Twin RSM 1000 Performance Certification
Instrument: Olis Twin RSM 1000 Spectrophotometer Toolbox
Certificate Number: PDB111413
Date Issued: October 14, 2013
Serial Number: OLIS-132915-RSMTB
Client: Juan Carlos Slebe
Certifier: Paul Boxrud
Summary of Results:
Holmium Oxide Spectrum – Test#1
Passed ______
Absorbance Stopped-flow – Test#2
Passed ______
CD d-camphorsulfonic acid spectrum – Test#3
Passed ______
Anisotropy Rhodamine spectrum–Test#4
Passed ______
Circularly Polarized Luminescence–Test#5
Passed ______
Fluorescence Detected Circular Dichroism – Test #6
Passed_______
Fluorescence Stopped-flow – Test #7
Passed _______
Synchronous Scanning – Test #8
Passed _______
Olis, Inc. certifies that the above instrument meets or exceeds published specifications
and has been calibrated with the following tests. Instrument tested and calibrated by
manufacturer. This certificate shall not be reproduced, except in full, without the written
approval of the manufacturer.
Signature of Certifier:___________________
Date:________________________
_______________________ certifies that the above calibrations have been performed on
the instrument and accepts instrument as installed in complete working order.
Signature of End User:___________________
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Date:________________________
Test #1: Wavelength calibration
Absorbance spectrum of holmium oxide standard with conventional or rapid
scanning.
Conventional Scan Protocol: Configure the instrument to measure Conventional
absorbance (Configuration #1A). Open the “Absorbance” instrument in the software.
Set the wavelength range to cover the 400 nm to 500 nm range. Set the integration time
to 0.2 sec. Enter 470 nm as the Current Wavelength. Click on Live Mode and set the
PMT HV to give raw signal values (Sam and Ref) between 8 and 9 V). Collect a
spectrum by clicking on Collect Data. Right-click on the dataset and choose Assign
Baseline from the right-click menu. Place the holmium oxide sample in the cuvette
holder. Begin the scan by clicking Collect Data.
Rapid Scanning Protocol: Configure the instrument to measure Rapid scanning
absorbance (Configuration #1B). Open the “RSM 1000 Stopped-flow Absorbance”
instrument in the software. Set the Center Wavelength to 450 nm. Set the Collection
Time to 1.0 second and the averaging mode to 1000 scans per second. With an empty
cuvette holder, click on the Apply Baseline check box. Place the holmium oxide sample
in the cuvette holder. Click on Collect Data. Click on the dataset, then right-click on the
icon in the Experiment Window. Choose Select from the right-click menu. Right-click
again and choose Average Scans Within Dataset.
Absorbance
2.0
1.6
1.2
0.8
0.4
0.0
400
420
440
460
480
Wavelength, nm
Expected and Measured results:
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Peak
Expected peak position
(nm)
1
418.7
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Measured peak position
(nm)
500
2
445.3
3
453.2
4
460.0
Comments: ________________________________________________
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Test #2: Absorbance Stopped-Flow
Measure the change in absorbance of the reduction of dichloroindolephenol (DCIP)
with ascorbate.
Single Wavelength Protocol: Configure the instrument for single wavelength stoppedflow (Configuration #2A). Open the “RSM 1000 Stopped-flow Absorbance” instrument
in the software. Prepare a 100 uM solution of DCIP in pH 7 buffer. Prepare a solution of
1 mM sodium ascorbate in pH 7 buffer (+/1 pH unit is ok). Click on the Pretrigger
checkbox to select the pre-trigger data. Add DCIP and the 1 mM ascorbate to the
stopped-flow. Set the Current Wavelength to 600 nm. Set the Collection Time to 1.0
second. Fire a few stopped-flow shots to flush the tubing. Click on Collect Data to fire
the stopped flow.
Rapid Scanning Protocol: Configure the instrument for rapid scanning stopped-flow
(Configuration #2B). Use the RSM 1000 Stopped-flow Absorbance” instrument in the
software. Use the above DCIP and ascorbate solutions for the rapid scanning
measurement. Set the Center wavelength to 600 nm and the averaging mode to 1000
scans per second. Click on the Assign Baseline check box to collect a baseline. Click
Collect Data to fire the stopped-flow.
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Test #3: Circular dichroism
Spectrum of 320-250 nm collected in a 1 mm pathlength cuvette containing a sample
of 1.0 mg/mL d-camphorsulfonic (dCSA) acid dissolved in water.
