A POWER POINT PRESENTATION ON

SPECTROMETER
• A spectrophotometer is an instrument used to measure the intensity of light as a function of its wavelength. The
basic principle of a spectrophotometer is based on the Beer-Lambert law, which states that the amount of light
absorbed by a solution is directly proportional to the concentration of the solution and the path length through which
the light travels.

• A typical spectrophotometer consists of three main parts: a light source, a monochromator, and a detector. The light
source emits a beam of light that is passed through a sample solution. The monochromator is used to select a
specific wavelength of light from the beam, and the detector measures the amount of light that passes through the
sample at that specific wavelength.

• The amount of light absorbed by the sample is determined by comparing the intensity of the light before and after it
passes through the sample. This is accomplished by using a reference sample, which is a solution with a known
concentration of the substance being analyzed. The reference sample is used to calibrate the instrument and
ensure accurate measurements.

• The spectrophotometer generates a graph known as a spectrum, which displays the amount of light absorbed by
the sample at each wavelength. The spectrum can be used to determine the concentration of the substance being
analyzed, as well as to identify the substance based on its characteristic absorption spectrum.
 Review on properties of light: photon
Light is energy in the form of electromagenetic field

• 1
Wavelength (): Crest-to-crest distance between waves
Frequency (): Number of complete oscillations that the wave makes each second
units: number of oscillations/sec or s-1 or Hertz |(Hz)

Light travelling speed:

in other media: c/n (n = refractive index, generally >1)
in a vacuum: c=2.998 x 108 m s-1 (n=1 exactly, in air n=1.0002926)

c/n= 

And of course, the relationship between energy and frequency:

E = h = hc/ = hc  ~
h = Planck’s constant (6.626 x 10-34 J s)
~= wavenumber (most common units = cm-1)

Therefore:
Energy is inversely proportional to wavelength
but proportional to wavenumber
 Frequency Scanning Techniques: a few
definitions
Emission method: source of light is sample

• 2
Absorption method: intensities of a source with and without the sample in place are
compared

Spectrum: a plot of intensity vs. frequency/wavelength

In quantitative analysis:
common to work at 1 wavelength
running a spectrum is an important initial step (to select best conditions)
Regions of Electromagnetic Spectrum-the “colour” of
light
 Electronic structures of simple molecule
Energy

Excited state
Singlet
S1

T1 Excited state
Vibration states

Triplet

D
Dissociated states

S0 Bond length

Ground state
Key concept from energy diagram
• Electronic structures

• Singlet and triplet

• Bond length for ground and excited states

• Vibrational structures-infrared absorption/transmission (FTIR)

• Internal conversion

• Intersystem crossing

• Photon adsorption excitation (Beer’s law, UV-vis)

• Frank Condon condition and The Stokes' shift

• Radionless relaxation and vibration relaxation

• Luminescence-fluorescence/phosphorescence
Type of optical spectroscopy

• UV-vis absorption spectroscopy (UV-Vis)

• FT-IR absorption/transmission spectroscopy (FTIR)
• Atomic absorption spectroscopy (AAS)
• Atomic fluorescence spectroscopy (AFS)
• X-ray fluorescence spectroscopy (XFS)
Optical spectrophotometer components
Optical spectrophotometer components
3. Excitation sources

Deuterium Lamp UV Detectors

Tungsten Lamp
UV-vis PMT
Laser X-ray, UV, vis, IR
Monochromators
CCD/CID
X-ray tube X-ray Filters
Mercury lamp Grating+slit Photodiode
UV-vis
prism Thermocouple
Xenon lamp UV-vis
Silicon carbide globar IR MCT
Flame Pyroelectric detector
Furnaces
Plasmas
Hollow-cathode lamp

What is the advantage and disadvantage?

 Design of optical spectrophotometers
Single Beam vs. Double Beam
Q: what’s the advantage of double beam spectrophotometer?
(a) single-beam design
(b) dual channel design with beams separated in space but
simultaneous in time
(c) double-beam design in which beams alternate between two
channels."

(a)

(c)

(b)

"Instrument designs for photometers and spectrophotometers”

 Light sources Brightness
Line width
What is the important properties of a source? Background
Black-body radiation for vis and IR but not UV Stability
- a tungsten lamp is an excellent source of black-body radiation Lifetime
- operates at 3000 K
- produces  from 320 to 2500 nm ( How much in cm-1, J, Hz and eV?)
For UV:
- a common lamp is a deuterium arc lamp
- electric discharge causes D2 to dissociate and emit UV radiation (160 – 325 nm)
- other good sources are:
Xe (250 – 1000 nm)
Hg (280 – 1400 nm)

Lasers:
- high power
- very good for studying reactions
- narrow line width
- coherence
- can fine-tune the desired wavelength (but choice of wavelength is limited)
- £££ expensive £££
 Sample a source containers:
for UV: quartz (won’t block out the light)
for vis: glass [ 800nm (red) to  400 nm (violet)]
for IR: NaCl (to or 15384 nm or 650 cm-1)
KBr (to 22222 nm or 450 cm-1)
CsI (to 50000 nm or 200 cm-1)

Best material: diamond, why?

