Macromolecular Chemistry

Transcription

1 Macromolecular Chemistry Lecture 8

2 Measuring Molecular Weight Membrane Osmometry Alfredo Vapor Phase Osmometry Linda Viscometry GW Gel Permeation Chromatography Size exclusion Chromatography Light Scattering MALDI GW Others GW End group analysis You

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4 Molecular Weight from [η][ Mark-Houwink-Kuhn-Sakurada equation [η]] = K M a a 0 a = a = 1.8

5 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-costyrene) mol% mol% Poly(ethylene terephthalate) Nylon 66 Solvent Cyclohexane Cyclihexane Benzene Decalin Benzyl alcohol Cyclohexanone Toluene Toluene DMF g DMF 1-Chlorobutane 1-Chlorobutane M-Cresol M-Cresol Temp o C Molecular Weight Range e e 3-61 f e 4-35 e 7-13 f 5-50 f 5-16 f 5-27 e f 5-55 e e f f K a

6 Mass Spectroscopy MALDI

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8 Typical Mass Spectrometer

9 Mass Spectrum But..how do you volatilize a polymer??

10 Answer: By ESI & MALDI!!

11 Electrospray Ionization ESI Atmospheric pressure ionization Enables MS detection of large, nonvolatile 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 ESI Electrospray Ionization: Formation of Charged Droplets Formation of multiply charged ions

13 Electrospray

14 ESI: the exact mechanisms are still debated

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

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

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

18 Matrices Matrix 1,8,9-Trihydroxyanthracen (Dithranol) OH OH OH polymers 2,5-Dihydroxy-benzoic acid (DHB) HO COOH OH proteins, peptides, polymers N C C CH COOH α-cyano-4-hydroxycinnamic acid peptides, (polymers) OH OH 4-Hydroxypicolinic acid oligonucleotides N COOH COOH Trans-Indol-3-acrylacid (IAA) N H polymers

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

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

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22 THE UNIVERSITY OF TEXAS AT AUSTIN PEG Diacrylate Synthesis: HO O n H + O Cl TEA THF M = (44)n + 18 M = (44)n O O O n O MALDI TOF Mass Spectrum (before acrylation) MALDI TOF Mass Spectrum (after acrylation) Austin AIChE-Fall 2004 WILLSON RESEARCH GROUP

23 THE UNIVERSITY OF TEXAS AT AUSTIN MALDI Spectra Austin AIChE-Fall 2004 WILLSON RESEARCH GROUP

24 Monoisotopic mass Monoisotopic mass corresponds to lowest mass peak When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

25 Mass spectrum of peptide with 94 C-atoms C (19 amino acid residues) Monoisotopic mass No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms

26 Mass spectrum of insulin 2 x 13 C 13 C 12 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 What if the resolution is not so good? At lower resolution, the mass measured is the average mass. Better resolution Poorer resolution Mass

28 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.

29 100 How is mass resolution calculated? ISO:CH3 M R = M/ M % Intensity FWHM = M Mass (m/z)

30 Mass measurement accuracy depends on resolution High resolution means better mass accuracy ppm error Resolution =18100 Counts ppm error Resolution = ppm error Resolution = Mass (m/z)

31 Mass Spectra of Synthetic Polymers CH 3 -(CHO)n-OH+Na x