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Experiment 2
UV-Visible Spectrophotometry
Object:
To determine phosphate composition in cola beverages by UV-visiblespectrophotometry and pH titrimetry
 Introduction:
Methods based on the absorption of radiation are powerful and useful tools for theanalytical chemist. The ultraviolet region (about 450-200 nm) is particularly important for the qualitative and quantitative determination of many organic compounds. In the visibleregion (about 450-700 nm), spectrophotometric methods are widely used for thequantitative determination of many trace substances, especially inorganic species.The basic principle of quantitative absorption spectroscopy lies in comparing theextent of absorption of a sample solution with that of a set of standards under radiation of aselected wavelength through the application of Beer's law.where A = absorbance 
 P 
0
= power of incident light 
 P 
= power of transmitted light= extinction coefficient (mol
-1
dm
3
cm
-1
) 
b
= path length of cell (cm) 
c
= concentration of absorbing species (mol dm
-3
)In many cases, the sample compound does not absorb radiation appreciably in thewavelength regions provided; it is then necessary to form an absorbing substance byreacting the compound in question with other reagents. Phosphoric acid is found to be present in small quantities in cola beverages. The amount of phosphoric acid in a givencola beverage can be determined by the vanadomolybdophosphoric acid colorimetricmethod. In this method, ammonium molybdate reacts with phosphoric acid to form aheteropoly acid, molybdophosphoric acid. In the presence of vanadium, yellow
1
 
vanadomolybdophosphoric acid is formed. The intensity of the yellow colour is proportional to phosphate concentration. Alternatively, the concentration of H
3
PO
4
can bedetermined, at least approximately, by titration with standard base. The equivalence pointsmust be determined by potentiometric methods, since the intense colour of the samples willobscure any acid-base indicator colour change. This part of the experiment points out animportant use of pH titration.
 Experimental Procedure Part I: UV-VIS analysis
1. Effervescence of carbon dioxide from the sample causes problems in pipetting and other volume measurements, and CO
2
must be absent for the titrimetric analysis. To removethe carbon dioxide, approximately 100 cm
3
of the cola is heated on a steam bath for 20min in a beaker covered with a watch glass.2. For the colorimetric analysis, the cooled decarbonated sample should be dilutedquantitatively 25-fold with distilled water in a 100-cm
3
volumetric flask.3. Prepare phosphate standards (KH
2
PO
4
) from the stock solution (1 ×10
-3
M KH
2
PO
4
):0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM by using 50-cm
3
volumetric flasks and glass pipettes for construction of a calibration curve.4. Both the samples and phosphate standards (KH
2
PO
4
or K 
2
HPO
4
) then can be preparedfor absorbance measurements at 400 nm using the ammonium molybdate / ammoniummetavanadate colour reagent. Place 25.0 cm
3
of the diluted sample solution in a 50-cm
3
volumetric flask. Add 10 cm
3
vanadate-molybdate reagent (which is a mixture of ammonium molybdate, (NH
4
)
6
Mo
7
O
24
•
4H
2
O and ammonium metavanadate NH
4
VO
3
in HCl solution) and dilute to the mark with distilled water.5. Prepare a blank (standard blank) in which 25 cm
3
of distilled water is substituted for the sample or phosphate standards.
2
 
 Part II: Titrimetric analysis
For the titrimetric analysis, the electrodes from a previously calibrated pH meter areimmersed in 25 cm
3
of the undiluted decarbonated cola in a 100 cm
3
beaker, and the pHis measured. The sample is stirred and titrated with standard 0.02 M KOH, measuringthe pH after the addition of each increment of titrant. Because this titration givesapproximate results only, operations need not be as painstaking as in the case whereone is attempting to determine acid dissociation constants.
 Data Treatment 
You may hand in this experiment at the next practical session. The following arerequired, and the marks for each are in brackets. Total marks = 10.1. Plot the calibration curve: Absorbance at 400 nm vs. [H
3
PO
4
] in mM. [2]2. From the calibration curve, determine the concentration of H
3
PO
4
in the diluted colasample [0.5], and hence the original undiluted sample. [0.5]3. Plot the pH titration curve for the titration of 25 cm
3
of decarbonated cola with 0.0200M KOH (pH vs volume of base (cm
3
)). [2]4. Determine the equivalence points of the titration from the pH titration curve. [1]5. From (4), calculate the concentration of H
3
PO
4
in the sample. [1]6. Compare the results obtained by the two different methods and explain any difference.[1]7. What advantages over the titrimetric method does the colorimetric method possess? [1]8. Give the reason for sample dilution in colorimetric analysis. [1]
Appendix: Introduction to Beer-Lambert Law
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below showsa beam of monochromatic radiation of radiant power 
 P 
0
, directed at a sample solution.Absorption takes place and the beam of radiation leaving the sample has radiant power 
 P 
.
3
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