2.2.49. Falling ball viscometer method EUROPEAN PHARMACOPOEIA 5.

0
Verification of the wavenumber scale. Verify the
wavenumber scale of the Raman shift (normally expressed
in reciprocal centimetres) using a suitable standard which
has characteristic maxima at the wavenumbers under
investigation, for example, an organic substance, an Ne lamp
or Ar
+
plasma lines from an argon-ion laser.
The calibration measurement should be matched to the
sample type, i.e. a solid calibration sample should be
used for solid samples and a liquid calibration sample
for liquid samples. Choose a suitable substance (e.g.
indene, cyclohexane or naphthalene) for which accurate
wavenumber shifts have been established. The indene
sample can favourably be placed in an NMR tube, evacuated
and sealed under inert gas, and stored cool in the dark to
avoid degradation of the sample.
Table 2.2.48.-1. Wavenumber shifts (and acceptable
tolerances) of cyclohexane, indene and naphthalene.
cyclohexane
A
indene
B
naphthalene
A
3056.4 ( 1.5)
2938.3 ( 1.5)
2923.8 ( 1.5)
2852.9 ( 1.5)
1609.7 ( 1.0) 1576.6 ( 1.0)
1444.4 ( 1.0) 1552.6 ( 1.0) 1464.5 ( 1.0)
1266.4 ( 1.0) 1205.2 ( 1.0) 1382.2 ( 1.0)
1157.6 ( 1.0) 1147.2 ( 1.0)
1028.3 ( 1.0) 1018.6 ( 1.0) 1021.6 ( 1.0)
801.3 ( 1.0) 730.5 ( 1.0) 763.8 ( 1.0)
533.9 ( 1.0) 513.8 ( 1.0)
A
Standard guide for Raman shift standards for spectrometer
calibration (American Society for Testing and Materials ASTM E 1840).
B
D. A. Carter, W. R. Thompson, C. E. Taylor and J. E. Pemberton,
Applied Spectroscopy, 1995, 49 (11), 1561-1576.
Verification of the response-intensity scale. The absolute
and relative intensities of the Raman bands are affected by
several factors including:
the state of polarisation of the irradiating light,
the state of polarisation of the collection optics,
the intensity of the irradiating light,
differences in instrument response,
differences in focus and geometry at sample,
differences in packing density for solid samples.
Appropriate acceptance criteria will vary with the application
but a day-to-day variation of 10 per cent in relative band
intensities is achievable in most cases.
Establishment of a spectral reference library. Record
the spectra of a suitable number of materials which have
been fully tested (e.g. as prescribed in a monograph) and
which exhibit the variation (manufacturer, batch, crystal
modification, particle size, etc.) typical of the material to be
analysed. The set of spectra represents the information that
defines the similarity border or quantitative limits, which
may be used, e.g. to identify the substance or control the
amount formed in a manufacturing process. The number
of substances in the database depends on the specific
application. The collection of spectra in the database may be
represented in different ways defined by the mathematical
technique used for classification or quantitation.
The selectivity of the database which makes it possible
to identify positively a given material and distinguish it
adequately from other materials in the database is to be
established during the validation procedure. This selectivity
must be challenged on a regular basis to ensure ongoing
validity of the database; this is especially necessary after any
major change in a substance (e.g. change in supplier or in
the manufacturing process of the material) or in the set-up of
the Raman instrument (e.g. verification of the wavenumber
and response repeatability of the spectrometer).
This database is then valid for use only with the originating
instrument, or with a similar instrument, provided the
transferred database has been demonstrated to remain valid.
Method. Prepare and examine the sample in the same
manner as for the establishment of the database. A suitable
mathematical transformation of the Raman spectrum may be
calculated to facilitate spectrum comparison or quantitative
prediction.
Comparison of the spectra or transforms of the spectra
or quantitative prediction of properties or amounts in
the material in question may involve the use of a suitable
chemometric or statistical classification or calibration
technique.
01/2005:20249
2.2.49. FALLING BALL VISCOMETER
METHOD
The determination of dynamic viscosity of Newtonian liquids
using a suitable falling ball viscometer is performed at
20 0.1 C, unless otherwise prescribed in the monograph.
The time required for a test ball to fall in the liquid to be
examined from one ring mark to the other is determined. If
no stricter limit is defined for the equipment used the result
is valid only if 2 consecutive measures do not differ by more
than 1.5 per cent.
Apparatus. The falling ball viscometer consists of : a glass
tube enclosed in a mantle, which allow precise control of
temperature; six balls made of glass, nickel-iron or steel
with different densities and diameters. The tube is fixed in
such a way that the axis is inclined by 10 1 with regard
to the vertical. The tube has 2 ring marks which define
the distance the ball has to roll. Commercially available
apparatus is supplied with tables giving the constants, the
density of the balls and the suitability of the different balls
for the expected range of viscosity.
Method. Fill the clean, dry tube of the viscometer, previously
brought to 20 0.1 C, with the liquid to be examined,
avoiding bubbles. Add the ball suitable for the range of
viscosity of the liquid so as to obtain a falling time not less
than 30 s. Close the tube and maintain the solution at
20 0.1 C for at least 15 min. Let the ball run through the
liquid between the 2 ring marks once without measurement.
