On Leave from: Department of Physics, Faculty of Science,

Helwan University, Cairo , Egypt

Gihan kamel

SESAME Infrared Beamline Scientist

gihan.kamel@sesame.org.jo

INTRODUCTION TO SYNCHROTRON INFRARED

MICROSPECTROSCOPY

(9:00-11:00 a.m.):

Introduction to the IR Microspectroscopy: Infrared microspectroscopy

technique, instrumentation, measuring modes.

Pieces of advice on samples’ preparation and handling.

General knowledge: (data collection, processing, analysis and interpretation).

The advantages of the synchrotron-based FTIR Microspectroscopy.

(12:20 – 13:00 p.m.):

SESAME IR beamline capabilities.

Practical session: Group 1- (14:00 – 15:50 p.m.):

Practical session: Group 1I - (16:10 – 18:00 p.m.):

IR MICROSPECTROSCOPY:

THURSDAY, JUNE 26, 2019.

Questions:

What is the history of the sample?

What is the reason for the analysis?

What information is needed?

Can the sample be moved? If yes, how much (which form)? If no, other

options?

Does the sample have a single component or is it multicomponent?

Are all components original to the piece?

Has any previous analysis been done?

WHY, HOW AND WHAT?

(HISTORY, DESIGN AND RESULTS)

WHAT DO YOU NEED TO KNOW: I

Infrared Absorption Theory

Electromagnetic Radiation

Molecular Absorptions

Infrared Spectra

Infrared Regions (Near-Infrared, Mid-Infrared, Far-Infrared)

Sample Collection and Preparation

Sampling Methodology (Design, Tools, Documentation and Storage)

Avoidance of Contamination

Sample Collection and Preparation\manipulation Procedures

Infrared Analysis Methods

Infrared Transmission Measurements (Infrared Window Materials)

Infrared Reflection Measurements (Specular Reflection, Reflection-

Absorption, Diffuse Reflection, Internal Reflection, etc)

Infrared Microspectroscopy

Microspectrometer (Design and Capabilities)

Microspectroscopic Accessories

WHAT DO YOU NEED TO KNOW: 1I

WHAT DO YOU NEED TO KNOW: III

Spectral Interpretation

Instrument Configuration

Qualitative Analysis (Spectral Quality, Visual Comparison, Spectral Libraries)

Spectral Region Examination

Spectra-Structure Correlations

Mathematical Manipulations of Spectra (Subtraction Techniques, Resolution

Enhancement Methods, etc.).

TO TAKE AWAY:

Scientific approach to the environmental science field.

(Why and how?)

Introduction to the IR Microspectroscopy Basics.

(EM radiation, Absorption theory, IR spectroscopy, ...)

The advantages of the SR-based FTIR Microspectroscopy.

(IR thermal sources are still great, but sometimes NOT

great enough!)

“Ideally”, a technique for a proper analysis should be*:

• Non-destructive (non-invasive) [rare/one of a kind items]

• Fast.

• Universal: different objects may be studied with minimal or no sample pre-

treatment.

• Versatile: local information of small areas and average composition to be

obtained (spatial resolution).

• Sensitive: able to detect trace quantities.

• Multi-elemental: simultaneously detect multiple components in a single

measurement.

* Lahanier et al. Nuc. Instrum. Meth. B14 (1986) 1-9.

IR spectroscopy

A COMPARISON OF COMMONLY USED CHEMICAL ANALYSIS

TECHNIQUES.

COMMON SR TECHNIQUES EMPLOYED IN THE FIELD OF

ENVIRONMENTAL SCIENCES

L. Bertrand et al., Heritage and archaeological materials studied by synchrotron methods, DOI 10.1007/s00339-011-6686-4, Appl Phys A.

Pharmaceuticals

Cultural heritage

Food

Disciplines.. or better to say DEMANDS?!

Chemistry

Physics Forensics

Cell biology

Ecology

Biology

Mineralogy

Agriculture

Geology

Archeology

Astronomy

Art restoration

Environment

Medicine

Forensics

Data Analysis

(Imaging)

POCKET: WORK FLOW FOR FTIR MICROSPECTROSCOPY

FTIR Data CollectionSamples preparation

Powder

Cross sections

Bulk Original as it is

IR reflective slides

BaF2 or CaF2 slides Globar

SR

Others

Transmission

Reflection

Data Analysis (Diagnosis)

ELECTROMAGNETIC RADIATION SPECTRUM

Electromagnetic radiation can be characterized by the number

of waves per unit length (wavenumber (cm-1))

The science mission in all synchrotron

facilities defines performance goals

reaching from:

- the mid-IR (2.5–25μm),

- and the far-IR (25–1000μm).

