CIRCULAR DICHROISM

Sample Considerations &

Parameter Optimization
LEAH PANDISCIA, PhD
JASCO Corporation
JASCO Corporation
Hachioji, Tokyo
Since 1958

JASCO, Inc
Easton, MD
JASCO: Our Products
Seminar Overview
I. Sample considerations
II. Measurement parameter optimization
III. Data processing functions
What is Circular Dichroism?
▪ Difference in absorption of left and right circularly polarized light

▪ CD signal observed when a chromophore is chiral (optically active)

▪ Intrinsically chiral
▪ Covalently linked to chiral center
▪ Placed in asymmetric environment

LH ∆𝐴 = 𝐴𝐿 − 𝐴𝑅

RH
Why use CD?
1. Uniquely sensitive to asymmetry.
2. Information on molecular and electronic structure.
3. Experiments are relatively quick and easy to perform.
4. Non-destructive (can recover most samples).
5. Solution phase.
▪ Crystallation process could change molecular structure.

6. Low concentrations (0.1 mg/mL).

▪ Doesn’t require a concentration which could change the system being studied.
Sample Considerations
 What factors affect a CD spectrum?
1. Sample concentration
2. Cell pathlength
3. Type of buffer and concentration
4. Solution conditions: pH, ionic strength, temperature

Beer’s Law

𝐴 =𝜀∙𝑙∙𝑐
e: molar absorptivity
l: pathlength
c: concentration
l

Norma J. Greenfield Nature Protocols 1. 6(2006).

What is the HT Voltage?

PMT PMT

Data points cannot be used at HTVs above 700 V

More absorbance = less light throughput = higher HTV = lower S/N

 Sample Preparation
▪ 95% purity (via SDS Page)
▪ Check for nucleic acid contaminants
▪ A280A260 ~1.7 for proteins, 0.6 for NAs

▪ Dialysis to remove protective agents or buffer ions

▪ Imidazoles (<1 mM)
▪ Chloride ions (<100 mM)

▪ Clear solution → centrifugation or filtration

▪ Differential light scattering
▪ Absorption flattening
 Differential light scattering
Is

I0 I
PMT

▪Detected by PMT as difference in absorption

▪ Increase in apparent absorbance due to light loss → Decrease in CD signal

▪Difference in extent of scattering of left and right circularly polarized light by chiral sample
▪Can redshift and change magnitude of peaks
▪Reduce particulate size (~1/10 of incident light)

: Chem. Soc. Rev., 2016, 45, 4859

 Absorption Flattening
▪Chromophores are closely packed → nonuniform distribution of proteins
▪Decrease of absorbance due to decrease in effective concentration of absorbers Beer’s Law

▪Absorption is function of extinction coefficient of sample at given wavelength → 𝐴=𝜀∙𝑙∙𝑐

spectrum is not uniformly flattened across wavelengths
▪Overall spectral amplitude reduced, as well as magnitude of different peaks to
different extents

I0 I I0 I
Sample exhibiting
Isotropic sample
absorption flattening

: Chem. Soc. Rev., 2016, 45, 4859

Concentration and pathlength
considerations
▪ OD in the proposed wavelength region should be ~1
 Cuvette selection
▪Quartz!
Demountable cell
holder
▪Cell types (z-height = 15 mm):

Cylindrical quartz cell

1 mm rectangular
quartz cell Submicro cell
Demountable cell
10 mm rectangular
quartz cell
 Microsampling Accessories
▪ Micro sampling Disc

2 mL: pathlength 0.2 mm

10 mL: pathlength 1 mm → 1 mg/sample

▪Capillary cell and jacket

Approximately 10 mL (0.5 mm pathlength) → ~2 mg/sample

Checking that the cuvette is clean
▪Washing and drying the cell
(water or solvent)
▪Hellmanex, 3% Conc. HNO3
for 10 min
Solvent selection
▪ Solubility

▪ Transparency in the measuring

wavelength region

▪ Optically inactive

▪ No sugars

▪ The sample does not denature

and decompose
Can I use ‘—’ as my buffer?
▪ Solvent HT should be less than 450 V

▪ Decrease buffer concentration 50 mM 50 mM 50 mM

CHAPS Citric acid Bicine
▪ Decrease cell pathlength

50 mM
Phosphoric acid
Measurement
Parameter Optimization
Nitrogen Purge
1. Oxidation of mirrors → less reflectance → reduction in S/N
2. Avoid absorption of oxygen in far-UV

▪ Flowrates: shorter wavelengths, higher flowrate

▪ >185 nm: 2 L/min
▪ <180 nm: 5 L/min
General Parameters
I. Photometric Mode
II. Data Pitch
III. Scanning Mode
IV. Scanning Speed
V. D.I.T.
VI. CD Scale
VII. Bandwidth
VIII. Accumulations
Photometric Modes
Data Pitch

Data pitch = 1/3 of bandwidth

 Spectral Bandwidth
▪ Spectral bandwidth is full width of the
Bandwidth Dl Gaussian distribution at half the peak
maximum (FWHM).
▪ Smaller spectral bandwidth, more peak

Intensity
Intensity

resolution, less light throughput.

