Principles and Applications of Vibrational Circular Dichroism ...VCD Spectra of Solvent 0 1 Abs...
Transcript of Principles and Applications of Vibrational Circular Dichroism ...VCD Spectra of Solvent 0 1 Abs...
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Principles and Applications of Vibrational Circular Dichroism (VCD) SpectroscopyDR. CARLOS MORILLO
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JASCO (Nihon Bunko)R&D and Manufacturing, Hachioji, Japan
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Founding MembersEstablished 1958 at the Optical Research Institute at Tsukuba University, Tokyo
Founding members include:
▪ World famous physicist Yoshio Fujioka
▪ Nobel Prize winner Shinichiro Tomonaga(1965 - Physics for QED with Richard Feynman)
JASCO in the USA, first incorporated in 1972.
Dr. Tomonaga
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JASCO: Our Products
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Content1. Basic principles
2. Instrumentation
3. Measurement procedures
4. Applications
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Infrared Spectroscopy
Wavelength
Since absorption is specific to each functional group, infrared spectroscopy is used for qualitative analysis
and molecular structure determination.
Irradiating a sample with infrared light and measuring its absorption spectrum
0
1
4000 400100020003000
Abs
Wavenumber [cm-1]
200 nm 400 nm700 nm 2500 nm
(4000 cm-1)
25000 nm
(400 cm-1)
UV Vis NIR IR FIR
n
c c
H H
H
Structure of polystyrene
c
H
H
Polystyrene
C-H bondsBenzene
Sample
Infrared light
AbsorptionWavenumber
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Circular Dichroism Spectroscopy
Sample
Definition
R L
R L
DAbs = AL - AR
Circularly polarized light
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What is VCD? VCD : Vibrational Circular Dichroism
ECD: Electronic Circular Dichroism circular dichroism in ultraviolet/visible region
Vibrational: absorption in the infrared region due to molecular vibrations
Circular dichroism: degree of absorption of left- and right-handed circularly
polarized light
VCD: difference in absorption of left- and right-
handed circularly polarized infrared light
Sample
R L
R L
Circularly polarized
infrared light
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VCD Spectrum D and L Alanine
-1x10-4
1x10-4
0
1550 1250130014001500
DAbs
Wavenumber [cm-1]
0
1
0.2
0.4
0.6
0.8
Abs
L-alanine
D-alanineIR
VCD
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Substances Exhibiting CD
C*H
NH2
CH3
COOH
*CH
2HN
CH3
COOH
L-Alanine D-Alanine
• Compounds with a combination of enantiomers that cannot be
superimposed on their mirror image
• Compounds consisting of just one of these enantiomers
Substances that exhibit CD are optically
active compounds
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Substances Exhibiting CD
Types of chirality (chiral: lack of symmetry)
Point chirality Planar chirality
Axial chirality Helical chirality
*Asymmetric carbon atom
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Causes birth defects
Effective against illness
Thalidomide
Importance of Absolute Configuration and
Stereochemistry
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What is Learned from VCD Spectroscopy?
1.All organic compounds absorb infrared light and can
be analyzed without the use of a chromophore
2. Chiral compounds can be identified
3. The absolute configuration of a sample can be determined by
comparing its spectrum to that calculated using molecular
orbital methods
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Determining Absolute Molecular Configuration
Procedure
VCD can determine the absolute configuration of a molecule.
