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emission of light by a compound after it has absorbed a particular wavelength of
light. Under most circumstances, the emission of light will occur at a longer
wavelength than the light used to excite it. The spectrometer comes with three
LEDs (375 nm, 450 nm, and 525 nm) that serve as the excitation wavelengths.
Additional excitation LEDs can be purchased separately.
There are three general types of data collection that measure fluorescence—
fluorescence
vs.
wavelength, which produces a spectrum, fluorescence
vs.
concentration, and fluorescence
vs.
time for kinetics experiments. Once the units
have been changed to Fluorescence from the Experiment menu, follow the
instructions in the section titled Collect Data with Logger
Pro
to collect these
types of data.
These are some additional features in fluorescence mode that may improve your
data quality:
Adjusting the LED Brightness
1. Open the Spectrometer dialog box to set the LED intensity. To display this box
choose Set Up Sensors ► Spectrometer from the Experiment menu in Logger
Pro
.
2. The LED Intensity is set to 50 by default. Adjust it between 0 and 100. A
setting of 0 turns the LED off while a setting of 100 is the maximum LED
intensity.
Note:
If you adjust this value during data collection, you may want
to recalibrate or perform a manual baseline adjustment with a calculated
column.
Adjusting the Sample Time
1. Open the Spectrometer dialog box to set the Sample Time. To display this box
choose Set Up Sensors ► Spectrometer from the Experiment menu in Logger
Pro
.
2. By default, this value is set to 100 ms. The Sample Time is the amount of time
the detector is exposed to the emission light. The longer the sample time, the
greater the signal, and the longer the time it takes to collect data. 100 ms is a
good starting point for data collection. You may adjust this value while data
collection is active. If you do this, the spectrum will update in real time.
Note:
If you adjust this value during data collection, you may want to recalibrate or
perform a manual baseline adjustment with a calculated column.
Measure Emission Spectra with Logger
Pro
You may use your Spectrophotometer to measure the visible emission spectrum of
a light source such as an LED or a gas discharge tube. To do so, you will need to
purchase an optical fiber assembly (order code: VSP-FIBER).
Measure Intensity of Light Emissions
1. Insert the Spectrophotometer Optical Fiber into the Fluorescence/UV-VIS
Spectrophotometer.
2. Aim the tip of the optical fiber cable at a light source. Click
. Click
to end data collection.
Note:
The Spectrophotometer is not calibrated
for measuring intensity.
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Option 1
The default option is to use a single 10 nm band. This measures
the average absorbance from ~5 nm on either side of the chosen
wavelength. You can change the center wavelength value by clicking on
the graph or by choosing a wavelength from the list.
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Option 2
If you wish to use the λ max chosen by Logger
Pro
, but you
want the absorbance to be measured only at that one wavelength, change
Single 10 nm Band to Individual Wavelengths. You may then select up to
ten wavelengths to measure at the same time.
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Option 3
If you wish to measure an average over a range of contiguous
wavelengths of your choice, change Single 10 nm Band to Individual
Wavelengths. Click
. Select boxes in the list or drag your cursor
on the graph to select up to ten contiguous wavelengths. Check Combine
Contiguous Wavelengths.
4. Click
to continue.
5. Click
. Place your first sample in the cuvette slot of the
Spectrophotometer. After the readings stabilize, click
. Enter the
concentration of the sample and click
.
6. Place your second sample in the cuvette slot. After the readings stabilize, click
. Enter the concentration of the second sample and click
.
7. Repeat Step 6 for the remaining samples. When finished, click
to end
data collection.
8. Click Linear Fit,
, to see the best fit line equation for the standard solutions.
9. If doing Beer’s law to determine the concentration of an unknown, place the
unknown sample in the cuvette holder. Choose Interpolation Calculator from
the Analyze menu. A helper box will appear, displaying the absorbance and
concentration of the unknown. Click
.
Measurement
vs.
Time (Kinetics)
1. Generate a spectrum as described above.
2. Click the Configure Spectrophotometer Data Collection button,
.
3. Select Absorbance
vs.
Time as the data-collection mode. The wavelength of
maximum absorbance will be selected. Click
to continue or select a
wavelength on the graph or in the list of wavelengths. See the previous section
for more details.
4. The default settings are 1 sample per second for 200 seconds. To change the
data-collection parameters for your experiment, choose Data Collection from
the Experiment menu and make the necessary changes. Click
.
5. Mix the reactants. Transfer ~2 mL of the reaction mixture to a cuvette and
place the cuvette in the Spectrophotometer. Click
. Click
if you
wish to end data collection early.
6. Click Curve Fit,
, to calculate a function for your data.
Measure Fluorescence with Logger
Pro
You may use your Spectrophotometer to measure the fluorescence spectrum of an
aqueous sample, such as chlorophyll, quinine, and fluorescein. Fluorescence is the
