A wavelength resolving device – Grating.
Design single beam or double-beam
spectrophotometers requirement
 A focussing lens or mirror
 Exit slit
Grating Monochromator
Focal plane λ2
λ1

22. UV-Vis Spectrophotometer
UV-VIS detectors:
 A detector produces an electric signal when it is struck by photons.
 Phototube emits electrons from a photosensitive, negatively charged
surface (the cathode) when struck by visible light or ultraviolet radiation.
The electrons flow through a vacuum to a positively charged collector
The electrons flow through a vacuum to a positively charged collector
whose current is proportional to the radiation intensity.
 Photoelectric detectors, are the most common. It generate an electric
current that is directly proportional to the intensity of the radiant energy
incident upon them.
 Photosensitive semiconductor devices, either discrete detectors, linear or
two-dimensional arrays, or photomultipliers or photodiodes.
Data processing: connected to Suitable computerised data processing &
evaluation systems.

23. Operation of the Spectrophotometer
 Set spectrum mode or Photometry mode .
 Select the scan range or wavelength max
 Zero correction before starting the analysis .
 Baseline line flatness : ±0.001abs or Photometric noise < 0.001
Baseline line flatness : ±0.001abs or Photometric noise < 0.001
 Select a suitable spectroscopic blank e.g. air, blank solvent, solid
material
 Blank run value may differ by NMT ± 2 nm
 Quantitative measurements: Absorption values less than 2.0
 Wavelength must not exceed 0.4 and is preferably < 0.2nm.
 To improve resolution or sensitivity, use derivative spectra

24. Operation of the Spectrophotometer
 Software used shall compliance with 21 CFR Part 11
 Ensure the calibration is performed and within validity date
 Use 1cm Cell unless otherwise stated.
 Cell used must be clean, no finger print, no deposit or contaminant
Cell used must be clean, no finger print, no deposit or contaminant
 Perform system suitability is required.
 Check the lamp energy
 Use only spectroscopic reagents / solvents

25. Qualification
Ensure the Qualification is performed and documented
before use of the Instrument.
 Design Qualification / URS
 Installation Qualification
 Installation Qualification
 Operational Qualification
 Performance Qualification
Reference guideline for qualification:
PA/PH/OMCL (07) 11 Def Corr: Qualification Of Equipment-
Annex 3: Qualification Of UV-Visible Spectrophotometers

26. Performance Check of Instrument
Method Wavelength
Accuracy
Absorbance
Accuracy
Photometric
Linearity
Stray
Light
Quantitative or limit test*
Based on measurement of the
absorbance at one or more
identified wavelengths (e.g. assay
or impurities test)
X X X X
or impurities test)
Identification test
Based on wavelength of
absorption maxima & minima X - - X
Based on absorption
measurement and wavelength
of absorption maxima
X X - X
Based on comparison of
spectrum with that of
reference substance
X X - -
*Resolution/spectral bandwidth: As required in the monograph

27. Control of Wavelength Accuracy
Material Wavelengths (nm)*
Solutions :
Didymium in Perchloric acid 511.8; 731.6; 794.2
Ensure the control the wavelength accuracy for appropriate number of
bands in the intended spectral range using below reference materials.
511.8; 731.6; 794.2
Holmium in Perchloric acid
(Generally used )
241.1; 287.2; 361.3; 451.4; 485.2; 536.6; 640.5
Solid filters :
Holmium Glass
(Generally used )
279.3; 360.9; 453.4; 637.5
Lamps : Deuterium 486.0; 656.1
Lamp : Mercury (low pressure)
184.9; 253.7; 312.5; 365.0; 404.7; 435.8; 546.1; 577.0;
579.1
Acceptance Limit: 200- 400 ± 1 nm & above 400 nm ± 3 nm .

28. Control of Absorbance
Absorbance Accuracy:
Nicotinic acid solution: Dissolve 57.0-63.0 mg of Nicotinic acid
(RS/CRS) in 0.1 M hydrochloric acid solution and dilute to 200mL.
Dilute 2.0mL of the solution to 50mL. (final conc- 2 mg/L).
 Measured the absorbance at 213nm & 261nm.
Acceptance criteria: The difference between the measured
absorbance and the absorbance of the standard value (RS) should
be ± 0.010 or ± 1 per cent, whichever is greater.
Photometric Linearity :
 Measure the Absorbance at 5.0 - 40.0 mg/L Nicotinic acid solution
of min 4 concentration and determine coefficient.
Acceptance criteria: The coefficient of determination (R²) is not
less than 0.999.