Optical transmission coefficient

Criteria
High transmission
Chemically inert
Mechanically strong
 Monochromators
Early spectrophotometers used prisms
- quartz for UV
- glass for vis and IR Why?

These are now superseded by:

http://www.ii.com/images/prism.jpg
Diffraction gratings:
- made by drawing lines on a glass with a diamond
stylus
ca. 20 grooves mm-1 for far IR
ca. 6000 mm-1 for UV/vis
- can use plastic replicas in less expensive
instruments
Think of diffraction on a CD

http://www.mrfiber.com/images/
cddiffract.jpg
10mx10m
 Monochromators: cont’d
What is the purpose of concave mirrors?
Polychromatic radiation enters
The light is collimated the first concave mirror
Reflection grating diffracts different
wavelengths at different angles

Second concave mirror focuses each wavelength at

different point of focal plane
Orientation of the reflection grating directs only one
narrow band of wavelengths to exit slit

http://oco.jpl.nasa.gov/images/grating_spec-br.jpg
 Interference in diffraction
d sin()+d sin()=n

d
Bragg condition

Phase relationship
>0 
<0  n=1, 2, 3 In-phase
n=1/2, 3/2, 5/2 out-phase
Monochromators: reflection grating
 Monochromators: reflection grating
Each wavelength is diffracted off the grating at a different angle

Angle of deviation of diffracted beam is wavelength dependent  diffraction grating

separates the incident beam into its constituent wavelengths components

Groove dimensions and spacings are on the order of the wavelength in question

In order for the emerging light to be of any use, the emerging light beams must be in phase
with each other

Resolution of grating:  n: diffraction order

=nN
 N: number of illuminated groves

Angular resolution:
As: d sin()+d sin()=n
So: n =d cos() 
Therefore: =n/[d cos()]
 Monochromators: slit
Bottom line:
- it is usually possible to arrange slits and
mirrors so that the first order (n = 1) reflection
is separated
- a waveband of ca. 0.2 nm is obtainable

However, the slit width determines the

resolution and signal to noise ratio

Large slit width: more energy reaching the

detector  higher signal:noise

Small slit width: less energy reaching the

detector BUT better resolution!
 Detectors : Radiation-----charger converter

Choice of detector depends upon what wavelength you are studying

Want the best response for the wavelength (or wavelength range) that you are studying
In a single-beam spectrophotometer, the 100% transmittance control must be adjusted
each time the wavelength is changed
In a double-beam spectrophotometer, this is done for you!
 Detectors : Radiation-----charger converter

Choice of detector depends upon what wavelength you are studying

Want the best response for the wavelength (or wavelength range) that you are studying
In a single-beam spectrophotometer, the 100% transmittance control must be adjusted
each time the wavelength is changed
In a double-beam spectrophotometer, this is done for you!
 Photomultiplier-single channel, but very high sensitivity

- Light falls on a photosensitive alloy

(Cs3Sb, K2CsSb, Na2KSb)
- Electrons from surface are
accelerated towards secondary
electrodes called dynodes and
gain enough energy to remove
further electrons (typically 4-12,
to 50 with GaP).

- For 9 stages giving 4 electrons for 1,

the amplification is 49 or 2.6 x 105)

- The output is fed to an amplifier

which generates a signal
- To minimise noise it is necessary to
operate at the lowest possible
voltage

What decide the sensitive wavelength?

 Photodiode Array-multiplex, but low sensitivity

Good for quick (fraction of a second) scanning of a full spectrum

Uses semiconductor material:
Remember: n-type silicon has a conduction electron – P or As doped
p-type silicon has a ‘hole’ or electron vacancy – Al or B doped

A diode is a pn junction:
under forward bias, current flows from
n-Si to p-Si
under reverse bias, no current flows
boundary is called a depletion layer or
region
 Photodiode Array
- Electrons excited by light partially discharge the condenser
- Current which is necessary to restore the charge can be detected
- The more radiation that strikes, the less charge remains
- Less sensitive than photomultipliers  several placed on placed on single crystal
- Different wavelengths can be directed to different diodes
- Good for 500 to 1100 nm
- For some crystals (i.e. HgCdTe) the response time is about 50 ns

Could you compare photodiode with CCD detector?

 Photodiode Array Spectrophotometer
- For photodiode array spectrophotometers, a white light passes through sample
- The grating polychromator disperses the light into the component wavelengths
- All wavelengths are measured simultaneously
- Resolution depends upon the distance between the diodes and amount of dispersion
 Photodiode Array Spectrophotometers
vs Dispersive Spectrophotometers

Dispersive Spectrophotometer:
- only a narrow band of wavelengths reaches the detector at a time
- slow spectral acquisition (ca. 1 min)
- several moving parts (gratings, filters, mirrors, etc.)
- resolution: ca. 0.1 nm
- produces less stray light  greater dynamic range for measuring high absorbance
- sensitive to stray light from outside sources i.e. room light

Photodiode Array
Spectrophotometer:
- no moving parts  rugged
- faster spectral acquisition (ca.
1 sec)
- not dramatically affect by room
light

What are the components 1 to 10?

