LOCALISATION,
IDENTIFICATION AND
SEPARATION OF MOLECULES
Gilles Frache
Materials Characterization Day
October 14th 2016
1
MOLECULAR ANALYSES
Which focus?
• LOCALIZATION of molecules by Mass Spectrometry Imaging Techniques
•
•
•
Time-Of-Flight Secondary Ion MS (TOFSIMS)
Matrix-Assisted Laser Desorption Ionization MS (MALDI-MS)
Applications & current developments
• IDENTIFICATION by High Resolution Mass Spectrometry
•
•
Accurate mass and structural analysis
Applications of (AP-MALDI) HRMS
• SEPARATION of molecules
•
•
According to their Polarity
According to their Molecular Weight
2
• LOCALIZATION of molecules by Mass Spectrometry Imaging Techniques
•
•
•
Time-Of-Flight Secondary Ion MS (TOFSIMS)
Matrix-Assisted Laser Desorption Ionization MS (MALDI-MS)
Applications & current developments
• IDENTIFICATION by High Resolution Mass Spectrometry
•
•
Accurate mass and structural analysis
Applications of (AP-MALDI) HRMS
• SEPARATION of molecules
•
•
According to their Polarity
According to their Molecular Weight
3
TOFSIMS
Time-Of-Flight Secondary Ion Mass Spectrometry
TOFSIMS
- Ionization by a pulsed, focused, highly energetic
primary ion bombardment (UHV conditions)
- Low primary ion dose
- Analysis of all secondary ions by a Time-Of-Flight
Mass spectrometer
Performances:
Max Mass Resolving Power : 10 000
Mass accuracy: 50 ppm
Lateral resolution : 2 um - 400 nm
Depth Resolution : 1 nm
- Primary ion guns:
Binm+ Analysis/Imaging ion gun
Cs, C60 Sputter/Analysis ion gun
Surface Spectroscopy
Surface Imaging
Depth Profiling
Retrospective Analysis
4
TOFSIMS Imaging
• Red ink on InP substrate :
Global Mass spectrum (Sx,y)
Primary
ion beam
PI Species:
PI Dose:
Raster Area:
Sample:
Comment:
Origin:
Bi5
Filename:
Date:
Polarity:
500 x 500 µm²
~tmpchqalp_0.ita
Tue Nov 12 16:11:01 2013
Positive
5
x10
m/z=443.23
5.0
4.0
In+
3.0
C28H31O3N2+
Intensity (counts)
2.0
256x256 pixels
Rhodamine 6G
1.0
150
200
250
300
350
400
450
Mass (u)
500
400
Specific 2D distribution
of 2 molecules of interest
(ink vs. substrate)
300
200
100
0
μm 0
Max. FoV
500 um x 500 um
200
400
Overlay of 443.23 u, In+,
5
Several modes of operations
6.0
• Large area TOFSIMS imaging
(up to several square cm2)
Lipid distribution in a
mouse brain cryosection
(6mm x 1 cm)
4.0
red : m/z 184 Lipid fragment,
PC headgroup, C5H15NO4P+,
green: m/z 369, Cholesterol
2.0
0.0
mm 0.0
2.5
5.0
7.5
10.0
Lateral Resolution :
2 um
(500um x 500 um)
• Standard Imaging mode
(500um x 500um)
• High resolution TOFSIMS imaging
(down to 400 nm spot size)
200
0.030
160
Lateral Resolution :
400 nm
(250um x 250 um)
0.020
120
80
0.010
40
0
μm 0
-0.000
100
200
6
Surface Spectroscopy
Surface Imaging
Elemental 3D TOFSIMS imaging
• Multi-layer on glass
• 5 pixels from a smartphone display
Depth Profiling
Retrospective Analysis
 Quick analysis 10-15 layers within 15 minutes
without any preliminary information
(screening technique)
7
TOFSIMS
Applications
• Surface contaminations/defects on industrial materials (glass, steel, automotive, packaging
…)
• Functional coatings on nanoparticles
• Identification of polymers
• Biological tissue imaging (brain, skin, eye, liver, hair, …) for endogenous or exogenous
molecular imaging
• …
Fingerprint on
silicon wafer
(fatty acids)
Defects on a
surface
Endogenous lipid in skin
cross-section overlaid on a
SEM-like image
Cosmetic ingredients on a
single hair fiber
8
Applications & current
developments
Current developments
• Limiting factor : Low ionization yield in TOFSIMS analysis / imaging
 Improvement by dedicated sample preparations
 Controlled deposition of a “matrix” (as in MALDI-MS) to tune the ionization yield
TOFSIMS signal enhancement by (MALDI) matrix deposition
x102
5.0
4.0
3.0
ME-TOFSIMS:
Signal increase by
a factor of 30
on a (PEG)34
oligomer at
m/z=1538
Intensity (counts)
2.0
Direct analysis:
very weak signal
of (PEG)34
oligomer at
m/z=1538
For TOFSIMS imaging : Need for a reproducible matrix
deposition device
 HTX MALDI matrix sprayer (evaluation period)
1.0
1530
1535
1540
1545
1550
Mass (u)
Matrix enhanced TOF-SIMS (manual deposition) :
- Very successful with 10x - 30x signal enhancement for intact PEG oligomers
- New molecules become visible !