29. Limit of Stray Light
 Stray light is determined at an appropriate wavelength
using suitable solid or Solution
 Use 1 cm cell and water as reference liquid.
 Absorbance of Potassium chloride 12 g/L solution at
 Absorbance of Potassium chloride 12 g/L solution at
198 nm is not less than 2.0
 Sodium iodide 10 g/L solution at 220nm
 Potassium iodide 10 g/L solution at 250 nm or
 Sodium nitrite - 50 g/L solution at 340 nm and 370 nm
Acceptance Limit: Absorbance must not Less than 3.0

30. Cuvettes /Cells
 Cell used should be 1 cm path length.
 Measured value obtained may not differ by ± 2 nm, unless
otherwise prescribed in monograph
 Quantitative measurements relying on absorption values above 2.0
should be avoided.
Acceptance criteria for Cuvettes as per Eu. Pharm
 The apparent absorbance is not greater than 0.093 for 1 cm quartz
cuvettes (UV region) and 0.035 for 1 cm glass cuvettes
(visible region);
 The absorbance measured after rotation (180°) does not differ by
more than 0.005 from the value previously obtained.

31. Control of Resolution
 Measure the resolution of the equipment as per
monograph using reference materials
 Alternately record the spectrum of a 0.02% (v/v) solution
of Toluene in Hexane or in Heptane.
 Use Hexane / Heptane as the compensation liquid.
Acceptance criterion:
 The minimum ratio of the absorbance at the maximum
(269 nm) to that at the minimum (266 nm) is stated in the
monograph.

32. Calibration
Spectrophotometer shall be Calibrated to ensure that Instrument
is performing well and measurement is accurate & reliable.
 Internal calibration
 Match pairing of cells (Cuvette qualification)
Control of wavelength
 Control of wavelength
 Control of absorbance
 Limit of Stray Light
 Resolution Power
 Linearity study
 Use reference standard for calibration is NIST traceable or certified
Refer for further detail Ph. Eur. 10 2.2.25 and USP 42-NF 37 GC <857>

33. Calibration: Match Pairing Cells
Internal Calibration of UV Spectrophotometer :
 Perform the internal Calibration as per manufacturer’s instruction
using software
Match Pairing of Cells (Cuvette Qualification):
 Fill the cell with distilled water and measure the absorbance
against air at 240 nm for quartz cells and 650 nm for glass cells.
 Absorbance should not be greater than 0.093 for 1 cm quartz cells
(UV region) and 0.035 for 1 cm glass cells (Visible region).
 Rotate the cell in its holder (180°) again check the absorbance
 Difference not greater than 0.005 from initial.

34. Calibration :Control of Wavelength:
 Take the UV spectrum of 4%w/v Holmium oxide in 1.4 M Perchloric
acid solution from 200 nm to 600 nm against the 1.4 M Perchloric
acid as a blank.
 Wavelength shall be check for the peak detection of Holmium
 Wavelength shall be check for the peak detection of Holmium
Oxide at 241.15 nm, 287.15 nm, 361.5 nm, 486.0 nm & 536.3 nm.
 The permitted tolerance limit shall be ± 1 nm for the range of 200
nm to 400 nm (UV range) and ± 3 nm for the range of 400 nm to
800 nm.(Visible range)

35. Calibration: Control of Absorbance:
 Take the spectrum of the Potassium dichromate (60ppm) solution between
200 nm to 400 nm using 0.005 M Sulfuric acid as a blank.
 Measure the absorbance of peak detection at 350 nm & 257 nm and Valley
detection at 313 nm & 235 nm.
 Absorbance of the Potassium dichromate 60ppm at 430 nm using 0.005
M Sulfuric acid as a blank in photometric mode
M Sulfuric acid as a blank in photometric mode
 Control of absorbance = (Absorbance X 10000 ) / Wt. Taken in mg.
 Control of absorbance (for λ 430 nm) = (Absorbance X 1000) / Wt (g)X100
Wavelength Maximum Tolerance
235 nm 122.9 nm to 126.2 nm
257 nm 142.8 nm to 146.2 nm
313 nm 47.0 nm to 50.3 nm
350 nm 105.6 nm to 109.0 nm
430 nm 15.7nm to 16.1nm

