Spectrophotometry

Nature of light:

Electromagnetic radiation includes radiant energy that

extends from cosmic rays with wave lengths as short
as 10-9nm (γ rays) up to wavelengths longer than
1000 Km (radio waves).

The term light is used to describe radiant energy with

wavelengths visible to the human eye and with
wavelengths bordering on those visible to the human
eye.

Light is responsible for the sense of sight.

In spectrophotometry, usually radiant energy from
visible and ultraviolet portions of spectrum (180-
800 nm) are used.
The wavelength of light is the distance between two
peaks as the light travels in a wavelike manner,
expressed in nanometers (nm).

1 nanometer (nm) = 1millimicron (mµ) = 10

Angstroms (Å) =10-9 meter (m)
Regions of the electromagnetic spectrum:
Wave length (nm) Color observed Region
name
< 380 ultraviolet invisible
380-440 violet visible
440-500 blue visible
500-580 green visible
580-600 yellow visible
600-620 orange visible
620-750 red visible
800-2500 near-infrared not visible
2500-15,000 mid-infrared not visible
15,000-1,000,000 far-infrared not visible
The ultraviolet (UV) portion of the spectrum is sometimes
further divided into near UV (200-380 nm) and far UV (< 200
nm).
Major components of a spectrophotometer:
Major components of a spectrophotometer:

1. Light source: made from tungsten (for visible

portion) or deuterium (for UV portion).
2. Entrance slit: to ensure the entrance of a narrow
beam of light.
3. Monochromator: made from a dispersion device
(e.g. prism), for selection of desired color or
wavelength.
4. Exit slit: for isolation of a narrow beam of light
5. Cuvette: made of plastic, glass, or quartz .To
contain the sample.
6. Detector: to convert the light into electrical
current.
7. Meter or Recorder: to record the electrical
current.

UV-visible spectrophotometer: uses light over

the ultraviolet range and visible range of
electromagnetic radiation spectrum.
Solutions required for spectrophotometry:

1. Test solution: made from serum or other unknown

specimen.
2. Standard solution: made from a known concentration of
a substance to be measured. Glucose standard contains
100 mg/dl.
3. Blank solution: a solution that contains all the reagents
used for measurement of a substance except the substance
to be measured, it is a compensate for non-specific color
such as the color of reagents.
4. Control solution: made sometimes in enzyme assay, no
chemical reaction take place.
5. Quality control solution: made from pooled serum of a
known chemical constituent.
Beer’s Law:
(the relation between concentration, absorbance and
transmittance)

Beer’s Law (also called Beer-Lambert’s Law) states

that the concentration of a substance is directly
proportional to the amount of absorbed light
(absorbance) or inversely proportional to the
logarithm of the transmitted light (transmittance).
Mathematically, Beer’s Law is expressed as:

A =abc
Where:
A = Absorbance
a = proportionality constant defined as absorptivity of the
substance
b = light path in cm
c = concentration of the absorbing compound, usually expressed in
g/l or mg/dl
We can say
Therefore, for two solutions of the same substance:
Light path (diameter of the cuvette) is constant : (= )

So
Suppose:
1 = solution of a substance in which concentration (C) is
unknown = test solution
2 = solution of the same substance in which concentration is
known = standard solution

The equation can be written as:

C test x A std = A test x C std

when using blank solution: (why ? refer to definition
of blank solution )
Absorbance is the amount of light absorbed by the solution.
It is equal to the negative logarithm of the transmittance.
Absorbance and transmittance bear an inverse relationship. So,
absorbance is directly proportional to concentration of the
substance in solution.
Transmittance is the ratio of the amount of light transmitted
through a solution of a substance to the amount of light that
initially fell on the solution. It is inversely proportional to the
concentration of that substance in solution.
Calibration Curve:

Is used to calculate the concentration of unknown solution.

From solution of potassium dichromate (10mg / ml), prepare different
concentrations:

1) mix 10ml of dichromate solution with 0 ml of water

2) mix 9ml of dichromate solution with 1ml of water
3) mix 8ml of dichromate solution with 2ml of water
4) mix 7ml of dichromate solution with 3ml of water
5) mix 6ml of dichromate solution with 4ml of water
6) mix 5ml of dichromate solution with 5ml of water
7) mix 4ml of dichromate solution with 6ml of water
8) mix 3ml of dichromate solution with 7ml of water
9) mix 2ml of dichromate solution with 8ml of water
10) mix 1ml of dichromate solution with 9ml of water
11) mix 0ml of dichromate solution with 10ml of water

Measure absorbance for each concentration. Plot the readings against

concentration, then calculate the concentration of unknown solution.