9
ANALYTICAL CAPABILITIES
Molecular Mass Spectrometry Imaging (MSI)
• Localization of molecules/elements of interest by Mass Spectrometry Imaging (MSI)
techniques :
•
Type of
information
Molecular MS Imaging by TOFSIMS and MALDI-MS
Intact + fragmented
molecules
(+ elements)
0.4 – 2 um
Intact small molecules
Accurate mass
and structural MSn
10 um
80 um
>100 um
Spot size
10
• LOCALIZATION of molecules by Mass Spectrometry Imaging Techniques
• Time-Of-Flight Secondary Ion MS (TOFSIMS)
• Matrix-Assisted Laser Desorption Ionization MS (MALDI-MS)
• Applications & current developments
• IDENTIFICATION by High Resolution Mass Spectrometry
• Accurate mass and structural analysis
• Applications of (AP-MALDI) HRMS
• SEPARATION of molecules
•
•
According to their Polarity
According to their Molecular Weight
11
Mass accuracy
• A TOF analyser is fast and sensitive and thus well suited for high rate acquisition as it
is required for imaging MS.
• Nevertheless, a TOF analyser is limited for unambiguous identification
C = 12.00000
H = 1.00783
N = 14.00307
O = 15.99491
195.09 +/- 0.01 Da for a TOF analyser
(+/-50ppm)
 13 chemically possible formulas
(C,H,N,O,S containing molecules)
195.0876 +/- 0.0002 Da for the
Orbitrap analyser (+/-1ppm)
 1 single chemically possible
formula : C8H11O2N4+
Caffeine
[C8H10O2N4 + H]+ = 195.08765
12
MS/MS and MSn capabilities
• MS/MS = Selection of an ion of interest in the spectrum, isolation, fragmentation at a given
dissociation energy
• MSn = n times MS/MS (fragmentation of a fragment of an ion of interest..)
C8H11O2N4+
 C6H8N3O+ (m/z=138)
High-energy Collision Dissociation of caffeine
Structural identification
Mass resolution
• A TOF analyser has a
mass resolving power
(m/Dm) of 5000,
• An orbitrap has a
mass resolving power
(m/Dm) of 240 000 .
Caffeine
[C8H10O2N4 + H]+
m/z = 195.08765
TOF
Orbitrap
14
Mass resolution
• A TOF distinguishes ions with mass differences of 0.08 amu (atomic mass unit), while
a Orbitrap distinguishes ions with mass differences of 0.00017 (0.17mDa)
Time-Of-Flight
Orbitrap
HRMS
Caffeine
[C8H10O2N4 + H]+
m/z = 195.08765
Methyl-α-D-galactose
[C7H14O6 + H]+
m/z = 195.08631
 Mass difference = 0.00134 Da
15
Application
• Plasma polymerization Glycidyl methacrylate (GMA) at various power (P1<P2<P3)
C7H10O3
C21H33O9+
C21H31O9+
C20H31O10+
C21H33O9+
Portion of the MALDI-Orbitrap spectra
16
Applications of MALDI HR-MS
• Reverse engineering on industrial materials (Polymer additives, identification of small
molecules/oligomers in complex mixtures, OLED ingredients …)
• Identification of degradation products from complex formulation (accelerated ageing of
cosmetics,…)
• Understanding of plasma chemistry
• Functional coatings (nanoparticles …)
• Complementary identification for TOFSIMS imaging
• …
Accurate identification for:
- most easy-to-ionize molecules
- most abundant molecules
Complex
mixture of
molecules
Ionization
(competitive
mechanism)
“Some” ions :
most easy-to-ionize
molecules ?