1. Calculate the optimized molecular geometry
2. Calculate the VCD spectrum for the optimized molecular structure
3. Measure the VCD spectrum of the sample
4. Compare the calculated and measured spectra to determine the
absolute sample configuration
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Structural Optimization Calculation
O
O
O
O
O
O O
O
MeMe
Me Me
ξ
ζ
ξ'
ζ' Form 1 Form 2
Form 4 Form 5
Form 3
B3LYP/6-31G*//B3LYP/6-31G*
A Menthol
A
A
A
A
By allowing these ester groups to rotate freely,
various conformations occur
The results of the structural optimization calculationMolecule in which four ester groups are bonded to
a benzene ring
Chiral symmetry across the benzene ring
(planar chirality)
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Calculated VCD Spectra for 5 Stable Conformations
Form 2
2000 1700Wavenumber /cm-1
1900 1800
Form 3
2000 1700Wavenumber /cm-1
1900 1800
Form 4
2000 1700Wavenumber /cm-1
1900 1800
Form 5
2000 1700Wavenumber /cm-1
1900 1800
Calculated based on carbonyl group
Form 1
2000 1700Wavenumber /cm-1
1900 1800
The spectrum varies depending on the molecular structure
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Comparison of Calculated and Measured Spectra
Measured
Form 1 2000 1700Wavenumber /cm-1
190
0
1800
1900 1600Wavenumber /cm-1
180
0
1700
Measured spectrum
Calculated spectrum
• Measured spectrum is similar to calculated spectrum of Form 1
• Form 1 is the most likely molecular conformation in this sample
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Considerations of VCD Measurement
1. A high sample concentration is needed, implying a large amount of sample
Concentration: approximately 10 to 100 mg/ml
2. VCD signals are weak, so a long accumulation time is required
VCD: DA/A = 10-3 to 10-5
ECD: DA/A = 10-1 to 10-3
Highly sensitive and stable instrument
is required
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JASCO VCD System
FVS-6000
FT/IR-4700VFT-4000
Dedicated spectrometer for VCD measurements
Accessories
• Highly sensitive system for
detecting weak VCD signals
• FTIR accessory
• Highly extendable
(ATR, etc.)
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FVS-6000 System
Detector
InterferometerLight source
Sample compartment
PEM
(Photoelastic Modulator)
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Block Diagram of the VCD system
1. Infrared light is transformed to the Fourier frequency domain using a Michelson
interferometer2. Only light in the measurement range is transmitted through the optical filter
3. Linearly polarized light is produced using a polarizer4. Left- and right-handed circularly polarized light is generated by the PEM
operating at 50 kHz.5. Infrared light is detected using a high-speed / high sensitivity detector
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Approaches for Measuring Weak VCD Signals using FVS-6000
1. Improved sensitivity using high-throughput interferometer
2. Combination of highly sensitive detector and optical filter
3. Symmetry and stability
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Effect of Interferometer Incidence Angle on IR Intensity
Increased amount of light by changing incidence angle of
interferometer leads to 2.2 times sensitivity improvement
Incidence angle: 28°Incidence angle: 45°
0
60
20
40
4000 40010003000
SB
Wavenumber [cm-1]
2000
Resolution: 4 cm-1
Detector: DLATGS
Dependence of IR light intensity on incidence angle of interferometer
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Advantages of FVS-6000 for Weak VCD Signals
1. Improving the sensitivity using high-throughput interferometer
2. Combination of highly sensitive detector and optical filter
3. Symmetry and stability
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• Liquid N2 cooling is needed for the MCT detector
• Compared with the DLATGS detector (operating at room
temperature) generally used in FTIR spectroscopy, the
sensitivity of the MCT detector is greater than an order of
magnitude higher
MCT Detector
• In the solution method (transmission method) used for
VCD measurements, the light is too intense to be directly
transmitted to the detector without saturating it
• A metal mesh or an optical filter is used so that the
detector is not saturated
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Metal Mesh and Optical Filter
Decreased sensitivity
High sensitivity
Metal mesh Optical filter
Infrared light intensity Infrared light intensity
Wavenumber cm-1 Wavenumber cm-1
• Metal mesh: decreases sensitivity because light at all wavenumbers is
attenuated
• Optical filter: does not attenuate light but passes only light in a certain
wavenumber range, allowing highly sensitive measurements
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Optical filter: 5 mm LPF
Resolution: 4 cm-1
No metal mesh
Metal mesh (70% cut)
Pseudo signal
20010003000 2000
Wavenumber [cm-1]
0
60
20
40
80
Pseudo signal
SB
Photoconductive MCT (PC-MCT) Detector
• If no metal mesh is used, the detector is saturated, and false signals appear
• The sensitivity decreases because the light is cut by about 70% with a metal mesh
Single beam spectrum using photoconductive MCT detector
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Optical filter: 5 mm LPF
Resolution: 4 cm-1
Photovoltaic MCT (PV-MCT)
20010003000 2000
Wavenumber [cm-1]
0
60
40
80
SB
20
100
No metal mesh
Single beam spectrum using photovoltaic MCT detector
• With a PV-MCT, the detector is not saturated even if no metal mesh is used.
• This provides a measurement sensitivity about 3 to 4 times higher than that for
a PC-MCT.
