photoelectrically

1. Spectrophotometry
Dr.Gurumeet C Wadhawa ,Assistant
Professor, Department of Chemistry.
Rayat Shikshan sansthas Veer Wajekar ASC
College,Phunde,Uran

2. Terms:/Parameters
Transmittance : The passing of light through a sample
Absorbance: Amount of light absorbed by a sample (the
amount of light that does not pass through or reflect off
a sample)
%Transmittance: The manner in which a
spectrophotometer reports the amount of light that
passes through asample
Absorbance units: A unit of light absorbance
determined by the decrease in the amount of light in a
light beam
Absorbance spectrum: A graph of a sample’s
absorbance at different wavelengths
Lambdamax The wavelength that gives
absorbancevalue for a sample
the highest

3. Absorption: The Beer-LambertLaw
Pierre Bouguer
Astronomer: Light
isdiminished asit
passesthrough the
atmosphere.
August Beer(1825-1863): Added
absorptionco-efficient and related to
conc.in solution.
JohanLambert
Mathematician, first to
prove that  isirrational.
No absorptioncoefficient
A  log(I1 / I0 )  cl
€: Extinctioncoefficient
c: Concentration l :Pathlength

4. BEER–LAMBERT’SLAW
(Beer–Lambert–Bouguer law)
• Relates the absorption of light to the properties of the material
through which the light istravelling.
BEER'SLAW
• When monochromatic light (light of aspecific wavelength) passes
through a solution there is usually a quantitative relationship
between the solute concentration and the intensity of the
transmitted light
The amount of light absorbed by the a medium ( solution/ sample)
is proportional to the concentration of the absorbing material or
solute present.
Thus the concentration of a coloured solute in a solution may
be determined in the lab by measuring the ABSORBANCY OF
LIGHTATAGIVEN WAVELENGTH
•
•

5. BEER–LAMBERT’SLAW(Beer–Lambert–Bouguer law)
….contd
LAMBERT'SLAW
Lambert described how intensity changes
with distance in an absorbingmedium.
o The amount of light absorbed by
medium ( solution/ sample) at a
the a
given
wavelength is proportional to thickness of the
absorbing layer: path length of thelight

6. Beer –Lambert Law
States that the Absorbance (O.D) of a solution is
directly proportional to the concentration of the
absorbing species in the solution and the path
length.
The fraction of the incident light absorbed by a solution at a given wavelength is
related to
a. thickness of the absorbing layer (path length) and
b. concentration of the absorbing species

7. intensity(power) of the incident light intensity(power) of the
transmitted light ; ℓ . thickness of the absorbing layer (path length) and
cross section of light absorption by a single particle;
Transmittance
Defined as the ratio of the intensity of light emerging from the
solution (I) to that of incident light entering (Io)
Thereis a logarithmic dependence between the transmission (or
transmissivity), T, of light through a substance and
The product of : the absorption coefficient of the substance, α,
and the distance the light travels through the material (i.e. the
path length), ℓ.
• The ABSORPTION COEFFICIENT: (α ) =
Molar absorptivity (extinction coefficient) of the absorber, (c)
the concentration (c) of absorbing species in the material

8. T- Transmittance
T =
I
0
I - Original lightintensity
I- Transmitted lightintensity
% Transmittance (T)= 100 x
I
Absorbance (A) = Log
(OPTICAL DENSITY)
= Log = KCL
I0
I0
I0
I
1
T
I0
I

9. By definition ofthe Beer - Lambert Law.
α =
A= α
A= ECL
A = Transmission/Transmissivity ;expressed interms
of Absorbance (numerical number only)- (OPTICAL
DENSITY)
E = Molar Extinction Coefficient of the absorber ( )-
Extinction Coefficient of a solution containing 1g
molecule of solute per 1 liter of solution
L= length of light path through the solution

10. IMPLICATIONSOFBEER-LAMBERTSLAW
• The absorbance (A) becomes linear with the
concentration ( C; number density of absorbers)
• Thus,if the path length and the Molar absorptivity
ae known; & the absorbance is
measured: The
concentration of the substance (or the number
density of absorbers) can beobtained.
As Concentration (C) increases, light Absorption
(A) increases, LINEARL
Y
As Concentration (C) increases, light
Transmission (T) decreases: EXPONENTIALL
Y
(INVERSLY)
•
•

