ASTM F2605-08
(Test Method)Standard Test Method for Determining the Molar Mass of Sodium Alginate by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
Standard Test Method for Determining the Molar Mass of Sodium Alginate by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
SIGNIFICANCE AND USE
The composition and sequential structure of alginate, as well as the molar mass and molar mass distribution, determines the functionality of alginate in an application. For instance, the gelling properties of an alginate are highly dependent upon the composition and molar mass of the polymer.
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 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 variation allows determination of size, branching, aggregation, and molecular conformation.
Size exclusion chromatography uses columns, which are typically packed with polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. While in the pores, molecules are effectively trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the size of the...
SCOPE
1.1 This test method covers the determination of the molar mass of sodium alginate 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 alginate has been published as Guide F 2064.
1.2 Alginate 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 alginate for their particular application. Alginate 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.
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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 2605 – 08
Standard Test Method for
Determining the Molar Mass of Sodium Alginate by Size
Exclusion Chromatography with Multi-angle Light Scattering
Detection (SEC-MALS)
This standard is issued under the fixed designation F 2605; 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
mass of sodium alginate intended for use in biomedical and
pharmaceutical applications as well as in tissue engineered
3. Terminology
medical products (TEMPs) by size exclusion chromatography
3.1 Definitions:
with multi-angle laser light scattering detection (SEC-MALS).
3.1.1 alginate, n—a polysaccharide substance extracted
Aguide for the characterization of alginate has been published
from brown algae, mainly occurring in the cell walls and
as Guide F 2064.
intercellular spaces of brown seaweed and kelp. Its main
1.2 Alginate used in TEMPs should be well characterized,
function is to contribute to the strength and flexibility of the
including the molar mass and polydispersity (molar mass
seaweed plant. Sodium alginate, and in particular calcium
distribution) in order to ensure uniformity and correct func-
cross-linkedalginategelsareusedintissueengineeredmedical
tionality in the final product. This test method will assist end
products (TEMPs) as biomedical scaffolds and matrices, for
users in choosing the correct alginate for their particular
immobilizing living cells (see Guide F 2315) and in drug
application. Alginate may have utility as a scaffold or matrix
delivery systems.
material for TEMPs, in cell and tissue encapsulation applica-
3.1.2 molar mass average, n—the given molar mass (Mw)
tions, and in drug delivery formulations.
of an alginate will always represent an average of all of the
1.3 This standard does not purport to address all of the
molecules in the population. The most common ways to
safety concerns, if any, associated with its use. It is the
–
responsibility of the user of this standard to establish appro-
expressthemolarmassareasthe number average(M )andthe
n
–
priate safety and health practices and determine the applica-
mass average (M ). The two averages are defined by the
w
bility of regulatory limitations prior to use.
following equations:
2. Referenced Documents
NM wM NM
(i i i (i i i (i i i
M 5 and M 5 5 (1)
2 n w
2.1 ASTM Standards: N w NM
(i i (i i (i i i
F 2064 Guide for Characterization and Testing ofAlginates
where:
as Starting Materials Intended for Use in Biomedical and
N = numberofmoleculeshavingaspecificmolarmass M,
i i
Tissue-Engineered Medical Products Application
and
F 2315 Guide for Immobilization or Encapsulation of Liv-
w = mass of molecules having a specific molar mass M.
i i
ing Cells or Tissue in Alginate Gels
3.1.2.1 Discussion—In a polydisperse molecular population
2.2 United States Pharmacopeia/National Formulary:
– – – –
the relation M > M is always valid. The coefficient M /M is
<621> Chromatography
w n w n
referred to as the polydispersity index, and will typically be in
the range 1.5 to 3.0 for commercial alginates.
This test method is under the jurisdiction ofASTM Committee F04 on Medical
NOTE 1—The term molecular weight (abbreviated MW) is obsolete and
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs. should be replaced by the SI (Système Internationale) equivalent of either
Current edition approved Feb. 1, 2008. Published May 2008.
relative molecular mass (M ), which reflects the dimensionless ratio of the
r
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
mass of a single molecule to an atomic mass unit (see ISO 31-8), or molar
contact ASTM Customer Service at service@astm.org. 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.
F2605–08
mass (M), which refers to the mass of a mole of a substance and is
min, and it is therefore in full equilibrium with the elution
typically expressed as grams/mole. For polymers and other macromol-
buffer when it reaches the MALS detector.
ecules, use of the symbols M , M , and M continue, referring to
w n z
mass-average molar mass, number-average molar mass, and z-average
5. Materials
molar mass, respectively. For more information regarding proper utiliza-
tion of SI units, see NIST SP811.
