LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES. Gilles Frache Materials Characterization Day October 14 th 2016

Transcription

1 LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES Gilles Frache Materials Characterization Day October 14 th

2 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

3 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

4 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 Surface Spectroscopy Surface Imaging Performances: Max Mass Resolving Power : Mass accuracy: 50 ppm Lateral resolution : 2 um nm Depth Resolution : 1 nm - Primary ion guns: Bi n m+ Analysis/Imaging ion gun Cs, C 60 Sputter/Analysis ion gun Depth Profiling Retrospective Analysis 4

5 Intensity (counts) TOFSIMS Imaging Red ink on InP substrate : Primary ion beam Global Mass spectrum (Sx,y) Sample: PI Species: Bi5 Filename: ~tmpchqalp_0.ita Comment: PI Dose: Date: Tue Nov 12 16:11: Origin: Raster Area: 500 x 500 µm² Polarity: Positive 5 x10 m/z= In C 28 H 31 O 3 N 2 + Rhodamine 6G 256x256 pixels Mass (u) Specific 2D distribution of 2 molecules of interest (ink vs. substrate) Max. FoV 500 um x 500 um μm Overlay of u, In+, 400 5

6 Several modes of operations 6.0 Large area TOFSIMS imaging (up to several square cm 2 ) Lipid distribution in a mouse brain cryosection (6mm x 1 cm) red : m/z 184 Lipid fragment, PC headgroup, C 5 H 15 NO 4 P +, green: m/z 369, Cholesterol mm Standard Imaging mode (500um x 500um) Lateral Resolution : 2 um (500um x 500 um) High resolution TOFSIMS imaging (down to 400 nm spot size) Lateral Resolution : 400 nm (250um x 250 um) μm

7 Surface Spectroscopy Surface Imaging Elemental 3D TOFSIMS imaging Multi-layer on glass 5 pixels from a smartphone display Depth Profiling Retrospective Analysis Quick analysis layers within 15 minutes without any preliminary information (screening technique) 7

8 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

9 Intensity (counts) 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 2 x10 TOFSIMS signal enhancement by (MALDI) matrix deposition Direct analysis: very weak signal of (PEG) 34 oligomer at m/z=1538 ME-TOFSIMS: Signal increase by a factor of 30 on a (PEG) 34 oligomer at m/z= Mass (u) Matrix enhanced TOF-SIMS (manual deposition) : - Very successful with 10x - 30x signal enhancement for intact PEG oligomers - New molecules become visible! For TOFSIMS imaging : Need for a reproducible matrix deposition device HTX MALDI matrix sprayer (evaluation period) 9

10 ANALYTICAL CAPABILITIES Molecular Mass Spectrometry Imaging (MSI) Localization of molecules/elements of interest by Mass Spectrometry Imaging (MSI) techniques : Molecular MS Imaging by TOFSIMS and MALDI-MS Type of information Intact + fragmented molecules (+ elements) Intact small molecules Accurate mass and structural MSn um 10 um 80 um >100 um Spot size 10

11 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

12 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 = H = N = O = / Da for a TOF analyser (+/-50ppm) 13 chemically possible formulas (C,H,N,O,S containing molecules) / Da for the Orbitrap analyser (+/-1ppm) 1 single chemically possible formula : C 8 H 11 O 2 N 4 + Caffeine [C 8 H 10 O 2 N 4 + H] + =

13 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..) C 8 H 11 O 2 N 4 + C 6 H 8 N 3 O + (m/z=138) High-energy Collision Dissociation of caffeine Structural identification

14 Mass resolution A TOF analyser has a mass resolving power (m/dm) of 5000, An orbitrap has a mass resolving power (m/dm) of Caffeine [C 8 H 10 O 2 N 4 + H] + m/z = TOF Orbitrap 14

15 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 (0.17mDa) Time-Of-Flight Orbitrap HRMS Caffeine [C 8 H 10 O 2 N 4 + H] + m/z = Methyl-α-D-galactose [C 7 H 14 O 6 + H]+ m/z = Mass difference = Da 15

16 Application Plasma polymerization Glycidyl methacrylate (GMA) at various power (P1<P2<P3) C 7 H 10 O 3 C 21 H 33 O 9 + C 21 H 31 O 9 + C 20 H 31 O 10 + C 21 H 33 O 9 + Portion of the MALDI-Orbitrap spectra 16

17 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

18 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

19 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

20 Signal LC/UV-vis Quantitative measurements Amount degradation of ingredients in formulations, loading and release of nanovectors, photocatalysis ) 20

21 LC/MS Chromatographic separation + High Resolution Mass Spectrometry + Retention time MS #1 MS #2 MS #3 MS #4 MS #5 MS #6 21

22 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

23 Signal (a.u.) HMW POLYMERS Gel Permeation chromatography (or Size Exclusion Chromatography) Principle Solvent rack LPG pump Auto-sampler / Fraction collector Column oven UV-visible detector Refractive index detector Retention time (min) or Log (Mw) 23

24 Application Gel Permeation chromatography of a polydisperse industrial polymer ( resin A ) (Mw ranging from 100s to s) 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

25 Complex polymer mixture GPC with fraction collection Applications & current developments Current methodological developments: Combination of techniques GPC with fractionation for subsequent analyses Fractions of different ingredient (separated MW fractions): / / / / FT-IR TOF SIMS MALDI TOFMS MALDI HRMS LC/MS 25

26 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: gilles.frache@list.lu jerome.bour@list.lu or mrt-platform@list.lu 26