1. Chemistry 367L/392N Macromolecular Chemistry Lecture 8

2. Chemistry 367L/392N Measuring Molecular Weight Alfredo Membrane Osmometry Alfredo Linda Vapor Phase Osmometry Linda GW Viscometry GW Gel Permeation Chromatography –Size exclusion Chromatography Light Scattering MALDI Others You –End group analysis You GW GW

3. Chemistry 367L/392N

4. Molecular Weight from [  ] Mark-Houwink-Kuhn-Sakurada equation [  ] = K M a [  ] = K M a a = 1.8 a ≈ 0 a = 0.5-0.8

5. Chemistry 367L/392N Representative Viscosity-Molecular Weight Constants a Polymer Polystyrene (atactic) c Polyethylene (low pressure) Poly(vinyl chloride) Polybutadiene 98% cis-1,4, 2% 1,2 97% trans-1,4, 3% 1,2 Polyacrylonitrile Poly(methyl methacrylate-co- styrene) 30-70 mol% 71-29 mol% Poly(ethylene terephthalate) Nylon 66 Solvent Cyclohexane Cyclihexane Benzene Decalin Benzyl alcohol Cyclohexanone Toluene DMF g DMF 1-Chlorobutane M-Cresol Temp o C 35 50 25 135 155.4 20 30 25 30 25 Molecular Weight Range  10 -4 8-42 e 4-137 e 3-61 f 3-100 e 4-35 e 7-13 f 5-50 f 5-16 f 5-27 e 3-100 f 5-55 e 4.18-81 e 0.04-1.2 f 1.4-5 f K  10 3 80 26.9 9.52 67.7 15.6 13.7 30.5 29.4 16.6 39.2 17.6 24.9 0.77 24.0 a0.50 0.599 0.74 0.67 0.50 1.0 0.725 0.753 0.81 0.75 0.67 0.630.95 0.61

6. Chemistry 367L/392N Mass Spectroscopy MALDI

7. Chemistry 367L/392N

8. Typical Mass Spectrometer

9. Chemistry 367L/392N Mass Spectrum But…..how do you volatilize a polymer??

10. Chemistry 367L/392N Answer: By ESI & MALDI !!

11. Chemistry 367L/392N Electrospray Ionization ESI  Atmospheric pressure ionization  Enables MS detection of large, non-volatile molecules (e.g., polymers) with no fragmentation (→Nobel Prize 2002)  Liquid elutes through a high voltage tip  Coulombic explosions yield a continuous mist of bare, gas-phase ions (positive or negative)  Conveniently coupled to liquid separations  Characterized by multiply charged ions Newobjective.com

12. Chemistry 367L/392N Electrospray Ionization: Formation of Charged Droplets Formation of multiply charged ions ESI

13. Chemistry 367L/392N Electrospray

14. ESI: the exact mechanisms are still debated

15. Chemistry 367L/392N Formation of singly charged ions Sample is co-crystallized with matrix (solid) Koichi Tanaka, Nobel Prize 2002 MALDI: Matrix Assisted Laser Desorption Ionization

16. Chemistry 367L/392N Drift region (D) MALDI Time-of-flight MALDI - TOF MALDI TOF Average time in TOF: 10 -7 sec : average speed 1-2 x 10 5 km/h

17. Chemistry 367L/392N h Laser 1. Sample is mixed with matrix e.g. dihydroxybenzoic acid and dried on plate. 2. Laser flash ionizes matrix molecules. MH + MALDI: Matrix Assisted Laser Desorption Ionization +/- 20 kV Grid (0 V) Sample plate

18. Chemistry 367L/392N Matrices Matrix 1,8,9-Trihydroxyanthracen (Dithranol)polymers 2,5-Dihydroxy­benzoic acid (DHB) proteins, peptides, polymers  -Cyano-4-hydroxycinnamic acid peptides, (polymers) 4-Hydroxypicolinic acidoligonucleotides Trans-Indol-3-acrylacid (IAA) polymers

19. Chemistry 367L/392N Time-of-flight (TOF) Mass Analyzer + + + + Source Drift region (flight tube) detector V Ions are formed in pulses. The drift region is field free. The drift region is field free. Measures the time for ions to reach the detector. Measures the time for ions to reach the detector. Small ions reach the detector before large ones. Small ions reach the detector before large ones.

20. Chemistry 367L/392N Calibration of the mass scale The mass-to-charge ratio of an ion is proportional to the square of its drift time. t=Drift time L=Drift length m=Mass K=Kinetic energy of ion z=Number of charges on ion 2 L22Kt z m 

21. Chemistry 367L/392N

22. T HE U NIVERSITY OF T EXAS AT A USTIN W ILLSON R ESEARCH G ROUP Austin AIChE-Fall 2004 MALDI TOF Mass Spectrum (before acrylation) MALDI TOF Mass Spectrum (after acrylation) PEG Diacrylate Synthesis: M = (44)n + 18M = (44)n + 126

23. T HE U NIVERSITY OF T EXAS AT A USTIN W ILLSON R ESEARCH G ROUP Austin AIChE-Fall 2004 MALDI Spectra

24. Chemistry 367L/392N Monoisotopic mass When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

25. Chemistry 367L/392N 1981.84 1982.84 1983.84 Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms “Monoisotopic mass”

26. Chemistry 367L/392N Mass spectrum of insulin 12 C : 5730.61 13 C 2 x 13 C Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.

27. Chemistry 367L/392N 6130 6140 6150 6160 6170 Poorer resolution Better resolution What if the resolution is not so good? At lower resolution, the mass measured is the At lower resolution, the mass measured is the average mass. Mass

28. Chemistry 367L/392N Average mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.

29. Chemistry 367L/392N M FWHM =  M How is mass resolution calculated? R = M/  M

30. Chemistry 367L/392N Mass measurement accuracy depends on resolution 0 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 15 ppm error 24 ppm error 55 ppm error High resolution means better mass accuracy

31. Chemistry 367L/392N CH 3 -(CHO)n-OH+Na + 1067 1111 1155 1199 1023 979 935 1243 15 + 44x+17 +23 Mass Spectra of Synthetic Polymers