BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an analysis method of low-substituted
hydroxypropyl cellulose. More specifically, the invention relates to an analysis
method suitable for use in the quality control and performance evaluation of low-substituted
hydroxypropyl cellulose by gel permeation chromatography (GPC) coupled with light
scattering such as GPC coupled with multiple angle laser light scattering (which
will hereinafter be called "GPC-MALLS") or GPC coupled with low angle laser light
scattering (which will hereinafter be called "GPC-LALLS").
2. Description of the related art
Low-substituted hydroxypropyl cellulose is a cellulose derivative
used as an additive to pharmaceuticals and mainly serves as a binder or disintegrant
In consideration of its use as such a material, quality control and
performance evaluation of it are indispensable. Accordingly, there is a demand for
the determination of the absolute molecular weight and molecular weight distribution
of low-substituted hydroxypropyl cellulose.
The molecular weight of low-substituted hydroxypropyl cellulose could
not be determined using GPC-MALLS, because low-substituted hydroxypropyl cellulose
is insoluble in various solvents except a special strong alkaline solvent such as
an aqueous solution of copper ethylenediamine or a cadmium ethylenediamine solution.
Moreover, such a strong alkaline solvent cannot be used for column to be used in
gel permeation chromatography. A method for determining the molecular weight distribution
of a polymer by a light scattering method is exemplified in K. Jumel et al., Carbohydrate
Polymers 29 (1996) 105-109.
The molecular weight of such a cellulose derivative insoluble in various
solvents is conventionally determined by converting it into a cellulose trinitrate,
dissolving the resulting product in tetrahydrofuran and subjecting the solution
to gel permeation chromatography, as exemplified in TAPPI standard method T-238
su-63. However, this method involves problems, for example, it causes deterioration
in polymerization degree, and it requires strict care because of use of a mixed
acid of sulfuric acid and fuming nitric acid ("Cellulose no Jiten (encyclopedia
of cellulose)", ed. by Cellulose Society of Japan, published by Asakura Shoten,
It is well-known that a method for determining the absolute molecular
weight and molecular weight distribution of a pulp, comprises converting the pulp
into tricarbanilated cellulose, dissolving the tricarbanilated cellulose in tetrahydrofuran,
and carrying out GPC-MALLS with the resulting solution as a sample solution and
with tetrahydrofuran as a mobile phase (see "Chemistry and Processing of Wood and
Plant Fibrous Materials", Woodhead Publishing Limited, Chapter 15, 1996).
SUMMARY OF THE INVENTION
The present invention relates to an analysis method of the absolute
molecular weight and molecular weight distribution of low-substituted hydroxypropyl
cellulose by using a light scattering method.
The present inventors have carried out an extensive investigation
with a view to overcoming the above-described problems. As a result, it has been
found that low-substituted hydroxypropyl cellulose which has been carbanilated is
soluble in a solvent, leading to the completion of the present invention.
In the present invention, there is thus provided an analysis method
of the absolute molecular weight and molecular weight distribution of low-substituted
hydroxypropyl cellulose, comprising steps of carbanilating a portion or all (the
whole) of the low-substituted hydroxypropyl cellulose, dissolving the carbanilated
cellulose in a solvent, and analyzing the resulting solution by subjecting it to
GPC coupled with light scattering such as GPC-MALLS or GPC-LALLS while using a solvent
identical or similar in kind to the solvent as a mobile phase.
According to the present invention, the absolute molecular weight
and molecular weight distribution of low-substituted hydroxypropyl cellulose can
be determined efficiently and accurately. Based on the result thus obtained, the
quality control and performance evaluation of low-substituted hydroxypropyl cellulose
can be carried out conveniently.
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
- FIG. 1 illustrates an apparatus for carrying out the method of the present invention;
- FIG. 2 illustrates one of the analysis results obtained using the method of
the present invention.
Low-substituted hydroxypropyl cellulose to be used in the present
invention is a cellulose having a hydroxypropoxyl group as a substituent and its
substitution degree may be preferably from 10 to 16 wt%. The substitution degree
can be measured based on the Japanese Pharmacopoeia.
Low-substituted hydroxypropyl cellulose is obtained by substituting
a predetermined portion of three hydroxyl groups per glucose unit of C6H10O5
with a hydroxypropoxyl group and the portion substituted with a hydroxypropoxyl
group also has a hydroxyl group derived therefrom. The term "carbanilation" as used
herein means the carbanilation of the hydroxyl group of this glucose unit and at
most three hydroxyl groups can be carbanilated (tricarbanilated) per glucose unit.