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Optical filter lineupStandard: 2000 to 850, 3200 to 2000 cm-1
Optional: 4000 to 2650, 1000 to 750, 1850 to 1550 cm-1, 3050 to 2800 cm-1
(C-C, C-O, C-N)
(C=C, C=N)
(C=O)
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 750
Wavenumber (cm-1)
±
±
Bending
Stretching
O-H, N-H
C-HーH
Absorption Wavenumbers for Filters and Functional Groups
etc.
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1010
4000
Wavenumber [cm-1]
109
108
1011
850
D*
(l,f)
(cm
Hz
1/2
W-1
)
2000
Sensitivity characteristics of detectors
MCT
InSb
• Using a MCT detector, the
sensitivity in the high-
wavenumber region
decreases
• An InSb detector is effective at
2000 cm-1 or higher.
Measurements at High Wavenumbers
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0
1
0.5Abs
-5x10-5
5x10-5
0DAbs
-5x10-5
5x10-5
0
3050 280029003000
DAbs
Wavenumber [cm-1]
InSb MCT (+)-Camphor
(-)- Camphor
No. of
accumulations: 500
-5x10-5
5x10-5
0DAbs
0
1
0.5Abs
-5x10-5
5x10-5
0
3050 280029003000
DAbs
Wavenumber [cm-1]
IR
VCD
VCD Noise
Comparison of InSb and MCT Detectors
An InSb detector is a better choice at high wavenumbers
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(R)-(+)-BINOL
(S)-(-)-BINOL
0
1
0.5Abs
-4x10-4
4x10-4
0
2000 85010001500
DAbs
Wavenumber [cm-1]
-4x10-4
4x10-4
0DAbs
0
1
0.5Abs
-4x10-4
4x10-4
0
4000 265030003500
DAbs
Wavenumber [cm-1]
-4x10-4
4x10-4
0DAbs
IR
VCD
VCD Noise
Choice of Detector
InSb MCT
Best to use an MCT detector at 2000 cm-1 or lower and
an InSb detector at high wavenumbers
*The noise is excessive due to solvent absorption.
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Advantages of FVS-6000 for Weak VCD Signals
1. Improved sensitivity using high-throughput interferometer
2. Combination of highly sensitive detector and optical filter
3. Symmetry and stability
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Instrument Stability
Light source
• Instrumental stability is important because VCD signals are weak
• The baseline variations and the sign of peaks becomes unclear due to thermal
variations in the light source
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Instrument Stability
By mounting the light source independently from the optical system, the
influence of thermal variations on the baseline is minimized.
Light source
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Temperature Stabilization of Optical System
Measurements were performed every hour for 6 hours after
turning on the instrument
Thermostat OFF Thermostat ON
1 hour
6 hours
Sample: CCl4Resolution: 8 cm-1
Accumulation time: 30 min
Detector: InSb
-3x10-5
3x10-5
DAbs DAbs
-3x10-5
3x10-5
Baseline stability (carbon tetrachloride)
Temperature stabilization of optical elements also
stabilizes the baseline
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Content
1. Basic principles
2. Instrumentation
3. Measurement procedures
4. Applications
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• Sample amount: 1 to 10 mg
• Sample volume: ~100 ml
• Sample concentration: 10 to 100 mg/ml
Liquid sample cell Window Range Remarks
BaF2 ~ 800 cm-1 Slightly soluble in water
CaF2 ~ 1100 cm-1 Insoluble in water
Sample Preparation
Aqueous solution samples CaF2
Non-aqueous solution samples BaF2
Solubility should be checked in advance due to the high sample concentration
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Choice of Solvent (IR Spectrum: 2000 to 850 cm-1 )
Better results are obtained with a solvent with less IR absorption
CH3CN
CH2Cl2
CCl4
THF
Toluene
Hexane
DMSO
CHCl3
CH3OHH2O ※ CaF2 50mm
Cell: BaF2 50 mm
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VCD Spectra of Solvent
0
1
0.5Abs
-2x10-4
2x10-4
0DAbs
-2x10-4
2x10-4
0
2000 85010001500
DAbs
Wavenumber [cm-1]
IR
VCD
VCD Noise
Chloroform: CHCl3
In strong IR absorption range,
impossible to measure a VCD
spectrum of a sample
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Solvents Deuterium Substitution (2000 to 850 cm-1)
DMSO
DMSO-d6
CHCl3
CDCl3
CH3CN
CD3CN
CH3OH
CD3OD
Cell: BaF2 50 mm
1. Use deuterated solvents to reduce the effects of
solvent absorption
Solutions for solvents with strong absorption
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Optical Path Length (2000 to 850 cm-1)
Cell: CaF2 50 mm
H2O
D2O
Cell: CaF2 4 mm
H2O
D2OD2O
2. Shortening the optical
path length reduces
solvent absorptionHowever, note that a higher
concentration sample is required
In the case of water, when
heavy water is used, the
absorption decreases but
remains strong
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Sample Concentration Adjustment
High quality VCD spectrum can be obtained when
IR absorbance is adjusted to approximately 0.7.
