EXAMPLE EXPERIMENT UV-vis SPECTROPHOTOMETER

CONTENTS

CONTENTS	PAGE
Abstract	2
Introduction	3
Literature review	4
Objectives	6
Methodology	7
Results	8
Discussion	9
Conclusion and recommendation	11
Reference	12
Appendix	13

Abstract

This experiment wasabout concentration dependent absorbance values, determined using a UV-vis spectrometer which gives the value of absorbance of a solution based on the amount of light absorbed by the solution. The primary objective of this experiment is to determine the absorbance of copper sulphate solutions of different concentrations. Besides, the absorbance of a sodium chloride sample solution was also determined by using the same method.

In this experiment, copper sulphate solutions of concentrations 28g/L, 14g/L, 7g/L, 3.5g/L, and 1.75g/L were prepared in volumetric flasks from solid copper sulphate. The five solutions were labeled c₁ to c₅ following the sequence. A solution of NaCl was also prepared and labeled as X. These solutions, and distilled water were placed into the UV-vis spectrometer to determine their absorbance. Before the measurement was taken, the UV-vis spectrometer was adjusted so that the wave length of the emitted light was at a wavelength of 800nm. The absorbance value of distilled water was recorded as reference.

While measuring the absorbance of the solutions, the quartz cells containing the solutions must be clear from fingerprints because the fingerprints might affect the amount of UV light that reached the detector in UV-vis spectrometer. Therefore, tissue paper is used to wipe off the fingerprints before inserting the quartz cells into the UV-vis spectrometer.

The experiment basically showed that absorbance increases with the concentration of a solution, obeying Beer-Lambert’s Law.

Introduction

A UV-vis spectrophotometer is a research instrument used to gather information about a chemical sample by determining the absorbtion or transmission of UV-vis light by the sample. It can also be used to measure the concentration of the absorbing materials based on the calibration curves produced.

A UV-vis spectrophotometer exposes a chemical solution to the ultraviolet and visible region of the electromagnetic spectrum when the chemical solution is placed in the UV-vis beam. Depending on the type of chemical, a certain amount of light gets absorbed by the chemical which causes electrons to be promoted from one energy level to another. The amount of light which is not being absorbed will pass through the chemical to the detector. The amount of light that reaches the detector is then recorded as a spectrum. A spectrum is a graphical representation of the amount of light absorbed or transmitted by matter as a function of the wavelength. Since the samples are prepared in known concentrations, the graphed results make a calibration curve from which the unknown concentration can be determined by its absorbance. 

A UV-visible spectrophotometer measures absorbance or transmittance from the UV range from which the human eye is not sensitive to the visible wavelength range to which the human eye is sensitive to.

Literature review

When light passes through a substance, light of certain wavelength is being absorbed by the substance, while the rest of the light will pass through the substance, or being reflected by the substance. For example, when light passes through a solution of copper sulphate, the copper ions in the solution absorbed the visible lights from the red end of the spectrum. The blue light reflects into our eyes and this is why the copper sulphate solution appears to be blue to our eyes.

However, substances do not only absorb lights from the visible region of the wavelength. They also absorb invisible light, for example UV rays, dependent on the type of substance.

Since different substances absorb light of different wavelength, this can be used to determine the type of substance in a sample.

A UV-visible spectrometer can be used to measure the absorbance of solutions. light beams of wavelength in the visible region, and the UV region are passed through the solutions, where light of certain wavelengths are absorbed, and the rest will reach the detector. The detector converts the incoming light into a current. The higher the current, the greater the intensity of the light.

For each wavelength of light passing through the spectrometer, the intensity of the light passing through the reference cell is measured. This is usually referred to as I_o, where I is the intensity. The intensity of the light passing through the sample cell is also measured for that wavelength, given the symbol I.

The relationship between absorbance,A and the two intensities is given by:

From the equation, it is shown that A is a value without unit.

The derived equation, A=ebc

Where

A is absorbance
e is the molar absorbivity
b is the path length of the sample
c is the concentration of the compound in solution

shows that concentration of a solution is directly proportional to its absorbance value.

A UV-vis spectrometer is generallly used in analytical chemistry, especially in the quantitativeanalysis of transition metal ions, highly conjugated organic compounds, and biological macromolecules. Determination is usually carried out in solutions.

Objectives

1.      To determine absorbance of solutions at different concentrations.

The absorbance values of copper sulphate solutions of different concentrations is to be determined using a UV-vis spectrometer.

