chem 395 olumide - rusling research group
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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
18/04/2007
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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3
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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5
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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7
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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8
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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12
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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13
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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14
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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15
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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16
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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18
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
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