 Photodiode Array Spectrophotometers
vs Dispersive Spectrophotometers

Dispersive Spectrophotometer:
- only a narrow band of wavelengths reaches the detector at a time
- slow spectral acquisition (ca. 1 min)
- several moving parts (gratings, filters, mirrors, etc.)
- resolution: ca. 0.1 nm
- produces less stray light  greater dynamic range for measuring high absorbance
- sensitive to stray light from outside sources i.e. room light

Photodiode Array
Spectrophotometer:
- no moving parts  rugged
- faster spectral acquisition (ca.
1 sec)
- not dramatically affect by room
light

What are the components 1 to 10?

 Property of luminescence spectrum

Fluorescence vs phosphorescence

1. Phosphorescence is always at longer wavelength compared with fluorescence

2. Phosphorescence is narrower compared with fluorescence
3. Phosphorescence is weaker compared with fluorescence
Why?

Absorption vs emission

1. absorption is mirrored relative to emission

2. Absorption is always on the shorter wavelength compared to emission
3. Absorption vibrational progression reflects vibrational level in the electronic excited
states, while the emission vibrational progression reflects vibrational level in the
electronic ground states
4. 0 transition of absorption is not overlap with the 0 of emission

Why?
Fluorescence spectroscopy
 Fluorescence spectroscopy
Light source Beam
splitter Q: why the emission is
measured at 90 relative to
Excitation
monochromator
sample the excitation?

ght
li
of Emission
8% Monochromator
Reference
diode

PMT Amplifier Computer

Emission spectrum: hold the excitation wavelength steady and measure the emission at
various wavelengths
Excitation spectrum: vary the excitation wavelength and vary the wavelength measured
for the emitted light
Fluorescence spectroscopy: well defined molecules
Summary of spectrophotometric techniques
• A spectrophotometer is made up of two instruments: a spectrometer and a photometer. The
spectrometer is to produce light of any wavelength, while the photometer is to measure the intensity of
light. The spectrophotometer is designed in a way that the liquid or a sample is placed between
spectrometer and photometer. The photometer measures the amount of light that passes through the
sample and delivers a voltage signal to the display. If the absorbing of light changes, the voltage signal
also changes. Spectrophotometers come in a variety of shapes and sizes and have multipurpose uses
to them. The different types of spectrophotometers available are all different from one another, based
on their application and desired functionality. The most popular spectrophotometers are 45 degrees,
sphere and multi-angle spectrophotometers. Another closely related concept is Spectroscopy, that
simply measures the absorption of light from its source and the intensity of light as well.
• The basic spectrophotometer instrument consists of a light source, a digital display, a monochromator, a wavelength
sector to transmit a selected wavelength, a collimator for straight light beam transmission, photoelectric detector and
a cuvette to place a sample.

• The intensity of light is symbolized as l0 measure the number of photons per second. When the light is passed
through the blank solution, it does not absorb light and is symbolized as (l). Other important factors are Absorbance
(A) and Transmittance (T).

•
• The number of protons transmit and absorb totally depending on the length of the cuvette and the
concentration of the sample.

• The transmittance and absorption relation is:

• Experimental application
• As described in the applications section, spectrophotometry can be used in both qualitative and quantitative analysis
of DNA, RNA, and proteins. Qualitative analysis can be used and spectrophotometers are used to record spectra of
compounds by scanning broad wavelength regions to determine the absorbance properties (the intensity of the
color) of the compound at each wavelength.[5] One experiment that can demonstrate the various uses that visible
spectrophotometry can have is the separation of β-galactosidase from a mixture of various proteins. Largely,
spectrophotometry is best used to help quantify the amount of purification your sample has undergone relative to
total protein concentration. By running an affinity chromatography, B-Galactosidase can be isolated and tested by
reacting collected samples with ONPG and determining if the sample turns yellow.[3]: 21–119  Following this testing
the sample at 420 nm for specific interaction with ONPG and at 595 for a Bradford Assay the amount of purification
can be assessed quantitatively.[3]: 21–119  In addition to this spectrophotometry can be used in tandem with other
techniques such as SDS-Page electrophoresis in order to purify and isolate various protein samples.
 EXPERIMENT ON
SPECTROPHOTOMETRY
• Materials Required:
•

• Spectrophotometer
• Cuvette
• Blank solution
•
• Reagents:
•

• Cobalt (II) chloride

• Hexaaquacobalt (II) ion
• Ferrocene
• Crystal violet
• Rose bengal
• Coumarin
• Procedure:
•

• Determination of Molar Absorption Coefficient:

•

• Select a blank cuvette and place it in the spectrophotometer. Close the lid.
• Click on 0 ABS 100%T button, the instrument now reads 0.00000 A.
• Choose a solution with known concentration and measure the absorbance between the wavelengths 350 nm to 700 nm.
• Record the wavelength at the maximum absorbance value.
• Calculate the value of molar absorption coefficient , using the equation «math
xmlns=¨http://www.w3.org/1998/Math/MathML¨»«mi»§#949;«/mi»«mo»=«/mo»«mi»A«/mi»«mo»/«/mo»«mi»c«/mi»«mi»l«/mi»«/
math».
•