Analytical chemistry

1. UV-VISIBLE
SPECTROPHOTOMETRY AND
INORGANIC PHOSPHATE
DETERMINATION
DR.MAGEJA,BVSc,UZ

2. Principles
 Light of specific wavelength passes through the sample and some of it is
absorbed and some is transmitted.
 The intensity of transmitted light reaching the detectoris measured.
 The spectrophotometermeasures the amount of light of particular
wavelength that a sample absorbs.
 The amount of light absorbed is directly proportionalto the number of
absorbing moleculesof the soluteor concentrationof the solution(Beer-
Lambert law)

3. UV-Visible Spectrophotometry
 Techniquebased on absorptionof light
 Sample (analyte) is exposed to a beam of light
 Sample absorbs light…
 Instrument measures transmitted light
 Concentration of analyte is proportional to the amount of light absorbed

4. Absorption/Electronic Transitions
 Atoms (and ions) have finite permissible electronictransitionsand
absorb/emit monochromaticradiation.
 Complex ions and molecules have multiplepossible electronictransitions
owing to many overlapping molecularorbitals.
 Complex ions and molecules absorb (or emit) light over a wider range of
wavelengths.
 This is known as “broad band”absorption(emission).

5. Beer Lambert Law
 States that absorbanceof electromagneticradiation by a given species is
directly proportionalto the concentrationof the analyte.
It is expressed as: A= εbC
 whereA is the absorbance, ε is the molar absorptivity, b is the path length
and C is the concentrationof analyte.
 Because ε and b are fixed under experimental conditionsthe result is a
linear relationship between absorbanceand concentration.

6. Basic UV-VIS instrument

7. Light
• A form of radiant energy
• Light and electromagnetic radiation behave as
waves in motion
• The distance between successive crests is a
measure of wavelength and this distance is
measured in nanometers

8. Electromagnetic spectrum

9. Basic components

10. Basic components
Lamp or light source – provides the radiant energy
Slits – isolate a narrow beam of the light and
improves is chromatic purity
Monochromator – selects the band of light that passes
to the cuvette
Detector – senses the radiant energy that is not
absorbed by the sample and converts light energy to
electrical energy that can be registered on a meter or
digital read out
Meter – provides a read out of the transmitted light or
in absorbance

11. Absorption Spectrum
 An unknown substancecan often be identified by constructingan
absorptionspectrum.
 This is a plot of the amount of light absorbed from an incident light beam at
various wavelengths .
 The wavelength of the incident light may be taken from the ultra-violet,
visible, or infra-red sections of the spectrumas appropriateand should
represent a series of wavelengths at consecutive intervals.
 A given substance has one or more absorptionpeaks at specific
wavelengths.
 This enables identification of unknown substances(qualitative analysis)

12. Absorption Spectrum
 For the spectrumon the left, a
(1.42 • 10-5 M) solutionthe
aldehydein 95% ethanolwas
placed in a 1 cm cuvette for
measurement.
 Using the Beer Lambert Law
formula,
 ε = 36,600for the 395 nm peak,
and 14,000for the 255 nm peak.

13. Measurement of Absorbance
 Absorbanceis not directly measurable
 Instead measure “transmittance”,the fraction of incident radiation
transmitted by the solution
T = I/Io
 Where T = transmittance, Io = Incident radiation,I= exiting (transmitted)
radiation
 Absorbanceis:
A = -log T = log (Io/I)

14. Processes affecting T
 Reflection loss at air/cuvette interface
 Scatteringlosses in solution
 Absorption by analyte
 Absorption by cuvette material
 Absorption by interfering species-Particlecontaminationin a sample causes
light scatter, preventing unabsorbedlight reaching the detector.
 Air bubblesin sample: air drops cause refraction of the light from its
normal path, which can in turn cause some unabsorbedlight not to reach
the detectorand thus increasing apparentabsorption.
 Particles in sample: Again this will increase the apparent absorption. Thus
affect absorbancereadings.
 The single beam spectrophotometer is less accurateas compared to the
doublebeam spectrophotometer.

