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Varian Cary Eclipse Spectrofluorometer: (Fluorescence, Phosphorescence, Chemiluminescence)

Table of Contents:

Chapter 1: Introduction…………………………………………………….3 a. General Characteristics…………………………………………...3 b. Signal Theory…………………………………………………….3/4 c. Sample Type……………………………………………………...5 d. Advantages……………………………………………………….5 e. Disadvantages/Limitations……………………………………….5

Chapter 2: Instrumentation………………………………………………....6 a. Block Diagram…………………………………………………...6/7 b. Internal Design…………………………………………………..6/7 c. External Design…………………………………………………6/7

Chapter 3: Operating Instructions………………………………………….8 a. Start Up/Shut Down…………………………………………......8 b. Precautions…………………………………………………….....8 c. Fluorescence Scan………………………………………………..9 d. Phosphorescence Scan…………………………………………..13 e. Chemiluminescence Scan………………………………………...11

Chapter 4: References……………………………………………………...15

Chapter 1: Introduction a. General Characteristics
Varian has been supplying essential tools for scientists engaged in drug discovery, life science, genetic engineering, neuroscience studies, health care, environmental analysis, and academic applications for over 50 years.
Cary has been associated with high performance spectrophotometers for over 50 years. The Cary Eclipse Fluorescence spectrophotometer fit well within the Cary range, offering the excellent specifications and modern software that has become the Cary standard.
The Spectrophotometer was designed from the ground up, incorporating the latest optical and electric technology.

b. Signal Theory

Molecular Fluorescence
AB(aq) + h1 AB*(aq)
AB* (aq) AB (aq) + h2
AB(aq) + h1 AB*(aq)
AB* (aq) AB (aq) + h2

Chemiluminescence Theory

Phosphorescence Theory
P = k P Po bC

P = k P Po bC

c. Sample Type
Rigid Molecular Structure
Absence of “Heavy” Atoms
Dissolved in a Viscous Solvent
Low Solvent Temperature
Absence of Paramagnetic Substances
Presence of e- donating groups (-OH, –NH2, -OCH3)
Rigid Molecular Structure
Absence of “Heavy” Atoms
Dissolved in a Viscous Solvent
Low Solvent Temperature
Absence of Paramagnetic Substances
Presence of e- donating groups (-OH, –NH2, -OCH3)
Aqueous and Liquid Solutions that Fluoresce

d. Advantages
The Varian Cary Eclipse Spectrofluorometer can be set to run on three different settings including chemiluminescence, fluorescence, and phosphorescence. Instead of scanning the wavelengths the wavelengths can be optimized to determine the optimized intensity. Since Simple Reads will help with the optimizing protocol the running the samples requires minimal preparation except for preparing the samples and standards. e. Limitations
Both internal and external factors can affect fluorescence measurements. Internal factors include those within the sample matrix, such as the structure and rigidity of the molecule, functional groups, solution pH, temperature, concentration, and internal scattering. External factors include instrument and method sensitivity.
Temperature variation has been known to affect the wavelength at which a molecule fluoresces and the width of the fluorescence spectra. Changes in temperature affect the viscosity of the medium and hence influence the number of collisions between fluorescing molecules and solvent molecules.

Chapter 2: Instrumentation
General Spectrofluorometer Design
Xe Flash Lamp
Xe Flash Lamp

Chapter 3: Operating Instructions a. Start-up Procedure 1. Turn on the computer 2. Log onto the computer 3. Double click on the Cary icon on the desktop 4. Choose desired application b. Shut-down Procedure 1. Close the Cary Eclipse application and shut down the computer (if you do not close the Cary software, the lamp will remain on and this will significantly decrease the life of the lamp) 2. Log off the computer c. Preventative Maintence * Check the fluid levels in the Cary Temperature Controller and the Neslab circulating bath (daily) * Replace the fluid in the Cary Temperature Controller and the Neslab circulating bath if algae growth is apparent (monthly) * Conduct validation tests to check that the instrument is functioning properly (three months) * Replace the xenon lamp (as required) d. Hazards
Chemical Hazards: * Use of the fluorometer and accessories may involve materials, solvents, and soltuions which are flammable, corrosive, toxic or otherwise hazardous.
UV Hazards: * Hazardous UV radiation is emitted by the xenon flash lamp in the instrument. NEVER look directly at the UV source lamp; the lid should always be shut during data acquisition

Fluorescence Scan:
This demonstrates how to perform a wavelength scan in fluorescence mode 1. Start the Scan Application
Step 1: * Click the Windows Start button, point to programs, Cary Eclipse and then select Scan. The Scan Application is displayed

2. Set Up Data Collection Parameters
Step 2: * Select the Setup button to display the Setup dialog and specify the method parameters for a new method

3. Set Up Instrument Parameters such as Data Mode and Scan Setup
Step 3: * Set the Data Mode to Fluorescence * Set the Scan Setup mode to Emission (during the emission scan, the excitation monochromator is set to a fixed wavelength and the emission monochromator is scanned over a wavelength range) * Set the X Mode to Wavelength (nm) * Enter an Excitation (nm) value that is within the region where the fluorescent molecule to be scanned will absorb light * Enter an Excitation slit (nm) value of 5 and an Emission slit (nm) value of 5. (slits determine the resolution of the spectrum and therefore are used in conjunction with the PMT Detector Voltage to determine concentration) * Enter a Start (nm) value and enter a Stop (nm) value (the start value should be set to the excitation value plus the sum of the slits and the stop value should be set to 150-200nm greater than the start value) * Clear the 3-D Mode check box * Set the Scan Control to Medium

