Chromatographic Fidelity and Matrix

/Analyte Solubility in Complex Polymer

Systems using HPLC-MALD/I TOF MS

Olumide Adebolu

CHEM 395

March 1st , 2007

Instructor : Prof J.Rusling

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2Overview

� Introduction to MALDI� Objective:

– To demonstrate compatibility of matrix/analyte mixtures and to also ascertain the Chromatographic fidelity in MALDI analysis of polymers.

� Approach:– Coupling of HPLC with MALD/I TOF MS……Hence the name “LC-MALDI”– LC-MALDI interfaced using LC-Transform Series 600 (LabConnections,

Inc.).– Selective use of solvents’ gradient over varying temperature range.

� Results: – Retention time/scan reproducibility over various samples analyzed and the

signal to noise ratio.– Capability of functional HPLC separation combined with automated MALDI

analysis is demonstrated using K-GH Polyol (thermal dye-receiver polymer).

– Demonstrated advanced capability for differentiating materials.

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Introduction to MALDI

� Since its discovery in the late 1980s,Matrix Assisted Laser Desorption Ionization (MALDI) has emerge as an important soft-ionization MS technique.

� Applications in many biologically related research areas (e.g.,protein,peptides,DNA,carbohydrates) and materials-related fields (e.g., synthetic polymers)

� The fundamental mechanisms still remain unknown despite its routine usage

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4Introduction Cont’d

MALDI experiment can be reduce to four different steps:�Sample Prep: Molar ratio of matrix to analyte is large (e.g. between 102 : 1 and 106 : 1)�Desorption: Pulsed laser beam (typically a Nitrogen laser,337nm)�Ionization: Gas phase ionization reactions(Cationized,protonated,de-protonated or radical species)�Mass Analysis: Time-of-flight (TOF),FTICR ,QIT etc.

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UV Laser Pulse

2) Desorption

1) Sample Preparation

3) Ionization+

Sample

Plate

- Individual solutions of matrix material and analyte

- Solutions are mixed (high matrix-to-analyte ratio)

- Mixture is deposited onto the sample plate (solvent evaporated)

- Analyte is homogeneously embedded within the matrix crystal

(solid solution)

Matrix

Analyte

Cation

- Typically nitrogen laser (337 nm)

- Matrix resonantly absorbs the laser energy

- Matrix is desorbed off the surface, transferring the analyte molecules

into the gas phase.

- A supersonic expansion from the surface - the velocities of various

components of the sample are found to be very similar

- Reactions occur in the dense plume - Gas-phase ionization

- Formation of ions: cationized species, protonated species, de-

protonated species, or radical cations

Sample

Plate

4) Mass Analysis - Time-of-Flight Mass Spectrometer…

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6

m

KEv

2=

In linear TOF instrument, ions drift through the flight tube until they reach the detector.

Because ions of different species in a mixture have different masses, they have different velocities based on the following equation:

TOF Cont’d

ν=velocity, KE =kinetic energy, and m=mass

DKE

mt

2= Where D is the flight distance

2

22

D

Ut

z

m= z=number of charge on an ion, U=accelerating voltage

t=flight time

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HPLC

PumpLC Column

Mass Spectrometer

Retention time

LC/MS

Liquid phase Gas Phase Ionization

HPLC

PumpLC Column

LC-MALDI

Mass Spectrometer

Retention time

Spray

interface

Liquid phase Gas Phase IonizationSolid phase

m/z

m/z

LCMS vs LC-MALDI

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Beginning

of separation

LC-MALDI Interface

Research & Development Laboratories, Eastman Kodak Company, Rochester, New York

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9LC-Interface Experimental Conditions

HPLCColumn: Zorbax Eclipse XDB-C8

2.1mm x 150mm; 5µmA: Water

B: ACN/IPA (1:1)

Gradient:

- 50% B → 100%B in 20 min. at 250uL/min

- Hold 100% B for 5 min at 250uL/min

- 100% B → 50% B in 0.1 min at 350uL/min

- Hold 50% B for 5 min at 350uL/min

Inj Vol.: 2uL

Detection: DAD (monitor at 210 nm)

Matrix PumpPump 1: CHCA matrix 1 mg /mL in ACN:IPA

Pump 2: ACN:IPA

Isocratic pumping of 50:50 (pump 1 : pump 2)

Flow Rate: 100 uL/min

SprayerTemperature gradient: 172 oC at initial conditions to 133 oC at final conditions N2 Gas Nebulization: 30 psi

6

OH

N

O

OH

α-cyano-4-hydroxycinnamic acid (CHCA)

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10Application

Example used for Demonstration of Capability

● K-GH Polyol (low MW polymer used in Thermal dye-receiver)

● Important to have correct end-group chemistry

● Multiple end-group combinations have been observed

● Interest in pursuing functional separation

CH3

CH3

O O

O

OO

O

O

n

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11Direct MALD/I TOF MS of K-GH Polyol (no separation)