36. Calibration: Stray Light
 Limit of Stray Light :
 Prepare Potassium chloride of 12,000 ppm in distilled water or Use
certified standard solution
 Measure the absorbance of the potassium chloride solution against
distilled water as a blank between 220 nm and 190 nm in scan mode.
distilled water as a blank between 220 nm and 190 nm in scan mode.
 Check absorbance at 198 nm by keeping cursor.
 Absorbance steeply increases between 220 nm to 200 nm and shall be
more than 2.0 at 200nm
 Resolution Power:
Measure Toluene solution in Hexane (0.02%v/v) refer performance check

37. Calibration :Linearity
Linearity:
 Prepare potassium dichromate using 0.005 M Sulfuric Acid at
20ppm, 40ppm, 60ppm, 80ppm & 100ppm
 Measure the absorbance of the solutions at 257 nm by using
 Measure the absorbance of the solutions at 257 nm by using
0.005 M sulfuric acid as a blank.
 Plot a graph of absorbance verses concentration.
 Co-relation co-efficient R2 shall be > 0.999.

38. Tips to Handle the Cells
 Carefully clean & store properly to avoids contamination.
 Clean Cells with high purity water, if required clean with 1% (v/v) nitric
acid, do not use chromic acid for cleaning.
 Do not use cracked or scratched cells.
 Wipe cells carefully with a soft, clean, lint-free cloth while use.
 Wipe cells carefully with a soft, clean, lint-free cloth while use.
 Contaminated cells are major source of error
 Cells should never be handled by the optical polished faces.
 Should rinse off residual or spilled solution.
 Do’t use Strong Acid or Alkaline solutions. Impact will be based on the pH
and contact time.
 Try to used in the cell same beam ref/ test by marking

39. Application
Method : External standard & Internal standard (calibration), Standard
addition and etc
 Enzymatic Analysis in biochemical and Clinical Lab. Eg Sugar, acids, or their
salts, alcohol etc
 Analysis of nucleic acids, proteins and bacterial cell cultures. To detremine
 Analysis of nucleic acids, proteins and bacterial cell cultures. To detremine
Concentration & Purity of nucleic acids – DNA and RNA
 Enzyme base test kits are readly available
 Food analysis
 Absorbance of co-enzyme NADH or NADPH (340nm)
 Chloesterol in mayonnaise by oxidation method

40. Application
Qualitative & Quantitative Analysis:
 Identification / characterizing aromatic compounds and conjugated
olefins.
 Detection of impurities in organic compound and solvents.
Detection of isomers are possible.
 Detection of isomers are possible.
 Determine of assay , molar concentration of the solute .
 Determination of pKa
 Determination of molecular weight using Beer’s law.
 Determination of most of metal ions, by preparing coloured complex with
suitable ligand. Eg. Iron – 1,10 Phenanthroline

41. Analysis of Pharmaceuticals
Analysis of Pharmaceutical ingredients
 Pseudoephedrine hydrochloride
 Triprolidine hydrochloride
 Codeine , Morphine
 Vitamins
Pharmaceutical dosage form
Pharmaceutical dosage form
 Simultaneous Equation method for determination of binary / ternary mixture in
 Rabeprazole sodium and Levosulpiride at 284 nm, 232 nm methanol
 Tenofovir, Efavirenz, and Lamivudine at 260 nm, 347 nm, 272 nm (methanol)
 Difference Spectrophotometry
 Pioglitazone and Metformin phosphate buffer (pH 9) and chloride buffer (pH 2) 228.1 nm
and 228.2 nm
 Zero crossing technique to analysis of binary mixtures
 Gatifloxacin and Prednisolone 348 and 263 nm

42. Reference
 Analytical Chemistry -7th Edition. Gary D. Christian, Purnendu K. (Sandy)
Dasgupta & Kevin A. Schug
 Quantitative Chemical Analysis. 7th Edition Daniel C. Harris
 Chemical Analysis: Modern Instrumentation Methods and Techniques
Francis and Annick Rouessac and Steve Brooks, 2007- John Wiley & Sons Ltd,.
Vogel’s – Quantitative Chemical Analysis- 6th edition
 Vogel’s – Quantitative Chemical Analysis- 6th edition
 PA/PH/OMCL (07) 11 Def Corr: Qualification Of Equipment-Annex 3:
Qualification Of UV-Visible Spectrophotometers
 European Pharmacopeia General Chapter 2.2.25. Absorption
Spectrophotometer, Ultraviolet And Visible
 USP General Chapter <857> and <1857> Ultraviolet-Visible Spectroscopy &
Theory And Practice
 Ultraviolet Spectroscopy and its Pharmaceutical Applications- A Brief review
Dipali M Atole et al ; Asian J Pharm Clin Res, Vol 11, Issue 2, 2018, 59-66