According to the present invention, low-substituted hydroxypropyl
cellulose becomes soluble in a solvent by being converted into carbanilated cellulose.
In low-substituted hydroxypropyl cellulose, all the substitutable
hydroxyl groups are not necessarily carbanilated, but the substitutable hydroxyl
groups may be carbanilated within a range necessary for obtaining a solution concentration
suitable for the determination by GPC with light scattering such as GPC-MALLS.
Although no particular limitation is imposed on the solvent insofar
as it can dissolve therein low-substituted hydroxypropyl cellulose, which has been
partially or wholly carbanilated, at a concentration suitable for the determination
by GPC with light scattering such as GPC-MALLS, ethers such as tetrahydrofuran (THF)
and amines such as dimethylformamide (DMF) are preferred.
The number of the hydroxyl groups to be carbanilated may differ depending
on a solvent. When THF is used, a preferable range is from 2.8 to 3.0, more preferably
There is no particular limitation imposed on a method of converting
low-substituted hydroxypropyl cellulose into carbanilated cellulose. The target
low-substituted hydroxypropyl cellulose which has been carbanilated is available
preferably by uniformly dispersing the low-substituted hydroxypropyl cellulose in
pyridine, adding excess phenyl isocyanate to the resulting dispersion, causing them
to react at 90°C for about 3 hours and re-precipitating the reaction mixture in
a mixed solvent of methanol-water.
No polymerization breakage occurs in the step of converting into carbanilated
cellulose and the converted cellulose is suitable for a light scattering method
because a coefficient of refractive index on basis of concentration (which will
hereinafter be abbreviated as "dn/dc") is high.
When the carbanilated, low-substituted hydroxypropyl cellulose is
added to a solvent (for example, THF), the cellulose dissolves therein. The resulting
solution is subjected to for example, GPC-MALLS. By using a known sample introducing
apparatus, a predetermined amount of the solution can be injected into GPC-MALLS.
The carbanilated, low-substituted hydroxypropyl cellulose in a sample
solution has any concentration insofar as it is suitable for the measurement by
GPC-MALLS. It may be preferably from 0.01 to 0.5 wt%, more preferably from 0.05
to 0.2 wt%. If necessary, impurities in the carbanilated, low-substituted hydroxypropyl
cellulose solution can be removed by filtering it through a membrane filter prior
to the measurement.
The solution injected into GPC-MALLS is subjected to a separation
column, with a solvent (for example, THF) identical or similar in kind to the solvent
of the sample solution as a mobile phase.
For GPC-MALLS, a separation column may include a column filled with
a general-purpose polymer gel such as a column preferably filled with polystyrene
as a base material.
A detecting unit of GPC-MALLS may include a combination of a multi-angle
light scattering detector and a differential refractometer detector. GPC-LALLS using
a low-angle light scattering detector and a differential refractometer detector
in combination can also be used for the determination, because the carbanilated,
low-substituted hydroxypropyl cellulose exhibits no absorption in the ultraviolet
region. The combined use can reveal adsorption condition of the sample to the column
or breakage condition of the molecular chain so that the absolute molecular weight
can be determined.
According to the present invention, the absolute molecular weight
and molecular weight distribution of the carbanilated, low-substituted hydroxypropyl
cellulose can be determined by subjecting the solution of the carbanilated, low-substituted
hydroxypropyl cellulose to GPC with light scattering such as GPC-MALLS and fractionally
collecting an eluate therefrom. The absolute molecular weight thus obtained is divided
by the molecular weight of the monomer of the carbanilated, low-substituted hydroxypropyl
cellulose to fmd the value of the polymerization degree. Then, the polymerization
degree is multiplied by the molecular weight of the monomer of the low-substituted
hydroxypropyl cellulose monomer so as to obtain the absolute molecular weight of
the low-substituted hydroxypropyl cellulose.
In the present invention, the absolute molecular weight is preferably
a weight average absolute molecular weight. It is not a relative value available
using another substance as a standard, but an average, by weight fraction, of the
absolute value obtained from a theoretical equation by the light scattering method.
The molecular weight distribution of the invention is a value obtained
by dividing a weight average molecular weight by a number average molecular weight.
