Circular Dichroism

2.2.41.
Circular dichroism
EUROPEAN PHARMACOPOEIA 5.0
for any analytical procedure. Specific acceptance criteria

for each validation parameter must be consistent with the
intended use of the method.
TRANSFER OF DATABASES
When databases are transferred to another instrument,
spectral range, number of data points, spectral resolution
Specificity. The relative discriminatory power and selectivity and other parameters have to be taken into consideration.
Further procedures and criteria must be applied to
for quantitative determination must be similar to those
demonstrate that the model remains valid with the new
mentioned under Qualitative analysis. The extent of
database or new instrument.
specificity testing is dependent on the application and the
risks being controlled. Variations in matrix concentrations
within the operating range of the method must not affect the DATA STORAGE
Store the electronic NIR spectra, libraries and data according
quantitative measurement significantly.
to the current regulations.
Linearity. The validation of linearity involves the correlation
Store the NIR spectra with the necessary data pre-treatment
of NIR results calculated from NIR responses within the
for the special use (for example identification, particle size
used algorithms to reference method results distributed
analysis, content of water etc.) according to the current
throughout the defined range of the calibration model.
specifications.
Actual NIR responses that are non-linear may still be valid.
Range. The range of analyte reference values defines the
range of the NIR method and quantitation limits of the
method. Controls must be in place to ensure that results
outside the validated range are not accepted.
Accuracy. This can be determined by comparison with
the validation method or with known samples (samples of
blank and added amounts of tested substance). Accuracy
can be indicated by the standard error of prediction (SEP)
of the NIR method that should be in close agreement with
the data of the validated method. The SEP is the standard
deviation of the residuals obtained from comparing the
NIR results with analytical reference data for the specified
samples. It is demonstrated by correlation of NIR results
with analytical reference data, by comparison of the SEP
to the reference method used for validation. Alternatively
statistical comparison methods may be used to compare NIR
results with reference values (paired t-test, bias evaluation).
01/2005:20241
2.2.41. CIRCULAR DICHROISM

The difference in absorbance of optically active substances
within an absorption band for left and right circularly
polarised light is referred to as circular dichroism.
Direct measurement gives a mean algebraic value :
circular dichroic absorbance,
AL
absorbance of left circularly polarised light,
AR
absorbance of right circularly polarised light.
Circular dichroism is calculated using the equation :

Precision. This expresses the closeness of agreement
between a series of measurements under the prescribed
conditions. It is assessed by a minimum of 6 measurements
performed according to the developed analytical method.
= molar circular dichroism or molar differential
Precision may be considered at 2 levels, repeatability
dichroic absorptivity expressed in litremole 1cm 1,
(replicate measurements of the same sample with or without
variation in sample positioning) and intermediate precision
L = molar absorptivity (2.2.25) of left circularly
(replicate measurements by different analysts, different days
polarised light,
of measurements).
R = molar absorptivity of right circularly polarised
Robustness. This includes the effects of variations of
light,
temperature, humidity, sample handling and the influence
c
concentration of the test solution in molelitre 1,
=
of instrument changes.
= optical path of the cell in centimetres.
Outliers. Outlier results from NIR measurements of a sample l
containing an analyte outside the calibration range indicates The following units may also be used to characterise circular
that further testing is required. If further testing of the
dichroism :
sample by an appropriate analytical method gives the analyte Dissymmetry factor :
content within the specifications, this may be accepted and
considered to have met the specifications. Thus an outlier
result generated by NIR measurements of the sample may
still meet specifications for the analyte of interest.
= molar absorptivity (2.2.25).

ONGOING MODEL EVALUATION
Molar ellipticity :
NIR models validated for use are subjected to ongoing
Certain types of instruments display directly the value of
performance evaluation and monitoring of validation
ellipticity , expressed in degrees. When such instruments
parameters. If discrepancies are found, corrective action will are used, the molar ellipticity [ ] may be calculated using
be necessary. The degree of revalidation required depends
the following equation :
on the nature of the changes. Revalidation of a qualitative
model will be necessary when a new material is added to
the reference library and may be necessary when changes
in the physical properties of the material occur and when
changes in the source of supply take place. Revalidation of a [ ] = molar ellipticity, expressed in
quantitative model is required on account of changes in the
degreescm2decimole 1,
composition of the finished product, in the manufacturing
= value of ellipticity given by the instrument,
process and in sources/grades of raw materials.
General Notices (1) apply to all monographs and other texts
63
2.2.42. Density of solids
EUROPEAN PHARMACOPOEIA 5.0
Figure 2.2.41.-1. Optical scheme of a dichrograph

