localisation, identification and separation of … · 2018-10-15 · •localization of molecules...

LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES

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Gilles Frache Materials Characterization Day October 14th 2016

• 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

Which focus?

MOLECULAR ANALYSES

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Time-Of-Flight Secondary Ion Mass Spectrometry

TOFSIMS

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Retrospective Analysis

Surface Spectroscopy Surface Imaging

Depth Profiling



Depth Profiling

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

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

• Red ink on InP substrate :

TOFSIMS Imaging

5

500

400

300

200

100

0

4002000μm

Overlay of 443.23 u, In+,

Primary ion beam

256x256 pixels

Max. FoV 500 um x 500 um

Mass (u)

150 200 250 300 350 400 450

5x10

1.0

2.0

3.0

4.0

5.0

Inte

nsity (

cou

nts

)

Sample:

Comment:

Origin:

PI Species: Bi5

PI Dose:

Raster Area: 500 x 500 µm²

Filename: ~tmpchqalp_0.ita

Date: Tue Nov 12 16:11:01 2013

Polarity: Positive

In+

Global Mass spectrum (Sx,y)

m/z=443.23

C28H31O3N2+

Rhodamine 6G

Specific 2D distribution of 2 molecules of interest

(ink vs. substrate)

Several modes of operations

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Lipid distribution in a

mouse brain cryo-

section

(6mm x 1 cm)

6.0

4.0

2.0

0.0

10.0 7.5 5.0 2.5 0.0 mm

red : m/z 184 Lipid fragment, PC headgroup, C5H15NO4P+, green: m/z 369, Cholesterol

• Large area TOFSIMS imaging

(up to several square cm2)

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

160

120

80

40

0

2001000μm

Sum of: 369.86 u, 385.52 u normalized to totalMC: 0; TC: 6.920e+002

0.030

0.020

0.010

-0.000

Lateral Resolution :

400 nm

(250um x 250 um)

• Multi-layer on glass

Elemental 3D TOFSIMS imaging

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Quick analysis 10-15 layers within 15 minutes without any preliminary information (screening technique)

• 5 pixels from a smartphone display



Depth Profiling



Depth Profiling

• 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

• …

Applications

TOFSIMS

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Endogenous lipid in skin cross-section overlaid on a

SEM-like image

Cosmetic ingredients on a single hair fiber

Fingerprint on silicon wafer (fatty acids)

Defects on a surface

• 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

Applications & current

developments Current developments

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For TOFSIMS imaging : Need for a reproducible matrix deposition device HTX MALDI matrix sprayer (evaluation period)

Matrix enhanced TOF-SIMS (manual deposition) : - Very successful with 10x - 30x signal enhancement for intact PEG oligomers - New molecules become visible !

TOFSIMS signal enhancement by (MALDI) matrix deposition

ME-TOFSIMS: Signal increase by a factor of 30 on a (PEG)34 oligomer at m/z=1538

Mass (u) 1530 1535 1540 1545 1550

2 x10

1.0

2.0

3.0

4.0

5.0

Inte

nsity (

counts

)

Direct analysis: very weak signal of (PEG)34 oligomer at m/z=1538

Molecular Mass Spectrometry Imaging (MSI)

ANALYTICAL CAPABILITIES

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• Localization of molecules/elements of interest by Mass Spectrometry Imaging (MSI)

techniques :

• Molecular MS Imaging by TOFSIMS and MALDI-MS

Spot size

0.4 – 2 um

Type of information

Intact + fragmented molecules

(+ elements)

>100 um

Intact small molecules Accurate mass

and structural MSn

80 um 10 um











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• 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

Mass accuracy

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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

MS/MS and MSn capabilities

C8H11O2N4+

• 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..)

C6H8N3O+ (m/z=138)

High-energy Collision Dissociation of caffeine Structural identification

• A TOF analyser has a

mass resolving power

(m/Dm) of 5000,

• An orbitrap has a

mass resolving power

(m/Dm) of 240 000 .

Mass resolution

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Caffeine [C8H10O2N4 + H]+

m/z = 195.08765

Orbitrap

TOF

• 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)

Mass resolution

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[C7H14O6 + H]+ m/z = 195.08631

Methyl-α-D-galactose Caffeine [C8H10O2N4 + H]+

m/z = 195.08765

Mass difference = 0.00134 Da

Time-Of-Flight

Orbitrap HRMS

• Plasma polymerization Glycidyl methacrylate (GMA) at various power (P1<P2<P3)

Application

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C7H10O3

C21H31O9+

C21H33O9+

Portion of the MALDI-Orbitrap spectra

C20H31O10+

C21H33O9+

Applications of MALDI HR-MS

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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

Accurate identification for: - most easy-to-ionize molecules - most abundant molecules

• 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

• …











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• Principle : LC is based on the affinity of molecules between a stationary phase and a

mobile phase

Liquid Chromatography for small molecules

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Stationary phase

Mobile phases

Complex mixture

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

Refractive index detector

UV-visible detector

Column oven

Auto-sampler / Fraction collector

LPG pump

Solvent rack

To HRMS (Orbitrap)

• Quantitative measurements

LC/UV-vis

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Amount Si

gnal

degradation of ingredients in formulations, loading and release

of nanovectors, photocatalysis…)

• Chromatographic separation + High Resolution Mass Spectrometry

LC/MS

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+

Retention time

MS #1 MS #2

MS #3 MS #4

MS #5 MS #6

• 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 ??

LC/MS applications:

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Gel Permeation chromatography (or Size Exclusion Chromatography)

Refractive index detector

UV-visible detector

Column oven

Auto-sampler / Fraction collector

LPG pump

Solvent rack

Retention time (min) or Log (Mw)

Sign

al (

a.u

.)

HMW POLYMERS

• Principle

Application

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• Gel Permeation chromatography of a polydisperse industrial polymer (“resin A”)

(Mw ranging from 100s to 100 000s)

• Application :

• Quantitative evaluation of the global degradation of polymers (by optical measurements using UV-Vis, RI)

• Molecular information ?

Determination of polymer characteristics (Mw, Mn, Polydispersity index)

GPC with fraction collection

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• Combination of techniques

GPC with fractionation for

subsequent analyses

Applications &

current

developments Current

methodological

developments:

Fractions of different ingredient (separated MW fractions):

LC/MS FT-IR TOF

SIMS MALDI HRMS

MALDI TOFMS

/ / / /

Complex polymer mixture

CONCLUSION

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• 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]

mailto:[email protected]





localisation, identification and separation of … · 2018-10-15 · •localization of molecules...

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