IR BEAMLINES UTILIZE THE INFRARED REGION ON THE

ELECTROMAGNETIC SPECTRUM

INTRODUCTION TO SR-IR BASICS

EFFECT OF ELECTROMAGNETIC RADIATION ON MOLECULES

Molecular spectroscopy uses EM energy, or radiation, as the physical stimulus

Infrared radiation is largely thermal energy. It induces stronger molecular

vibrations in covalent bonds as springs holding together two masses..

THE ORIGINS OF THE INFRARED SPECTRUM

The IR spectrum is formed as a consequence of the absorption of EM

radiation at frequencies that correlate to the vibration of specific

sets of chemical bonds within a molecule.

The energy distribution possessed by a molecule at any given moment, is

defined as the sum of the contributing energy terms.

Etotal = Eelectronic + Evibrational + Erotational + Etranslational (Eq. 1)

The vibrational energy: corresponds to the absorption of energy by

a molecule as the component atoms vibrate about the mean center

of their chemical bonds.

DO ALL MOLECULES INTERACT WITH IR-EM

FIELD?

Fundamental rule: “There MUST be a

net change in dipole moment

during the vibration for the

molecule or the functional group

under study.”

• Not all covalent bonds display bands in the IR

spectrum. Only polar bonds do so. These are

referred to as IR active.

• The intensity of the bands depends on the

magnitude of the dipole moment associated

with the bond in question:

• Strongly polar bonds such as carbonyl groups

(C=O) produce strong bands.

• Medium polarity bonds and asymmetric bonds

produce medium bands.

• Weakly polar bond and symmetric bonds

produce weak or non observable bands.

DEGREES OF FREEDOM IN A MOLECULE

The atoms are constrained by molecular bonds to move together in certainspecified ways: Degrees of Freedom (DoF).

For a molecule with N atoms, the total number of coordinates specified will be 3N.These 3N coordinates are the maximum number of potential transitionspossessed by that molecule. (assigned to the translational, rotational, andvibrational motions of the molecule.):

- All molecules have 3 translational DoF.- Nonlinear molecules have 3 rotational DoF and linear molecules have only 2.- The total of translational and rotational degrees of freedom is 6 (or 5 forlinear molecules).- All remaining motions are vibrational (3N-6) DoF for nonlinear and (3N -5) forlinear molecules. All vibrational motions of the atoms can be described completelyas 3N-6 or 3N-5 fundamental vibrations (normal modes of vibration) for themolecule.

POCKET:

NORMAL MODES

EXAMPLE...

The typical IR absorption range

for covalent bonds is 600 - 4000

cm-1. The graph shows the

regions of the spectrum where

the following types of bonds

normally absorb.

IR ABSORPTION RANGE

Two types of interactions- absorption and transmission- are

important in the typical IR experiment:

When the molecule in the sample compartment of the spectrometer

is exposed to a source of continuous IR radiation, the photons of

discrete energy units that are absorbed by the molecule do not reach

the detector. Photons that are not absorbed by the sample are

transmitted to the detector essentially unaltered.

The IR spectrum reveals these “missing photons”, or absorptions,

as a series of well-defined, characteristic, and reproducible

absorption bands.

For a given wavelength or frequency of IR radiation striking a

sample, these two interactions are inversely related where: A:

absorbance and T: transmittance (% T/l00).

The presence or the absence of specific functional groups.

The molecular fingerprint that can be used when comparing samples. (If two pure

samples display the same IR spectrum it can be argued that they are the same

compound.)

The molecular information on both organic and inorganic species without inducing

any beam damage nor suffering from fluorescence effects often encountered with the

UV/visible excitation in Raman spectroscopy.

The information regarding the chemical structure, as well as, quantitative information

in specific conditions.

INFORMATION OBTAINED FROM IR SPECTRA

POCKET: FTIR SPECTROSCOPY

Alvise Vianello, Jes Vollertsen Aalborg University, Denmark

Fourier Transform Infrared Spectroscopy (FT-IR)

Michelson Interferometer

Michelson interferometerPractical sessions

cooled or RT detectors

TE Cooled DLaTGS Detector with KBr Window (12,500-350 cm-1)

DLaTGS Detector with Polyethylene Window (700-50 cm-1)

Room Temperature InGaAs Detector for NIR (12,000-3,800 cm-1)

50um MCT-A Detector (11,400-700 cm-1)

MCT-B Detector (11,700-450 cm-1)

IR DETERCTORS

SAMPLING TECHNIQUES

Depends on:

- Sample form (solid, liquid, powder, film),

- what to mantain? (it is possible to use different sampling techniques,

destructive or non destructive?)