An example of an apparatus for carrying out the method of the invention
will next be described based on a drawing, but the invention is not limited to or
FIG. 1 illustrates one example of an apparatus for carrying out the
invention method, comprising a mobile phase reservoir 1, an on-line degasser 2,
a feed pump 3, a manual injector 4, a column 5, a column oven 6, a multi-angle light
scattering detector 7, a difference refractometer detector 8 and a data processing
Upon analysis, a mobile phase is fed from the mobile phase reservoir
1 by the pump 3 in advance so as to reach a steady state. A sample is then injected
from the manual injector 4. The sample thus injected is fed with the mobile phase
to the column 5 in the column oven 6 and its absolute molecular weight and molecular
weight distribution are detected by the multi-angle light scattering detector 7
and difference refractometer detector 8. The data thus obtained is processed in
the data processing unit 9, at which the molecular weight is calculated.
Analysis was made using the above apparatus under the following analysis
<Pretreatment of sample>
The 170 mg of Low-substituted hydroxypropyl cellulose (product of
Shin-Etsu Chemical Co., Ltd.) was dispersed in 15 ml of pyridine by stirring overnight.
To the resulting dispersion was added 2 ml of phenyl isocyanate (product of Wako
Pure Chemical Industries, Ltd.), followed by heating under reflux at 90°C. After
heating under reflux for 3 hours, 1 ml of methanol (product of Wako Pure Chemical
Industries, Ltd.) was added to decompose excess phenyl isocyanate. The resulting
solution was gradually added dropwise to a mixed solution of 70 ml of methanol,
30 ml of distilled water and 2 ml of glacial acetic acid to cause reprecipitation.
The solution was then subjected to suction filtration through a glass filter having
a pore size of from 40 to 50 µm. The precipitate thus obtained was dispersed, stirred
and washed in 100 ml of methanol. The dispersion was subjected again to suction
filtration through a glass filter having a pore size of from 40 to 50 µm. By repeating
these operations twice or thrice, pyridine was completely removed. The residue was
then dried, whereby the target tricarbanilated, low-substituted hydroxypropyl cellulose
The 10 mg of the resulting tricarbanilated, low-substituted hydroxypropyl
cellulose was dissolved in 10 ml of tetrahydrofuran, followed by filtration through
a membrane filter (pore size: 0.45 µm, product of ADVANTEC).
It was confirmed by an elementary analysis that the molar substitution
of the carbanilate group was 3, indicating that the resulting low-substituted hydroxypropyl
cellulose had been tricarbanilated accurately.
<Coefficient of refractive index on basis of concentration (which will hereinafter
be abbreviated as "dn/dc")>
Low-substituted hydroxypropyl cellulose (product of Shin-Etsu Chemical
Co., Ltd.) was tricarbanilated as described above and dissolved in tetrahydrofuran
to prepare five solutions different in concentration (from 0.05 W/V% to 0.3 W/V%).
The dn/dc was measured in accordance with the static light scattering method by
using a high-sensitivity differential refractometer ("DRM-1021", product of Otsuka
Electronics Co., Ltd.).
- Column: "TSK guard column Super H-L" (product of TOSOH Corporation)
"TSK gel Super H 2000" (product of TOSOH Corporation)
"TSK gel Super H 3000" (product of TOSOH Corporation)
"TSK gel Super H 4000" (product of TOSOH Corporation)
"TSK gel Super H 5000" (product of TOSOH Corporation)
- Mobile phase: tetrahydrofuran
- Degasser: "ERC-3115" (product of ERC Inc.)
- Flow rate: 0.4 ml/min
- Temperature: 40°C
- Injector: "Model 7125" (product of Rheodyne LLC)
Multi-angle light scattering detector: "DAWN DSP" (product of Wyatt Technology
Differential refractometer detector: "RI-71" (product of Showa Denko K.K.) dn/dc:
As a result of analysis under the above-described conditions, the
tricarbanilated, low-substituted hydroxypropyl cellulose had a weight average molecular
weight of 360,000 g/mol and the molecular weight distribution (weight average molecular
weight divided by number average molecular weight) was 1.70. The molecular weight
of the converted monomer was 532.6 g/mol and the monomer of the low-substituted
hydroxypropyl cellulose before the conversion was 176.32 g/mol. Based on them, the
weight average molecular weight of the low-substituted hydroxypropyl cellulose before
the conversion was 119,000 g/mol and its molecular weight distribution was 1.70.
The results are illustrated in FIG. 2. The peak of FIG. 2 is that
of the tricarbanilated, low-substituted hydroxypropyl cellulose. Thus, according
to the present invention, the tricarbanilated low-substituted hydroxypropyl cellulose
can be analyzed with good sensitivity. In FIG. 2, a light scattering intensity and
a signal intensity of refractive index are plotted along the ordinate, while the
outflow (mL) is plotted along the abscissa.