ASTM F2602-08

Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)

SIGNIFICANCE AND USE
The degree of deacetylation of chitosan, as well at the molar mass and molar mass distribution, determines the functionality of chitosan in an application. For instance, functional and biological effects are highly dependent upon the composition and molar mass of the polymer.
This test method describes procedures for measurement of molar mass of chitosan chlorides and glutamates, and chitosan base, although it in principle applies to any chitosan salt. The measured molar mass is that for chitosan acetate, since the mobile phase contains acetate as counter ion. This value can further be converted into the corresponding molar mass for the chitosan as a base, or the parent salt form (chloride or glutamate).
Light scattering is one of very few methods available for the determination of absolute molar mass and structure, and it is applicable over the broadest range of molar masses of any method. Combining light scattering detection with size exclusion chromatography (SEC), which sorts molecules according to size, gives the ability to analyze polydisperse samples, as well as obtaining information on branching and molecular conformation. This means that both the number-average and mass-average values for molar mass and size may be obtained for most samples. Furthermore, one has the ability to calculate the distributions of the molar masses and sizes.
Multi-angle laser light scattering (MALS) is a technique where measurements of scattered light are made simultaneously over a range of different angles. MALS detection can be used to obtain information on molecular size, since this parameter is determined by the angular variation of the scattered light. Molar mass may in principle be determined by detecting scattered light at a single low angle (LALLS). However, advantages with MALS as compared to LALLS are: (1) less noise at larger angles, (2) the precision of measurements are greatly improved by detecting at several angles, and (3) the ability to detect angular variati...
SCOPE
1.1 This test method covers the determination of the molar mass of chitosan and chitosan salts intended for use in biomedical and pharmaceutical applications as well as in tissue engineered medical products (TEMPs) by size exclusion chromatography with multi-angle laser light scattering detection (SEC-MALS). A guide for the characterization of chitosan salts has been published as Guide F 2103.
1.2 Chitosan and chitosan salts used in TEMPs should be well characterized, including the molar mass and polydispersity (molar mass distribution) in order to ensure uniformity and correct functionality in the final product. This test method will assist end users in choosing the correct chitosan for their particular application. Chitosan may have utility as a scaffold or matrix material for TEMPs, in cell and tissue encapsulation applications, and in drug delivery formulations.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 31-Jan-2008
• ICS: 11.120.10 - Medicaments
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.42 - Biomaterials and Biomolecules for TEMPs

Relations

• Replaced By: ASTM F2602-08e1 - Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
• Effective Date: 01-Feb-2008
• Referred By: ASTM F2103-18 - Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
• Effective Date: 01-Feb-2008

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F 2602 – 08
Standard Test Method for
Determining the Molar Mass of Chitosan and Chitosan Salts
by Size Exclusion Chromatography with Multi-angle Light
Scattering Detection (SEC-MALS)
This standard is issued under the fixed designation F 2602; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 2.3 National Institute of Standards and Technology:
NIST SP811 Special Publication: Guide for the Use of the
1.1 This test method covers the determination of the molar
International System of Units (SI)
mass of chitosan and chitosan salts intended for use in
biomedical and pharmaceutical applications as well as in tissue
3. Terminology
engineered medical products (TEMPs) by size exclusion chro-
3.1 Definitions:
matography with multi-angle laser light scattering detection
3.1.1 chitosan, n—a linear polysaccharide consisting of
(SEC-MALS).Aguideforthecharacterizationofchitosansalts
b(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose
has been published as Guide F 2103.
(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).
1.2 Chitosan and chitosan salts used in TEMPs should be
Chitosan is a polysaccharide derived by N-deacetylation of
well characterized, including the molar mass and polydisper-
chitin.
sity(molarmassdistribution)inordertoensureuniformityand
3.1.2 degree of deacetylation, n—the fraction or percentage
correct functionality in the final product. This test method will
of glucosamine units (GlcN: deacetylated monomers) in a
assist end users in choosing the correct chitosan for their
chitosan polymer molecule.
particular application. Chitosan may have utility as a scaffold
3.1.3 molar mass average, n—the given molar mass (M) of
or matrix material for TEMPs, in cell and tissue encapsulation
a chitosan will always represent an average of all of the
applications, and in drug delivery formulations.
molecules in the population. The most common ways to
1.3 This standard does not purport to address all of the
–
safety concerns, if any, associated with its use. It is the
expressthemolarmassareasthe number average(M )andthe
n
responsibility of the user of this standard to establish appro- –
mass average (M ). The two averages are defined by the
w
priate safety and health practices and determine the applica-
following equations:
bility of regulatory limitations prior to use.