2.      To determine the concentration of given samples.

Methodology

Reagents and equipment

Solid CuSO_4, distilled water, solid NaCl, 5 100ml volumetric flasks, measuring cylinder, dropper, 200ml beaker, glass rod, 10-mm path length quartz cells, UV-vis spectrometer.

Method

1.      A CuSO₄  stock solution of 2.8g is prepared in 100ml distilled water.

2.      Four sample solutions is prepared in the 100ml volumetric flasks by diluting the stock solution, followed by each previously prepared solution, using sample concentrations of 14g/L, 7g/L, 3.5g/L and 1.75g/L.

3.      A small amount of NaCl (,0.1mg) is added in a 100 ml volumetric flask and the flask is filled with the stock solution. This sample is labeled as X.

4.      The absorbance of distilled water is measured and used as a reference.

5.      The absorbance values of the stock solution and of each of the other solutions are measured at 800nm.

Results

List of concentrations of the solutions:

C₁   = 28g/L

C₂     = 14 g/L

C₃   = 7g/L

C₄   = 3.5g/L

C₅   = 1.75g/L

Sample solution	Absorbance (AU)
C1	2.276
C2	1.029
C3	0.463
C4	0.192
C5	0.126
Cx	0.460

Table 1: measured absorbance values at different concentrations

Absorbance of distilled water = 0

Discussion

Plot absorbance of C₁, C₂, C₃, C₄ and C₅ as a function of concentration. Explain the relationship between the absorbance and the concentration.

Figure 1

From the graph shown in Figure 1, it is shown that, the higher the concentration of CuSO₄, the higher its absorbance value.

The graph of absorbance versus concentration is a straight line graph.

It was determined that the absorbance of distilled water, which concentration is g/L, is 0 AU. The graph, therefore, if plotted to concentration=0g/L, is a straight line graph which passes through the origin.

According to Beer-Lambert Law, 

A=ebc

Where A is absorbance (no unit)
e is the molar absorbivity with units of L mol^-1 cm^-1
b is the path length of the sample (cm, since the unit of length used in e is cm)
c is the concentration of the compound in solution (mol L^-1)

Note that the units of the equation cancel off each other, thus absorbance, A has no unit.

In the experiment, the value of e is constant because solution of a same substance is used throughout the experiment. Each and every compound or substance has its own molar absorbivity.

Value of b is also a constant as the sizes of the quarts cells used are the same. Thus, the path length of the sample is also a constant.

The concentration, c is the only variable in the equation. Therefore, in this experiment, A=ebcis a straight line equation where concentration, c is directly proportional to A.

Discuss the cause of differences in the absorbance values for c₁ and c_x.

C₁ has an absorbance value of 2.276 while c_x has an absorbance value of 0.460. Even though the two sample solutions have different concentrations, the difference in their absorbance values is mainly caused by the different substance they are containing. C₁ is copper sulphate solution, containing copper ions and sulphate ions. Meanwhile, c_x is sodium chloride solution, containing sodium ions and chloride ions. The ions present in the solutions absorb light rays of different wavelength (which also explains the colour difference of the solutions), and thus having different absorbance values.

Conclusion and Recommendation

Conclusion

For the same solution of different concentrations, the absorbance value is directly proportional to the concentration.

Absorbance value is dependent on the type of substance. Different substances have different absorbance values when the other conditions are kept constant.

Recommendation

The absorbance value can be measured using UV-vis spectrometer for a few times and obtain the average reading to avoid errors.

Before the quartz cells are placed into the UV-vis spectrometer, fingerprints should be wiped off from the surface to obtain accurate results, because the natural secretions contained in the fingerprint will absorb some region of the light as well, thus affecting the reading for the solutions.

Reference

·         Zitzewitz, Paul W. (1999). Glencoe physics. New York, N.Y, Glencoe/McGraw-Hill.

·         Skoog, et al. Principles of Instrumental Analysis. 6th ed. Thomson Brooks/Cole. 2007

·         Ó. G. Björnsson, R. Murphy and V. S. Chadwick,Physicochemical studies of indocyanine green (ICG): absorbance/concentration relationship, pH tolerance and assay precision in various solvents, volume 38, number 12.

·         Dawson, R.M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M., eds., Data for biochemical research, 2nd edn, p. 502. Clarendon Press, Oxford 1978.

·         B. S. Gilfedder, F. Althoff, M. Petri and H. Biester,A thermo extraction–UV/Vis spectrophotometric method for total iodine quantification in soils and sediments, volume 389, number 7-8.

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