5.1 Chemicals:
5.1.1 Alginate sample.
4. Significance and Use
5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-
4.1 The composition and sequential structure of alginate, as
tivity < 10 µS/cm).
wellasthemolarmassandmolarmassdistribution,determines
5.1.3 Na SO (sodium sulfate).
2 4
the functionality of alginate in an application. For instance, the
5.1.4 EDTA (ethylene diamine tetraacetic acid).
gelling properties of an alginate are highly dependent upon the
5.1.5 NaOH (1 mol/L).
composition and molar mass of the polymer.
5.1.6 Pullulan standards. See Note 2.
4.2 Lightscatteringisoneofveryfewmethodsavailablefor
NOTE 2—A series of linear homopolysaccharides with sufficiently
the determination of absolute molar mass and structure, and it
narrow dispersity to be suitable for utilization as molar mass calibration
is applicable over the broadest range of molar masses of any
standards in aqueous eluent.
method. Combining light scattering detection with size exclu-
5.2 Mobile Phase:
sion chromatography (SEC), which sorts molecules according
5.2.1 For SEC-MALS of alginate, a mobile phase of 0.05
to size, gives the ability to analyze polydisperse samples, as
mol/L Na SO /0.01 mol/L EDTA in deionized water is used.
well as obtaining information on branching and molecular 2 4
5.2.2 Mobilephaseshouldbepreparedasastocksolutionof
conformation. This means that both the number-average and
0.10 mol/L Na SO /0.02 mol/L EDTA in deionized water,
mass-average values for molar mass and size may be obtained 2 4
which can be stored cool (3 to 8°C) for 6 months. Before use
for most samples. Furthermore, one has the ability to calculate
as a mobile phase, the stock solution is diluted 1:1 (v/v) with
the distributions of the molar masses and sizes.
deionized water and passed through a 0.22 µm filter.
4.3 Multi-angle laser light scattering (MALS) is a technique
5.3 Instruments:
where measurements are made simultaneously over a range of
5.3.1 Analytical balance (0.1 mg).
different angles. MALS detection can be used to obtain
5.3.2 Shaking device.
information on molecular size, since this parameter is deter-
5.3.3 pH meter.
mined by the angular variation of the scattered light. Molar
5.3.4 HPLC system with injector, pump, degassing unit.
mass may in principle be determined by detecting scattered
5.3.5 Size exclusion columns: TSK-Gel PW columns
lightatasinglelowangle(LALLS).However,advantageswith
XL
from Tosoh Biosep., for example, PW -guard column +
MALS as compared to LALLS are: (1) less noise at larger
XL
G6000 PW + G5000 PW + G3000 PW (last in the
angles, (2) the precision of measurements are greatly improved
XL XL XL
series), or equivalent.
by detecting at several angles, and (3) the ability to detect
5.3.6 Refractive Index (RI) detector, with a known calibra-
angular variation allows determination of size, branching,
tion constant (dn/dV).
aggregation, and molecular conformation.
5.3.7 MultipleAngle Laser Light Scattering (MALS) detec-
4.4 Sizeexclusionchromatographyusescolumns,whichare
tor, with known calibration constant.
typically packed with polymer particles containing a network
5.3.8 Computer with suitable software.
of uniform pores into which solute and solvent molecules can
diffuse. While in the pores, molecules are effectively trapped
6. Procedure
and removed from the flow of the mobile phase. The average
residence time in the pores depends upon the size of the solute
6.1 Preparation of Standards and Alginate Samples for
molecules. Molecules that are larger than the average pore size
SEC-MALS:
of the packing are excluded and experience virtually no
6.1.1 Samples are prepared at a concentration suitable for
retention; these are eluted first, in the void volume of the
injection of 200 µL of sample.
column. Molecules, which may penetrate the pores will have a
6.1.2 Dissolve all samples in deionized water at twice the
larger volume available for diffusion, they will suffer retention
required concentration for molar mass determination by shak-
-1
depending on their molecular size, with the smaller molecules
ingatabout100min overnightatcooltemperature(3to8°C).
eluting last.
6.1.3 Dilute samples 1+1 with stock solution of mobile
4.5 For polyelectrolytes, dialysis against the elution buffer
phase and shake gently for a few seconds.
has been suggested, in order to eliminate Donnan-type artifacts
6.1.4 Pass all samples through a 0.45 µm filter, and transfer
in the molar mass determination by light scattering (1, 2).
to HPLC vials.
However, in the present method, the size exclusion chroma-
6.1.5 Final concentration of pullulan standards of known
tography step preceding the light scatter detection is an
–
M values of approximately 11 800, 47 300, 112 000, 212 000,
w
efficient substitute for a dialysis step. The sample is separated
and 404 000 g/mol should be approximately 4, 3, 2, and 1.5
onSECcolumnswithlargeexcessofelutionbufferfor30to40
mg/mL, respectively.