NM wM NM
( ( (
i i i i i i i i i
M 5 and M 5 5 (1)
n w
2. Referenced Documents
N w NM
( ( (
i i i i i i i
2.1 ASTM Standards:
where:
F 2103 Guide for Characterization and Testing of Chitosan
N = numberofmoleculeshavingaspecificmolarmass M,
i i
Salts as Starting Materials Intended for Use in Biomedical
and
and Tissue-Engineered Medical Product Applications
w = mass of molecules having a specific molar mass M.
i i
2.2 United States Pharmacopeia/National Formulary:
3.1.3.1 Discussion—In a polydisperse molecular population
<621> Chromatography
– – – –
the relation M > M is always valid. The coefficient M /M is
w n w n
referred to as the polydispersity index, and will typically be in
the range 1.5 to 3.0 for commercial chitosans.
NOTE 1—The term molecular weight (abbreviated MW) is obsolete and
1 should be replaced by the SI (Système Internationale) equivalent of either
This test method is under the jurisdiction ofASTM Committee F04 on Medical
relative molecular mass (M ), which reflects the dimensionless ratio of the
and Surgical Materials and Devices and is the direct responsibility of Subcommittee r
mass of a single molecule to an atomic mass unit (see ISO 31-8), or molar
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Feb. 1, 2008. Published May 2008. mass (M), which refers to the mass of a mole of a substance and is
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. Available from National Institute of Standards and Technology (NIST), 100
Available from United States Pharmacopeia and National Formulary, U.S. Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://physics.nist.gov/cuu/
Pharmaceutical Convention, Inc. (USPC), Rockville, MD. Units/bibliography.html.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2602–08
typically expressed as grams/mole. For polymers and other macromol-
4.6 For polyelectrolytes, dialysis against the elution buffer
ecules, use of the symbols M , M , and M continue, referring to
w n z
has been suggested, in order to eliminate Donnan-type artifacts
mass-average molar mass, number-average molar mass, and z-average
in the molar mass determination by light scattering (1, 2).
molar mass, respectively. For more information regarding proper utiliza-
However, in the present method, the size exclusion chroma-
tion of SI units, see NIST SP811.
tography step preceding the light scatter detection is an
efficient substitute for a dialysis step. The sample is separated
4. Significance and Use
onSECcolumnswithlargeexcessofelutionbufferfor30to40
4.1 The degree of deacetylation of chitosan, as well at the
min, and it is therefore in full equilibrium with the elution
molar mass and molar mass distribution, determines the
buffer when it reaches the MALS detector.
functionality of chitosan in an application. For instance,
5. Materials
functional and biological effects are highly dependent upon the
composition and molar mass of the polymer. 5.1 Chemicals:
5.1.1 Chitosan or chitosan salt sample.
4.2 This test method describes procedures for measurement
5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-
of molar mass of chitosan chlorides and glutamates, and
tivity < 10 µS/cm).
chitosan base, although it in principle applies to any chitosan
5.1.3 CH COONH (ammonium acetate).
3 4
salt. The measured molar mass is that for chitosan acetate,
5.1.4 Pullulan standards. (See Note 2.)
since the mobile phase contains acetate as counter ion. This
NOTE 2—A series of linear homopolysaccharides with sufficiently
value can further be converted into the corresponding molar
narrow dispersity to be suitable for utilization as molar mass calibration
massforthechitosanasabase,ortheparentsaltform(chloride
standards in aqueous eluent.
or glutamate).
