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Ultraviolet-Visible (UV-Vis) Spectroscopy Background Information
Instructions for the Operation of the Cary 300 Bio
UV-Visible Spectrophotometer
See the Thermo OMNIC Help reference on page 49.
Ultraviolet-Visible (UV-Vis) Spectroscopy
Background Information
The ultraviolet-visible (UV-Vis) spectrophotometer is an instrument commonly
used in the laboratory that analyzes compounds in the ultraviolet (UV) and visible (Vis)
regions of the electromagnetic spectrum. Unlike infrared spectroscopy (which looks at
vibrational motions), ultraviolet-visible spectroscopy looks at electronic transitions. It
allows one to determine the wavelength and maximum absorbance of compounds. From
the absorbance information and using a relationship known as Beer’s Law (A = εbc,
where A = absorbance, ε = molar extinction coefficient, b = path length, and c =
concentration), one is able to determine either the concentration of a sample if the molar
extinction coefficient is known, or the molar absorptivity, if the concentration is known
(Wade 676-681). Molar extinction coefficients are specific to particular compounds,
therefore UV-Vis spectroscopy can aid one in determining an unknown compound’s
identity. Furthermore, the energy of a compound can be ascertained from this technology
by using the equation E = hc/λ (where E = energy, h = Planck’s constant, c = speed of
light, and λ = wavelength). Since photons travel at the speed of light, and h and c are
constants, one can find the energy (Wade 501).
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Cary 300 Bio UV-Visible Spectrophotometer
Be sure to turn on the UV-Vis at least 20 minutes before you intend on running
samples; it needs to warm up properly in order to function accurately.
Selecting a Wavelength to Run Your Experiment At
If your instructor has not given you a specific wavelength to run your samples at, you
must determine the appropriate wavelength by performing a scan.
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From the “Start” menu, click on “Programs” → “Cary WinUV” → “Scan”.
On the top toolbar options, click on the “Setup” tab.
o Under the “Cary” tab, type in your start and stop parameters (what
region you want the instrument to scan). Click OK.
o Under the “Options” tab, select whether you want to scan in the UV,
Vis, or UV-Vis range.
Use a Kimwipe to wipe down the sides of the cuvette and be careful to avoid
touching the sides of the cuvette when putting it in the instrument.
Zero the instrument by placing blanks (your solvent) in cells 1 and 7. On the
cuvette, the side with the arrow should be facing the left (there is a yellow
arrow on the top of the UV/Vis indicating this) when you insert them in the
cells. Having the cuvettes facing the same direction will ensure that the path
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lengths are the same. Close the lid on the UV-Vis and hit the Zero button on
the left-hand side of the screen.
o The box in the upper left-hand corner will show that the instrument has
been zeroed by displaying an absorbance of 0.000. The absorbance
reading may vary slightly (bounce above and below 0.000); this is due
to noise and is normal.
• To run a sample, take the blank cuvette out of cell 1 and replace it with the
sample to be run. Leave the other blank in cell 7 for all subsequent runs.
Click Start.
Saving/Retrieving Data
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To save data, go to “File” → “Save Data As”. Enter a filename and click
“Save”. Your file will be saved in the .BSW format.
To open saved data, go to “File” → “Open Data” → select the desired file.
Analysis
Once a plot has been drawn, you can use the several buttons on the toolbar to manipulate
the graph.
“Cursor mode”
• “Free mode”: the cursor (which appears as a +) can be moved in any
direction without any restrictions.
• “Track mode”: along with the cursor, a set of intersecting lines will
appear. As you drag the cursor to the left or right, the horizontal line rides
along the line produced by the data points. You can monitor the X and Y
values that result from specific data points by looking in the right-hand
corner beneath the graph.
“Track Preferences”
• This displays the names of the lines generated from your data points and
their corresponding colors and filenames.
“Graph Preferences”
• This allows you to change the color and width of the axes, as well as the
font and the way the data is plotted (i.e. dots or solid lines).
“Scale Graph”
• This allows you to change the scale of the graph by typing in the area you
would like to focus on.
“Add Label”
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This feature allows you to add labels to your graph.
Unless otherwise instructed, when running samples do not use the baseline
correction feature. This uses an already-stored file as the baseline for your sample.
Simple Reads
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This selection allows you to take a measurement at one wavelength.
Enter “Setup” and type in the wavelength you desire to read your sample
at.
Normally, the UV-Vis is setup to read in absorbance; if you wish to
change the setting to percent transmittance, click the “%T” button.
Running the sample:
o Zero the instrument, then place your sample in the UV-Vis and
press the “Read” button.
Running Multiple Samples in Scan Mode
Cell changer
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From the “Start” menu, click on “Programs” → “Cary WinUV” →
“Scan”.
Under the “Setup” tab select the following settings:
o “Cary” → set the start/stop wavelengths → OK.
o “Options” → UV-Vis
o “Baseline” → “Correction” → None
o “Accessory 1” → “Use Cell Changer” → “Select Cells”. Check
the boxes of the cells you want to use → OK. Do not check the
cell box that you have your blanks in or they will be used as
samples.