▪ SBW = 1/10 of natural bandwidth

Wavelength Wavelength
Complete Monochromatic light Light from Monochromator
 Bandwidth
▪ ACS Spectrum: ▪ Lysozyme Spectrum:
▪ Bands are far apart, separated by > 20 nm ▪ Bandwidth of 1 nm is more suitable
▪ Bandwidth of 2 nm is more than suitable
2 0 7

1 . 5

1st derivative
5

C D [ m d e g ]

C D [ m d e g ]
0
0
C D [ m d e g ]

- 2 0

- 1

- 5
1 7 5 2 0 0 2 2 0 2 4 0 2 5 0

W a v e l e n g t h [ n m ]
- 6

- 4 0 1 7 5 2 0 0 2 2 0 2 4 0 2 5 0

1 8 0 2 0 0 2 5 0 3 0 0 3 5 0
W a v e l e n g t h [ n m ]

W a v e l e n g t h [ n m ]
Scanning Modes
▪ Continuous Scan
▪ Measurement time does not depend on step Continuous Scan
resolution
Step Scan
▪ Suitable for obtaining data with better S/N in Difference between the two obtained spectra
short time

▪ Step Scan
▪ Stops wavelength based on data pitch
▪ Not used to improve S/N or high speed scan

▪Auto-step Scan
▪ Performs step scan while automatically
adjusting between upper and lower D.I.T. limits
D.I.T. and Scanning Speed
▪ D.I.T.: Time which the data obtained is integrated over.
▪ S/N ~ DIT1/2

▪ Scanning Speed: How quickly the monochromator moves to obtain data points.

𝐷. 𝐼. 𝑇. × 𝑆𝑐𝑎𝑛𝑛𝑖𝑛𝑔 𝑠𝑝𝑒𝑒𝑑 < 𝐹𝑊𝐻𝑀ൗ10

Response-wavelength-width
D.I.T. 1 sec
Scanning speed: 100 nm/min = 1.67 nm/sec

𝑥 = 1.67 𝑛𝑚ൗ𝑠𝑒𝑐 ∙ 1 𝑠𝑒𝑐 = 1.67 𝑛𝑚

Response-wavelength-width: 1.67 nm

× × × × × × × × × × × × × × × × × × × × ×

200 205 210 215 220

Wavelength [nm]
Determining the D.I.T. and scanning speed for
maintaining resolution
D.I.T. and scanning speed should be set so that the response-wavelength-width is 1/3
or less of the full width at half maximum (FWHM) of a target peak in the spectrum.

FWHM= 28.1nm

28.1/3 = 9.4 nm
D.I.T. and Scanning Speed
Determining the D.I.T. and Scanning Speed

Prioritize S/N ratio

D.I.T. large noise small
Scanning speed low measurement time long

Prioritize measurement time

D.I.T. small noise large
Scanning speed high measurement time short
D.I.T.
1 sec DIT 1 sec DIT
4 sec DIT 4 sec DIT

a-helical b-sheet
Accumulations
𝑆ൗ = 𝐷. 𝐼. 𝑇. ∙ 𝑁
𝑁
N = number of accumulations

1 accumulation
3 accumulations

*data offset
CD Scale
To Summarize…..
▪ Increase the CD signal
1. Increase the concentration
2. Increase the pathlength
▪ Decrease the HTV/absorption
1. Decrease the buffer concentration (remove salts, imidazole, etc)
2. Decrease the sample concentration
3. Decrease the cell pathlength
4. Open the bandwidth up
▪ Increase the S/N
1. Increase the DIT (possibly have to decrease the scanning speed)
2. Increase the number of accumulations
3. Open the bandwidth up
▪ Shorten measurement time
1. Increase scanning speed (possibly have to decrease DIT)
2. Reduce number of accumulations if more than 1
Troubleshooting
▪HTV at 1000 V → something blocking the detector
▪ Is the spacer upside down?

▪HTV at 0 V
▪ Shutter is closed
▪ Sample compartment lid is open
Data Processing Functions
Baseline and Sample Measurement

Baseline
measurement

Solvent

Sample
measurement

Sample
Subtraction
CD Units
∆𝐴 𝜃
∆𝜀 = 𝜀𝐿 − 𝜀𝑅 = =
𝑐 ∙ 𝑙 𝑐 ∙ 𝑙 ∙ 3298 ΔA: Delta OD/CD Absorbance
θ: ellipticity (mdeg)
100 ∙ 𝜃 c: molar concentration
𝜃 = = 3298 ∙ ∆𝜀 l: pathlength (cm)
𝑐∙𝑙
[θ]: molar ellipticity

𝜃 𝑀𝑅 = 𝜃 ∙ 𝑐 ∙ 𝑙ൗ𝑅
Mean Residue Molar Ellipticity
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