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Units of CD Measurement, Schemes and Mind Maps of Biochemistry

Units of CD Measurement. CD is reported in units of absorbance or ellipticity. Each of these can be normalized for molar concentration of the sample.

Typology: Schemes and Mind Maps

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Units of CD
Measurement
Units of CD Measurement
CD is reported in units of absorbance or ellipticity.
Each of these can be normalized for molar
concentration of the sample. The most direct
data from the Olis CD instrument is absorbance
(Abs(L)- Abs(R)). This value is often reported in
milliabsorbance units (mA), which are a thousandth
of an absorbance unit.
which is related to absorbance by a factor of 32.98
(θ = 32.98 ΔAbs). Ellipticity is usually reported in
millidegrees (mdeg or m
ο
), which are a thousandth
of a degree.
Molar ellipticity ([θ]) is CD corrected for
concentration. The units of molar ellipticity are
historical (deg×cm
2
/dmol). Conversion from molar
extinction (absorbance corrected for concentration)
to molar ellipticity uses a factor of 3298 ([θ] =
3298Δε). To calculate molar ellipticity, the sample
concentration (g/L), cell pathlength (cm), and the
molecular weight (g/mol) must be known.
If the sample is a protein, the mean residual
weight (average molecular weight of the amino
acids it contains) is used in place of the molecular
weight, essentially treating the protein as a
solution of amino acids.
From
To
Absorbance1Milliabsorbance2Molar Extinction3Degrees4Millidegrees5Molar Ellipticity6
(A)
A
A*1000
A*M/(C*L)
A*32.98
A*32980
A*M*3298/(L*C)
(mA)
mA/1000
mA
A*M/(C*L*1000)
mA*0.03298
mA*32.98
mA*M*3.298/
(L*C)
(
)
*C*L/M
*C*L*1000/M
*C*L*32.98/M
*C*L*32980/
M
*3298
(
°
)
°
/32.98
°
/0.03298
°
*M/(C*L*32.98)
°
°
*1000
°
*M*100/(L*C)
(m
°
)
m
°
/32980
m
°
/32.98
m
°
*M/(C*L*32980)
m
°
/1000
m
°
m
°
*M/(10*L*C)
[
Θ
]
[Θ]
*C*L/(3298*M)
[Θ]
*C*L/(3.298*M)
[Θ]
/3298
[Θ]
*C*L/(100*M)
[Θ]
*C*L*10/M
[Θ]
C is concentration in g/L
M is average molecular weight (g/mol)
L is path length of cell (cm)
1
Units are Absorbance (Abs)
2
Units are milliabsorbance (mAbs)
3
Units are A*L/mol*cm
4
Units are degrees (
º
)
5
Units are millidegrees (m
º
)
6
Units are deg*cm2/dmol
pf2

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Units of CD

Measurement

Units of CD Measurement

CD is reported in units of absorbance or ellipticity.

Each of these can be normalized for molar

concentration of the sample. The most direct

data from the Olis CD instrument is absorbance

(Abs(L)- Abs(R)). This value is often reported in

milliabsorbance units (mA), which are a thousandth

of an absorbance unit.

CD data are commonly reported as ellipticity (θ),

which is related to absorbance by a factor of 32.

(θ = 32.98 ΔAbs). Ellipticity is usually reported in

millidegrees (mdeg or mο), which are a thousandth

of a degree.

Molar ellipticity ([θ]) is CD corrected for

concentration. The units of molar ellipticity are

historical (deg×cm^2 /dmol). Conversion from molar

extinction (absorbance corrected for concentration)

to molar ellipticity uses a factor of 3298 ([θ] =

3298Δε). To calculate molar ellipticity, the sample

concentration (g/L), cell pathlength (cm), and the

molecular weight (g/mol) must be known.

If the sample is a protein, the mean residual

weight (average molecular weight of the amino

acids it contains) is used in place of the molecular

weight, essentially treating the protein as a

solution of amino acids.

From

To 

Absorbance^1 Milliabsorbance^2 Molar Extinction^3 Degrees^4 Millidegrees^5 Molar Ellipticity^6

(A) A A1000 AM/(CL) A32.98 A32980 AM3298/(LC)

(mA) mA/1000 mA AM/(CL1000) mA0.03298 mA32.98 mAM3.298/ (LC) () CL/M CL1000/M  CL32.98/M CL*32980/ M

(°) °/32.98 °/0.03298 °M/(CL32.98) ° °1000 °M100/(L*C)

(m°) m°/32980 m°/32.98 m°M/(CL32980) m°/1000 m° m°M/(10LC) [Θ] [Θ]CL/(3298M) [Θ]CL/(3.298M) [Θ]/3298 [Θ]CL/(100M) [Θ]CL10/M [Θ]

C is concentration in g/L

M is average molecular weight (g/mol)

L is path length of cell (cm)

(^1) Units are Absorbance (Abs) (^2) Units are milliabsorbance (mAbs) (^3) Units are AL/molcm (^4) Units are degrees (º) (^5) Units are millidegrees (mº) (^6) Units are deg*cm2/dmol

Sample

Concentration Effects

CD signals obey Beer’s law – CD intensity is

proportional to the concentration of the active species

  • so it is a tempting to increase the concentration of

the sample to improve the signal to noise ratio. This

strategy is not always useful, as the signal to noise

is a function of the signal strength and the overall

light intensity passing through the sample to the

detectors. Since absorbance must occur at the CD

active wavelengths, increasing the concentration

also increases the overall absorbance, thus reducing

the amount of light reaching the detectors. This

necessitates the need for higher PMT high volts,

which, in turn, increases the noise. The relationship

between sample absorbance and signal to noise ratio

is illustrated in Figure 3^1.

The optimal protein concentration is a function of

the pathlength of the cuvette. Figure 4 shows a plot

of the protein concentration required to produce an

absorbance of 0.5 2. This is lower than the optimal

0.9 to account for absorbing buffer components. This

plot indicates an optimal protein concentration of

approximately 0.1 mg/mL for a protein solution, if the

absorbance due to the buffer itself is minimized.

(^1) 1 Johnson C. W. (1996) in Circular Dichroism and the Conformational Analysis of Biomolecules. Fasman G.D., Editor. Plenun Press, New York pp 635- (^2) Sutherland, J.C. (1996) in Circular Dichroism and the Conformational Analysis of Biomolecules. Fasman G.D., Editor. Plenun Press, New York pp 599-

Figure 3

Figure 4

There is an optimum absorbance

to use (Abs = 0.89). For a 1 mm

pathlength cell, this absorbance is

achieved with a protein concentration

of about 0.1-0.3 mg/mL.

In the Olis dual-beam CD, no

concentration calibration is

required because the CD signal is

directly digitally derived from the

two photodetectors signals.