Chem 302 Lab Instrumental Analysis Laboratory

AY 2016-2017

Ultraviolet and Visible Spectroscopy

Model Number: LAMBDA 25
Manufacturer: Perkin-Elmer
Local Distributor: Perkin-Elmer Instruments (Philippines) Corporation
Contact Number: (02) 637 0180

Prepared by:
GROUP 4
SULO, Ericsson C.
TADLE, Marjorie Hayes D.G.
TOLENTINO, Celine E.
VARGAS, Louise Erika Z.
VILLANUEVA, Matthew L.
3Chemistry

I. Introduction
Ultraviolet-visible spectroscopy or UV-Vis is a very vital part in the quantitative
analysis of liquid solutions. The UV-Vis spectrometry is known for being efficient,
flexible, and cost-effective. One of its uses is regulatory testing. It is one of the chosen
mode of analysis because of the information that it can provide.
The principle behind the UV-Vis spectroscopy is the UV-Vis spectrum. UV-Vis
spectroscopy uses the concept of the electromagnetic spectrum, mainly the ultraviolet
and visible region. The wavelength range of this spectroscopy is 200-380 nm (near
ultraviolet) and 380-780 nm (visible). The visible spectrum pertains to the different
colors and the corresponding wavelength range. Some of organic molecules undergo
electronic transitions upon interaction with the types of wavelength mentioned making
an electron to jump from a lower energy to a higher. Shorter wavelengths corresponds
to higher energy radiation.
The types of analytes that are subjected to analysis are but not limited to metal
ions, organic compounds with conjugated pi electrons and other biological organic
compounds. All samples must be in the form of solution and must fall into the
specification of the wavelength range. Common samples are dissolved in solvents like
water, ethanol and hexane. Concentration of species can also be identified using this
spectroscopy. For solutions containing metal ions, the response of the instrument is by
absorbance meaning if the concentration increases, the absorbance also increases
having a directly proportional relationship between the two values. The relationship of

this can be explained through the fundamental equation of Beer-Lambert Law is

followed. The law is expressed to this equation:

A=log

( PP )=abc
0

Wherein A is absorbance, I0 represents the intensity of light incident upon sample

cell, I is the intensity of light leaving sample cell, b is the path length of the absorbing
medium, c is the concentration of the absorbing species and a is the proportionality
constant called the absorptivity. The intensity of light leaving must be lower than the light
incident. It is because the light passing the sample is the one responsible for absorption.
The general mechanism of the instrument can be seen on the block and
schematic diagram. The schematic diagram prefers to the double beam type UV-Vis
spectrometer wherein light is being subjected to splitting. A UV-Vis is typically comprises
of a light source, container of sample, a diffraction grating in a form of monochromator
and a detector diode. First, there is a light source that will pass through a filter which
only a certain type of light will be permitted. The light source consists of deuterium or
tungsten lamp. Then the light will be subjected to the monochromator, a device wherein
a certain narrow range of wavelength can be isolated. It will then go through a series of
mirrors so that both, in respective cuvettes, the reference and the sample can be
passed by the light. A photo diode will detect the corresponding absorbance of the
sample and it can be seen through the graph with a tabulated data or a graph that can
be seen in the screen.

II. Block Diagram

III. Schematic Diagram

Source: https://upload.wikimedia.org/wikipedia/commons/9/95/Schematic_of_UV-_visible_spectrophotometer.png

IV. Operating Instructions

i.

Start-up procedure
A. Instrument initialization
1. Turn the instrument and the computer on.

2. The instrument should be warmed up for at least 30 minutes for best

results.
3. In the computer, open the software for the UV-Vis spectrometer.
4. Wavelength, number of samples, and other specifications should be set up
before the running of samples.
B. Sample running
1. Acquire the cuvettes from the refrigerator. The cuvettes are in a reagent
bottle submerged with methanol.
2. The cuvettes must be suited for the right experiment and the right
instrument.
3. Wash the cuvettes with distilled water twice or thrice before filling any
sample solution.
4. The frosted side must be the ones that is touching the fingertips of the
analyst. Fingerprints and any other types of dirt may affect the readings.
5. Cuvette containing samples should be at least filled.
6. Wipe the outside of the cuvettes with soft tissue or Kim Wipes.
7. Autozero the instrument by putting a reagent blank.
8. After the autozero, place your samples accordingly. Be sure to check if the
position of the cuvettes are right.
C. Post run procedures
1. Empty the cuvettes and clean it with distilled water thrice.
2. Place to the reagent bottle and return to the refrigerator.
3. Be sure that the working area is clean and take out the used tissues.
ii.

Shut-down procedure.

1. Close the software used.

2. Be sure that the UV-Vis spectrometer is close and turn it off before unplugging.
3. Shut-down the computer used in the analysis.
iii.

Precautions/ Hazards

1. Do not place anything on top of the instrument. Be sure that the solutions used
will not spill over the instrument.
2. The cuvettes must not be over or under filled.
3. Be sure the cuvettes are clean before and after the analysis.
4. During the sample run, the cuvettes should be clean in the opaque side and must
not be wet.
5. Instrument must be closed in the sample running.
6. Be sure to check if the computer and instrument used are properly shut down.
V. References
1. GE Healthcare Life Sciences. Spectrophotometer Health and Safety Document
including

General

Operating

Instructions.

https://promo.gelifesciences.com/gl/spectrophotometers/misc/Health_and_Safety_m
anual.pdf
2. Perkin

Elmer

Inc.

Lambda

25,

35,

45:

Users

guide.

http://people.bath.ac.uk/gp304/uv/PerkinElmer_Lambda35_manual_EN.pdf
3. Sheffield Hallam University. UV-Vis Absorption Spectroscopy: Theoretical principles.
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm
4. William

Reusch.

Visible

and

Ultraviolet

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uvvis/spectrum.htm

Spectroscopy.