- Transmission: liquids, powders, thin sections...

- Specular reflection: crystals, polished sections...

- Diffuse reflectance: powders...

- Attenuated Total Reflection (ATR): thick samples, non reflecting surfaces...

FTIR SAMPLES HANDLING (SAMPLE TYPE, METHOD, RATING)

WHAT IF THE SAMPLE IS REALLY REALLY SMALL?!

MICROSCOPE

IR Microspectroscopy

WHY SR-IR BEAMLINES?

• Non-destructive technique that exhibits a strong interest at various worldwide

synchrotron facilities.

• Combining the spatial resolution of a the IR-visible microscope with the high chemical

sensitivity of the FTIR spectrometer.

• SR-IR source broad spectral emission and wavelength characteristics,

• SR-IR sources provide extremely valuable information in its brightness/brilliance (about

1000 times brighter) with a signal-to-noise ratio that cannot be achieved by the

conventional sources.

• The mid-infrared region (2.5-25 µm) is very informative on the microscopic scale (a few

tens of micron resolution). The spatial resolution is no longer controlled by the

geometrical aperture size, but rather by the numerical aperture of the optical system and

the wavelength of the light. Therefore, the spot size is set to diffraction limit (3 to 10 µm)

in confocal geometry.

The high brightness of the SR-IR enables rapid acquisition of spectra at

high spatial resolution for static or time-resolved measurements of

dynamic properties.

The high degree of the SR optical polarization makes it the perfect tool for

many customized/tailored industrial applications.

Small samples studies are accessible thanks to SR-IR microscopic power.

Hot filament

Flux: radiated in all directions

Flux ∝Temperature

Accelerated charged particles

Flux: radiated in a tangential, well

defined cone

Flux ∝ beam current

Conventional IR SR-IR

IR SOURCES (LAB-BASED)

Adapted from S. Lupi

IR MICROSPECTROSCOPY BASICS

MS: coupling of a microscope to the IR spectrometer.

First made commercially in the 1950s. (satisfactory design, but

costly, limited by the low energy throughput and

corresponding low signal-to-noise ratios)

FT-IR advantages + advances in IR detector technology fueled

the reemergence in 1983.

Recent success in application of this instrumentation to many

areas of research has established the technique of IR

microspectroscopy as a powerful tool in the analysis of small

samples in so many applications..

The microscope contained all the necessary transfer optics to

direct the IR beam from the spectrometer source to the sample

positioned on the stage of the microscope.

The optics used in IR microspectrometers are reflecting

optics. Since both glass and quartz absorb IR light over much of

the region of interest, the IR microspectrometers are unable to

employ standard, visible-light refracting (lens) optics but rather

must use reflecting (mirror) optics.

MICROSPECTROMETER DESIGN

The next wave of improvements came when IR spectrometers were built

with the ability to direct the IR source beam external to the instrument.

This flexibility allowed modifications, redesign, and production of new

spectrometers with optimal geometry for coupling to IR microscopes.

Since the IR microscope had its own onboard detector, it was a complete

system, minus the IR source and data processing/computer

system. With this new design, the microscope no longer occupied the

sample compartment of the spectrometer, so that conventional methods

could still be used to analyze macro samples.

OPTIMUM COUPLING

The detector of choice is (MCT) detector.

The MCT detector element is cryogenically cooled to liquid

nitrogen temperature, thus providing high sensitivity and signal-to-

noise values necessary for the low energy levels and small

sampling areas.

As the microscope designs changed, a major improvement in

signal-to-noise levels was achieved when the MCT

detector was repositioned from the spectrometer bench

to the microscope apparatus.

IR SIGNAL DETECTION

Minimizing the IR beam path after interaction with the sample..

MICROSPECTROMETER VS. OPTICAL MICROSCOPE

First: All IR microspectrometers have visible-light imaging

available. Provide photomicroscopy and/or videomicroscopy.

Second: the ability to isolate a particular area of the sample

optically by the use of movable apertures [fixed-diameter-circle or

a variable-circle iris, or an adjustable knife-edge rectangle.].