12. As Concentration
LINEARL
Y
(C) increases, light Absorption (A) increases,
.
·As Concentration (C) increases, light Transmission (T) decreases,
EXPONENTIALL
Y

14. PRINCIPLESANDAPPLICATIONOFSPECTROPHOTOMETRYIN DISEASEDIAGNOSIS
Absorption : UV/Visible/IR
• Certain molecules absorb light in a characteristic way: helps
to identify and quantify biologicalmolecules
Absorption occurs when the energy contained in a photon is
absorbed by an electron resulting in a transition to an excited
state
•
• The absorption efficiency of an analyte is affected by: The
nature of the analyte, number of available microstates, The
solvent
Absorption spectroscopy: Bioanalytical methods; signal
intensity is directly proportional tothe concentration
•

16. E
ntra
nceslit
E
xitslit
D
e
te
cto
r
C
u
ve
tte
R
e
d
V
io
le
t
P
r
ism
M
o
n
ochr
o
m
ato
r
L
ig
h
tsou
r
ce
I0
I
R
e
a
d
o
u
t
d
e
vi
ce

17. COMPONENTSOFSPECTROPHOTOMETER
1. Light source(UV and visible)
2. Optical system/Wavelength selector
(Monochromator)
3. Samplecontainers
4. Detector
5. Output: Signalprocessor andreadout

18. • Pigment Chlorophyll- which absorbs light;
in the blue and red region of the visible light
spectrum.
• For this reason, leaves are- green (because
they reflect green).
• If Leaf is extracted in an organic solvent, the
leaf extract (containing the solute chlorophyll)
with a high chlorophyll content will produce:
dark green colour
• A leaf extract with a low chlorophyll content
will yield a pale green extract.
Spectrophotometry is
• a mean of measuring how densely green
the solution is.(concentration)

19. The study how the chemical compound interacts with
different wavelenghts in a given region of electromagnetic
radiation
Spectrophotometry
reflection or transmission properties of a material as a
function of wavelength.; Involves the use of a
spectrophotometer.
SPECTROPHOTOMETER :
•A device that is used to measure intensity of light as a function
of the wavelength oflight.
•An instrument that measures the amount of light of a specified
wavelength that passes through (is transmitted through) a sample
( )
SPECTROSCOPY / SPECTROCHEMICAL ANALYSIS.

20. DIAGRAMOFSPECTROPHOTOMETER

21. TheSpectrophotometer
Single-beam
Double-beam

22. Entranceslit Detector
Cuvette
Red
Violet
Pr
ism
Monochromator
Lightsour
ce
I0
I
Readout
device
producing light of any selected wavelength or color
Spectrometer: for
Photometer: used for measuring the intensity of light.
Thetwo devices are placed at either side of acuvette filled withaliquid
Exitslit

23. Colors & Wavelengths
COLOR WAVELENGTH (λ in nm)
Violet 380 – 435
Blue 436 – 480
Greenish-blue 481 – 490
Bluish-green 491 – 500
Green 501 – 560
Yellowish-green 561 – 580
Yellow 581 – 595
Orange 596 – 650
Red 651 – 780
si
V

24. SPECTROPHOTOMETRY
• A photometer (a device for
measuring light intensity)
• Measure intensity as a
function of the color, or more
specifically, the wavelength of
light
Tungsten or xenon flashlamp
as the source of white light
•
• Tungsten
measurements
lamp for
in visible
region(360-900nm)
Hydrogen /deuterium lamp
for UV region(200-380nm)
•
COLORIMETRY
•
•
T hemeasurement of color
Any technique used to evaluate an
unknown color in reference to
known colors
• It determines color based on the red,
blue, and green components of light
absorbed by the object or sample,
•
optical filter, which transmits
Colored light beam through an
only
one particular color / band of
wavelengths of light to the
photodectector

25. Spectroscopy and Spectrophotometry
• Light can either be transmitted or absorbed by dissolved
substances
• Presence & concentration of dissolved substances is analyzed
by passing light through thesample
• Spectroscopesmeasure electromagnetic emission
•
•
Spectrophotometers measure electromagnetic absorption
Principle:BasedonBeerLambert’s LAW