most abundant
molecules ?
No information for:
- difficult-to-ionize molecules
- low abundant molecules
No quantitative information
17
• LOCALIZATION of molecules by Mass Spectrometry Imaging Techniques
• Time-Of-Flight Secondary Ion MS (TOFSIMS)
• Matrix-Assisted Laser Desorption Ionization MS (MALDI-MS)
• Applications & current developments
• IDENTIFICATION by High Resolution Mass Spectrometry
•
•
Accurate mass and structural analysis
Applications of (AP-MALDI) HRMS
• SEPARATION of molecules
• According to their Polarity
• According to their Molecular Weight
18
Liquid Chromatography for small molecules
• Principle : LC is based on the affinity of molecules between a stationary phase and a
mobile phase
Complex
mixture
Mobile
phases
Liquid chromatography (HPLC or LC/MS):
Mobile phase : different solvent composition
Stationary phase : e.g. C18-coated silica, HILIC,
bare silica, biphenyl-coated silica
Detection: UV-visible, refractive index, HRMS
Solvent rack
Stationary
phase
LPG pump
Auto-sampler / Fraction collector
Column oven
UV-visible detector
Refractive index detector
To HRMS (Orbitrap)
19
LC/UV-vis
Signal
• Quantitative measurements
Amount
 degradation of ingredients in formulations, loading and release
of nanovectors, photocatalysis…)
20
LC/MS
• Chromatographic separation + High Resolution Mass Spectrometry
+
Retention time
MS #2
MS #1
MS #4
MS #3
MS #5
MS #6
21
LC/MS applications:
•
High resolution LCMS of complex mixtures :
•
•
•
•
•
industrial formulations,
natural products,
organic chemistry syntheses,
kinetic studies of degradation products (photo-catalysis, ageing)
Investigation of Low MW copolymers
And for High Molecular Weight or highly polydisperse polymers ??
22
HMW POLYMERS
Gel Permeation chromatography (or Size Exclusion Chromatography)
• Principle
Solvent rack
LPG pump
Auto-sampler /
Fraction collector
Column oven
Signal (a.u.)
UV-visible detector
Refractive index detector
Retention time (min) or Log (Mw)
23
Application
• Gel Permeation chromatography of a polydisperse industrial polymer (“resin A”)
(Mw ranging from 100s to 100 000s)
 Determination of
polymer characteristics
(Mw, Mn, Polydispersity
index)
• Application :
• Quantitative evaluation of the global degradation of polymers
(by optical measurements using UV-Vis, RI)
• Molecular information ?
24
Applications &
current
developments
GPC with fraction collection
 Current
methodological
developments:
• Combination of techniques
 GPC with fractionation for
subsequent analyses
Complex
polymer
mixture
Fractions of different ingredient (separated MW fractions):
/
FT-IR
TOF
SIMS
/
/
MALDI
TOFMS
/
MALDI
HRMS
LC/MS
25
CONCLUSION
• LOCALIZATION, IDENTIFICATION and SEPARATION of molecules
• 3 main techniques (TOFSIMS, HRMS, LC)
• 4 additional instruments (GC/MS, FT-IR, MALDI-TOF MS, MALDI-LTQ).
• On-purpose methodological developments to be defined according to your
requirements
• Worflow:
•
•
•
•
•
•
Problem definition
Sample preparation
Method development
Data acquisition
Data evaluation
Reporting
Contact: [email protected]
[email protected]
or [email protected]
26