EXPERIMENT: ULTRA VIOLET- VISIBLE (UV-Vis)

SPECTROSCOPY FOR QUANTITATIVE ANALYSIS

OBJECTIVE:

To perform quantitative analysis of organic and inorganic samples using Ultraviolet-Visible

spectroscopy.

ULTRA VIOLET- VISIBLE SPECTROSCOPY

INTRODUCTION

The Ultraviolet -Visible spectroscopy is considered an important tool in analytical chemistr y.

In fact, this is one of the most commonly used techniques in clinical as well as chemica l
laboratories. This tool is used for the qualitative analysis and identification of chemica ls.
However, its main use is for the quantitative determination of different organic and inorganic
compounds in solution.

Basically, spectroscopy is related to the interaction of light with matter. As light is absorbed
by matter, the result is an increase in the energy content of the atoms or molecules. The
absorption of visible light or ultraviolet light by a chemical compound will produce a distinct
spectrum.

When ultraviolet radiations are absorbed, this results in the excitation of the electrons from the
ground state towards a higher energy state. The theory revolving around this concept states that
the energy from the absorbed ultraviolet radiation is actually equal to the energy differe nce
between the higher energy state and the ground state.

The Basic Principle of Ultraviolet -Visible spectroscopy:

The UV spectrophotometer principle follows the Beer-Lambert Law. This law states that
whenever a beam of monochromatic light is passed through a solution with an absorbing
substance, the decreasing rate of the radiation intensity along with the thickness of the
absorbing solution is actually proportional to the concentration of the solution and the incide nt
radiation.

This law is expressed through this equation:

A = log (I0 /I) = ECI

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where, A stands for the absorbance, I0 refers to the intensity of light upon a sample cell, l refers
to the intensity of light departing the sample cell, C stands for the concentration of the solute,
L stands for the length of the sample cell and E refers to the molar absorptivity.
Basing from the Beer-Lambert law, it has been established that the greater the number of the
molecules that are capable of absorbing light at a certain wavelength, the greater the extent of
the absorption of light. The main parts of UV spectrophotometer are given below.

Applications of Ultraviolet -Visible spectroscopy:

The concept and principle of UV spectrophotometer have several applications. For instance,
this is used to detect a functional group. It can be used to detect the absence or the presence of
chromophore in a complex compound.
This can also be used to detect the extent of conjugation in polyenes. When there is an increase
in double bonds, the absorption shots to the longer wavelength. In addition, UV spectroscopy
may be used to identify unknown compounds. The spectrum of an unknown compound is going
to be compared with the spectrum of a reference compound. If both spectrums coincide, this
unknown compound will be successfully identified.
UV spectroscopy can also help determine the configurations of a geometrical isomer. It has
been established that cis-alkenes are absorbed at a different wavelength compared to trans-
alkenes. If one of the isomers comes with non-coplanar structure, it can still be determined by
UV spectroscopy.
Lastly, this tool can determine the purity of a substance. To do this, the absorption rate of the
sample solution is going to be compared with the absorption rate of the reference solution. The
intensity of the absorption can be used to calculate the purity of a substance.

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APPARATUS AND CHEMICAL USED

Apparatus

1) Glassware
2) UV-Visible Unit

Chemicals:

Part 1:

1) Urea
2) Distilled water

Part 2:

3) Copper (II) Chloride

4) Distilled water

METHOD

1. Preparation of standard samples

Prepare from the given standard solution to obtain 100, 200, 300, 400 and 500ppm of
standard solutions.
2. Procedure of UV-Vis measurement
Measure all the standard solutions and establish the calibration curve.
3. Measurement of sample
Determine the concentration of unknown sample.

PROCEDURE:

Measurement of samples

Start-up UV-Vis

1. Connect the power supply to the instrument

2. Switch on Instrument and PC.

Part 1: UV range analysis

1. Click on the UV Probe software. Once the software open, click CONNECT. A series of
test will be automatically conducted which takes about five minutes. Make sure all
checklist is green.
2. Before perform the analysis, set the method according to you requirement. To do so, Click
button M. Check on this parameter:

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 Method: Liquid (absorbance) Wavelength: 800-0.0

3. Then save the method to your name. Click FILE menu. Select Save as = method file (must
be chosen). Create your own folder.
4. Calibration must be performed using solvent for liquid (Distilled water) containing the
analyte (five concentration urea solution plus blank i.e. without sample) as the standard
calibration. Place the solvent for liquid (containing sample or without analyte for blank)
cell at sample holder. Then click BASELINE. Wait until the calibration finish, then click
AUTOZERO.
5. Place your sample (A/B/C or D) at the sample holder and click button START.
Measurement will start. Wait until you are instructed to save the file. Click OK.
6. Click on OPERATION menu, choose manipulate button. Select equation type according to
your requirement. Then, Save all.
7. To save the graph, move your cursor on the graph and right click on your mouse, select
Copy. Then open Paint, paste in the new file and save.
8. You can also copy raw data to construct the graph using Microsoft Excel. To do so, click
on OPERATION menu, go to Data Print. Raw data table will be displayed on the right of
the monitor. Select and copy all the data, paste into a new text document then save.
9. Once you have finish with your current sample, click FILE menu, select Properties and
delete the current sample file. The new page will be displayed.

Part 2: Visible Analysis

Chemicals: Copper ( II) Chloride, Distilled water
1. Repeat steps 1-4 from Part 1 using Copper (II) Chloride solution.
2. Place your sample (E/F/G or H) at the sample holder and click button START.
3. Repeat steps 5-9 from Part 1 to analyze the next sample.
4. Determine the Copper (II) Chloride concentration found in the unknown.
5. Summarize your finding based on the spectrum and related information derived from your
experiment.
6. After completing all analysis, click button DISCONNECT.
Shut Down
1. Make sure the software is already disconnected with the instrument.

2. Switch OFF Instrument and PC.

QUESTIONS:
1. Explain which sample is measure under UV and which sample for Visible. Explain the
reason.

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2. Measure the concentration of unknown samples A/B/C/D for part 1(urea solution) and
E/F/G/H for part 2 (Copper (II) Chloride solution).
3. Run 3 repetitions for each of the samples and calculate;
a. Mean
b. Standard Deviation
c. Variance
4. Discuss the possible sources of systematic and random errors in the above experiments

5. How are systematic methods and instrument errors detected?

6. Discuss the results in term of accuracy and precision.

7. 10. Determine the λ max from the peaks recorded in the graph.
8. 11. Determine the concentration of the unknown A, B, C and D.

9.

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