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November 6, 2012 1
CAG
Presented by Mark Fisher
Applications Engineer
November 6, 2012
Optimizing Your Absorption or
Fluorescence Thermal Melt Run
Thermal Melt Application
• Typically used to follow a change in signal as temperature is
changed
• Absorption or Fluorescent Intensity can be used to monitor
system as temperature is ramped
• Single Wavelength monitoring is generally done
• Scan of a Spectral Region may be desired
• How can you increase Sample throughput ?
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Cary 100
UV-Vis NIR Spectroscopy Solutions
The Agilent Cary Family
Cary 300
Cary 4000
8453
Cary 5000/6000i
Cary 60
Cary
Eclipse
Perf
orm
ance
Routine to Research
“A word from our sponsors”
Thermal Melts using UV-Vis Absorption
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Instrumentation Needed for a Thermal Melt
Collection
Instrument typically Cary 100 or 300 (8453 is a
possibility as well)
Multi-cell Changer with Temperature Control
Temperature Probe
Purge Gas if Initial or Final Temperature 15 deg C or less
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Typical Thermal Melt Curve
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Thermal Melt Curve Expanded
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SetUp Dialog Box and Setting Up Simple Collection
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Using Multiple Wavelengths in a Single Melt
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User Collect Function
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Typical Simple Single Ramp
• 0.1 degrees/minute
• Collect data every 0.1 degrees or 0.5 degrees
• 20-90 degrees C
• Time Required to Finish Data Collection on a Sample
70 deg / 0.1 deg per min = 700 minutes or 11
hours and 40 min
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Advance Collect using Multiple Temperature
Collection Stages
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Optimizing Data Collection Using Multi-Stage
Ramp • 20 to 50 degrees Ramp at 1.0 deg/min
• 50 to 70 degrees Ramp at 0.1 deg/min
• 70 to 90 degrees Ramp at 1.0 deg/min
• Collect Data every 0.1 deg or 0.5 deg
• Time Required to Finish Data Collection
20 to 50 deg 30 deg/ 1 deg per min = 30 min
50 to 70 deg 20 deg/ 0.1 deg per min = 200 min
70 to 90 deg 20 deg/ 1 deg per min = 20 min
Total Time 250 min or 4 hours and 10 min
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Double Beam SetUp
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Where and When Should the Temperature be
Measured
Choices for Monitoring Temperature
Block
Probe inserted into Cuvette
Can Sacrifice one cuvette for data collection if you do not want sample in
contact with probe
Temperature measured before each measurement
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QNW T2 Single Cell Peltier Accessory
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QNW T2 Temperature Controller
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Temperature Probe In Semi-Micro Cell
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Temperature Lag from Block to Solution Heating
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Temperature Lag from Block to Solution Cooling
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Effect of Signal Averaging Time on Noise
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Additional Capabilities for Collecting Temperature
Related Data
Possible to Collect a Scan at User Define Temperatures
While at each User Defined Temperature, it is possible to
collect a number of scans at a user define time interval
before going to the next temperature
These capabilities are possible through ADL, which is the
controlling language of the software. User have access to
an editor to able to write/edit macros
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Initial Screen after Program Started
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Temperature SetUp for Scans at Temperature
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Trace Preferences Dialogbox Showing Trace
Names
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Scanning at Temperatures
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Thermal Melts using Fluorescence
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Cary Eclipse Fluorescence Spectrometer
The Power of Xenon…
• Unique Xe flashlamp technology
• Measure small volume samples
• Fiber optics
• Room light immunity: unique,
Varian patented technology
• Eliminates photo-degradation
• Long lamp lifetime
Application focus
• Biochemical applications
• Academia
• Industrial chemistry
Fluorescent Thermal Melt Curve
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20 40 60 80
20
40
60
80
100
Temperature (°C)
Inte
nsity (
a.u
.)
DABCYL Labelled PNA and
Fluoreceine Labelled DNA
Two Ramps Each
Simple Single Ramp Collection
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Sample Cell Selection and Temperature Monitoring
Choice
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Advance Collection Choices
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Multiple Wavelength Pairs
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Advance Collection SetUp for Fluorescence
Thermal going from High to Low Intensity
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Example of Fluorescence Thermal Melt going from
High to Low Intensity
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50 60 70 80 90
0
100
200
300
400
Temperature (°C)
Inte
nsity (
a.u
.)
Importance of Choice of Cuvette
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40 60 80
0
20
40
60
80
100
Temperature (°C)
Inte
nsity (
a.u
.)
40 60 80
0
20
40
60
80
100
Temperature (°C)
Inte
nsity (
a.u
.)
Plastic Cuvette Black
Quartz Cuvette Red
Temperature Monitor Block
Importance of Choice of where Temperature is
Monitored
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20 40 60 800
20
40
60
80
100
120
Temperature (°C)
Inte
nsity (
a.u
.)
Quartz Cuvette Block Monitor RedQuartz Cuvette Probe Monitor Purple
Overlay of Cuvette and Temperature Monitor
Choices
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40 60 800
20
40
60
80
100
120
Temperature (°C)
Inte
nsity (
a.u
.)
Black Plastic CuvetteBlue Quartz Cuvette Block as monitor
Red Quartz Cuvette Probe as monitor
Determination of Tm
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40 60 80
0
2
4
6
Temperature (°C)
Deriv1
Ist Derivative Tm Calculation RedOriginal Data Black
Calculated Tm’s
Cuvette Type Calculated Tm (deg C)
Plastic 72.34
Quartz (Block used as Temperature
Monitor)
67.00
Quartz (Probe used as Temperature
Monitor
64.00
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Effect of Noise on Tm Calculation
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40 60 80
0
2
4
6
Temperature (°C)
Deriv1
Tm Calculation with and without Smoothing
Purple Original Temperature Data
Olive Green 1st derivative no smoothing
Red 1st derivative with smoothing
Effect of Noise on Tm Calculation
Cuvette Type and Calculation
Conditions
Calculated Tm (deg C)
Quartz Cuvette using Probe as monitor
no smoothing
64.00
Quartz Cuvette using Probe as monitor
with smoothing
65.00
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Example of Optimization of Temperature Ramp and
Data Interval
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40 60 80
20
40
60
80
100
Temperature (°C)
Inte
nsity (
a.u
.)
40 60 80
50
100
Temperature (°C)
Inte
nsity (
a.u
.)
Probe Monitor Red
Block Monitor Black
Additional Capabilities in Collecting Temperature
Related Data
Like UV-Vis, it is possible to Collect a Scan at a series of
user define temperatures with a user define equilibration
time
While not yet available, it is possible to control the
autopolarizers in addition to the temperature to
automatically collect data for anisotropy calculations for a
set of user define temperatures
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Questions
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Thanks For Your Patience And Good Humor
Shortly You Will Receive A Link
To Down Load The Presentation
My Contact is [email protected]
Thanks Again