26. Spectrometer produces the light of desired wavelength and it
passesthrough the tube and reaches photometer that measures its
intensity.
Then the photometer produces a voltage signal to a display
device, usually agalvanometer.
As the amount of light absorbed by the liquid changes; the
signal also changes.
The concentration of a substance in solution can be measured by
calculating the amount of absorption of light at the appropriate
wavelength or aparticular colour
Readingof Spectrophotometer: (Number)-Absorbancethat is
directly proportional to the color intensity, and also the
concentration of the species responsible for the color.

27. • To use absorbance for analytical purposes, a calibration
curve must be generated by measuring the absorbance
of several solutions that contain known concentrations of
analyte.
If development of color is linked to the concentration of
a substance in solution then: That concentration can be
measured by determining the extent of absorption of
light at the appropriatewavelength.
Forexample: Hemoglobin appears red
•
• Hemoglobin absorbs blue and green light rays much
more effectively than red.)
Thus, Thedegree of absorbance of blue or green light is
proportional to the concentration of hemoglobin.

28. 1) Wavelength Selection Using Filters The simplest method for isolating a
narrow band of radiation is to use an absorption or interference filter.
Absorption filters work by selectively absorbing radiation from a narrow region
of the electromagnetic spectrum. Interference filters use constructive and
destructive interference to isolate a narrow range of wavelengths. A simple
example of an absorption filter is a piece of colored glass. A purple filter, for
example, removes the complementary color green from 500–560 nm.
Commercially available absorption filters provide effective bandwidths
from 30–250 nm.
The maximum throughput for the smallest effective bandpasses, however, may
be only 10% of the source’s emission intensity over that range of wavelengths.
Interference filters are more expensive than absorption filters, but have
narrower effective bandwidths, typically 10–20 nm, with maximum throughputs
of at least 40%.

29. Wavelength Selection Using Monochromators One limitation of an absorption
or interference filter is that they do not allow for a continuous selection of
wavelength. If measurements need to be made at two wavelengths, then the
filter must be changed in between measurements. A further limitation is that
filters are available for only selected nominal ranges of wavelengths. An
alternative approach to wavelength selection, which provides for a continuous
variation of wavelength, is the monochromator.

30. WORKINGOFSPECTROPHOTOMETER
• White light radiation source that passes through a MONOCHROMATOR ( prism
or a diffraction grating that separates the white light into all colors of the
visible spectrum) .
After the light is separated, it passes through a FIL
TER (to block out unwanted
light, sometimes light of a different color) and a SLIT (to narrow the beam of
light).
Next the beam of light passes through the SAMPLE that is in the sample
holder.(cuvette)
The light passes through the sample and the unabsorbed portion (reflected)
strikes a PHOTODETECTOR that produces an electrical signal which is
proportional to the intensity of the light.
The signal is then converted to A READABLE OUTPUT (absorbance )that is
usedin the analysis of thesample.
•
•
•
•
• Calibration curve : generated by measuring the absorbance of several
solutions that contain known concentrations ofanalyte.

31. • Deuterium Lamps － Continuous spectrum in the ultraviolet region is
produced by electrical excitation of deuterium at low pressure. (160nm-
375nm)
Tungsten Filament Lamps － the most common source of visible and
near infrared radiation ( at wavelength 320 to 2500 nm)
Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from
200 to 800 nm)- in UV Spectrophotometer
Silicon Carbide (SiC) Rod : Radiation at wavelengths:1200 -40000
nm
•
•
•
•
• NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm)
– for IR Region:
Laser: Used when high intensity line source is required
COMPONENTS OF SPECTROPHOTOMETER
1. LIGHTSOURCE

