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ADVANCED BIO-FRIENDLY POLYMERS
Igor Lacík
Gel permeation chromatography – a tool for determination of molar mass of polymers
Molecular weight: characterization techniques
Mn techniques related to colligative properties (dependence on the number of molecules)
membrane osmometry (> 25 000 g/mol) vapor pressure osmometry (< 25 000 g/mol)
mass spectrometry electrospray ionization mass spectrometry (ESI-MS) matrix-assisted laser desorption/ionization (MALDI)
size exclusion chromatography
Mw static laser light scattering analytical ultracentrifuge size exclusion chromatography
Mz static laser light scattering analytical ultracentrifuge size exclusion chromatography
M viscometry size exclusion chromatography
Miw
i
Mn
Mw
Mz
Molecular weight: characterization techniques
Mn techniques related to colligative properties (dependence on the number of molecules)
membrane osmometry (> 25 000 g/mol) vapor pressure osmometry (< 25 000 g/mol)
mass spectrometry electrospray ionization mass spectrometry (ESI-MS) matrix-assisted laser desorption/ionization (MALDI)
size exclusion chromatography
Mw static laser light scattering analytical ultracentrifuge size exclusion chromatography
Mz static laser light scattering analytical ultracentrifuge size exclusion chromatography
M viscometry size exclusion chromatography
Miw
i
Mn
Mw
Mz
1. Column separation technique 2. Based on enthalpy-free partitioning of analyzed solutes, most often the polymer
chains, of different molecular weight / length (size) between mobile and stationary phases
Size-exclusion chromatography – principle
Separation mechanism is based on the size
Stationary phase• pore size• pore size distribution• particle size
Mobile phase • flow rate in ml/min providing “elution volume, Ve” or elution time, te ”
Separation mechanism is based on the size
Time 1 < Time 2 < Time 3
Ve,1 < Ve,2 < Ve,3
Time 1
Time 2
Time 3
1. Column separation technique 2. Based on enthalpy-free partitioning of analyzed solutes, most often the polymer
chains, of different molecular weight / length (size) between mobile and stationary phases
Velution
Ve - elution volume for given size
V0 - interstitial volume (between particles of column packing)
Vt - total available volume of column (interstitial and pore volumes)
Kd - partition coefficient 0 Kd 1
solute exluded
solute permeated
)( 00 VVKVV tde
Size-exclusion chromatography – principle
Size-exclusion chromatography – setup
Pump
Commercial columns of defined characteristics for column
packing:diameter, exclusion
limit, pore-size distribution
Injection of polymer of unknown molecularweight characteristics
eluent
signal = f(elution volume)Detector
calibration curvewith standards:elution volume = f(MW)
molecular weight characteristics
(MWD, Mw, Mn) of polymer
sign
al
elution volume
detector
sign
al
elution volume
sign
al
elution volume
detector
elution volume
log
M
calibration curve
elution volume
log
M
elution volume
log
M
calibration curve molecular weight distribution
log M
sign
alseparation
exclusion
permeation
)()10ln()()10ln()(log 2 MfMMfMMwsignalGPC nw
PumpWaters 515
MALLSSLD7000
WinGPC®7.4 Software
Columns
Rheodyne inj. or Waters Autosampler
Degasser DRI DetectorWaters 2410
PUMP INJECTOR COLUMNS
MALLS
RI
DEGASSER
SOFTWAREMAREK
Size-exclusion chromatography – hardware
SEC is an indirect method needs RELATION of elution volume (volume slice) to molecular
weight = calibration• several types of calibration
1. narrow distributed standards of the same polymer 1.1 2. universal calibration []1 M1 = []2 M2 ~ size of coil ~ Velution (Benoit et al, 1966)
note: a) Mark-Houwink equations is related to MW range b) careful in low MW range where a ~ 0.5 J.Polym.Sci. 6, 1759 (1968)
3. absolute molecular weight detectors (LS, viscosity)
4. effective calibration (no match between standards and analyzed polymer)
decrease in accuracy
nw M/M
Size-exclusion chromatography – calibration
Remember suppress interactions to avoid non-SEC phenomenaRemember suppress interactions to avoid non-SEC phenomena
(1) polymer - polymer (aggregation) – earlier elution, artificially increased molecular weight
(2) polymer - column packing (adsorption) - delayed elution, artificially decreased molecular weight
(3) eluent-column packing (repulsion) – earlier elution, artificially increased molecular weight
(4) polymer - eluent (aggregation) - earlier elution, artificially increased molecular weight
(5) etc.