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Zero the instrument.
Click the “Start” button. A “Save As” box appears; create a filename and
click “Save”. The “Cell Loading Guide” box will appear indicating the
active cells; clicking in the desired cell box will allow you to change the
name of the sample.
Traces will be drawn starting with the first cell clicked. With varying
concentrations you will be able to see the various intensities corresponding
to the concentrations. Moving the cursor over the desired trace and
clicking the left mouse button will indicate the sample number or name
that you have designated.
Concentration
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Setup
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From the “Start” menu, click on “Programs” → “Cary WinUV” →
“Concentration”.
Go “Setup”; a “Setup” box then appears with tabs. To set up the
parameters for your experiment, fill in the appropriate tabs.
“Cary” tab:
o “Wavelength (nm)” – type in the desired wavelength.
o “Ave. Time (sec)” – a good starting point is 0.100.
o “SBW (nm)” – this is the spectral bandwidth. It is automatically
set at 1.5; this value may need to be adjusted.
o “Replicates” – set this value at 1 unless otherwise instructed.
o “Sample/Std Averaging” should not be clicked unless instructed.
o “Y Mode” – “Abs” should be clicked.
o The “Y Min” and “Y Max” fields need to be set.
“Standards” tab:
o “Standards” box:
ƒ The “Calibrate during run” box should be checked.
ƒ Change “Units” to the appropriate labeling by the
drop-down menu.
ƒ From the “Standards” drop-down menu, adjust
accordingly.
™ The box below should then change to the
correct number of standards as you
indicated.
ƒ In the “Std/Conc” box, change the concentration so
that it correctly corresponds to your standards.
o “Fit Type” box:
ƒ Select the appropriate curve fitting for your method.
ƒ “Min R2” should be set at 0.9500 to start.
“Samples” tab:
o “Sample Names” box:
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“Number of Samples” can be adjusted with the
toggle; the box below should then change
accordingly. You can change the name of the
sample by clicking in the box and typing.
o “Weight/Volume Corrections”
ƒ Enter values as needed.
“Options” tab:
o In the “Source” box:
ƒ The “Autolamps off” box should be unchecked.
ƒ The “UV-Vis” button should be selected.
ƒ “Source Changeover (nm)” is automatically set at
350 nm. Ask your instructor if this needs
adjustment.
o In the “Beam Mode” box:
ƒ The “Normal” button should be clicked.
ƒ “Double Beam” should be toggled on.
“Accessories 1” tab
o “Cells” box – this allows you to use a cell changer or well plate.
ƒ If you do not want to run multiple samples at the
same time, the “Use Cell Changer” box should not
be checked.
ƒ If you do want to run multiple samples, check the
“Use Cell Changer” box and refer to the instructions
for this feature as previously mentioned.
o “Temperature” box:
ƒ The “Automatic Temperature Setting” box should
not be checked unless the method you are using
requires the samples to be above room temperature.
o “RBA” box
ƒ The “Use RBA” box should not be checked.
“Accessories 2” tab:
o None of the boxes in this tab should be selected.
“Samplers” tab:
o Nothing should be checked.
o The drop-down box should read “None”.
“Reports” tab:
o This will probably not be used unless instructed by your professor.
“Autostore” tab:
o The “Storage on (Prompt at end)” toggle should be highlighted.
After all of the parameters have been set, press OK.
Running Standards/Samples
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Zero the instrument as previously indicated.
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Put the first standard in the cell holder and press “Start”. A “Standard/Sample
Selection” will appear. Select the standards and samples for your calibration so
that they are in the “Selected for Analysis” portion of the box. Move any other
solutions over to the “Solutions Available” box by using the arrows.
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will move the selected standards or samples to the left side
(Solutions Available) and right side (Selected for Analysis), respectively.
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will move all standards and solutions to the left and right
side, respectively.
When the standards and samples are in the correct positions, press OK.
o A “Present Standard” box will appear. Press OK if it indicates that the
correct standard is selected. Once a reading has completed, the “Present
Standard” box will appear again indicating the next standard. Repeat until
all standards have been run.
After the standards have been run, a calibration curve (or line) will be drawn
and a correlation coefficient will be displayed. In some cases, however, a box
labeled “Concentration” will appear saying, “Min R2 test failed”. This means
that your data did not fit with the indicated correlation coefficient (i.e. you had
bad data). Proceed by pressing OK. A “Standards” box will appear saying,
“There is no valid calibration. Proceed in Abs?” By pressing OK, the
absorbance of subsequent samples will be measured, but no concentration will
result since the calibration failed.
A “Present Sample” box then appears. Follow the instructions as with the
standards.
Closing Down
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Save your data as needed and exit out of the program.
Log-off.
Turn off the instrument.
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Absorbance reading
Toolbar for analysis
Changes to a Start
button when online
Wavelength