Protocol: Configure the instrument for circular dichroism (Configuration #3). Open the
“Circular Dichroism” instrument in the software. Prepare a solution of 1.0 mg/mL dCSA
(Aldrich catalog # C2107) in water. Check concentration by absorbance at 285 nm (1% at
285 nm=1.49). Place sample in a 1 mm pathlength cuvette. From the Live Display screen,
change the Scan Range to 320 nm to 250 nm, the Integration Time to 0.5 sec, and the
Number of Intervals to 70. With an empty chamber, click on Collect Data to collect a
baseline. After the scan has finished, click on the data and then right-click again.
Choose Apply Baseline from the pop-up menu. Insert the sample into the sample
chamber and click on Collect Data.
Expected result: CD intensity at 290.5 nm = between 31 and 34 millidegrees
Actual result: ________________________________________________
Comments: ________________________________________________
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Test #4: Anisotropy of Rhodamine acrylic block or Rhodamine/glycerol
solution
Fluorescence anisotropy excitation spectrum of Rhodamine acrylic block or
Rhodamine glycerol solution
Protocol: Configure the instrument for fluorescence anisotropy (Configuration #4).
Open the “gPC 74” instrument in the software Place a Rhodamine acrylic block in the
cuvette holder. Set the emission wavelength (Mono B) to 575 nm. Set the excitation
(Mono A) scan range 350 nm to 550 nm. Set the Integration Time to 1 sec. Click Collect
Data to start the spectrum.
Expected result: Excitation spectrum dips below 0 at low wavelengths and plateaus at
around 0.3 (0.34 for Rhodamine/glycerol) above 500 nm.
Actual result: ________________________________________________
Comments: ________________________________________________
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Test #5: Circularly Polarized Luminescence (CPL)
CPL Spectrum Eu(facam)3 in DMSO
Protocol: Configure the instrument for CPL (Configuration #5). Open the Circularly
Polarized Luminescence instrument in the software. Place a cuvette containing
Eu(facam)3 in DMSO. Set the excitation wavelength (Mono A) to 385 nm and the
emission scan range (Mono B) to 550 nm to 620 nm. Set the Integration Time to 2.0
seconds. Click Collect Data to start the scan.
Expected result: Minimum peak at 595 nm of approximately -0.77.
Actual result: ________________________________________________
Comments: ________________________________________________
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Test #6: Fluorescence Detected Circular Dichroism (FDCD)
FDCD Spectrum of d-camphorsulfonic acid (dCSA)
Protocol: Configure the instrument for CPL (Configuration #6). Open the
“Fluorescence Detected Circular Dichroism” instrument in the software. Prepare a 1
mg/mL dCSA solution in water. Place a cuvette containing the CSA in the sample holder
of the instrument. Set the excitation wavelength scan range (Mono A) to 250 nm to 350
nm nm and the emission wavelength to 427 nm. The Integration Time is set to 2 seconds.
Ensure the emission filter in the PMT is appropriate for the measurement (e.g. high pass
400 nm)
Expected result: Peak at 290 nm excitation.
Actual result: ________________________________________________
Comments: ________________________________________________
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Test #7: Fluorescence Stopped-flow
Single wavelength and rapid scanning fluorescence stopped-flow
Single Wavelength Protocol: Configure the instrument for stopped-flow fluorescence
(Configuration #7A). Open the “RSM 1000 Stopped-flow Fluorescence” in the software.
Prepare a 500 µM N-acetyl-tryptophanimide (NATA) and 800 µM N-bromosuccinimide
in pH 7 buffer. Set the excitation wavelength (Mono A) to 290 nm and the emission to
350 nm. Fill one of the stopped-flow syringes with NATA and fill the other syringe with
buffer. Push a few shots of NATA to flush the tubing. Set the PMT HV to a value to
give a fluorescence signal of over 1 V.
Expected result: Minimum peak at 595 nm of approximately -0.77.
Actual result: ________________________________________________
Comments: ________________________________________________
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Test #8: Synchronous Scanning
Scatter spectrum of scattering sample
Protocol: Configure the instrument to measure synchronous rapid scanning
(Configuration #8). Open the “Scatter” instrument in the software. Place a scattering
sample (or mirror) in the sample position. Set the Center Wavelengths of each
monochromator to 450 nm. Note that if fluorescence is to be measured, the Mono B
center wavelength should be set higher than the Mono A center wavelength. Set the
Average Mode to 1 scan per second and adjust the PMT HV to give a signal of > 0.5 V if
possible. Note the two possible positions for the PMT. Set the Collection time to 10
seconds. Click Collect Data to begin the scan. After data collection, right-click on the
icon in the Experiment Window. Choose ‘select’ and right-click again. Choose Average
Scans within the Dataset.