Size-exclusion chromatography – hints/experience
water-soluble polymers (polyelectrolytes)
Problem Cure
•H-bonding•hydrophobic interactions•polyelectrolyte effect•adsorption (ion inclusion)•repulsion (ion exclusion)
•calibration
•concentration effect
•resolution (eluent viscosity)
Eluent composition &
column selection
Direct calibration with standards of well-defined Mp
Optimized solute concentration
Temperature of analysis
PSS Suprema HEMA GRAM (DMAc)TosoH Biosep TSK PWXL Polym. Laboratories Waters Ultrahydrogel
Remember suppress interactions to avoid non-SEC phenomenaRemember suppress interactions to avoid non-SEC phenomena
Size-exclusion chromatography – hints/experience
sample
EG
system peaks- salt excluded from PE region
eluent: 0.1M LiNO 3 , 0.04 M phosphate bufer, pH 8.0, 0.01M NaN 3
columns: Suprema 100, 1000, 3000, 10 00 0 Å (8 x 300 mm, 10 m)
flow rate 0.6- 1.0 ml/min (ethylene glycol – internal standard)
temperature 60-80ºC
calibration p(NaA) standards 1 250 – 1 100 00 0 g/mol (PSS, Mainz, Germany)
sample: concentration 0.5 - 2 mg/ml, injection volume 200 L
data acquisition and evaluation WinGPC 7 (PSS, Mainz, Germany)
or0.1M Na2HPO4 (pH 9)or other compositions
or0.1M Na2HPO4 (pH 9)or other compositions
water-soluble polymers (polyelectrolytes like poly (acrylic acid))Remember suppress interactions to avoid non-SEC phenomenaRemember suppress interactions to avoid non-SEC phenomena
Size-exclusion chromatography – hints/experience
Calibration curve (polyacrylic acid)
Guide-lines:Guide-lines:
• no upward curvature at high M.w, i.e. no adsorption of PAA to column packing (solute-gel interactions) pronounced at M.w.• variation of ionic strength - test to estimate the interactions ionic strength too low: elution volumes increase due to electrostatic interactions ionic strength too high: elution volumes increase due to hydrophobic interactions remove charged monomer by dialysis: as it adds to the ionic strength of eluent
a suitable ionic strength is in case of the lowest elution volumes
Remember suppress interactions to avoid non-SEC phenomenaRemember suppress interactions to avoid non-SEC phenomena
Size-exclusion chromatography – hints/experience
decrease in hydrodynamic volume of polymer coil at higher polymer concentration leads to the artificially lower M.w. lowering the sample concentration until the constant molar mass is reached yet detector sensitivity is sufficient
Concentration effect (polyacrylic acid)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Mol mass
10 3 10 4 10 5 10 6 g/mol
rel.
W(lo
g M
)
PS
S W
inG
PC
sci
entif
ic
V 4
.02
0.3
0.7
1.0
3.0
5.0PAA concentration in mg/ml
0 1 2 3 4 5 6
20.0
21.0
22.0
2E+5
4E+5
6E+5
8E+5
Elu
tion
vol
ume
at p
eak
[ml]
Mol
ar m
ass
at p
eak
[g/
mol
]
Polymer concentration [ml/g]
Remember suppress interactions to avoid non-SEC phenomenaRemember suppress interactions to avoid non-SEC phenomena
Size-exclusion chromatography – hints/experience
Elution volume of
internal standard
[ml]
Difference
[%]
Mp
[g/mol]
Difference in Mp
[%]
36,525 0 57 700 0
36,342 - 0,5 55 000 - 4,7
36,160 - 1,0 50 700 -12,1
35,794 - 2,0 42 600 -26,2
36,707 + 0,5 64 300 11,4
36,890 + 1,0 69 300 20,1
37,256 + 2,0 80 000 38,6
! EXTREME SENSITIVITY TO VARIATION IN THE FLOW RATE!
Accuracy of the flow rate (polyacrylic acid)
sample
EG
it is essential flow rate is controlled by the flowmarker (ethylene glycol) which also controls the pore volume and any changes on columns
analysisdards
ncalibratiodardsanalysis
sampleadjusted
sample V
VVV
tan.int
tan.int
Size-exclusion chromatography – hints/experience
the partitioning of the solute between mobile and stationary phases depends on viscosity of mobile phase: improved partitioning improved resolution viscosity of aqueous mobile phase can be adjusted to that of the organic one by the temperature of analysis
Effect of temperature of analysis (polyacrylic acid)
0 20 40 60 80Temperature [ C]
0.0
0.5
1.0
1.5
2.0V
isco
sity
[m
Pa.
s]W ater
Toluene
TH F
room Troom T
Size-exclusion chromatography – hints/experience
Final hints with special emphasis to the aqueous-phase SEC
• fast advancing in the last years with strong achievements(columns and new experience)
• there exist more and more examples of very precise analysis with high resolution (a strong improvement in aqueous-phase SEC)
• patience in thorough testing after thinking of all the possibilities which may hamper the experiment
• column history may play a role (more in aqueous-phase SEC than in aqueous-phase SEC)
Size-exclusion chromatography – hints/experience