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000m/z0

100

%

0

100

%

2371-06938-1a_raster 4 (0.503) Sb (30,30.00 ); Cm (1:49) TOF LD+ 1.06e5901.4

1287.5

902.4

1288.5

1673.7

1289.5

2060.8

1675.72446.8

2834.03220.1

3607.0 3993.2 4380.1

2371-06937-1raster 41 (5.436) Sb (30,30.00 ); Cm (1:49) TOF LD+ 9.87e4650.1

901.3

1287.4

902.31288.4

1673.61305.4

1307.5

2060.7

1693.5 2061.7 2446.82833.9

3220.1 3607.0 3993.2

“In-Spec” Material

“Out of Spec” Material

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1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050m/z0

100

%

0

100

%

2371-06938-1a_raster 4 (0.503) Sb (30,30.00 ); Cm (1:49) TOF LD+ 9.41e41287.5

1277.3

1288.5

1673.7

1289.5

1419.6

1675.7

1806.7

2371-06937-1raster 41 (5.436) Sb (30,30.00 ); Cm (1:49) TOF LD+ 5.63e41287.4

1277.2

1288.4

1673.6

1305.4

1306.4

1307.5

1541.61323.4 1386.6 1567.6

1691.6

1693.5

1694.5 1928.71806.6

386 DaA

A

Z

BB

Z

XX D

D

A

A

Y Y

CC

BB

WW

DDC C

“In-Spec” Material

“Out of Spec” Material

Direct MALD/I TOF MS of K-GH Polyol (no separation)

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Andrew, J.H.; William, J.E.; Robert, J.T.; Kelvin, G.O.; Anal.Chem. 2004, 76, 5157-5164

Spectral of PMMA 6300 using NaTFA as the cationization reagent with six matrixes (a) RA (b) DCTB (c) IAA (d) CHCA (e ) DHB (f) DHBQ

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Andrew, J.H.; William, J.E.; Robert, J.T.; Kelvin, G.O.; Anal.Chem. 2004, 76, 5157-5164

Spectral of PS 7000 using AgTFA as the cationization reagent with six matrixes (a) RA (b) DCTB (c) IAA (d) CHCA (e ) DHB (f) DHBQ

Results

DHBQ 2,5-dihydroxy-p-benzoquinoneDHB 2,5-dihydroxybenzoic acidCHCA a-cyano-4-hydroxycinnamic acidIAA trans- indoleacrylic acidDCTB 2-[(2E)-3-(4-tert-butylphenyl)-2methylpropenylidene] malanonitrileRA all-trans-retinoic acid

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Andrew, J.H.; William, J.E.; Robert, J.T.; Kelvin, G.O.; Anal.Chem. 2004, 76, 5157-5164

Results Cont’d

Spectral of PEG 10000 using NaTFA as the cationization reagent with six matrixes (a) RA (b) DCTB (c) IAA (d) CHCA (e ) DHB (f) DHBQ

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Conclusion� LC-MALDI retention time reproducibility is within a factor of 3,

compared with conventional LC-DAD, despite the solid state transition. (Within a factor of 2 compared with conventional LC/MS).

� Demonstrated chromatographic fidelity for LC-MALDI compared to LC-DAD and LC-ELSD.

� Matrix-addition solvent effects were observed which compromised chromatographic fidelity. 3D MALDI Imaging being used to investigate the dynamics of this effect.

� Matching the RTs of the matrix and analyte consistently producedthe best MALDI spectra with regard to S/N.

� The results presented here suggest that a good starting point for choosing the appropriate matrix for an “unknown” sample would be to match its relative polarity with that of the matrix.

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Acknowledgements

Andrew Hoteling & Bill Nichols – Eastman Kodak Co.

CHEM 395 Class of Spring ’07 !!

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References

Karas, M. Hillenkamp,F. Anal. Chem. 1988, 60, 2299-2301

Tanaka, K. Waki, H.; Ido, Y.; Akita, S.; Yoshido, Y,; Yoshido, T.; Rapid Commun.Mass Spectrom. 1988,2,151

Gidden, J.; Wyttenbach,T.; Jackson,A.T.; Scrivens, J.H.; Bowers,M.T.; J. Am. Chem. Soc. 2000,122,4692-4699

Gidden, J.; Bowers, M.T.; Jackson, A.T.; Scrivens, J.H.; J. Am. Soc. Mass Spectrom. 2002,13,499-505

Andrew, J.H.; William, J.E.; Robert, J.T.; Kelvin, G.O.; Anal.Chem. 2004, 76, 5157-5164

Cotter R.J.; 1997,Washington D.C.; ACS Professional Reference Books Opsal, R.B.; Owens, K.G.; Reilly, J.P.; Anal. Chem. 1985 57 1884-1889

Andrew J.H. PhD Thesis 2003