ADVANCED BIOFRIENDLY POLYMERS Gel permeation chromatography a tool

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

Molecular weight: characterization techniques Mn q techniques related to colligative properties (dependence on the number of molecules) § membrane osmometry (> 25 000 g/mol) § vapor pressure osmometry (< 25 000 g/mol) q mass spectrometry § electrospray ionization mass spectrometry (ESI-MS) § matrix-assisted laser desorption/ionization (MALDI) q size exclusion chromatography Mw q static laser light scattering q analytical ultracentrifuge q size exclusion chromatography M Mw wi Mz Mn q static laser light scattering q analytical ultracentrifuge q size exclusion chromatography Mz Mi q viscometry q size exclusion chromatography

Size-exclusion chromatography – principle 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

Separation mechanism is based on the size Stationary phase • pore size distribut • particle size Mobile phase • flow rate in ml/min providing “elution volume, Ve” or elution t

Separation mechanism is based on the size Time 3 Time 2 Time 1 < Time 2 < Time 3 Ve, 1 < Ve, 2 < Ve, 3

Size-exclusion chromatography – principle 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 Kd - partition coefficient 0 Kd 1 solute exluded solute permeated Ve - elution volume for given size Velution V 0 - interstitial volume (between particles of column packing) Vt - total available volume of column (interstitial and pore volumes)

Size-exclusion chromatography – setup Injection of polymer of unknown molecular weight characteristics Pump eluent Commercial columns of defined characteristics for column packing: diameter, exclusion limit, pore-size distribution Detector signal = f(elution volume) calibration curve with standards: elution volume = f(MW) molecular weight characteristics (MWD, Mw, Mn) of polymer molecular weight distribution calibration curve separation signal exclusion log M sign al detector permeation elution volume log M

Size-exclusion chromatography – hardware DEGASSER MALLS MAREK PUMP INJECTORCOLUMNS SOFTWARE RI Rheodyne inj. or Waters Autosampler Degasser Pump Waters 515 Columns MALLS SLD 7000 DRI Detector Waters 2410 Win. GPC® 7. 4 Software

Size-exclusion chromatography – calibration 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 M 1 = [ ]2 M 2 ~ 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

Size-exclusion chromatography – hints/experience Remember 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 Remember suppress interactions to avoid non-SEC phenomena water-soluble polymers (polyelectrolytes) Problem • H-bonding • hydrophobic interactions • polyelectrolyte effect • adsorption (ion inclusion) • repulsion (ion exclusion) • calibration • concentration effect • resolution (eluent viscosity) Cure Eluent composition & column selection PSS Toso. H Biosep Polym. Laboratories Waters Direct calibration with standards of well-defined Mp Optimized solute concentration Temperature of analysis Suprema HEMA GRAM (DMAc) TSK PWXL Ultrahydrogel

Size-exclusion chromatography – hints/experience Remember suppress interactions to avoid non-SEC phenomena water-soluble polymers (polyelectrolytes like poly (acrylic acid)) eluent: 0. 1 M Li. NO 3 , 0. 04 M phosphate bufer, p. H 8. 0, 0. 01 M Na. N columns: Suprema 100, 1000, 3000, 10 00 flow rate 0. 6 -1. 0 ml/min (ethylene glycol temperature 60 -80ºC calibration p(Na. A) standards 1 250 sample: concentration 0. 5 - 2 mg/ml, injection volume 200 L data acquisition and evaluation 0 Å (8 x 300 mm, 10 3 m) – internal standard) – 1 100 00 0 g/mol (PSS, Mainz, Germany) Win. GPC 7 (PSS, Mainz, Germany) or 0. 1 M Na 2 HPO 4 (p. H 9) or other compositions EG sample system peaks - salt excluded from PE region

Size-exclusion chromatography – hints/experience Remember suppress interactions to avoid non-SEC phenomena Calibration curve (polyacrylic acid) 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

Size-exclusion chromatography – hints/experience Remember suppress interactions to avoid non-SEC phenomena Concentration effect (polyacrylic acid) 3. 0 1. 0 0. 7 0. 3 22. 0 8 E+5 6 E+5 21. 0 4 E+5 20. 0 0 1 2 3 4 Polymer concentration [ml/g] 5 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 6 Molar mass at peak [g/mol] 5. 0 Elution volume at peak [ml] PAA concentration in mg/ml

Size-exclusion chromatography – hints/experience Accuracy of the flow rate (polyacrylic acid) ! EXTREME SENSITIVITY TO VARIATION IN THE FLOW RATE! EG sample it is essential flow rate is controlled by the flowmarker (ethylene glycol) which also controls the pore volume and any changes on columns

Size-exclusion chromatography – hints/experience Effect of temperature of analysis (polyacrylic acid) room T 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

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 – final slide This is also the common SEC analysis