0
1
Abs
-2x10-4
2x10-4
0DAbs
-2x10-4
2x10-4
0
1500 850
DAbs
Wavenumber [cm-1]
Most appropriate
0
0.1
Abs
-2x10-5
2x10-5
0DAbs
-2x10-5
2x10-5
0
1500 850
DAbs
Wavenumber [cm-1]
Low concentration
0
5
Abs
-1x10-3
1x10-3
0DAbs
-1x10-3
1x10-3
0
1500 850
DAbs
Wavenumber [cm-1]
High concentration
IR
VCD
VCD Noise
(+)-Camphor
Concentration dependence for VCD spectra
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Sample Concentration Adjustment with Wavenumber
Sample: Camphor
Cell: BaF2 50 mm
0
1
0.5
3200 8502000
Abs
Wavenumber [cm-1]
90 mg/ml 30 mg/ml 300 mg/ml
If the absorbance for a sample differs greatly for different target peaks, when
measurements are performed on samples with the most appropriate concentration for
each peak, high-quality VCD spectra can be obtained.
0
1
0.5
3200 8502000
Abs
Wavenumber [cm-1]
30 mg/ml
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Contents
1. Basic principles
2. Instrumentation
3. Measurement procedures
4. Applications
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Measurement Examples using Autosampler
Autosampler (TAS-FVS)
• Up to 3 samples such as D-form, L-form and solvents can be measured sequentially.
• The influence of baseline drift with time can be reduced by changing a sample in a short time. (shuttle effect)
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With shuttle effect
(autosampler use)
Without shuttle effectDAbs
-4x10-5
4x10-5
0
3040 280029003000
Wavenumber [cm-1]
0
0.6
Abs
-4x10-5
4x10-5
0DAbs
(+) – Camphor
(-) – CamphorIR
VCD
VCD
Shuttle Effect
By changing a sample in a short time, the baseline of the VCD spectrum
becomes close to zero due to the shuttle effect.
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Measurement Example using ThermostattedCell Holder
Thermostatted cell holder (TCH-FVS)
• Spectral change with temperature can
be obtained from -5 to 90oC
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Protein spectra (room temperature)
-1x10-4
1x10-4
0
1700 16001650
DAbs
Wavenumber [cm-1]
0
0.5
Abs
IR
VCD
-1x10-4
1x10-4
0
1700 16001650
DAbs
Wavenumber [cm-1]
0
0.5
Abs
IR
VCD
Wavenumber [cm-1]
-1x10-4
1x10-4
0
1700 16001650
DAbs
0
0.5
Abs
IR
VCD
Hemoglobin
a- Helix rich
Lysozyme
Mixture of a- helix and b-sheet
Concanavalin
b- Sheet rich
Measurement Examples for Proteins
Secondary structure of proteins can be evaluated based on
VCD spectra
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R. T.
71.7°C
76.1°C
80.6°C
84.8°C
1700 16001650
DAbs
Wavenumber [cm-1]
Abs
0.2
5x10-5
VCD
IR
Spectra for lysozyme
Evaluation of Thermal Denaturation of Lysozyme
IR spectrum shape does not greatly change, but VCD
spectrum shape changes starting at 76.1 oC
Suggests protein denaturation between 71.7 and 76.1 oC
Disappearance of peaks derived from α-helix means that
the α-helix structure has been lost by heating lysozyme.
IR peak near 1650 cm-1 associated with α-helix or random
coil, but difficult to distinguish higher order structure of
proteins.
In VCD spectrum, negative peak due to α-helix observed
at wavenumber higher than 1650 cm-1.
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Temperature Dependence of Enzymatic Carbohydrate Reaction
Carbohydrate
Maltohexaose
Enzyme
Amyloglucosidase
Glucose
• When maltohexaose is hydrolyzed to glucose, the glucosidic bond in the
carbohydrate weakens.