32. E
n
t
ra
n
c
es
lit
E
x
it
s
lit
D
e
t
e
c
t
o
r
C
u
v
e
t
te
R
e
d
V
io
le
t
P
ris
m
M
o
n
o
c
h
ro
m
a
to
r
L
ig
h
ts
o
u
rc
e
I0
I
R
e
a
d
o
u
t
d
e
v
i
c
e
OPTICALSYSTEM/WAVELENGTHSELECTOR
MONOCHROMATOR
•
•
•
•
Opticaldevice
Dispersesabeam of
light into its
component
wavelengths
Allows only anarrow
band of wavelengthsto
pass
Blocksall other
wavelengths
1. Anentrance slit
2. A collimating
lens (concave)
3. A dispersing device
(usually a prism or a
grating)
4. Afocusinglens
5. Anexit slit

33. MONOCHROMATOR
•Czerny-Turner setup
•AS A FILTER: It will select a narrow portion of the spectrum
(the bandpass) of agivensource.
•IN ANAL
YSIS:the monochromator will sequentially select for
the detector to record the different components
(spectrum) of any source or sample emittinglight.
• Mirror collimates light (parallelrays)
• Gating disperses light ( Prismswere formerlyused)
• Light coming through entrance slit ispolychromatic
• Light out ofexit slit is monochromatic

34. CUVETTES ( SAMPLE CONTAINERS)
• The containers for the sample- usuallyplastic or quartz:
•Reference solution must be transparent to the radiation which will
pass through them.
• Quartz or fused crystalline silica cuvettes for UV spectroscopy .
• Glass cuvettes for Visible Spectrophotometer
• NaCl and KBr Crystals for IR wavelengths

35. CellTypesI AND II

36. A photoemissive c
a
t
h
Do
d
ee
t(
eac
c
a
t
t
h
oo
r
d
e
swhich emits electrons
•
when struck by photons)
• S
everal dynodes (which emit several electrons for each
electron striking them)
An anode.
•
• Produces an electric signal proportional to the radiation
intensity
Signalis amplified and made available for directdisplay
Asensitivity control amplifies the signal
•
•
• Examples: Phototube (UV); Photomultiplier tube (UV-Vis);
Thermocouple (IR); Thermister (IR)

38. 5. OUTPUT:SIGNALPROCESSORANDREADOUT
(DISPLAYDEVICE)
DISPLA
YDEVICE(Output device)
• Consist of a moving–coil meter or a pen
recorderdisplaying %transmission (%T).
• At present: Instrument control, operation,
standardization and data processing or
storage: carried out by a microcomputer or
microprocessorbuilt in or interfaced toit.

39. Steps in working with spectrophotomoter
When warming up the spectrophotometer, there should be no cuvettes in the
machine
Preparation of samples
Aseries of standard solutions of known concentration
of maximum lightabsorption
•
Set spectrophotometer to wavelength
Measure light absorbance of standards
Setthe %transmittance of light as0%
In the sample space,lodge acuvette, filled with solvent and close the sample
space.
Setthe transmittance at 100%
For comparing, fill the cuvette with sample and place it in sample spaceand
close the sample space.
Note down the reading on the Photometer for calculations.
Plot standard curve: Absorbance vs.Concentration
Calculating the concentration of sample using Beer Lambert Equation:
A =ECL

40. Measuring the Absorbance

41. MEASURINGTHECONCENTRATION USINGSTANDARD

42. DIFFERENTTYPESOFSPECTROPHOTOMETERS
ClassificationBased on:
Different measurement techniques Differ with respect to the
speciesto be analysed (such asmolecular or atomicspectroscopy)
The sources of intensity variation: Type of radiation-matter
interaction to be monitored (such as absorption, emission, or
diffraction)
The region of the electromagnetic spectrum (The wavelengths
they work with )used in the analysis
· Based on the absorption or emission of radiation, in the
ultraviolet (UV), visible (Vis), infrared (IR), and radio (nuclear
magnetic resonance, NMR) frequency ranges are most commonly
encountered

43. TYPES AND APPLICATIONS OF SPECTROPHOTOMETER
• Primarily used for QUANTITATIVE Analysis
of Known Compounds

44. Tissueabsorption
Major tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water,
proteins, blood components, body fluids
Oxy and deoxy hemoglobin have distinct spectra. Optical measurements can provide
information on tissue oxygenation, oxygen consumption, blood hemodynamics

