Spectrophotometry

Muhammad Abdullah
Lecturer BioMedical Engineering
Department
 Instrumentation
• The main component parts of a
spectrophotometer are:

1. Light source
2. Monochromator
3. Sample holder
4. Detector
5. Readout device
 Light source
• Tungsten filament lamp: 360 to 2500 nm
– It can be used for NIR and UV

• Halogen lamp made with quarts diode: 290-

800nm

• Hydrogen or deuterium lap: 200-375 nm

– For work in lower UV ranges
Intensity of radiation as a function of wave-length for a typical tungsten bulb at 3000 K
 Monochromator (1)
• Monochromator isolates the desired wavelengths and
excludes all others

• In its simplest form, it is a filter that allows only discrete

wavelengths to pass

• An instrument that uses filters is called photometer

• There are 2 types of filters:

– Absorptive or Glass filters: use two pieces of colored glass to
absorb wavelengths on either side of the selected wavelength
– Interference filters
 Monochromator (2)
Absorptive or Glass filters
• Colored glass or a dye that absorbs the wavelength we wish to reject
• Bandwidths are extremely large (30 to 250 nm)
• Combining two filters of different wavelengths can yield a B.P filter
 Monochromator (2)
• Interference filters are made up of multiple
layers of reflective and transparent materials
with selective refractive indices

• Light reaching the filters bounces off the

reflective surfaces and refracted by the
transparent material many times before
exiting
 Monochromator (3)
Interference filters :
• The peak wavelength of the exit beam can be
calculated as:

2dN sin
 m  Eq.1
Where;
• m=order of diffraction pattern (1, 2, 3…)
• d=thickness of transparent layer
• N=refractive index of transparent layer
• Θ=angle of incidence (usually 90o)
 Monochromator
• What if

1.d  / 2
2.d  / 4
and Θ= 90o
 Working principle of interference
filters
• Spacer: the gap between the reflecting surfaces is
a thin film of dielectric material, with a thickness
of one-half wave at the desired peak transmission
wavelength. (d = λ/2). This is where the
constructive interference will occur

• Reflection layers: consist of several film layers,

each of which is a quarter wave thick (d = λ /4).
This is where the destructive interference will
occur
 Interference Vs Absorption Filters
• Interference filter can provide superior band width
definition over an absorption filter (Looking from the graph, how is it better)

• The greater the bandwidth definition the lower the

%transmittance through that filter
 Monochromator (4)
• In contrast to filters, which blocks out
unwanted wavelengths, Prisms and gratings
provide a continuous range of wavelengths

• Polychromatic light is disperse by refraction or

diffraction

• Instruments that use prisms and gratings are

called spectrophotometer
 Monochromator (4)
Prisms:
• Prism disperse white light into a series of
wavelengths by refraction
• The angle of deviation depends on
wavelength
• Red deviates the least
• Violet deviates the most

• Prisms are rotated to allow the choice of wavelength

to pass through
 Monochromator (5)
Diffraction gratings:
• It consists of many equally spaced parallel
grooves etched on a glass or aluminum plate
• The general equation
For the wavelength of
Light diffracted is:

n  d sin
 Eq.2
d= Thickness of the grating
n= repetition of the pattern
θ =angle of diffraction d= hyp
θ
beam(deviation)
λ= perpendicular
 Sample holder
• Most sample for photo/spectrometric
instruments are solutions
• Therefore, most sample holders are built to
hold cuvettes or flow cells (for continuous
measurement)
 Sample holder (2)
• Some instruments have two holders; one for the sample and one
for the blank

• The holder must be transparent through out the wavelength region

being measured

• Glass cuvettes are used between 320 and 950 nm and quartz
cuvettes are used below 320 nm (due to the absorption of UV light by the glass below
320 nm)

• To avoid stray light, flat surface cuvettes are used

• Sample holders must be clean and clear of finger prints and

scratches
 Detector
• There are two most commonly used detectors:
1. Photomultiplier tube
2. Barrier layer cells (photocells)
Photomultiplier tube
• They are electron tubes that amplify current
proportional to the energy of the photons
• The emitted electrons jump to a positively charged
dynode
• The dynode then emits 4 to 6 electrons for every
electron striking it
 Detector (2)
• These emitted electrons are passed to the
next dynode, which is more positively charged
than the first
• The number of emitted electrons is multiplied
by each dynode
• Finally the electrons are collected at the
anode
• PMT are more sensitive and have fast
response time than photocells
 Detector (3)
• Photomultiplier tube (continued)
 Detector (4)
• Photomultiplier tube (continued)
 Readout device
• The analogue photometer readout device
shows both absorbance and percent
transmittance

• Many instruments have digital readout allow

the operator to select absorbance,
transmittance or concentration units
75 % Transmittance
100 50 25 10 1 0.1

0 0.3 0.6 1.0 2.0 3.0

0.125 Absorbance
 For further study
• http://www.analiticaweb.com.br/newsletter/

02/AN51721_UV.pdf
 For further study
• http://www.analiticaweb.com.br/newsletter/

02/AN51721_UV.pdf

• http://www.microscopyu.com/articles/polariz
ed/interferenceintro.html