6.1.6 Guidelines for final concentration of alginates for
molar mass determination are given in Table 1. If SEC-MALS
The boldface numbers in parentheses refer to a list of references at the end of
this standard. data display poor reproducibility with respect to replicates, this
F2605–08
TABLE 1 Suggestions for Concentration and Injected Mass of
(2) After a collection delay of 10 mL(20 min), data should
Alginate Samples for SEC-MALS
becollectedfrombothdetectorsevery2secondsfor40mL(80
Apparent Concentration for Injected
–
min).
M A
w
Viscosity Injection Mass
(g/mol) (3) Use dn/dc = 0.148 mL/g and 0.150 mL/g for pullulans
(mPas) (mg/mL) (mg)
and alginates, respectively (relevant only for calculations).
<50 000 <10 4 0.8
-4 -2
50 000–75 000 10–20 3 0.6 (4)Useasecondvirialcoefficientof2*10 mol.mL.g and
75 000–100 000 20–40 2 0.4 -3 -2
5*10 mol.mL.g for pullulans and alginates, respectively
100 000–150 000 40–100 1.5 0.3
(relevant only for calculations).
150 000–250 000 100–300 1 0.2
>250 000 >300 0.5 0.1
6.2.4 After all samples have been run, purge the injector
A
Injected mass = Concentration*200 µL.
with deionized water to wash off remaining salt from the
valves.
6.3 Data Analysis:
might be an indication of column overload. In this case, less
6.3.1 Data analysis follows closely recommended proce-
sample should be injected.
dures for SEC-MALS data. Generally, the chromatograms are
6.2 Chromatography and Data Collection:
divided into a number of volume elements, defined by the peak
6.2.1 The complete experimental setup of the SEC-MALS
width, the rate of data collection and the flow rate. Concentra-
system is shown in Fig. 1. The refractive index detector is
tion of sample in each volume element (c) is determined from
i
placed at the end of the solvent/sample line as it is highly
the RI-detector response using known values of dn/dc and
sensitive to pressure changes.
dn/dV (the RI-detector calibration constant). Furthermore,
6.2.2 Pullulan standards should be injected and analyzed
LS-detector response is divided by c, the molar mass in each
i
with 2 replicates before and after all alginate samples (total of
volumeelement(M)isconsideredmonodisperse,andthemass
i
4 replicates). 3 replicates should be injected for alginates.
is determined from a Zimm representation of a Debye plot by
6.2.3 A procedure for setting up the chromatography run
extrapolation to zero angle (which is essentially a solution to
and collecting the data is given below:
Eq X2.1 in X2.2). Once the values of c and M are known,
i i
6.2.3.1 Use a flow rate of 0.5 mL/min.
calculation of the various average molar masses is straightfor-
6.2.3.2 Purge the injector with mobile phase before the
ward.
sample set is run.
6.3.2 In detail, the above procedure consists of the follow-
6.2.3.3 Purge the RI-detector for at least 30 min (at 0.5
ing operations to be performed in a suitable software:
mL/min) before start of the run.
6.3.2.1 Define baselines for signals from both detectors.
6.2.3.4 Confirm that both the MALS detector and RI detec-
6.3.2.2 Calculate inter-detector delay volume using a mono-
tor has a stable and low baseline level.
disperse low-molar mass pullulan standard.
6.2.3.5 Define the collection set-up as follows:
(1) Inject 200 µL of sample. 6.3.2.3 Define the peak area of interest.
NOTE—Solid lines indicate solvent/sample flow, dashed lines indicate cabling for data transfer.
FIG. 1 Complete SEC-MALS Set-Up
F2605–08
NOTE—Solid line: RI detector; dashed line: MALS detector; (L) molar mass for each chromatographic data point.
–
FIG. 2 A Chromatogram of Sodium Alginate (M 160 000 g/mol)
w
–
6.3.2.4 Normalize LS-detector responses to correct for dif-
7.2.1 Condition 1—M of pullulan standards (using at least
w
ferent sensitivity at different angles. Normalization is per-
3 replicates) should be within 610 % of the stated value from
formedonanisotropicscatterer(lowmolarmasscompound)in
the manufacturer.
the sample set, and is saved with the data file. For the other
7.2.2 Condition 2—Relative standard deviation (RSD, for
samples,onereadsthenormalizationperformedonanisotropic
example, standard deviation divided by mean value) for
scatterer from file.
pullulan standards should be less than 610 %.
6.3.2.5 Check the goodness-of-fit of the LS-detectors using
7.2.3 Condition 3—Reproducibility in the detector re-
a 3D-representation of the data or a Debye-plot (in Zimm
sponses for the 3 replicates of alginate samples should be
representation). Do not use LS-detector responses that are
manually eva
...
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