45. APPLICATIONS OF SPECTROPHOTOMETER
Forensic sciences.
Molecular biology: in measuring the growth of micro
organisms like bacteria.
UV-Vis : Most Popular in Pharmaceutical, Foods and
Paints Industries, Water Laboratories
In Disease diagnosis/ Pathological states (changes):
detected by the analysis of various samples.,taken from
the body : are analyzed in three different areas –
Chemistry, Hematology and Microbiology section
Blood (the blood plasma, and the formed elements – the
bloodcells)- Themost common substance for analysis

46. TYPESANDAPPLICATIONOFSPECTROSCOPY…contd
Types of Spectroscopy
AbsorptionSpectroscopy :
The power of a beam of light measured before and after
interaction with asample iscompared.
Specific absorption techniques tend to be referred to by the
wavelength of radiation measured such as ultraviolet, infrared or
microwaveabsorptionspectroscopy
Absorption occurs when the energy of the photons matches the
energy difference between two states of the material.
The absorption of ultraviolet radiation by molecules is
dependent upon the electronic structure of the molecule. Sothe
ultraviolet spectrum is called electronicspectrum

47. Ultraviolet Spectroscopy
All atoms absorb in the Ultraviolet (UV) region because these
photons are energetic enough toexcite outer electrons
Used in quantifying protein and DNA concentration, the ratio of
protein to DNAconcentration in a solution Amino Acids (aromatic),
Pantothenic Acid, Glucose Determination and Enzyme Activity
(Hexokinase)
Several amino acids usually found in protein, such as tryptophan,
absorb light in the 280 nm range and DNAabsorbs light in the 260
nm range. (Ratio of 260/280 nm absorbance- general indicator of
the relative purity ofasolution)
Used as a detector for high performance liquid chromatography
(HPLC). The presence of an analyte gives a response which can be
assumedto be proportional tothe concentration

48. VisibleSpectroscopy
Many atoms emit or absorb visiblelight.
In order to obtain afine line spectrum, the atoms must be in
agas phase.
Thismeans that the substance hasto bevaporised.
Thespectrum is studied in absorption oremission.
Often combined : UV absorption spectroscopy in UV/Vis
spectroscopy.
Applications- Estimation of : Niacin, Pyridoxine, Vitamin
B12, Metal Determination (Fe), Fat-quality Determination
(TBA)and EnzymeActivity (glucose oxidase)

49. Infrared Spectroscopy
• The IR spectral region Further subdivided into ; near-infrared
(NIR), mid-infrared (MIR), and far-infrared (FIR) based on
wavelength.
: most familiar to the organic chemist as
offers the possibility to measure different types of
interatomic bond vibrations at differentfrequencies.
In organic chemistry the analysis of IR absorption spectra
shows types of bonds are present in thesample.
•
• : Most common clinical analytical tests,
those involving serum, wholeblood, and urine.; fluids that
are lesscommonly assayed (e.g. saliva and amniotic fluid)

50. Near /Mid InfraredSpectroscopy
• Near Infrared Spectroscopy : immediately beyond the
visible wavelength range, -Much greater penetration depth into the
sample than in the caseof mid IRspectroscopyrange.
Allows large samples to be measured in each scan
•
Practical applications : Medical diagnosis,,
pharmaceuticals/medicines, biotechnology, genomics analysis,
proteomic analysis, interatomics research, inline textile monitoring,
food analysis and chemical imaging/hyperspectral imaging of intact
organisms, agricultural: rapid grain analysis; insectdetection
• Forensic lab application, crime detection and various military
applications.
To identify changes in biofluid metabolite concentrations reflecting
site and mechanism-specific toxicity, to define novel indices of toxic
insult, to evaluate control data, to monitor disease progression and
response to therapeutic intervention and to track progression and
regression of toxin-induced lesions over atimeperiod
•

52. X-Ray Spectroscopy
• When X-rays of sufficient frequency (energy) interact with a
substance, inner shell electrons in the atom are excited to
outer empty orbitals, or they may be removed completely,
ionizing the atom.
The inner shell "hole" will then be filled by electrons from
outer orbitals.
The absorption or emission frequencies (energies) are
characteristic of the specificatom.
Used in chemistry and material sciences to determine
elemental composition and chemicalbonding.