5.2 Mobile Phase:
4.3 Lightscatteringisoneofveryfewmethodsavailablefor
5.2.1 For SEC-MALS of chitosan and chitosan salts, a
the determination of absolute molar mass and structure, and it
mobile phase stock solution of 0.40 mol/L CH COONH in
3 4
is applicable over the broadest range of molar masses of any
deionized water is prepared.
method. Combining light scattering detection with size exclu-
5.2.2 The stock solution can be stored cool (3 to 8°C) for 6
sion chromatography (SEC), which sorts molecules according
months. Before use as a mobile phase, the stock solution is
to size, gives the ability to analyze polydisperse samples, as
diluted 1:1 (v/v) with deionized water and passed through a
well as obtaining information on branching and molecular
0.22 µm filter.
conformation. This means that both the number-average and
5.3 Instruments:
mass-average values for molar mass and size may be obtained
5.3.1 Analytical balance (0.1 mg).
for most samples. Furthermore, one has the ability to calculate
5.3.2 Shaking device.
the distributions of the molar masses and sizes.
5.3.3 pH meter.
4.4 Multi-angle laser light scattering (MALS) is a technique 5.3.4 HPLC system with injector, pump, degassing unit.
5.3.5 Size exclusion columns: TSK-Gel PW columns
where measurements of scattered light are made simulta- XL
from Tosoh Biosep., for example, PW -guard column +
neously over a range of different angles. MALS detection can XL
G6000 PW + G5000 PW + G3000 PW (last in the
XL XL XL
be used to obtain information on molecular size, since this
series), or equivalent.
parameter is determined by the angular variation of the
5.3.6 Refractive Index (RI) detector, with a known calibra-
scattered light. Molar mass may in principle be determined by
tion constant (dn/dV).
detecting scattered light at a single low angle (LALLS).
5.3.7 MultipleAngle Laser Light Scattering (MALS) detec-
However, advantages with MALS as compared to LALLS are:
tor, with known calibration constant.
(1) less noise at larger angles, (2) the precision of measure-
5.3.8 Computer with suitable software.
ments are greatly improved by detecting at several angles, and
(3) the ability to detect angular variation allows determination
6. Procedure
of size, branching, aggregation, and molecular conformation.
6.1 Preparation of Standards and Chitosan Salt Samples for
4.5 Sizeexclusionchromatographyusescolumns,whichare
SEC-MALS:
typically packed with polymer particles containing a network
6.1.1 Samples are prepared at a concentration suitable for
of uniform pores into which solute and solvent molecules can injection of 200 µL of sample.
diffuse. While in the pores, molecules are effectively trapped 6.1.2 Dissolve all samples in deionized water at twice the
required concentration for molar mass determination by shak-
and removed from the flow of the mobile phase. The average
-1
ingatabout100min overnightatcooltemperature(3to8°C).
residence time in the pores depends upon the size of the solute
6.1.3 Dilute samples 1+1 with stock solution of mobile
molecules. Molecules that are larger than the average pore size
phase and shake gently for a few seconds.
of the packing are excluded and experience virtually no
6.1.4 Pass all samples through a 0.45 µm filter, and transfer
retention; these are eluted first, in the void volume of the
to HPLC vials.
column. Molecules, which may penetrate the pores will have a
larger volume available for diffusion, they will suffer retention
depending on their molecular size, with the smaller molecules 5
The boldface numbers in parentheses refer to a list of references at the end of
eluting last. this standard.
F2602–08
TABLE 2 Suggestions for Concentration and Injected Mass of
6.1.5 Final concentration of pullulan standards of known
Chitosan Glutamate Samples for SEC-MALS
–
M values of approximately 11 800, 47 300, 112 000, 212 000,
w
Apparent Viscosity Concentration for
A
Injected Mass
and 404 000 g/mol should be approximately 4, 3, 2, and 1.5
as Chitosan Glutamate Injection
(mg)
(mPas) (mg/mL)
mg/mL, respectively.
6.1.6 Guidelines for final concentration of chitosans for <10 1.5 0.3
10–50 1 0.2
molar mass determination are given in Table 1. If SEC-MALS
>50 0.75 0.15
data display poor reproducibility with respect to replicates, this
A
Injected mass = Concentration*200 µL.
might be an indication of column overload. In this case, less
sample should be injected.
