Macromolecular Chemistry Lecture 8
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
Molecular Weight from [η][ Mark-Houwink-Kuhn-Sakurada equation [η]] = K M a a 0 a = 0.5-0.8 0.8 a = 1.8
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) 30-70 mol% 71-29 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 35 50 25 135 155.4 20 30 30 25 25 30 30 25 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 a 0.50 0.599 0.74 0.67 0.50 1.0 0.725 0.753 0.81 0.75 0.67 0.63 0.95 0.61
Mass Spectroscopy MALDI
Typical Mass Spectrometer
Mass Spectrum But..how do you volatilize a polymer??
Answer: By ESI & MALDI!!
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
ESI Electrospray Ionization: Formation of Charged Droplets Formation of multiply charged ions
Electrospray
ESI: the exact mechanisms are still debated
MALDI: Matrix Assisted Laser Desorption Ionization Sample is co-crystallized with matrix (solid) Formation of singly charged ions Koichi Tanaka, Nobel Prize 2002
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
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)
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
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.
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
THE UNIVERSITY OF TEXAS AT AUSTIN PEG Diacrylate Synthesis: HO O n H + O Cl TEA THF M = (44)n + 18 M = (44)n + 126 O O O n O MALDI TOF Mass Spectrum (before acrylation) MALDI TOF Mass Spectrum (after acrylation) Austin AIChE-Fall 2004 WILLSON RESEARCH GROUP
THE UNIVERSITY OF TEXAS AT AUSTIN MALDI Spectra Austin AIChE-Fall 2004 WILLSON RESEARCH GROUP
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.
Mass spectrum of peptide with 94 C-atoms C (19 amino acid residues) Monoisotopic mass 1981.84 1982.84 No 13 C atoms (all 12 C) One 13 C atom 1983.84 Two 13 C atoms
Mass spectrum of insulin 2 x 13 C 13 C 12 C : 5730.61 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.
What if the resolution is not so good? At lower resolution, the mass measured is the average mass. Better resolution Poorer resolution 6130 6140 6150 6160 6170 Mass
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.
100 How is mass resolution calculated? ISO:CH3 M 15.0229 100 90 80 70 R = M/ M % Intensity 60 50 40 FWHM = M 30 20 10 0 15.01500 15.01820 15.02140 15.02460 15.02780 15.03100 Mass (m/z)
Mass measurement accuracy depends on resolution High resolution means better mass accuracy 8000 15 ppm error Resolution =18100 Counts 6000 4000 24 ppm error Resolution = 14200 2000 55 ppm error Resolution = 4500 0 2840 2845 2850 2855 Mass (m/z)
Mass Spectra of Synthetic Polymers 979 1067 11111155 1199 1023 935 1243 CH 3 -(CHO)n-OH+Na + 15 + 44x+17 +23