ASTM D5296-97
(Test Method)Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography
Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography
SCOPE
1.1 This test method covers the determination of molecular weight (MW) averages and the distribution of molecular weights for linear, soluble polystyrene by liquid high performance size-exclusion chromatography (HPSEC). This test method is not absolute and requires the use of commercially available narrow molecular weight distribution (MWD) polystyrene standards for calibration. This test method is applicable for samples containing molecular weight components that have elution volumes falling within the elution volume range defined by polystyrene standards (that is, molecular weights generally from 2000 to 2000000 g[dot]mol -1 ).
1.2 HPSEC is differentiated from traditional size-exclusion chromatography SEC (also referred to as gel permeation chromatography (GPC)) in that the number of theoretical plates generated per second with an HPSEC system is about ten times greater than that for traditional SEC (see Terminology D883 and Practice D3016). HPSEC systems employ low-volume liquid chromatography components and columns packed with relatively small (generally 3 to 20 [mu]m) microporous particles. High performance liquid chromatography instrumentation and automated data handling systems for data acquisition and processing are required.
1.3 The values stated in SI units are to be regarded as the standard.
1.4 This standard does not purport to address all of the safety problems, 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. Specific precautionary statements are given in Section 9.
General Information
Relations
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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:D5296–97
Standard Test Method for
Molecular Weight Averages and Molecular Weight
Distribution of Polystyrene by High Performance Size-
Exclusion Chromatography
This standard is issued under the fixed designation D5296; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
NOTE 1—There is no similar or equivalent ISO standard.
1. Scope *
1.1 This test method covers the determination of molecular
2. Referenced Documents
weight (MW) averages and the distribution of molecular
2.1 ASTM Standards:
weights for linear, soluble polystyrene by liquid high-
D883 Terminology Relating to Plastics
performance size-exclusion chromatography (HPSEC). This
D2857 Test Method for Dilute Solution Viscosity of Poly-
test method is not absolute and requires the use of commer-
mers
cially available narrow molecular weight distribution (MWD)
D3016 Practice for Use of Liquid Exclusion Chromatogra-
polystyrene standards for calibration. This test method is
phy Terms and Relationships
applicable for samples containing molecular weight compo-
E685 Practice for Testing Fixed-Wavelength Photometric
nents that have elution volumes falling within the elution
Detectors Used in Liquid Chromatography
volume range defined by polystyrene standards (that is, mo-
E691 Practice for Conducting an Interlaboratory Test Pro-
−1
lecular weights generally from 2000 to 2000000 g·mol ).
gram to Determine the Precision of Test Methods
1.2 The HPSEC is differentiated from traditional size-
exclusion chromatography SEC (also referred to as gel perme-
3. Terminology
ation chromatography (GPC)) in that the number of theoretical
3.1 Definitions—For definitions of technical terms pertain-
plates per metre with an HPSEC system is about ten times
ingtoplasticsusedinthistestmethodseeTerminologyD883.
greater than that for traditional SEC (see Terminology D883
and Practice D3016). The HPSEC systems employ low-
4. Summary of Test Method
volume liquid chromatography components and columns
4.1 In this test method a dilute solution of a polystyrene
packed with relatively small (generally 3 to 20 µm) mi-
sample is injected into a liquid mobile phase containing the
croporous particles. High-performance liquid chromatography
samesolventusedtopreparethepolymersolution.Themobile
instrumentation and automated data handling systems for data
phase transports the polymer into and through a chromato-
acquisition and processing are required.
graphic column (or set of columns connected in series) packed
1.3 The values stated in SI units are to be regarded as the
with a solid or semirigid, porous substrate which separates the
standard.
polymer molecules according to their size in solution. Starting
1.4 This standard does not purport to address all of the
frominjection,adetectorcontinuouslymonitorstheeluateasa
safety concerns, if any, associated with its use. It is the
function of elution volume (or time). Upon emerging from the
responsibility of the user of this standard to establish appro-
column(s), the size-separated molecules are detected and
priate safety and health practices and determine the applica-
recordedaccordingtotheirconcentration.Throughcalibration,
bility of regulatory limitations prior to use. Specific precau-
the elution volumes (or times) are converted to molecular
tionary statements are given in Section 9.
weights, and various molecular weight parameters for the
samplearecalculatedfromthemolecularweight/concentration
data.
ThistestmethodisunderthejurisdictionofASTMCommitteeD-20onPlastics
and is the direct responsibility of Subcommittee D20.70 on Analytical Methods.
Current edition approved Nov. 10, 1997. Published April 1998. Originally Annual Book of ASTM Standards, Vol 08.01.
published as D5296–92. Last previous edition D5296–92. Annual Book of ASTM Standards, Vol 08.02.
