Chemistry 367L/392N

Macromolecular ChemistryMacromolecular Chemistry

Lecture 8 Lecture 8

Chemistry 367L/392N

Measuring Molecular WeightMeasuring Molecular Weight Membrane Osmometry AlfredoAlfredo Vapor Phase Osmometry LindaLinda Viscometry GWGW Gel Permeation Chromatography

– Size exclusion Chromatography Light Scattering MALDI Others

– End group analysis YouYou

GWGW

GWGW

Chemistry 367L/392N

Chemistry 367L/392N

Molecular Weight from [Molecular Weight from []]

Mark-Houwink-Kuhn-Sakurada equation

[[] = K M] = K Maa

a = 1.8a = 1.8

a ≈ 0a ≈ 0

a = 0.5-0.8a = 0.5-0.8

Chemistry 367L/392N

Representative Viscosity-Molecular Weight Constantsa

PolymerPolymer

Polystyrene(atactic)c

Polyethylene(low pressure)Poly(vinyl chloride)

Polybutadiene98% cis-1,4, 2% 1,297% trans-1,4, 3% 1,2Polyacrylonitrile

Poly(methyl methacrylate-co-styrene)30-70 mol%71-29 mol%Poly(ethylene terephthalate)Nylon 66

SolventSolvent

CyclohexaneCyclihexaneBenzeneDecalin

Benzyl alcoholCyclohexanone

TolueneTolueneDMFg

DMF

1-Chlorobutane1-ChlorobutaneM-CresolM-Cresol

Temp Temp ooCC

355025135

155.420

30302525

30302525

Molecular WeightMolecular WeightRange Range 10 10-4-4

8-42e

4-137e

3-61f

3-100e

4-35e

7-13f

5-50f

5-16f

5-27e

3-100f

5-55e

4.18-81e

0.04-1.2f

1.4-5f

KK 10 1033

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

aa

0.500.500.5990.740.67

0.501.0

0.7250.7530.810.75

0.670.630.950.950.61

Chemistry 367L/392N

Mass SpectroscopyMass Spectroscopy

MALDIMALDI

Chemistry 367L/392N

Chemistry 367L/392N

Typical Mass SpectrometerTypical Mass Spectrometer

Chemistry 367L/392N

Mass SpectrumMass Spectrum

But…..how do you But…..how do you volatilize a volatilize a polymer??polymer??

Chemistry 367L/392N

Answer: Answer:

By ESI & MALDI !!By ESI & MALDI !!

Chemistry 367L/392N

Electrospray Ionization ESIElectrospray 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

Chemistry 367L/392N

Electrospray Ionization: Formation of Charged Droplets

Formation of multiply charged ions

ESIESI

Chemistry 367L/392N

ElectrosprayElectrospray

Chemistry 367L/392N

ESI: the exact mechanisms are still debatedESI: the exact mechanisms are still debated

Chemistry 367L/392N

Formation of singly charged ions

Sample is co-crystallized with matrix (solid)

Koichi Tanaka, Nobel Prize 2002Koichi Tanaka, Nobel Prize 2002

MALDI: MALDI: MMatrix atrix AAssisted ssisted LLaser aser DDesorption esorption IIonizationonization

Chemistry 367L/392N

Drift region (D)

MALDI Time-of-flight

MALDI - TOFMALDI - TOF

MALDI TOF

Average time in TOF: 10-7 sec : average speed 1-2 x 105 km/h

Chemistry 367L/392N

h LaserLaser

1. Sample is mixed with 1. Sample is mixed with matrixmatrix e.g. dihydroxybenzoic acid and e.g. dihydroxybenzoic acid and dried on plate.dried on plate.

2. Laser flash ionizes matrix 2. Laser flash ionizes matrix molecules.molecules.

MHMH++

MALDI: MALDI: MMatrix atrix AAssisted ssisted LLaser aser DDesorption esorption IIonizationonization

+/- 20 kV+/- 20 kV Grid (0 V)Grid (0 V)

Sample plateSample plate

Chemistry 367L/392N

MatricesMatrices

Matrix

1,8,9-Trihydroxyanthracen(Dithranol) polymers

2,5-Dihydroxy benzoic acid(DHB)

proteins, peptides, polymers

-Cyano-4-hydroxycinnamic acid

peptides, (polymers)

4-Hydroxypicolinic acid oligonucleotides

Trans-Indol-3-acrylacid(IAA)

polymers

OH OH OH

OH

OH

COOH

C CH

OH

CN COOH

N

OH

COOH

NH

COOH

Chemistry 367L/392N

Time-of-flight (TOF) Mass AnalyzerTime-of-flight (TOF) Mass Analyzer

+

+

+

+

SourceSource Drift region (flight tube)Drift region (flight tube)

dete

ctor

VV

• Ions are formed in pulses.Ions are formed in pulses.

• The drift region is field free.The drift region is field free.

• Measures the Measures the timetime for ions to reach the detector. for ions to reach the detector.

• Small ions reach the detector before large ones.Small ions reach the detector before large ones.

Chemistry 367L/392N

Calibration of the mass scaleCalibration of the mass scaleThe mass-to-charge ratio of an ion is proportional to the square of its drift time.

t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion

22LL

2222 KKtt

zz

mm

Chemistry 367L/392N

THE UNIVERSITY OF TEXAS AT AUSTIN

WILLSON RESEARCH GROUPAustin AIChE-Fall 2004

OHHO

n

+

Cl

O

OO

nO

O

TEATHF

MALDI TOF Mass Spectrum (before acrylation)

MALDI TOF Mass Spectrum (after acrylation)

PEG Diacrylate Synthesis:PEG Diacrylate Synthesis:PEG Diacrylate Synthesis:PEG Diacrylate Synthesis:

M = (44)n + 18 M = (44)n + 126

THE UNIVERSITY OF TEXAS AT AUSTIN

WILLSON RESEARCH GROUPAustin AIChE-Fall 2004

MALDI Spectra

Chemistry 367L/392N

Monoisotopic massMonoisotopic massMonoisotopic masscorresponds tolowest mass peak

When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

Chemistry 367L/392N

1981.84

1982.84

1983.84

Mass spectrum of peptide with 94 C-atoms Mass spectrum of peptide with 94 C-atoms

(19 amino acid residues)(19 amino acid residues)

No 13C atoms (all 12C)

One 13C atom

Two 13C atoms

“Monoisotopic mass”

Chemistry 367L/392N

Mass spectrum of insulinMass spectrum of insulin

12C : 5730.61

13C

2 x 13C

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.

Chemistry 367L/392N

6130 6140 6150 6160 6170

Poorer resolution

Better resolution

What if the resolution is not so good?What if the resolution is not so good?At lower resolution, the mass measured is theAt lower resolution, the mass measured is the average mass.

Mass

Chemistry 367L/392N

Average massAverage massAverage 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.

Chemistry 367L/392N15.01500 15.01820 15.02140 15.02460 15.02780 15.03100

Mass (m/z)

100

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

yISO:CH3

15.0229M

FWHM = M

How is mass resolution calculated?

R = M/M

Chemistry 367L/392N

Mass measurement accuracy depends on resolutionMass measurement accuracy depends on resolution

0

2000

4000

6000

8000

Co

un

ts

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

Chemistry 367L/392N

CH3-(CHO)n-OH+Na +10671111

11551199

1023

979

935 1243

15 + 44x+17 +23

Mass Spectra of Synthetic Mass Spectra of Synthetic PolymersPolymers