Principles and Applications of Vibrational Circular Dichroism (VCD) SpectroscopyDR. CARLOS MORILLO

JASCO (Nihon Bunko)R&D and Manufacturing, Hachioji, Japan

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

JASCO: Our Products

Content1. Basic principles

2. Instrumentation

3. Measurement procedures

4. Applications

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

Circular Dichroism Spectroscopy

Sample

Definition

R L

R L

DAbs = AL - AR

Circularly polarized light

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

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

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

Substances Exhibiting CD

Types of chirality (chiral: lack of symmetry)

Point chirality Planar chirality

Axial chirality Helical chirality

*Asymmetric carbon atom

Causes birth defects

Effective against illness

Thalidomide

Importance of Absolute Configuration and

Stereochemistry

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

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

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)

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

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

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

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.)

FVS-6000 System

Detector

InterferometerLight source

Sample compartment

PEM

(Photoelastic Modulator)

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

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

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

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

• 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

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

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

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.

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.

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

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

(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.

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

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

Instrument Stability

By mounting the light source independently from the optical system, the

influence of thermal variations on the baseline is minimized.

Light source

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

Content

1. Basic principles

2. Instrumentation

3. Measurement procedures

4. Applications

• 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

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

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

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

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

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

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

Contents

1. Basic principles

2. Instrumentation

3. Measurement procedures

4. Applications

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）

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.

Measurement Example using ThermostattedCell Holder

Thermostatted cell holder (TCH-FVS)

• Spectral change with temperature can

be obtained from -5 to 90oC

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

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.

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

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)

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

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

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

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

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

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

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

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

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