Linearity of modulation. Dissolve 10.0 mg of
(1S)-(+)-10-camphorsulphonic acid R in water R and dilute
to
10.0 ml with the same solvent. Determine the exact
c
= concentration of the solution to be examined
concentration of camphorsulphonic acid in the solution by
in g/ml,
ultraviolet spectrophotometry (2.2.25), taking the specific
absorbance to be 1.49 at 285 nm.
l
= optical path of the cell in centimetres.
Record the circular dichroism spectrum between 185 nm
Molar ellipticity is also related to molar circular dichroism by
and 340 nm. Measured at the maximum at 290.5 nm, is
the following equation :
+ 2.2 to + 2.5. Measured at the maximum at 192.5 nm,
is 4.3 to 5.
(1S)-(+)- or antipodal (1R)-()-ammonium
10-camphorsulphonate R can also be used.
Molar ellipticity is often used in the analysis of proteins
and nucleic acids. In this case, molar concentration is
01/2005:20242
expressed in terms of monomeric residue, calculated using
the expression :
2.2.42. DENSITY OF SOLIDS
M
relative molecular mass of the substance to be

examined,
The mean relative molecular mass of the monomeric residue

is 100 to 120 (generally 115) for proteins and about 330 for
nucleic acids (as the sodium salt).
Apparatus. The light source (S) is a xenon lamp
(Figure 2.2.41.-1) ; the light passes through a double
monochromator (M) equipped with quartz prisms (P1, P2).
The linear beam from the first monochromator is split
into 2 components polarised at right angles in the second
monochromator. The exit slit of the monochromator
eliminates the extraordinary beam.
The polarised and monochromatic light passes through
a birefringent modulator (Cr) : the result is alternating
circularly polarised light.
The beam then passes through the sample to be examined (C)
and reaches a photomultiplier (PM) followed by an amplifier
circuit which produces 2 electrical signals : one is a direct
current Vc and the other is an alternating current at the
modulation frequency Vac characteristic of the sample to be
examined. The phase gives the sign of the circular dichroism.
The ratio Vac/Vc is proportional to the differential absorption
A which created the signal. The region of wavelengths
normally covered by a dichrograph is 170 nm to 800 nm.
Calibration of the apparatus
Accuracy of absorbance scale. Dissolve 10.0 mg of
isoandrosterone R in dioxan R and dilute to 10.0 ml with
the same solvent. Record the circular dichroism spectrum of
the solution between 280 nm and 360 nm. Measured at the
maximum at 304 nm, is + 3.3.
The solution of (1S)-(+)-10-camphorsulphonic acid R may
also be used.
64
The density of solids corresponds to their average mass

per unit volume and typically is expressed in grams per cubic
centimetre (g/cm3) although the International Unit is the
kilogram per cubic meter (1 g/cm3 = 1000 kg/m3).
Unlike gases and liquids whose density depends only on
temperature and pressure, the density of a solid particle also
depends on its molecular assembly and therefore varies with
the crystal structure and degree of crystallinity.
When a solid particle is amorphous or partially amorphous,
its density may further depend upon the history of
preparation and treatment.
Therefore, unlike fluids, the densities of two chemically
equivalent solids may be different, and this difference
reflects a difference in solid-state structure. The density of
constituent particles is an important physical characteristic
of pharmaceutical powders.
The density of a solid particle can assume different values
depending on the method used to measure the volume of the
particle. It is useful to distinguish three levels of expression
of density :
the crystal density which only includes the solid fraction
of the material ; the crystal density is also called true
density ;
the particle density which also includes the volume due
to intraparticulate pores,
the bulk density which further includes the
interparticulate void volume formed in the powder bed ;
the bulk density is also called apparent density.
CRYSTAL DENSITY
The crystal density of a substance is the average mass
per unit volume, exclusive of all voids that are not a
fundamental part of the molecular packing arrangement. It
is an intrinsic property of the substance, and hence should
See the information section on general monographs (cover pages)

Circular Dichroism

Uploaded by

Copyright:

Available Formats

Circular Dichroism

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Circular Dichroism

Uploaded by

Copyright:

Available Formats

2.2.41.

EUROPEAN PHARMACOPOEIA 5.0

for any analytical procedure. Specific acceptance criteria

2.2.41. CIRCULAR DICHROISM

circular dichroic absorbance,

absorbance of left circularly polarised light,

absorbance of right circularly polarised light.

Circular dichroism is calculated using the equation :

= molar absorptivity (2.2.25).

General Notices (1) apply to all monographs and other texts

2.2.42. Density of solids

EUROPEAN PHARMACOPOEIA 5.0

Figure 2.2.41.-1. Optical scheme of a dichrograph

relative molecular mass of the substance to be

The mean relative molecular mass of the monomeric residue

The density of solids corresponds to their average mass

See the information section on general monographs (cover pages)

You might also like