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UV-VIS photometry
1. Identification by UV-VIS photometry
2. Effect of pH on absorption spectrum
of acid-base indicators
1. Theory
Molecules can absorb photons of suitable wavelength λ,
i. e. those corresponding to the energy of electronic
transition. In UV-VIS the photons exhibit energy
sufficient for outer electrons to be excited into higher
energy levels (200-600 kJ/mol).
Parts of the molecules where this effect occurs are called
chromophores. Absorption of UV-radiation occur by
transitions into antibonding * or * orbitals in compounds with ,  and n (nonbonding)
electrons (single or multiple bonds including nonbonding electron pairs). Absorption maxima
are shifted into visual (VIS) region if higher number of -* transitions is possible (e.g. due
to conjugation of double bonds). Visual absorption (transition to *) can occur with π or
nonbonding electrons.
Note: this text will not deal with energy transitions into d nor f orbitals (coordinate-covalent
bonding).
FIG. 1 Wavelengths of VIS electromagnetic radiation
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FIG.2 Maxima shifts of a conjugated system
FIG.3 En example of absorption spectrum with two maxima in VIS range
Definition of Transmitance, Absorbance and Lambert-Beer´s law
b=thickness of the cuvette [cm], =molar absorption coefficient
[l/mol.cm], c=molar concentration [mol/l], I0 and I=intensity of
the incident light and the transmitted light, respectively.
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INSTRUMENTATION
Classical Double-Beam Photometer with Monochromator
Monochromator selects (filtrates) from the polychromatic source the wavelength that is in use.
In double beam photometers there are two cuvettes (cells) so that blank sample can be always
measured.
Multichannel Spectrophotometer (single beam)
There is grating that decomposes a polychromatic beam after it passes through the cuvette!
The transmitted light is dispersed and measured “all wavelengths at ones”. This principle is
used at the photometer HP 8543 utilized in this exercise.
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2. Acid-base indicators
What is an acid-base indicator?
An acid-base indicator is a weak acid or a weak base. The undissociated form of the indicator
is a different color than the iogenic form of the indicator. An indicator does not change color
from pure acid to pure alkaline at specific hydrogen ion concentration, but rather, color
change occurs over a range of hydrogen ion concentrations. This range is termed the color
change interval. It is expressed as a pH range:
Indicator pH Range Quantity per 10 ml Acid Base
Thymol Blue 1.2-2.8 1-2 drops 0.1% soln. in aq. red yellow
Pentamethoxy red 1.2-2.3 1 drop 0.1% soln. in 70% alc. red-violet colorless
Tropeolin OO 1.3-3.2 1 drop 1% aq. soln. red yellow
2,4-Dinitrophenol 2.4-4.0 1-2 drops 0.1% soln. in 50% alc. colorless yellow
Methyl yellow 2.9-4.0 1 drop 0.1% soln. in 90% alc. red yellow
Methyl orange 3.1-4.4 1 drop 0.1% aq. soln. red orange
Bromphenol blue 3.0-4.6 1 drop 0.1% aq. soln. yellow blue-violet
Tetrabromphenol blue 3.0-4.6 1 drop 0.1% aq. soln. yellow blue
Alizarin sodium sulfonate 3.7-5.2 1 drop 0.1% aq. soln. yellow violet
-Naphthyl red 3.7-5.0 1 drop 0.1% soln. in 70% alc. red yellow
p-Ethoxychrysoidine 3.5-5.5 1 drop 0.1% aq. soln. red yellow
Bromcresol green 4.0-5.6 1 drop 0.1% aq. soln. yellow blue
Methyl red 4.4-6.2 1 drop 0.1% aq. soln. red yellow
Bromcresol purple 5.2-6.8 1 drop 0.1% aq. soln. yellow purple
Chlorphenol red 5.4-6.8 1 drop 0.1% aq. soln. yellow red
Bromphenol blue 6.2-7.6 1 drop 0.1% aq. soln. yellow blue
p-Nitrophenol 5.0-7.0 1-5 drops 0.1% aq. soln. colorless yellow
Azolitmin 5.0-8.0 5 drops 0.5% aq. soln. red blue
Phenol red 6.4-8.0 1 drop 0.1% aq. soln. yellow red
Neutral red 6.8-8.0 1 drop 0.1% soln. in 70% alc. red yellow
Rosolic acid 6.8-8.0 1 drop 0.1% soln. in 90% alc. yellow red
Cresol red 7.2-8.8 1 drop 0.1% aq. soln. yellow red
-Naphtholphthalein 7.3-8.7 1-5 drops 0.1% soln. in 70% alc. rose green
Tropeolin OOO 7.6-8.9 1 drop 0.1% aq. soln. yellow rose-red
Thymol blue 8.0-9.6 1-5 drops 0.1% aq. soln. yellow blue
Phenolphthalein 8.0-10.0 1-5 drops 0.1% soln. in 70% alc. colorless red
-Naphtholbenzein 9.0-11.0 1-5 drops 0.1% soln. in 90% alc. yellow blue
Thymolphthalein 9.4-10.6 1 drop 0.1% soln. in 90% alc. colorless blue
Nile blue 10.1-11.1 1 drop 0.1% aq. soln. blue red
Alizarin yellow 10.0-12.0 1 drop 0.1% aq. soln. yellow lilac
Salicyl yellow 10.0-12.0 1-5 drops 0.1% soln. in 90% alc. yellow orange-brown
Diazo violet 10.1-12.0 1 drop 0.1% aq. soln. yellow violet
Tropeolin O 11.0-13.0 1 drop 0.1% aq. soln. yellow orange-brown
Nitramine 11.0-13.0 1-2 drops 0.1% soln in 70% alc. colorless orange-brown
Poirrier's blue 11.0-13.0 1 drop 0.1% aq. soln. blue violet-pink
Trinitrobenzoic acid 12.0-13.4 1 drop 0.1% aq. soln. colorless orange-red
Because of the color change, the indicators are utilized for visual indication of pH change,
typically at acidobasic titrations.