4. Set Up Visual System Monitoring
Step 4: * Select the Status Display check box

5. Set Up the Scan Options
Step 5: * Select the Options tab to display the Options page * In the Display Options group, select the Overlay traces check box to overlay the results of the traces on one graph * Clear the CAT or S/N Mode, Cycle Mode and Smoothing check boxes * Set the Excitation filter to Auto * Set the Emission filter to Open in order to minimize steps in the spectra * Set the PMT Detector Voltage to Medium (if signal is too high decrease the PMT Detector Voltage or if signal is too low increase the PMT Detector Voltage)

6. Finish Setup
Step 6: * Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog

7. Zero the Instrument
Step 7: * Place the blank solution in the sample compartment of the instrument * Click the Zero button to zero the system

8. Start the Scan Run
Step 8: * Select the Start button to commence a data collection * Place the sample compartment * In the Sample Name dialog, enter the appropriate name for you sample and select OK
Chemiluminescence Scan
This demonstrates how to perform a wavelength scan in chemiluminescence mode 1. Start the Scan Application
Step 1: * Click the Windows Start button, point to programs, Cary Eclipse and then select Scan. The Scan Application is displayed

2. Set Up Data Collection Parameters
Step 2: * Select the Setup button to display the Setup dialog and specify the method parameters for a new method

3. Set Up Instrument Parameters such as Data Mode and Scan Setup
Step 3: * Set the Data Mode to Chemiluminescence * Select the Options button to display the Chemiluminescence Options dialog. Enter the Gate time to 200ms. * Select OK to clear the Chemiluminescence Options dialog and return to the Cary page. * Set the X Mode to Wavelength (nm) * Enter a Start (nm) value and enter a Stop (nm) value (the start value should be set to the excitation value plus the sum of the slits) * Enter an Emission slit (nm) value of 10 (slits determine the resolution of the spectrum and therefore are used in conjunction with the PMT Detector Voltage to determine concentration) * In the Scan Control group, set the Averaging time to 0.20000s and the Data Interval to 1.0000nm

4. Set Up Visual System Monitoring
Step 4: * Select the Status Display check box

5. Set Up the Display and Filter Options
Step 5: * Select the Options tab to display the Options page * In the Display Options group, select the Overlay traces check box to overlay the results of the traces on one graph * Clear the CAT or S/N Mode, Cycle Mode and Smoothing check boxes * Set the Emission filter to Open in order to minimize steps in the spectra * Set the PMT Detector Voltage to High (if signal is too high decrease the PMT Detector Voltage)

6. Finish Setup
Step 6: * Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog

7. Zero the Instrument
Step 7: * Place the blank solution in the sample compartment of the instrument * Click the Zero button to zero the system

8. Start the Scan Run
Step 8: * Select the Start button to commence a data collection * Place the sample compartment * In the Sample Name dialog, enter the appropriate name for you sample and select OK

Phosphorescence Scan:
This demonstrates how to perform a wavelength scan in phosphorescence mode 1. Start the Scan Application
Step 1: * Click the Windows Start button, point to programs, Cary Eclipse and then select Scan. The Scan Application is displayed

2. Set Up Data Collection Parameters
Step 2: * Select the Setup button to display the Setup dialog and specify the method parameters for a new method

3. Set Up Instrument Parameters such as Data Mode and Scan Setup
Step 3: * Set the Data Mode to Phosphorescence * Select the Options button to display the Phosphorescence Options dialog. The values you enter for the options depend upon the sample you are scanning * Select OK to clear the Phosphorescence Options dialog and return to the Cary page. * Set the Scan Setup mode to Emission (during the emission scan, the excitation monochromator is set to a fixed wavelength and the emission monochromator is scanned over a wavelength range) * Set the X Mode to Wavelength (nm) * If selected, clear the 3-D Mode check box

4. Set Up Visual System Monitoring
Step 4: * Select the Status Display check box

5. Set Up the Display and Filter Options
Step 5: * Select the Options tab to display the Options page * In the Display Options group, select the Overlay traces check box to overlay the results of the traces on one graph * Clear the CAT or S/N Mode, Cycle Mode and Smoothing check boxes * Set the Excitation filter to Auto * Set the Emission filter to Open in order to minimize steps in the spectra * Set the PMT Detector Voltage to High (if signal is too high decrease the PMT Detector Voltage)

6. Finish Setup
Step 6: * Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog

7. Zero the Instrument
Step 7: * Place the blank solution in the sample compartment of the instrument * Click the Zero button to zero the system

8. Start the Scan Run
Step 8: * Select the Start button to commence a data collection * Place the sample compartment * In the Sample Name dialog, enter the appropriate name for you sample and select OK

Chapter 4: References
Wilber, DJ. Cary Eclipse Fluorescence Spectrometer: Basic Operation. Tuffs
University, Department of Chemistry. http://chem.tufts.edu/instrumentation/Brief%20Directions/Cary%20Eclipse%20brief%20directions.doc. Accessed May 1, 2014.

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