Expected result: Scatter spectrum
Actual result: ________________________________________________
Comments: ________________________________________________
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Additional Information for Circular Dichroism Calibration
d-Camphorsulfonic acid1
F.W. = 232.3
g/mol
 = 2.36 @ 290.5 nm for circular dichroism
 = 34.5 @ 285 nm for absorbance
CD/Abs = 33/ = 2.26
1 mg/mL in 5 cm pathlength = 0.743 AU
d-Camphorsulfonic acid2
 = 2.37 @ 290.5 nm for circular dichroism
 = 34.5 @ 285 nm for absorbance
CD/Abs = 33/ = 2.27
Pantolactone2
F.W. = 130.1 g/mol
 = -4.90 @ 220 nm
 ~90 @ 215 nm
CD/Abs = 33/ = -1.77
1
Johnson C. W. (1996) in Circular Dichroism and the Conformational Analysis of Biomolecules. Fasman,
G. D. Ed. Plenum Press, New York, pp. 635-652.
2
Schippers, P. H. and Dekkers, H. P. J. M. (1981) Anal. Chem. 53, 778.
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Configuration 1A (Single Wavelength Absorbance):
Entrance Slit
(0.6 mm)
150 W xenon
lamp (ozone)
Emission
RSM
Mono B
(not used)
Beam
splitter
PEM, not
used
RSM (400 lines per
mm blazed at 500
nm; Mono A)
Sam
PMT
Fixed Slit
Cartridge
(0.12 mm)
Four cell
turret
Reference
PMT
Exit slit (0.12 mm)
Instrument: Absorbance
Data Reduction Mode: Absorbance
Data Collection Mode: Scan
Scan Mode: Fixed Slitwidth and PMT HV
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Configuration 1B (Rapid Scanning Absorbance):
Entrance slit
(0.6 mm)
150 W xenon
lamp (ozone)
Emission
RSM
Mono B
(not used)
Beam
splitter
PEM, not
used
RSM (400 lines per
mm, blazed at 500
nm; Mono A)
Sam
PMT
ScanDisk
(0.2 mm)
Four cell
turret
Exit slit
(0.12 mm)
Instrument: RSM 1000 Stopped-flow Absorbance
Data Reduction Mode: Absorbance
Data Collection Mode: Rapid Scanning
Scan Mode: Fixed Slitwidth and PMT HV
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Reference
PMT
Configuration 2A (Stopped-flow Absorbance, single wavelength):
Emission
RSM
(not used)
Entrance slit
(0.6 mm)
Beam
splitter
150 W xenon
lamp (ozone)
PEM, not
used
RSM (blazed at 500
nm)
Sam
PMT
Fixed Slit
Cartridge
(0.12 mm)
Stoppedflow cell
Reference
PMT
Exit slit
(0.12 mm)
Instrument: RSM 1000 Stopped-flow Absorbance
Data Reduction Mode: Absorbance
Data Collection Mode: Stopped-flow
Scan Mode: Fixed Slitwidth and PMT HV
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4 x 4 mm stopped-flow
(replaces sample holder)
Configuration 2B (Stopped-flow absorbance, rapid scanning):
Entrance slit
(0.6 mm)
150 W xenon
lamp (ozone)
Emission
RSM
(not used)
Beam
splitter
PEM, not
used
RSM (blazed at 500
nm)
Sam
PMT
ScanDisk
(0.2 mm)
Stoppedflow cell
Reference
PMT
Exit slit
(0.12 mm)
Instrument: RSM 1000 Stopped-flow Absorbance
Data Reduction Mode: Absorbance
Data Collection Mode: Rapid Scanning + SF
Scan Mode: Fixed Slitwidth and PMT HV
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4 x 4 mm stopped-flow
(replaces sample holder)
Configuration 3 (Circular Dichroism scan):
Entrance slit
(1.24 mm)
150 W xenon
lamp (ozone)
Emission RSM B (not used)
RSM (2400 line
blazed at 250 nm;
Mono A)
CD
PMT
Fixed
ScanDisk
(1.24 mm)
Four cell
turret
CD polarizer PEM
MgF2
Exit slit
(1.24 mm)
Instrument: Circular Dichroism
Data Reduction Mode: Circular Dichroism
Data Collection Mode: Scan
Scan Mode: Fixed Slitwidth
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Configuration 4 (Anisotropy/Polarization):
