ADVANCED BIO-FRIENDLY POLYMERS Igor Lacík Gel permeation chromatography – a tool for determination of molar mass of polymers

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



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


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



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]



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


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


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 – final slide

This is also the common SEC analysis

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ADVANCED BIO-FRIENDLY POLYMERS Igor Lacík Gel permeation chromatography – a tool for determination of molar mass of polymers