Instrument, principles & usage

 Spectrophotometry is the study of

quantifiable electromagnetic spectra.
 Light can either be absorb or transmitted
by dissolved substance
 Presence & concentration of dissolved
substance is analyzed by passing light
through the sample
 Spectrophotometry is a science of
measure quantity or quality of dissolved
substance using spectrophotometer
 A spectrophotometer generally consist of a
 Spectrometer
 Photometer
 Cuvette holder
 Galvanometer
 Filter/entry slit
 Exit slit
 A spectrometer is used to produce light of any
selected colour by adjusting their wavelength
 A photometer is a device for measuring light
intensity
 It can measure intensity as a function of color, or
the wavelength of light.
 Any of various instruments for producing a
spectrum and measuring the wavelengths,
energies, etc., involved.
 Usually it is equipped with a calibrated scale
allowing wavelengths to be read off or
calculated
 A photometer is an instrument for
measuring light intensity or optical
properties of solutions or surfaces
 Photometers are used to measure:
 Illuminance
 Irradiance
 Light absorption
 Scattering of light by media
 Reflection of light
 Fluorescence
 Phosphorescence
 Luminescence
 The phenomena on which Spectrophotometry is
based mainly absorption and transmission
 To understand how the process works, we need
to understand Beer Lambert's law
 Beer’s law - Stating that the quantity of light
absorbed by a substance dissolved in a non-
absorbent solvent is directly proportional to the
concentration of the substance and the path
length of the light through the solution
 Lambert’s law states that when a ray of
monochromatic light passes through an absorbing
medium, its intensity as the length of the
medium increases
 Beer – Lambert's law states that absorbance is
directly proportional to concentration
 The liquid in a cuvette are placed between the
spectrometer beam and the photometer.
 The amount of light passing through the tube is measured
by the photometer.
 The photometer delivers a voltage signal to a display
device, normally a galvanometer.
 The signal changes as the amount of light absorbed by the
liquid changes.
 If development of colour 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 appropriate wavelength.
 For example haemoglobin appears red because the
haemoglobin absorbs blue and green light rays much more
effectively than red.
 The degree of absorbance of blue or green light is
proportional to the concentration of haemoglobin.
 The light source shines through the
sample.
 The sample absorbs light.
 The detector detects how much light
the sample has absorbed.
 The detector then converts how much
light the sample absorbed into a
number.
 The numbers are either plotted straight
away, or are transmitted to a computer
to be further manipulated (e.g. curve
smoothing, baseline correction)
 Scattering of light due to particulates in the
sample.
 Fluorescence or phosphorescence of the
sample.
 Stray light (i.E. Light other than light of the
selected wavelength reaching the detector).
 Non-monochromatic radiation.
 Changes in refractive index at high analyte
concentration.
 Shifts in chemical equilibria as a function of
concentration.
 Very large and complex molecules.
 Can be used to identify matters depending
on their absorption and transmission of
certain wavelength
 Can be used to quantify dissolved substance
 Used in chemistry, biology, medical research
and also physics
Reflectance spectrophotometry
Flame emission photometry
• Definition: Measurement of the ratio of
spectral radiant flux reflected from a light-
diffusing specimen to that reflected from a
light-diffusing standard substituted for the
specimen.
• Reflectance spectrophotometers measure the
amount of light reflected by a surface as a
function of wavelength to produce a
reflectance spectrum
• The reflectance spectrum then will be
compared with known sample in order to
identify the material
• Based on the interaction of light and matter
• Absorption
• Scattering
• The sample is illuminated with white light and
the amount of light reflected by the sample is
calculated.
• Data are measured for 31 wavelength intervals
centered at 400nm, 410nm, 420nm, ..., 700nm.
• This is done by passing the reflected light though
a monochromating device that splits the light up
into separate wavelength intervals.
• The instrument is calibrated using a white tile
whose reflectance at each wavelength is known
compared to a perfect diffuse reflecting surface.
 Result obtained is expressed between 0 and 1
(as a fraction) or between 0 and 100 (as a
percentage)
 The result can be then interpreted as
percentage of components in sample.
 Determine absorbance of semi solid
matters/solid matters
 Identify reflectance of matters – leads to
identification of matter
 Flame emission photometry or flame atomic
emission spectrometry is a branch of
spectroscopy where the sample is examined
in the in the form of atoms
 atoms under investigation are excited by
light
 Uses absorption and emission of light when
interacting with matter
 Absorption measure the absorbance of light
due to the electrons going to a higher energy
level.
 Emission techniques measure the intensity of
light that is emitted as electrons return to
the lower energy levels
 Utilizes flame to evaporates the solvent
and also sublimates and atomizes the
metal and then excites a valence electron
to an upper energy state.
 Light is emitted at certain wavelengths
for each metal as the electron returns to
the ground state that makes qualitative
determination possible
 No need for source of light, since it is the
measured constituent of the sample that
is emitting the light
 energy that is needed for the excitation is
provided by the temperature of the flame
(2000-3000 ºC), produced by the burning of
acetylene or natural gas (or propane-butane
gas) in the presence of air or oxygen.
 By the heat of the flame and the effect of
the reducing gas (fuel), molecules and ions
of the sample are decomposed and reduced
to atoms
 The monochromator selects the suitable
(characteristic) wavelength of the emitted
light
 Result obtained in form of absorbance
 Using calculations and also reference sample,
identification of matter can be done
 Usually in form of percentage of matter in
given sample (provided user know the
wavelength of the given species)
 Qualitative and quantitative determination
of several cations, especially for metals that
are easily excited to higher energy levels at a
relatively low flame temperature (mainly Na,
K, Rb, Cs, Ca, Ba, Cu)
 Atomic identification of sample (comparison
to known sample or reference sample)
William James Durant (5 November
1885 – 7 November 1981)