2 5
Seealso AMD Bibliography and Bibliography SupplementsAMD 40-S1, 40-S2, Annual Book of ASTM Standards, Vol 14.01.
and 40-S3 on Size Exclusion Chromatography,availablefromASTMHeadquarters. Annual Book of ASTM Standards, Vol 14.02.
*A Summary of Changes section appears at the end of this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5296–97
5. Significance and Use 7.2.1 The interrelationships of the components are shown
schematically in Fig. 1. For instruments that have injector,
5.1 General Utility—The molecular weight (MW) and mo-
column(s), detector, or other components operated above
lecular weight distribution (MWD) are fundamental character-
ambient temperature, the use of a degasser located in the
istics of a polymer sample. They may be used for a wide
solventreservoirorbetweenthereservoirandpumpingsystem
variety of correlations for fundamental studies, processing, or
is recommended to remove air from the solvent. Typical
productapplications.Forexample,theobservedMWDmaybe
laboratoryglasswareandananalyticalbalancearealsoneeded.
compared to one predicted from assumed kinetics or mecha-
nisms for a polymerization reaction. Differences between the
NOTE 2—AnumberofsystemsandcomponentsforperformingHPSEC
values will allow alteration of theory or experimental design.
are available commercially.
Similarly, the strength, melt flow, and other properties of a
7.3 Solvent Reservoir—The solvent reservoir must hold
polymer sample may be dependent on MW and MWD.
sufficient solvent to ensure consistency of composition for a
Determinations of MW and MWD are used for quality control
number of runs or analyses. The reservoir should isolate the
of polymers.
solvent from the atmosphere, permit control of the environ-
5.2 Limitations—Because of the need for specific calibra-
mentincontactwiththesolvent,andbecompletelyinerttothe
tion of the polymer type under study, and because of the
solvent employed. In addition, some means of agitation (for
specific nature of polymer/solvent/column-packing interac-
example,magneticstirring)isrecommendedtoensureuniform
tions, this test method is valid only for polystyrene and
composition.
non-exclusion effects are to be avoided. However, many of the
7.4 Solvent Pumping System—The principal requirement of
principles of the method may be applied in generating HPSEC
a pumping system is production of a relatively constant and
methods for other polymer systems, for example, using the
pulseless flow of solvent through the columns. In general, the
principles of universal calibration. (see Practice D3016).
rate of flow should be adjustable between 0.1 and 5.0 mL/min
and back pressures should not exceed limits specified by the
6. Units and Symbols
column manufacturer (for example, 28 MPa). If the elution
6.1 UnitsandsymbolsrelatedtofunctionaregiveninTable
volumeisnotbeingmeasureddirectlyorcorrectedforsystem-
1.
atic changes, the precision in the flow rate must be at least
6.2 Equivalencies used in this test method are as follows:
60.3% as measured under the conditions and over the time
Common Unit/Symbol SI Unit or Symbol
interval required for running a typical analysis.
−1 −8 3 −1
7.5 Sample Injector—The purpose of an injection system is
1 mL·min = 1.667 3 10 m ·s¯
7 −2
1 3 10 dyn·cm = 145 psi = 1 MPa
to generate a sharply defined zone of solution containing the
sample when introducing the sample into the flow stream. A
7. Apparatus
valve and loop assembly or any of a number of commercially
7.1 Introduction—Liquid high-performance size-exclusion
available high-performance liquid chromatography automatic
chromatography (HPSEC) is a specific form of liquid chroma-
injection systems can be used for this purpose. Requirements
tography and is differentiated from traditional SEC in that
includeminimalcontributiontobandspreading,injectorability
HPSEC uses columns with about ten times the number of
to operate at the back pressure generated by the columns,
theoretical plates per metre. The principal distinguishing fea-
repeatability of injection volume, and no carryover.
tureofHPSECisthecolumnpackingmaterialthatisdiscussed
7.6 Columns—Stainless steel columns with uniform and
as follows.
highly polished inside walls are usually selected for HPSEC.
7.2 Essential Components—The essential components of
Columns with lengths ranging from 15 to 50 cm and special
instrumentation are a solvent reservoir, solvent pumping sys-
end fittings, frits, and connectors designed to minimize dead
tem, sample injector, packed column(s), solute detector, low
volumeandmixingarerecommended.Micro-particulate,semi-
dead-volume liquid chromatography tubing and fittings, waste
rigidorganicgels,andrigidsolid,porouspackingmaterialsare
container, recorder, and an automated data-handling system.
usedforHPSEC.Generally,thepackingmaterialshavenarrow
Any component may be used that meets safety and perfor-
particle size distributions with particle sizes in the range from
mance requirements specified as follows.
