4
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 LabQuest 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. To set the LED intensity, tap on the red meter and select Set LED.
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. On the Meter screen, tap Mode to set the Sample Time.
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.
Calibrate Fluorescence
1. Fill a cuvette about 3/4 full with distilled water (or the solvent being used in
the experiment) to serve as the blank.
2. To calibrate the spectrophotometer, choose Calibrate ► Spectrometer from the
Sensors menu.
3. Follow the instructions in the dialog box to complete the calibration, and then
click
.
Measure an Emission Spectrum with LabQuest
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 at a light source. Start data collection. Tap the
Stop button to end data collection.
Note:
The Spectrophotometer is not
calibrated for measuring intensity.
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 sample time (see Change the Settings in LabQuest below).
Measurement
vs.
Concentration (Beer’s Law Studies)
1. Generate a spectrum as described above. On the Meter screen, tap Mode.
Change the mode to Events with Entry.
2. Enter the Name (e.g., Concentration) and Units (e.g., mol/L). Select OK.
3. A message will appear warning you to either save or discard the full spectrum
run. Make your choice and proceed with data collection.
4. Place your first Beer’s law standard solution in the Spectrophotometer. Start
data collection. After the absorbance reading stabilizes, tap Keep. Enter the
concentration of the solution and select OK.
5. Place your second standard sample in the Spectrophotometer. After the
absorbance readings stabilize, tap Keep. Enter the concentration of the second
sample and select OK.
6. Repeat Step 5 for the remaining standard samples. After you have tested the
final standard, tap the Stop button to end data collection.
7. To calculate a best fit line equation for your standards, choose Curve Fit from
the Analyze menu. Select Linear for the Fit Equation, and then select OK. The
graph screen will appear again with the linear regression equation displayed.
8. Place a cuvette containing an unknown sample of solution in the
Spectrophotometer. Tap the Meter tab and write down the displayed
absorbance value. Tap the Graph tab and choose Interpolate from the Analyze
menu. Trace the linear regression equation to determine the concentration of
the unknown.
Measurement
vs.
Time (Kinetics)
1. Generate a spectrum as described above. On the Meter screen, tap Mode.
Change the data-collection mode to Time Based.
2. You can change the rate, interval, and/or duration of time of data collection, if
desired. Select OK when you are ready to proceed.
3. A message will appear warning you to either save or discard the full spectrum
run. Make your choice and proceed with data collection.
4. Mix the reactants, transfer ~2 mL of the reaction mixture to a cuvette and
place the cuvette in the Spectrophotometer. Start data collection. You may tap
the Stop button to end data collection early.
5. To calculate a function for your data, choose Curve Fit from the Analyze
menu. Select the Fit Equation, and then select OK. The graph screen will
appear again.
Measure Fluorescence with LabQuest
You may use your Spectrophotometer to measure the fluorescence spectrum of an
aqueous sample, such as chlorophyll, quinine, 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 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.