15. Application of Beer’s Law to Mixtures
 Beer’s law also applies to solutionscontainingmore than one absorbing
species
 In such cases the total absorbanceis the sum of individual component
absorbances
 AT = A1 + A2 + A3 + A4 + …. An

16. Limitations to the Applicability of
Beer’s Law
 Few exceptions to generalization that A is linearly related to path length
 Deviations from direct proportionality between A and C at fixed b are frequent
 Some of these are fundamental and represent real limitations of the law
 Others are a consequence of how the measurements were made…
(instrumental)
 Others include chemical changes associated with concentration changes
(chemical deviations)
 Beer’s law typically adhered to if C < 0.01M
 Limitations also depend on value of ε
 High ε will limit applicability to very low conc.
 Low ε will allow application to higher conc.
 Part of concentration limitation is due to potential for species (at high
concentration) to interact in solution and change how they interact with light
 Applies to same species and to others (electrolytes)
 Interaction between different species also impacts applicability

17. Use of analytical standards
Standard curve – at least three preferably four or
more dilutions of concentrated standard. The results
are plotted on a graph to produce a calibration curve
that is used to extrapolate the concetration of the
unknown
Beer’s law formula used,
absorbance of std As = absorbance of unknown Au
concetration of std Cs concetration of unknown Cu

18. Applications of UV-VIS Spectrometry
to Nutrient Analysis

19. Determination of inorganic phosphate.
 Phosphatechemistry is based on formation of a phospho-molybdateblue
complex
 Also use ascorbic acid/ferrous sulphatereduction
 Heating sample increases rate of color development
 Absorption maximum at 880nmthough there is also a peak at 720 nm
 Need to prepare blanks by mixing water with colourreagent(mix
everything else except phosphate)
 Inorganic phosphatereacts with ammonium molybdateto form
phosphomolybdatewhich is then reduced to form molybdenumblue.
 In this procedureferrous sulphate/ascorbicacid serves as the reducing
agent.
 Molybdenumblue is a coloured compound whose absorbancecan be
measured in the spectrophotometer.

20. Determination of inorganic phosphate.
 Information on the stability of the colouredcompoundsis important.
 The substanceto be measured is normally colourless but can be converted
to a colouredproduct.
 The stability of molybdenumblue can be determined by taking absorbance
measurements at various time intervals after addition of the colourreagent.
 In biological preparationsphosphatemay exist both as inorganic phosphate
and organic phosphate, e.g. sugar phosphateand nucleicacids.
 To measure organic phosphate,it is necessary to digest the organic portion
of the various moleculeswith sulphuricor perchloricacids.
 Organic phosphateis converted to inorganic phosphatewhich may then be
measured by the above procedure.
 Phosphateis measured before and after acid digestion and the difference
between the two values representsthe amount of organic phosphate.

21. Determination of inorganic phosphate
1.Prepare10ml of each the following potassium phosphatestandards10, 20
,40, 60, and 80 µg phosphate/ml from the given stock solution(100 µg/ml)-use
the dilution formula 𝐶1𝑉1 = 𝐶2𝑉2
2. Obtain a sample containingan unknown concentrationof phosphate.
3.Preparethe reagent blank by mixing 1ml of distilled water and 5ml of the
molybdate/FeS𝑂4 colourreagent.
4.Prepareduplicatesof the unknown solution.
5.Mix 1ml of each of the samples containingphosphate/standardswith 5ml of
the molybdate/FeS𝑂4 colorreagent in a test tube. If the colouris too dark to
be accuratelyread in the spectrophotometer(an absorbancereading above 0.9)
quantitatively dilutethe original sample with distilled water and repeat the
determination.
6.Mix well and leave the samples to stand at room temperaturefor 30 minutes

22. Determination of inorganic phosphate
7.Put each of the solutionsin 1cm cuvette(use the same cuvette) and take
absorbancesat 720nmin a spectrophotometerby first zeroing with the blank
reagent.
8..Plot a graph of absorbanceagainst the concentrationof the standards(a
standard curve).This should be a straight line positive slope passing through
the origin.use this standard curve to determine the pi conc.in the unknown
sample.
9.Record your results as mg of phosphateper 100ml of the original
solution(1mg=1000μg).

23. Standard curve or calibration curve

24. Absorption spectrum of molybdenum
blue
 A spectrophotometeris used to measure the absorbanceof Molybdenum
blueat different wavelengths.
 A graph of absorbanceagainst wavelength is drawn which is the absorption
spectrum.
 The absorptionspectra is the peak with the greatest absorbancewhich
correspondsto the wavelength at which the Molybdenumblue absorbs at
maximum.
 The sketch below is an expected absorptionspectrum
 The spectrumhas two absorptionpeaks,at 720nmand 880 nm and
maximum absorbanceis obtained at 880 nm but in this practical we used
720nmthe first peak only

25. Absorption spectrum of molybdenum blue

26. Absorption spectrum
N.B:Note that absorbance increases
with concentration thus at higher
concentration the absorption spectra
shifts upwards.
 Figure on the right shows the
absorption spectrum of solutions of
potassium permanganate (KMnO4)
at two different concentrations.
 The solution for curve 1 has a
higher concentration than that for
curve 2.

27. Clinical correlations