UV-VISIBLE SPECTROPHOTOMETRY AND INORGANIC PHOSPHATE DETERMINATION.pdf

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 detector is measured.
The spectrophotometer measures the amount of light of particular
wavelength that a sample absorbs.
The amount of light absorbed is directly proportional to the number of
absorbing molecules of the solute or concentration of the solution(Beer-
Lambert law)

3. UV-Visible Spectrophotometry
Technique based on absorption of 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 electronic transitions and
absorb/emit monochromatic radiation.
Complex ions and molecules have multiple possible electronic transitions
owing to many overlapping molecular orbitals.
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 absorbance of electromagnetic radiation by a given species is
directly proportional to the concentration of the analyte.
It is expressed as: A= εbC
where A is the absorbance, ε is the molar absorptivity, b is the path length
and C is the concentration of analyte.
Because ε and b are fixed under experimental conditions the result is a
linear relationship between absorbance and 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 substance can often be identified by constructing an
absorption spectrum.
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 spectrum as appropriate and should
represent a series of wavelengths at consecutive intervals.
A given substance has one or more absorption peaks at specific
wavelengths.
This enables identification of unknown substances(qualitative analysis)

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

13. Measurement of Absorbance
Absorbance is 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
Absorbance is:
A = -log T = log (Io/I)

14. Processes affecting T
Reflection loss at air/cuvette interface
Scattering losses in solution
Absorption by analyte
Absorption by cuvette material
Absorption by interfering species-Particle contamination in a sample causes
light scatter, preventing unabsorbed light reaching the detector.
Air bubbles in sample: air drops cause refraction of the light from its
normal path, which can in turn cause some unabsorbed light not to reach
the detector and thus increasing apparent absorption.
Particles in sample: Again this will increase the apparent absorption. Thus
affect absorbance readings.
The single beam spectrophotometer is less accurate as compared to the
double beam spectrophotometer.

15. Application of Beer’s Law to Mixtures
Beer’s law also applies to solutions containing more than one absorbing
species
In such cases the total absorbance is 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.
Phosphate chemistry is based on formation of a phospho-molybdate blue
complex
Also use ascorbic acid/ferrous sulphate reduction
Heating sample increases rate of color development
Absorption maximum at 880nm though there is also a peak at 720 nm
Need to prepare blanks by mixing water with colour reagent(mix
everything else except phosphate)
Inorganic phosphate reacts with ammonium molybdate to form
phosphomolybdate which is then reduced to form molybdenum blue.
In this procedure ferrous sulphate/ascorbic acid serves as the reducing
agent.
Molybdenum blue is a coloured compound whose absorbance can be
measured in the spectrophotometer.

20. Determination of inorganic phosphate.
Information on the stability of the coloured compounds is important.
The substance to be measured is normally colourless but can be converted
to a coloured product.
The stability of molybdenum blue can be determined by taking absorbance
measurements at various time intervals after addition of the colour reagent.
In biological preparations phosphate may exist both as inorganic phosphate
and organic phosphate, e.g. sugar phosphate and nucleic acids.
To measure organic phosphate, it is necessary to digest the organic portion
of the various molecules with sulphuric or perchloric acids.
Organic phosphate is converted to inorganic phosphate which may then be
measured by the above procedure.
Phosphate is measured before and after acid digestion and the difference
between the two values represents the amount of organic phosphate.

21. Determination of inorganic phosphate
1.Prepare 10ml of each the following potassium phosphate standards 10, 20
,40, 60, and 80 µg phosphate/ml from the given stock solution (100 µg/ml)-use
the dilution formula ??????
1??????
1=??????
2??????
2
2.Obtain a sample containing an unknown concentration of phosphate.
3.Prepare the reagent blank by mixing 1ml of distilled water and 5ml of the
molybdate/FeS??????
4
colour reagent.
4.Prepare duplicates of the unknown solution.
5.Mix 1mlof each of the samples containing phosphate/standards with 5ml of
the molybdate /FeS??????
4color reagent in a test tube. If the colour is too dark to
be accurately read in the spectrophotometer (an absorbance reading above 0.9)
quantitatively dilute the original sample with distilled water and repeat the
determination.
6.Mix well and leave the samples to stand at room temperature for 30 minutes

22. Determination of inorganic phosphate
7.Put each of the solutions in 1cm cuvette(use the same cuvette) and take
absorbances at 720nm in a spectrophotometer by first zeroing with the blank
reagent.
8..Plot a graph of absorbance against the concentration of 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 phosphate per 100ml of the original
solution(1mg=1000μg).

23. Standard curve or calibration curve

24. Absorption spectrum of molybdenum
blue
A spectrophotometer is used to measure the absorbance of Molybdenum
blue at different wavelengths.
A graph of absorbance against wavelength is drawn which is the absorption
spectrum.
The absorption spectra is the peak with the greatest absorbance which
corresponds to the wavelength at which the Molybdenum blue absorbs at
maximum.
The sketch below is an expected absorption spectrum
The spectrum has two absorption peaks,at 720nm and 880 nm and
maximum absorbance is obtained at 880 nm but in this practical we used
720nm the 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