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

2. Molecular weight: characterization techniques MnMn  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 MwMw  static laser light scattering  analytical ultracentrifuge  size exclusion chromatography MzMz  static laser light scattering  analytical ultracentrifuge  size exclusion chromatography MM  viscometry  size exclusion chromatography MiMi wiwi MnMn MwMw MzMz

3. Molecular weight: characterization techniques MnMn  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 MwMw  static laser light scattering  analytical ultracentrifuge  size exclusion chromatography MzMz  static laser light scattering  analytical ultracentrifuge  size exclusion chromatography MM  viscometry  size exclusion chromatography MiMi wiwi MnMn MwMw MzMz

4. 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

5. 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, V e ” or elution time, t e ”

6. Separation mechanism is based on the size Time 1 < Time 2 < Time 3 V e,1 < V e,2 < V e,3 Time 1 Time 2 Time 3

7. 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 V elution V e - elution volume for given size V 0 - interstitial volume (between particles of column packing) V t - total available volume of column (interstitial and pore volumes) K d - partition coefficient 0  K d  1 solute exluded solute permeated Size-exclusion chromatography – principle

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

9. Pump Waters 515 MALLS SLD7000 WinGPC®7.4 Software Columns Rheodyne inj. or Waters Autosampler DegasserDRI Detector Waters 2410 PUMPINJECTORCOLUMNS MALLS RI DEGASSER SOFTWARE MAREK Size-exclusion chromatography – hardware

10. 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 ~ V elution (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 – calibration

11. 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

12.  water-soluble polymers (polyelectrolytes) ProblemCure 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 M p Optimized solute concentration Temperature of analysis PSSSuprema HEMA GRAM (DMAc) TosoH BiosepTSK PWXL Polym. Laboratories Waters Ultrahydrogel Remember  suppress interactions to avoid non-SEC phenomena Size-exclusion chromatography – hints/experience

13. 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 000Å (8 x 300 mm, 10  m)  flow rate0.6-1.0 ml/min (ethylene glycol– internal standard)  temperature60-80ºC  calibration p(NaA) standards 1 250– 1 100 000 g/mol (PSS, Mainz, Germany)  sample: concentration 0.5 - 2 mg/ml, injection volume 200  L  data acquisition and evaluationWinGPC7 (PSS, Mainz, Germany) or 0.1M Na 2 HPO 4 (pH 9) or other compositions or 0.1M Na 2 HPO 4 (pH 9) or other compositions  water-soluble polymers (polyelectrolytes like poly (acrylic acid)) Remember  suppress interactions to avoid non-SEC phenomena Size-exclusion chromatography – hints/experience

14. 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 Remember  suppress interactions to avoid non-SEC phenomena Size-exclusion chromatography – hints/experience

15.  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) 0.3 0.7 1.0 3.0 5.0 PAA concentration in mg/ml 0123456 20.0 21.0 22.0 2E+5 4E+5 6E+5 8E+5 Elution volume at peak [ml] Molar mass at peak [g/mol] Polymer concentration [ml/g] Remember  suppress interactions to avoid non-SEC phenomena Size-exclusion chromatography – hints/experience

16. ! 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 Size-exclusion chromatography – hints/experience

17.  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) room T Size-exclusion chromatography – hints/experience

18. 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

19. Size-exclusion chromatography – final slide This is also the common SEC analysis