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PROTOCOL FOR SPECTROFLUOROPHOTOMETER SHIMADZU RF-5301PC There are 3 data acquisition modes: Spectrum, Quantitative and Time Course.

The Spectrum mode is used to acquire a spectrum. The Quantitative mode is used to analyze a sample quantitatively. The Time Course mode is used to acquire the fluorescence intensity at a specific wavelength over a period of time. This protocol describes how to use the Spectrum mode. See the Instruction Manual for details describing how to use the Quantitative and Time Course modes. 1. Power on the RF-5301PC unit. 2. Power on the computer. 3. Click on the RF-5301PC icon. The system will initialize. This will take approx 5 minutes. If the system fails the initialization process, ask ACCBR personnel for help. 4. Allow 15 minutes for the lamp to warm up before taking the first reading. 5. Select File > Acquire Mode> Spectrum to enter the Spectrum mode. 6. Select Configure > Parameters to display the Spectrum Parameters dialogue box. Spectrum Parameters Spectrum type: EX Wavelength: EM Wavelength Range: Recording Range: Scanning Speed: Sampling Interval [nm]: Slit Width [nm]: Sensitivity: Response Time [sec]: Repeat Scan Excitation 350 nm 350 Start Low 350 0.000 nm End High 450 50.000 Emission Synchronous

Medium 0.2 EX High Auto Auto File OK Cancel 5 EM Low 5

7. After specifying all parameters, click the [OK] button to enable the settings. During the parameter set-up procedure, the message Setup is displayed in the status bar at the bottom right corner of the RF-5301PC window. When the wavelength and fluorescence intensity reappear on the screen, the settings are complete. 8. Place a cuvette containing distilled water in the holder, close the spectroflurophotometer lid, and click the [START] button to start scanning. During scanning, the current spectral pattern is displayed on the screen, and the current wavelength and fluorescence intensity are indicated on the status bar. Load a cuvette with your blank (buffer, deionized water, or whatever your sample is dissolve in) Put the cell into the holder and close the spectroflurophotometer lid Click on the [Go To WL] (Go To Wavelength) button to ensure that your excitation and emission wavelengths are correct. Change the wavelengths, if necessary. Click [Set] to update the system to the desired wavelengths. Click on AUTOZERO to zero the reading (fluorescence intensity) on your blank 9. If using the Quartz cuvette, empty and rinse the cuvette with DI water. Pipette your sample into the cuvette, place the cuvette into the holder and close the lid. Click [START] if you are in the Spectrum Mode screen, or [Read], if you are in the Wavelength Selection dialogue box, to begin the scanning. 10. Upon completion of the scanning, the File Name dialogue box will appear. Type the file name for the spectrum measured using up to eight alphanumeric characters. Do not enter a file name extension. The default extension .SPC will be added to the file name automatically. When all details have been entered, click the [Save] button to save the data in the channel. If the [Discard] button is clicked, the dialogue button will close and all data entered will be lost. 11. You can set the graph limits by following these steps: a. Right clicking the mouse will cause a menu to appear. Select [Radar] from the menu, and then [Both Axes] from the subsequent submenu. New graph limits will be set up automatically. b. OR select [Limits] from the same submenu. Enter the upper and lower limits for the X and Y axis in the dialogue box that appears. 12. To enlarge a specific area of a spectrum, point to the left top corner of that area with the mouse and drag to define a rectangular box surrounding the intended area. 13. To read the wavelength and fluorescence intensity at the position of the mouse pointer, right click the mouse. Then select [Cross Hair] and then [Display] command from the submenu. Move the pointer to the desired position To erase the mouse pointer, select the [Display] command again.

14. To save the obtained data, select File > Save. 15. Disposable cuvettes should be discarded into the Biohazard waste. Quartz cuvettes should be rinsed first with DI water and then with methanol. If necessary, use KimWipes to dry the exterior of the cuvette. Use gloves when handling quartz cuvettes! ********************************************************************** If you are unsure of the optimal emission and excitation wavelengths you can use the [SEARCH] button to find the optimal wavelengths.

Search Optimal Wavelength EX Range: EM Range: EX Interval: 240 240 10 to to 450 650

Optimal Wavelengths: Excitation: Emission: nm nm

Search

Save

Cancel

Enter values for the [EX search] range, the [EM search] range and the [EX search interval]. Click the [Search] button. The search process will begin. Other than the peak resulting from the true fluorescence, peaks may also be obtained in a data acquisition run from scattered light, secondary light, or Raman scattering of the excitation light. These undesirable peaks will shift when the excitation wavelength varies, and can be discriminated from true excitation wavelengths to detect the true fluorescence peak that gives the maximum fluorescence intensity. The search program at first analyzes the fluorescence spectrum at varying excitation wavelengths to detect the true fluorescence peak that gives the maximum fluorescence intensity. Then, it adjusts the emission monochromator to this peak wavelength, analyzes the excitation spectrum, and then determines the excitation wavelength that gives the maximum fluorescence intensity. Upon

completion of the search process, the obtained wavelength set will appear in the [Optimum Wavelength] column. During the search process, the slit width is not altered. To change the slit width, use the [Parameters] command.

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