3
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Set the excitation wavelength.
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Click or tap on the gear to set the excitation wavelength so it appears
correctly in column headers and graph axes.
Note:
The default excitation wavelength is 375 nm. The app cannot
automatically detect the wavelength of the LED you are using. Be sure to
update the value from the drop down list to match your LED.
Using the Spectrophotometer with LabQuest 2 or LabQuest 3
Select the Type of Data (or Units) You Want to Measure
There are three general types of data collection that measure absorbance or
transmittance—absorbance (or %T)
vs.
wavelength, which produces a spectrum,
absorbance (or %T)
vs.
concentration for Beer’s law experiments, and absorbance
(or %T)
vs.
time for kinetics experiments.
The default data type is absorbance. If you want to measure the absorbance of a
solution, proceed directly to the Calibrate section.
If you want to measure %T, fluorescence, or intensity, do the following:
1. From the Sensors menu, choose Change Units ► USB: Spectrophotometer.
2. Select the unit or data type you wish to measure.
Calibrate the Spectrophotometer (Not Required if Measuring Intensity or
Fluorescence)
1. Choose Calibrate ► USB: Spectrophotometer from the Sensors menu.
Note:
For best results, allow the Spectrophotometer to warm up for a minimum of
five minutes.
2. Fill a cuvette about 3/4 full with distilled water (or the solvent being used in
the experiment) to serve as the blank. After the Spectrophotometer has warmed
up, place the blank cuvette in the Spectrophotometer.
3. Follow the instructions in the dialog box to complete the calibration, and then
tap OK.
Collect Data with LabQuest
Measurement
vs.
Wavelength (Generate a Spectrum)
1. Fill a cuvette about 3/4 full of the solution to be tested and place it in the
Spectrophotometer.
2. Start data collection by tapping on the Start button in the lower left corner of
the screen. Tap the Stop button to end data collection.
3. Select wavelength.
Note:
The wavelength of maximum absorbance (λ max)
selected will be used for any subsequent data collection, such as a Beer’s law
experiment (abs
vs.
conc.) or a kinetics experiment (abs
vs.
time). You can tap
on the graph to select a wavelength. Another way to change the wavelength is
to navigate to the Meter screen, tap on the meter, and select Change
Wavelength. Enter the wavelength of your choice and select OK. If the
wavelength you type in is not measured by the unit, LabQuest will
automatically choose the wavelength closest to your choice.
4. To store the spectrum data, tap on the file cabinet icon in the upper right of
your screen.
1. Connect the spectrometer following the steps in the Getting Started section of
this user manual.
2. Open Spectral Analysis.
3. Select the appropriate Emissions experiment from the listed options and follow
the prompts in the app. Intensity is a relative measure with a range of 0–1.
Note:
The Spectrophotometer is not calibrated for measuring intensity.
4. Aim the tip of the optical fiber at a light source. Start data collection. Click or
tap the Stop button to end data collection.
If the spectrum maxes out (flat and wide peaks at a value of 1), increase the
distance between the light source and the tip of the optical fiber cable or reduce
the integration time (see the Change the Settings in Spectral Analysis section).
To adjust the integration time, click or tap the gear. Set the integration time to a
suitable value.
Measure Fluorescence with Spectral Analysis
You may use your spectrophotometer to measure the fluorescence spectrum of an
aqueous sample, such as chlorophyll, quinine, riboflavin, and fluorescein.
Fluorescence is the emission of light by a compound after it has absorbed a
particular wavelength of light. Under most circumstances, the emission of light
after excitation 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. These were chosen specifically for common
fluorescent compounds such as quinine, fluorescein, DAPI, and tryptophan.
Additional 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. To collect these
types of data after the experiment type has been selected as Fluorescence, follow
the instructions in the Spectral Analysis user manual at
.
Note:
You may need to change the integration time to get accurate peak
intensities in fluorescence mode. To adjust the integration time click or tap on the
gear. Set the integration time to a suitable value. Temporal averaging is another
important value to adjust to get stronger peak intensities.
These are some additional features in fluorescence mode that may improve your
data quality:
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Adjust the LED brightness.
1. Click or tap on the gear to adjust the LED intensity.
2. Adjust the LED intensity between 0% and 100%. A setting of 0% turns the
LED off, while a setting of 100% is the maximum LED intensity.
Note:
The LED intensity is set to 50% by default.
Note:
If you adjust this value during data collection, you may want to
recalibrate or perform a manual baseline adjustment with a calculated column.