53. AtomicAbsorptionSpectroscopy -
Uses a pre-burner nebulizer (or nebulizing chamber) to
create a sample mist and a slot-shaped burner that
gives alonger path length flame.
The nebulizer and flame are used to desolvate and
atomize the sample, but the excitation of the analyte
atoms is done by the use of lamps shining through the
flame at various wavelengths for each type of analyte.
Theamount of light absorbed after going through the
flame determines the amount of analyte in the sample.
Agraphite furnace for heating the sample to desolvate
and atomize is commonly used for greatersensitivity.
Used for trace
elements in aqueous (and other liquid)samples.

54. PhotoEmissionSpectroscopy
Photoelectron spectroscopy
Refers to energy measurement of electrons emitted
from solids, gases or liquids by the photoelectric effect,
in order to determine the binding energies of electrons in
a substance.
Various techniques, depending on whether the
ionization energy is provided by an

55. MassSpectroscopy
•
•
Unique among the various techniques
Mass spectrometry: Highly sensitive detection and
identification technique, allowing determination of molecular
structures, and thus of asample’scomposition
, it can only determine mass (mass-to-
charge ratio (M/Z) for aparticle ingasphase.)
.For most massspectrometers, Zis equal to 1 sothat mass
can be determined
Involves the interaction of electromagnetic radiation or some
form of energy withmolecules.
•
•
•
• The molecules absorb the radiation and produce a spectrum : during
absorption processor asthe excited molecules return to the groundstate.

56. Mass Spectrometry
The Components of a Mass Spectrometer
1. Ion Source
2. Analyser
3. Detector
4. Data

57. Mass Spectrometry
Provides Information on
1. Molecular Mass
2. Molecular Structure(fragmentation)
3. Elemental composition

58. MSApplications
Non-biomedical
•
•
•
•
•
•
•
Pollutant Analysis
TraceMetal Analysis
ExplosiveAnalysis
Illegal DrugDetection
AlcoholAnalysis
OrganicChemicalAnalysis
Inorganic ChemicalAnalysis
Biomedical
•
•
•
•
•
•
ProteomicAnalysis
DNAsequencing
DNAfingerprinting for ForensicApplications
Biomolecule structure analysis
PolysaccharideAnalysis
Metabolomic Analysis and PharmacologicalApplications

59. Mass Spectrometry
Excellent in separation and quantitation
Poor in identification
M S (Mass Spectrometer)
Excellent in identification and quantitation
Poor in separation
Excellent in separation, identification and
quantitation!
Hyphenated techniques; GC-MS
G C (Gas Chromatograph)

60. RamanSpectroscopy
• Interactions between matter and electromagnetic radiation also
give rise to scattering processes, such as elastic scattering, and
inelastic scattering
It relies on inelastic scattering, or Raman scattering, of
monochromatic light, usually from a laser
•
• The laser light interacts with molecular vibrations, phonons or
other excitations in the system, resulting in the energy of the
laser photons being shifted up or down.
The shift in energy gives information about the phonon modes
in the system.
Thisprocess,it takes place with no change infrequency
for the radiation forming the beam involved.
To study vibrational, rotational, and other low-frequency
•
•
•
•

61. NuclearMagnetic ResonanceSpectroscopy
• Analyses the magnetic properties of
certain atomic nuclei to determine different
electronic local environments of hydrogen,
carbon, or other atoms in an organic
compound or other compound
• Used to determine the structure of the
compound.

62. APPLICATIONSOFNMRIN MEDICINE
BRAIN
Distinguishing gray matter & whitematter
Imaging posterior fossae, brain stem, spinal cord
Detect demyelinating lesions, tumors, hemorrhages, infarctions
ABDOMEN
Distinguishing renal cortex & medulla
Toevaluate transplanted kidney
Differentiates between BPH& prostatic carcinoma
Detects bladder tumours
1. Metabolic liver disease
2. Measures liver iron over load inhemochromatosis
3. Focalareasof inflammation in chronic activehepatisis
KIDNEYS
PELVIS
HEART
o
o
Tomographic imagesof heart muscle, chambers, valvular structures
Discrimination between infarcted, ischemic & normalmyocardium
o