• Since the VCD spectrum of carbohydrate contains a negative peak at 1149 cm-1
associated with glucosidic bonds, it is possible to analyze the enzyme reaction
in real time by monitoring the intensity of this peak.
Hydrolysis of Carbohydrate
K. Monde, T. Taniguchi, N.Miura, S. Nishimura, Specific Band Observed in VCD Predicts the
Anomeric Configuration of Carbohydrates, J. Am. Chem. Soc., 2004, 126 (31), pp 9496–9497
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Temperature Dependence of Enzymatic Reaction
-5x10-5
5x10-5
0
1200 1100
DAbs
Wavenumber [cm-1]
-5x10-5
-5x10-5
0
1200 1100
DAbs
Wavenumber [cm-1]
-5x10-5
-5x10-5
0
1200 1100
DAbs
Wavenumber [cm-1]
-5x10-5
5x10-5
0
1200 1100
DAbs
Wavenumber [cm-1]
44.3oC
-5x10-5
5x10-5
0
1200 1100
DAbs
Wavenumber [cm-1]
48.9oC 58.0
oC 62.5
oC53.4
oC
0.5h
1.0h
1.5h
2.0h
2.5h
3.0h
3.5h
4.0h
4.5h
5.0h
The change in peak height with time depends on the
temperature
Temporal change in the VCD spectrum for maltohexaose (0.5 M) and
amyloglucosidase 6.0 units/ml)
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Temperature Dependence of Enzymatic Reaction
Temporal change in intensity of VCD
peak due to glucosidic bonds at
different temperatures
44.3℃48.9℃53.4℃58.0℃62.5℃
Optimum temperature for
amyloglucosidase activity
is expected to be 55 to 60oC
Time / hour
Temperature /oC
DA
bs/
ho
ur
DA
bs
Change in glycosidic bonds
by enzymatic reaction is
temperature dependent
Change in the initial enzyme
velocity with temperature
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Conformation for Carnitine Family
L-Carnitine: Nutrients used for lipid metabolism. Used for
diet foods and food additives
D-Carnitine: toxic
Carnitine
• Synthesis of enantiopure carnitine and analysis of excess enantiomeric
percentage are important
• Analysis of carnitine by ECD and OR has been reported, but there are almost
no examples of VCD measurements that obtained useful information
• VCD measurement of carnitine family and conformation estimation by
calculationCarnitinenitrile chloride
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Conformation of Carnitine Family
Most common conformersComparison of experimental and
calculated VCD spectra
Since the information obtained from the VCD spectrum is abundant, in addition to chirality recognition,
determination of absolute configuration and examination of conformational characteristics can be
performed.
cm-1
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Summary
1. Basic principles
• No chromophores required because all organic compounds
absorb in the IR region
• The absolute configuration of a sample can be determined
by comparing the measured spectrum with that obtained
using molecular orbital calculations
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Summary
2. Instrumentation
•Approaches for measuring weak VCD signals using FVS-6000
Use a high-speed, and high-sensitivity liquid nitrogen cooled
detector, and limit the measured wavenumber range using an
optical filter
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Summary3. Measurement procedures
•The solubility should be checked in advance due to the high
sample concentration
•It is desirable to select a solvent with low absorption, and in
some cases it is necessary to use a deuterated solvent or to
adjust the optical path length
•The sample concentration should be adjusted so that the
absorbance in the IR region is approximately 0.7
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Summary
4. Applications
•When using an autosampler, VCD spectra with a flat baseline
can be obtained by the shuttle effect
•Various applications involving heating measurements were
described
•Molecular conformation analyses based on the VCD
spectrum were introduced
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JASCO Educational ResourcesUpcoming Webinars:• Circular Dichroism Part 2
• FTIR Theory, Instrumentation, and Techniques
• Raman Microscopy and Imaging
• SFC Theory and Applications
E-books and/or Tips and Tricks Posters• Raman
• Fluorescence
• FTIR
• CD
KnowledgeBase
ResearchGate• Fundamental theory and application of circular
dichroism spectroscopy
NEXT WEBINAR WILL BE ON
Circular Dichroism Part 2
Dr. Leah Pandiscia
TUESDAY APRIL 28TH AT 2:00 PM EDT
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Thanks for joining us!! Questions?