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CH 4212 Instrumental Analysis Fall, 2012
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UV-VIS Absorption Spectrophotometry
Background:
This exploration will introduce you to the process of taking
UV-Vis spectra using the Perkin-
Elmer Lambda 35 Spectrophotometer in Room 408.
Fundamentally, this instrument is no different from the Ocean
Optics Spectrophotometers thatyou used in Quantitative Analysis.
The same principles hold: Beers Law determines theabsorbance at any
wavelength. You get the same information (absorption spectra,
quantitativemeasurements, etc.) What is different is that this
Lambda 35 has powerful (and complicated)software attached to it
that requires you to learn specific operations to perform what
should be asimple task collecting spectra.
Because the learning curve for the software is so steep, your
actual laboratory work is reduced tothe preparation of a simple
dilution series of caffeine in water, from which you will generate
the
associated UV spectra. Using the same solutions, you will use
the Lambda 35 to generate acalibration curve using absorbance
measurements obtained at a single analytical wavelength.
Exploration:
Familiarization with the Lambda 35
You will each have a copy of the UV-Vis SOP written for the
Instrumental Analysis lab.
You will learn to
Perform pre-analysis cleanup and system checks Turn on the
instrument Handle quartz cuvettes Create a personal folder on the
instrument computer Generate spectrum; preparation (caffeine in
water); store Set up a data table in the software Generate a
calibration plot; store; print Shut down procedure
Cuvette CareThe quartz cuvettes you will be using are expensive
(~$350 for a matched set) so be careful
when handling the cuvettes. Keep them in the poly cuvette rack
between measurements andreturn them cleaned to the storage box when
you are through for the day. Clean the outer opticalfaces with lens
paper (not Kimwipes or paper towels). Rinse the inside with water
and dry witha Q-tip.
Preliminary spectrum and solution prepYour instructor will
provide you with a stock solution of caffeine in water. Make a
roughdilution using a graduated cylinder and a beaker. Obtain the
absorbance spectrum from 310 to240 nm. (You may have to do this
more than once.) From the absorbance at the wavelength of
maximum absorbance (max), calculate the solution concentrations
needed to make up a series of
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CH 4212 Instrumental Analysis Fall, 2010
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five solutions that will give you absorbances at max in the
range of 0.2 to 1.0 AU. Prepare thesesolutions with volumetric
glassware using proper lab technique learned in Quantitative
Analysis.All volumes should individually be less than 100 mL.
Solution spectraObtain the absorbance spectra (310 240 nm) of
the solutions you prepared, using the Scan
method on the Lambda 35. If the maximum absorbances fall outside
the AU range specified,you must prepare new solutions. Store each
of the spectra according to the SOP and thenproduce a plot of all
the spectra on one graph.
Calibration plotUsing the solutions prepared above, switch to
Wavelength method and generate a calibration plot
from the absorbance at max.
Moving filesCreate a personal folder in the CH 4212 folder. Move
your Method and Task files out of thecommon directory to your
personal folder.
Shut downPay special attention to the shut down procedure in the
UV-Vis SOP. There is a specific order inwhich operations must be
performed to protect the source.
Ending the exploration
Have your notebook signed by your instructor. Sign the log
book.
Lab Practicum:
At some point, you will schedule your lab practicum to prove
your competence on UV-Vis.From this exploration, you should know
how to do the following:
1. Instrument turn-on2. Care, handling of quartz cuvettes3.
Create personal folder; move Method and Task files to this folder4.
Calculation of needed solution concentration from initial
spectrum5. Generate spectra; store; overlay in printout6.
Generation of calibration curve7. Shut down procedure8.
Post-analysis cleanup; storing of materials; proper disposal of
waste