Let it run again and measure with a stop-watch, to the
nearest one-fifth of a second, the time required for the ball
to roll from the upper to the lower ring mark. Repeat the
test run at least 3 times.
Calculate the dynamic viscosity in millipascal seconds
using the formula:
k = constant, expressed in millimeter squared per
second squared,
1 = density of the ball used, expressed in grams per
cubic centimetre,
80 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0 2.2.54. Isoelectric focusing
2 = density of the liquid to be examined, expressed
in grams per cubic centimetre, obtained by
multiplying its relative density by 0.9982,
t
= falling time of the ball, in seconds.
01/2005:20254
2.2.54. ISOELECTRIC FOCUSING
GENERAL PRINCIPLES
Isoelectric focusing (IEF) is a method of electrophoresis
that separates proteins according to their isoelectric point.
Separation is carried out in a slab of polyacrylamide
or agarose gel that contains a mixture of amphoteric
electrolytes (ampholytes). When subjected to an electric
field, the ampholytes migrate in the gel to create a pH
gradient. In some cases gels containing an immobilised pH
gradient, prepared by incorporating weak acids and bases to
specific regions of the gel network during the preparation of
the gel, are used. When the applied proteins reach the gel
fraction that has a pH that is the same as their isoelectric
point (pI), their charge is neutralised and migration ceases.
Gradients can be made over various ranges of pH, according
to the mixture of ampholytes chosen.
THEORETICAL ASPECTS
When a protein is at the position of its isoelectric point,
it has no net charge and cannot be moved in a gel matrix
by the electric field. It may, however, move from that
position by diffusion. The pH gradient forces a protein to
remain in its isoelectric point position, thus concentrating
it ; this concentrating effect is called "focusing". Increasing
the applied voltage or reducing the sample load result in
improved separation of bands. The applied voltage is limited
by the heat generated, which must be dissipated. The use
of thin gels and an efficient cooling plate controlled by a
thermostatic circulator prevents the burning of the gel
whilst allowing sharp focusing. The separation is estimated
by determining the minimum pI difference (pI), which is
necessary to separate 2 neighbouring bands:
D = diffusion coefficient of the protein,
= pH gradient,
E = intensity of the electric field, in volts per
centimetre,
= variation of the solute mobility with the pH in
the region close to the pI.
Since D and for a given protein cannot be altered,
the separation can be improved by using a narrower pH
range and by increasing the intensity of the electric field.
Resolution between protein bands on an IEF gel prepared
with carrier ampholytes can be quite good. Improvements
in resolution may be achieved by using immobilised pH
gradients where the buffering species, which are analogous
to carrier ampholytes, are copolymerised within the gel
matrix. Proteins exhibiting pIs differing by as little as
0.02 pH units may be resolved using a gel prepared with
carrier ampholytes while immobilised pH gradients can
resolve proteins differing by approximately 0.001 pH units.
PRACTICAL ASPECTS
Special attention must be paid to sample characteristics
and/or preparation. Having salt in the sample can be
problematic and it is best to prepare the sample, if possible,
in deionised water or 2 per cent ampholytes, using dialysis
or gel filtration if necessary.
The time required for completion of focusing in thin-layer
polyacrylamide gels is determined by placing a coloured
protein (e.g. haemoglobin) at different positions on the gel
surface and by applying the electric field: the steady state is
reached when all applications give an identical band pattern.
In some protocols the completion of the focusing is indicated
by the time elapsed after the sample application.
The IEF gel can be used as an identity test when the
migration pattern on the gel is compared to a suitable
standard preparation and IEF calibration proteins, the IEF
gel can be used as a limit test when the density of a band
on IEF is compared subjectively with the density of bands
appearing in a standard preparation, or it can be used as
a quantitative test when the density is measured using a
densitometer or similar instrumentation to determine the
relative concentration of protein in the bands subject to
validation.
APPARATUS
An apparatus for IEF consists of :
a controllable generator for constant potential, current
and power; potentials of 2500 V have been used and
are considered optimal under a given set of operating
conditions; a supply of up to 30 W of constant power is
recommended;
a rigid plastic IEF chamber that contains a cooled plate,
of suitable material, to support the gel ;
a plastic cover with platinum electrodes that are
connected to the gel by means of paper wicks of suitable
width, length and thickness, impregnated with solutions
of anodic and cathodic electrolytes.
ISOELECTRIC FOCUSING IN POLYACRYLAMIDE GELS:
DETAILED PROCEDURE
The following method is a detailed description of an IEF
procedure in thick polyacrylamide slab gels, which is used
unless otherwise stated in the monograph.
PREPARATION OF THE GELS
Mould. The mould (see Figure 2.2.54.-1) is composed of a
glass plate (A) on which a polyester film (B) is placed to
facilitate handling of the gel, one or more spacers (C), a
second glass plate (D) and clamps to hold the structure
together.
Figure 2.2.54.-1 Mould
General Notices (1) apply to all monographs and other texts 81