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FIG. 4 Sheme of Hewlett Packard 8453
http://www.p-forster.com/english/themes/Spectroscopy/BASICS/
Instrument
Single-beam photometer HP8453
Chemicals and dish
Acids: benzoic a., caffeic a., p-cumaric a., gentisic a., cinnamic a., gallic a.
Methyl orange / phenolphthalein / bromthymol blue (or other indicators)
6x25 ml, 6x100 ml volumetric flasks, ethanol / methanol.
Quartz cuvette
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http://www.unco.edu/nhs/chemistry/Instrument/uvvis.html
Operating Instructions of Diode Array Spectrophotometer HP8453
Turning on the Instrument:
1. Switch on PC. Wait until CAG-Bootp window appears
2. Turn on the HP 8453 Diode Array. (Power button directly
below model plate.) Wait for the yellow light on the front of the
instrument to turn green. (This takes a few minutes.) CAGBootp
window shows some lines like this:
3. Double click on the HP Diode Array icon (on-line). When the window
UV-Visible ChemStation appears, click on Cancel, and the software program
will continue to load.
Taking Spectrophotometric Data:
1. For data to be taken, a task must be selected. Your selections
are found by clicking on the down arrow u of the Task box: (The
following selections appear.)
Fixed Wavelengths (Default)
Spectrum/Peaks
Ratio/Equation
Quantification
Determination of max (Spectrum/Peaks Task)
1. To determine a solution's wavelength of maximum absorption (max), click on
Spectrum/Peaks. The Spectrum/Peaks Parameters window will appear.
2. For normal use, the following Spectrum/Peaks parameters should be utilized:
Peak/Valley find
Find and annotate up* to 1 peaks (default is on X and 3)
Find and annotate up* to 3 valleys (default is on X and 3)
Prompt for sample information* (default is off)
*(to turn parameter off _ or on X click on parameter)
Data type* Display spectrum**
Absorbance From: 190 nm To: 1100 nm
3. Click on OK when finished, if you need to change any parameters click on Setup, and
enter the new parameters.
4. You are now ready to take data.
a. Taking a spectrum of your blank: Place your blank in a clean cuvette,
inspect for bubbles, and then carefully wipe the exterior surface. Place the cuvette
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into the holder in the diode array 8453 (make sure cuvette is positioned correctly), and
secure the locking mechanism by gently pressing down on the lever. Locate the Sampling
box (left side of screen), and confirm the Manual setting. Click on Blank. This spectrum
displays where the cuvette or blank is absorbing.
b. Taking a spectrum of your sample: Rinse the cuvette well, and fill it with sample.
Then, inspect for bubbles, and carefully wipe the exterior surface (with a Kimwipe). Place the
cuvette into holder, and secure it. Click on Sample .
c. Printing your sample spectrum: Locate the print icon (upper left corner of screen),
and click on it.
Shutting Down the Instrument:
1. Click on File, and then click on Exit ChemStation.
2. When the window, Close HP 845x UV-Visible, appears, make sure you DO NOT Save
Configuration, and then click on OK.
3. Click on CAG Bootp Server (lower left corner of screen), click on File, then Exit.
4. Click on Start (lower left corner of screen), then Shut Down. When the window Shut
Down Windows appears click on Yes .
6. Turn off the HP 8453 Diode Array.
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Sample preparation and measurement
1. Qualitative analysis of spectra
 Set the spectrum range to 210-410 nm
 Prepare solutions of standards of suitable concentration (20 mg/l)
 Measure blank
 Take spectra of all the standards and record the spectra characteristics (record
maxima and minima wavelengths, maxima intensities)
 Take a spectrum of an unknown sample
2. Spectral change of an acid-base indicator
 Set the spectrum range to 210-700 nm
 Prepare a solution of a selected indicator(s) – find a suitable concentration by stepwise
dilution so that the absorbance does not exceed 2
 Measure (adjust) pH of the solution and take the spectrum
 Adjust pH (addition of a drop of a strong acid or a strong base) until a visual color
change occurs and take the spectrum again
Evaluation of the data
 Comparing the spectrum of the unknown sample to spectra of standards confirm the
unknown
 After exporting the data from your experiments (USB-flash), construct the first
derivative spectrum and decide what an advantage the derivative spectrum brings
 Describe a color change of the indicator by shifts of absorption maximum. Draw the
indicator formula in both the colored forms and note the indicator pH-range.