3 to 20 µm. Packing materials also are available in a variety of
shapes and pore sizes. Columns may be packed with particles
TABLE 1 Units and Symbols Related to Function
of relatively uniform pore size or with a “mixed bed” of
Common Unit/ SI Unit/
particles to produce a broad range of pore sizes for polymer
Function
Symbol Symbol
separation. If a set of columns is used, it is recommended that
−1
Basic property definition Molecular weight (Daltons) g·mol
the columns be connected starting from the injector outlet in
−1 3 −1
Solvent flow rate mL·min m ·s
A
Sample weight (mass) mg
A
Sample solution volume µL, mL
A
Pore size Å
A
Particle Size µm
A
Elution volume µL, mL
A
Elution time s
A
Chromatogram peak mm
heights
−2 −2
Column back pressure dyn·cm (psi) N·m or pascal (Pa)
A
Same as common unit. FIG. 1 Schematic of an HPSEC System
D5296–97
order of columns having the smallest to those having the at pressures up to 42 MPa. Connecting column tubing should
largest packing pore size. bekeptasshortaspossibleandallfittingsandconnectorsmust
be designed to prevent mixing and have low dead volumes.
NOTE 3—Column packing materials and packed HPSEC columns are
7.9 Recorder/Plotter—Either a recording potentiometer
availablecommerciallyfromanumberofmanufacturers.Usersofthistest
with a full-scale response of at least2sora printing device
method are advised to follow manufacturers’ guidelines and recommen-
dations for the care and use of their HPSEC columns. For example, connected to a data handling system may be used to plot the
manufacturers’ guidelines may override the preceding recommendation
chromatographic data. Pen response and signal-to-noise ratio
fororderingtheplacementofcolumnsinacolumnsetbecauseofconcern
should be chosen so that the concentration signal is not
about the fragility of smaller pore size packing materials.
appreciably perturbed.
7.7 Detectors—The purpose of the detector is to continu-
7.10 Data Handling Systems—Means must be provided for
ously monitor the concentration of solute eluting from the determining chromatographic peak heights or integrated area
chromatographic column(s). Consequently, the detector must
segments at prescribed intervals under the HPSEC chromato-
be sufficiently sensitive and respond linearly to the solute gram and for handling and reporting the data. This can best be
concentration. Additionally, the detector must not appreciably
accomplished by means of a computer or a real-time data
distort the concentration gradient in the emerging stream. This
acquisition system with either off-line or on-line data process-
requirement imposes severe limitations on the volume of ing.
solution available for detection. For example, use of detectors
NOTE 6—Data acquisition and handling systems for HPSEC have not
with cell volumes greater than 15 µL generally will not be
been standardized. However, it is noted that a number of different
accepted with this test method. Most detectors employed for
manufacturers now provide chromatography data systems which include
HPSEC are based upon photometric measurements (refractive HPSEC software.Also, some users have developed their own specialized
HPSEC computer software.
index,UV-visible,fluorescenceandinfraredabsorbance).Prac-
tice E685 serves as a guide for testing the performance of
7.11 Other Components—Special solvent line filters, pres-
photometric detectors used in high-performance liquid chro-
suremonitors,pulsedampers,flowmeters,thermostatedovens,
matographic systems. Other detectors also may be used. The
syphon counters, plotters, raw data storage systems, software,
differential refractometer has moderate sensitivity and general
and so forth are oftentimes incorporated with the essential
utility. It provides a signal proportional to the difference in
components previously listed.
refractive index (DRI) between the solvent and the column
7.12 HPSEC System—Any satisfactory combination of the
−7 −8
eluate. The detector should respond to 10 to 10 DRI unit
above components that will meet the performance require-
with cell volumes# 10 µL.
ments of Section 12.
NOTE 4—The change in the specific refractive index increment (dn/dc)
8. Reagents and Materials
of polystyrene is negligible at molecular weights greater than about 5000
−1 8.1 Solvent—Tetrahydrofuran(THF)isrecommendedasthe
g·mol . No appreciable error in molecular weight averages will be
solvent for this test method. However, any solvent that is
introducedwiththisdetectorforpolystyreneaslongasitsnumber-average
−1
compatible with the HPSEC system components and column
molecular weight, M , is greater than 5000 g·mol . The principal
n
disadvantage of the differential refractometer is that precise control of
packing materials and is considered to be a good solvent for
temperature, pressure, and flow rate is required to maintain a stable signal
polystyrene may be used. To a certain extent, the choice of
for an appropriate level of sensitivity. For example, most organic liquids
solvent dictates the operating temperature, as well as the
−4
have a temperature coefficient of 10 RI units per K. Consequently, the
detector,selectedfortheHPSECsystem.Thetemperaturemust
−4
temperature within the RI detector cell must be controlled to within 10
be sufficiently high to keep the eluent viscosity low (usually 1
°C.
cp or less) and yet not too high to cause eluate to boil or degas
NOTE 5—Benzoyl peroxide is commonly used as a free radical initiator
for styrene in the synthesis of polystyrene. The presence of small
in the detector cell. Considering detector limitations, solvents
concentrations of initiator fragments containi
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.