TABLE 3 Suggestions for Concentration and Injected Mass of
6.2 Preparation of Chitosan Base Samples for SEC-MALS:
Chitosan Base Samples for SEC-MALS
6.2.1 Samples are prepared at a concentration suitable for
Apparent Viscosity Concentration for
A
injection of 200 µL of sample. Injected Mass
as Chitosan Acetate Injection
(mg)
6.2.2 Dissolve the chitosan base in 1 % acetic acid to a 1 %
(mPas) (mg/mL)
-1
solution by shaking at about 100 min overnight at cool
<100 0.75 0.15
temperature (3 to 8°C). 100–500 0.5 0.1
>500 0.375 0.075
6.2.3 Dilute samples mobile phase (Note—2 mol/L ammo-
A
Injected mass = Concentration*200 µL.
nium acetate, not stock solution) to the required concentration
(Table 3) and shake gently for a few seconds.
6.2.4 Filter all samples through a 0.45 µm filter, and transfer
to HPLC vials. 6.3.4 After all samples have been run, purge the injector
6.3 Chromatography and Data Collection:
with deionized water to wash off remaining salt from the
6.3.1 The complete experimental setup of the SEC-MALS valves.
system is shown in Fig. 1. The refractive index detector is
6.4 Data Analysis:
placed at the end of the solvent/sample line as it is highly
6.4.1 Data analysis follows closely recommended proce-
sensitive to pressure changes.
dures for SEC-MALS data. Generally, the chromatograms are
6.3.2 Pullulan standards should be injected and analyzed
divided into a number of volume elements, defined by the peak
with 2 replicates before and after all chitosan samples (total of
width, the rate of data collection and the flow rate. Concentra-
4 replicates). Three (3) replicates should be injected for
tion of sample in each volume element (c) is determined from
i
chitosans.
the RI-detector response using known values of dn/dc and
6.3.3 A procedure for setting up the chromatography run
dn/dV (the RI-detector calibration constant). Furthermore,
and collecting the data is given below:
LS-detector response is divided by c, the molar mass in each
6.3.3.1 Use a flow rate of 0.5 mL/min.
volumeelement(M)isconsideredmonodisperse,andthemass
i
6.3.3.2 Purge the injector with mobile phase before the
is determined from a Zimm representation of a Debye plot by
sample set is run.
extrapolation to zero angle (which is essentially a solution to
6.3.3.3 Purge the RI-detector for at least 30 min (at 0.5
Eq X2.1 in X2.2). Once the values of c and M are known,
i i
mL/min) before start of the run.
calculation of the various average molar masses is straightfor-
6.3.3.4 Confirm that both the MALS detector and RI detec-
ward.
tor has a stable and low baseline level.
6.4.2 In detail, the above procedure consists of the follow-
6.3.3.5 Define the collection set-up as follows:
ing operations to be performed in a suitable software:
(1) Inject 200 µL of sample.
6.4.2.1 Define baselines for signals from both detectors.
(2) After a collection delay of 10 mL(20 min), data should
6.4.2.2 Calculate inter-detector delay volume using a mono-
becollectedfrombothdetectorsevery2secondsfor40mL(80
disperse low-molar mass pullulan standard.
min).
6.4.2.3 Define the peak area of interest.
(3) Use dn/dc = 0.148 mL/g and 0.142 mL/g for pullulans
6.4.2.4 Normalize LS-detector responses to correct for dif-
and chitosans, respectively (relevant only for calculations).
-4 -2
ferent sensitivity at different angles. Normalization is per-
(4)Useasecondvirialcoefficientof2*10 mol.mL.g and
-3 -2
formedonanisotropicscatterer(lowmolarmasscompound)in
5*10 mol.mL.g for pullulans and chitosans, respectively
the sample set, and is saved with the data file. For the other
(relevant only for calculations).
samples,onereadsthenormalizationperformedonanisotropic
scatterer from file.
TABLE 1 Suggestions for Concentration and Injected Mass of
6.
...