Connect the Spectrophotometer following the steps in the Getting Started section of this user manual.
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 below.
If you want to measure %T, fluorescence, or intensity, do the following:
- Choose Change Units ► Spectrophotometer from the Experiment menu.
- Select the unit or data type you wish to measure.
Calibrate (Optional if Measuring Fluorescence or Intensity)
- To calibrate the Spectrophotometer, choose Calibrate ► Spectrophotometer from the Experiment menu. Note: For best results, allow the Spectrophotometer to warm up for a minimum of ten minutes.
- 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. Align the cuvette so the clear side of the cuvette is facing the light source.
- Follow the instructions in the dialog box to complete the calibration, and then click .
Collect Data with Logger Pro
Measurement vs. Wavelength (Generate a Spectrum)
- Fill a cuvette about 3/4 full of a sample of the solution to be tested. Place the sample in the Spectrophotometer and click . Click to end data collection.
- To store the spectrum data, choose Store Latest Run from the Experiment menu.
Measurement vs. Concentration (Beer’s Law Studies)
- Generate a spectrum as described above.
- Click the Configure Spectrophotometer Data Collection button, .
There are three regions in this box:
- Collection Mode The three options for data collection are offered. If the measurement (Absorbance in this example) vs. Time or vs. Concentration is selected, a wavelength or wavelengths will need to be chosen.
- Graph The graph displays a full-spectrum analysis of the sample in the cuvette holder. By default, the wavelength with the maximum measured value will be selected. You may wish to choose a different wavelength by tapping on the graph or selecting wavelength(s) from the list.
- List of wavelength options This column lists all the available wavelengths. It becomes active when either the Concentration or Time mode is selected.
Configure Spectrometer data-collection dialog box
- Select Absorbance (or %T) vs. Concentration as the data-collection mode. The wavelength with the maximum value from the spectrum (λ max) will be automatically selected. There are three options when choosing a wavelength (or wavelengths) for subsequent measurements.
- 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.
- 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.
- 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.
- Click to continue.
- 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 .
- Place your second sample in the cuvette slot. After the readings stabilize, click . Enter the concentration of the second sample and click .
- Repeat Step 6 for the remaining samples. When finished, click to end data collection.
- Click Linear Fit, , to see the best fit line equation for the standard solutions.
- 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)
- Generate a spectrum as described above.
- Click the Configure Spectrophotometer Data Collection button, .
- 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.
- 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 .
- 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.
- 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 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
- 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.
- 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
- 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.
- 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.
- Fill a cuvette about 3/4 full with distilled water (or the solvent being used in the experiment) to serve as the blank.
- To calibrate the spectrophotometer, choose Calibrate ► Spectrometer from the Experiment menu. The calibration is done automatically.
Measure Emission Spectra with Logger Pro
You may use your Spectrophotometer to measure the 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
- Insert the Spectrophotometer Optical Fiber into the Fluorescence/UV-VIS Spectrophotometer, lining up the white triangles.
- 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.
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 Logger Pro).
To increase the sample time, or if data collection is unusually slow, choose Set Up Sensors ► Spectrophotometer: 1 from the Experiment menu. Set the Sample Time (begin with 75 ms, with subsequent reductions by 20 ms) to a suitable value and decrease the Samples to Average to 1.
Use the Stored Emissions Files in Logger Pro
Logger Pro contains a folder of emissions graphs from selected discharge tubes, including: argon, helium, hydrogen, mercury, oxygen, sodium, and xenon. You can display and analyze these graphs without a spectrometer connected to your computer. Follow these steps to view one of these graphs.
- Choose Open from the File menu.
- Open the Sample Data folder.
- Inside the Sample Data folder, open the Physics folder.
- Inside the Physics folder, open the Gas Discharge Spectra. Open the desired file.
You can use the mercury emissions graph to test fluorescent lighting for the presence of mercury.
Change the Settings in Logger Pro
Spectrophotometer Dialog Box
The Spectrophotometer dialog box lists all the settings for the device. To display this box choose Set Up Sensors ► Spectrophotometer from the Experiment menu.
Spectrometer dialog box
For most experiments, the default settings work well.
There are five parameters listed in the dialog box.
- Sample Time: This is similar to the shutter speed of a camera. Logger Pro automatically selects the proper sample time during calibration. Note: For emission studies, you may need to change the sample time manually.
- Wavelength Smoothing: This is the number of adjacent readings on either side of a given value that is used to calculate an average value. Note: Be careful adjusting this parameter as it may shift your wavelength values slightly.
- Samples to Average: This is the number of readings taken at a given wavelength to calculate an average reading.
- Wavelength Range: The range is determined by the type of Spectrophotometer in use.
- LED Intensity: This allows you to change the intensity of the excitation LED.
By clicking on the picture of the Spectrophotometer in this dialog box, you will gain access to four options: calibrate, configure data collection, go to the support website, and units of measure. Click on an item to select it.