Entrance slit
(0.6 mm)
Fixed Scan
Cartridge
(0.6 mm)
Entrance slit
0.6 mm)
PEM
150 W xenon
lamp (ozone)
Emission
RSM (400 lines per mm,
500 nm Mono B)
Emission
polarizer,
Horizontal
RSM (400 lines
per mm, 500 nm,
Mono A)
Unused
PMT
Fixed Scan
Cartridge
(0.6 mm)
Four cell
turret
Exit slit
(0.6 mm)
Instrument: gPC 74
Data Reduction Mode: Polarization 74
Data Collection Mode: Scan
Scan Mode: Fixed Slitwidth and PMT HV
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Photon
counter
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Excitation polarizer,
Vertical
Configuration 5 (Circularly Polarized Luminescence):
Entrance slit
(1.24 mm)
Fixed Scan
Cartridge
(1.24 mm)
Emission
RSM (400 line,
500 nm, Mono B)
Photon
counter
PEM
150 W xenon
lamp (ozone)
Emission
polarizer,
horizontal
RSM (400 lines per
mm, blazed at 500
nm, Mono A)
Unused
PMT
Fixed Scan
Cartridge
(1.24 mm)
Four cell
turret
Exit slit
(1.24 mm)
Instrument: Circularly Polarized Luminescence
Data Reduction Mode: CPL
Data Collection Mode: Scan
Scan Mode: Fixed Slitwidth and PMT HV
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Configuration 6 (Fluorescence Detected Circular Dichroism):
Entrance slit
(1.24 mm)
150 W xenon
lamp (ozone)
Fixed Slit
Cartridge
(3.32 mm)
Unused
CD
PMT
RSM (2400 line,
blazed at 250 nm;
Mono A)
Fixed Scan
Cartridge
(1.24 mm)
Four cell
turret
CD polarizer PEM
MgF2
Exit slit
(0.6 mm)
Instrument: Fluorescence Detected Circular Dichroism
Data Reduction Mode: FDCD
Data Collection Mode: Scan
Scan Mode: Fixed Slitwidth and PMT HV
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Photon
counting
module
Emission RSM B
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Configuration 7A (Fluorescence Stopped-flow, single wavelength):
Entrance slit
(0.6 mm)
Fixed Scan
Cartridge
(0.6 mm)
Emission
RSM (400 line, 500 nm
blaze, Mono B)
PMT
150 W xenon
lamp (ozone)
RSM (Mono A)
Unused
CD PMT
Fixed
ScanDisk
(0.6 mm)
Stoppedflow cell
Unused PEM
positions
Exit slit
(0.6 mm)
Instrument: RSM 1000 Stopped-flow Fluorescence
Data Reduction Mode: Fluorescence
Data Collection Mode: Stopped-flow
Scan Mode: Fixed Slitwidth and PMT HV
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4 x 4 mm stopped-flow
(replaces sample holder)
Configuration 7B (Fluorescence stopped flow, rapid scanning):
1 .0 mm
ScanDisk
Emission
RSM (400 line, 500 nm
blaze, Mono B)
Entrance slit
(0.6 mm)
PMT
150 W xenon
lamp (ozone)
RSM (Mono A)
Unused
CD PMT
Fixed
ScanDisk
(0.6 mm)
Stoppedflow cell
Unused PEM
positions
Exit slit
(0.6 mm)
Instrument: RSM 1000 Stopped-flow Fluorescence
Data Reduction Mode: Fluorescence
Data Collection Mode: Rapid Scanning + SF
Scan Mode: Fixed Slitwidth and PMT HV
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4 x 4 mm stopped-flow
(replaces sample holder)
Configuration 8 (Synchronous scanning):
PMT optional
position
Synchronous Scanning Configuration:
Entrance slit
(1.24 nm)
150 W xenon
lamp (ozone)
StepDisk
(1.0 mm)
Emission
RSM (Mono B)
Exit slit
(1.24
mm)
Entrance slit
(1.24 nm)
PMT,
optional
position
RSM (400 line, 500
nm Mono A)
PMT not
used
StepDisk
(1.0 mm)
Four cell
turret
PEM (not
used)
Exit slit
(1.24 mm)
Instrument: Scatter
Data Reduction Mode: Fluorescence
Data Collection Mode: Rapid Scanning
Scan Mode: Fixed Slitwidth and PMT HV
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