Name __Bellerry C.

Bernante_____ Section _BSMT 3C_ Date _November 1, 2022_

Activity 4

COLORIMETRIC TECHNIQUE

Objective: At the end of the activity, the learner should be able to:

1. Identify the parts of a spectrophotometer

2. Explain the use of each part of a spectrophotometer

Colorimetry depends on the quantitative comparison of the amount of color developed in unknown
solutions with the amount of pure substance present. This method uses the fact that a particular
molecular structure will absorb light of a specific wavelength and that the degree of absorption has
some relationship with the concentration of a solution.

Some solutions have the property of absorbing a specific wavelength of light and transmitting others.
For example, a particular solution appears red because the solution absorbs all colors except red, which
is transmitted. The visible spectrum, the color visible to the human eye, runs from purple (340 – 430
nm) to red (620 – 700 nm). wavelength below 340 nm are ultraviolet while those above 700 are infrared,
both of which are invisible to the human eye.

Two primary considerations in the colorimetric analysis are:

a. Quality of Light

b. Intensity of color

Measurement is based on Beer's Law which states that the amount of light absorbed by a colored
solution is proportional to the concentration of the colored material present.

Principle of Photoelectric Colorimetry

Selected light passing through a solution to a greater or lesser extent strikes a photocell or
phototube, generating a current registered by a galvanometer.

Essential Parts of a Photoelectric Colorimeter

1. Power source

2. Light source

3. Wavelength selector

4. Electrical measuring system

5. Sample container
Questions:

1. Define the following terms

a. Transmittance – it is the quantity of light that passes through a solution.

b. Absorbance – it is the quantity of light absorbed by a solution.

c. Reagent blank – it is a sample that contains everything except for the analyte of interest.

2. What are the types of Photoelectric Colorimetry?

The following are the different types of Photoelectric Colorimetry:

 Single beam filter photometer - has only one beam of light; less reproducible

 Double beam photometer - has two beams of light, one passing through a reference solution
and one passing through the sample; highly reproducible because electronic and mechanical
effects on both sample and reference beams are equal

 Probe-type photometer - measurements of this type are made with a new thin-probe pulsed
photometer sensitive to 0.01 fL and versatile to use.

 Multi-channel photometer - an instrument for monitoring either transmitted or scattered light

intensity, or both, simultaneously on up to eight channels. The use of a laser light source (at
632.8-nm wavelength) provides high accuracy and dynamic range.

 Portable photometer - based on light-emitting-diodes (LED) for the determination of light

absorbance and transmittance of light in a liquid.

2. Name and give the principle of other analytical methods and quantitative methods used in

Clinical Chemistry. Give an example.

For analytical methods:

 Electrochemistry- is the study of electron movement in an oxidation or reduction reaction at a
polarized electrode surface.

 Electrophoresis- separate DNA, RNA or protein molecules based on their size and electrical
charge.

 Chromatography- an analytical technique commonly used for separating a mixture of chemical

substances into its individual components.

 Mass spectrometry- an analytical tool useful for measuring the mass-to-charge ratio (m/z) of
one or more molecules present in the sample.

 Spectroscopy- study of the absorption and emission of light and other radiation by matter.
For quantitative analysis:
 Titration- or volumetric analysis is a quantitative method wherein one solution is added to
another solution such that it reacts under conditions in which the added volume may be
accurately measured. Titration determines an unknown concentration of an identified

 Gravimetric analysis- a technique through which the amount of an analyte (the ion
analyzed) can be determined through the measurement of mass.