Heterogeneous samples

Third: all IR microspectrometers have the ability to collect IR

reflectance spectra. Reflection IR is useful for highly absorbing

samples that do not transmit IR well, as well as, for providing

information on the surface composition of a material. >> the

ability to perform reflection analysis increases the versatility of the

IR microspectrophotometer. Practical sessions

POCKET: FTIR MICRSCOPE IS BEAM CONDENSER

AlviseVianello, JesVollertsen Aalborg University, Denmark

Optical lens that renders a divergent beam from a point source into a parallel or

converging beam to illuminate an obejct.

Upper Aperturesize settings

Lower Aperturesize settings

Lateral resolution ~l/2

SR-IR MICROSCOPE WORKS IN CONFOCAL GEOMETRY

Adapted from P. Dumas

TRANSMISSION MODE

Sample must be thin.

Embedding (the medium may contribute to the spectra)

Requires IR transparent windows ( CaF2, BaF2, ZnSe, ZnS, diamond!)

Detector

REFLECTION MODE

Sample must be flat or flattened

Signal is weaker

Adapted from P. DumasPractical sessions

POCKET: FTIR SPECTROSCOPY

Adapted from: AlviseVianello, JesVollertsen Aalborg University, Denmark

POCKET: FTIR MICROSPECTROSCOPY

cooled or RT detectors

cooled or RT detectors

The heterogeneity and low amount of cultural heritage materials led to

seeking to attain higher lateral resolution with imaging capabilities. >>

Microscopy + SR.

Synchrotron-based FT-IR microscopy with a confocal arrangement made it

possible to analyse and even map very small samples with high signal-to-

noise ratio (S/N) at the diffraction limit. The diffraction limit is proportional

to the wavelength and inversely proportional to the numerical aperture of

the optics.

Q: Definition of: Diffraction limit?

Q: Definition of the Lateral resolution?

Q:What is the highest lateral resolution obtainable at 1000cm−1

and at 3000cm−1 in reflection and transmission?

WHY SR-BASED FTIR MICROSPECTROSCOPY?

SESAME SR-IR Emission Source: Bending magnet

Edge Radiation (ER): Emitted at the

entrance (exit) of the bending

magnet (BM) due to the rapid

variation of the magnetic field (B)

Standard Bending Magent Radiation:

Emitted during the circular trajectory

in the bending magnet (BM) due to the

constant magnetic field (B)

Vertical collection angle = 15 mrad

Horizontal collection angle = 39 mrad

Opening angles require

modifying the dipole chamber! More on the IR beamline.. Later today

Beginning of 1990 : 5 facilities

Beginning of 2000 : 14 facilities

In 2017: 30+ facilities

The high brightness of the synchrotron IR light (100–1000 times > conventional

sources) has allowed important development in both the mid- and far-IR regions.

S/N RATIO? INFORMATION?

P. Dumas

The coupling of SR-FT-IR microspectroscopy with other synchrotron-based

techniques (XRF, XRD, ...) or laboratory FT-IR imaging techniques (Globar) is

an interesting approach that is increasingly evolving.

The combination of SR-FTIR microspectroscopy with FT-IR imaging: provides

respectively the very high signal-to-noise ratio data on local areas together

with the spatial distribution of species over large areas in a short time.

Mid-IR microspectroscopy together with Raman spectroscopy is commonly

applied to identify the compounds or at least the functional groups present.

SR-IR MICROSPECTROSCOPY +

Synchrotron and thermal IR sources play complementary roles

• With the thermal source: many millimeters can be surveyed

quickly and offer excellent performance down to about 10μm

spatial resolution.

• With the synchrotron: the resolution limit may be extended

down to around 1μm, but over a much more limited area.

Something to remember..

POCKET: ADVANTAGES OF SR-FTIR MICROSPECTROSCOPY

(MICROSCOPY + SPECTROSCOPY)

Broadband

Brightness

Linear & Circular

Polarization

Pulsed Emission

Diffraction limited

spatial resolution

Better S/N ratio

Faster Data Collection

Spectroscopy

Polarized

Microspectroscopy

Time resolved

Studies

Microscopy

Broadband

Brightness

Adapted from Lisa Miller

Dr. MARTIN, Michael (Lawrence Berkeley National Laboratory)

While using FTIR spectroscopy appears straightforward for well handled samples,

the evaluation becomes significantly more complicated when probing very

small and/or heterogeneous samples.

: because it monitors the global chemical composition in the probed volume, the

vibrational signatures recorded are a superposition of the spectra of thousands

of constituents.

.. Yet despite the complexity, it has been demonstrated that the technique is highly

sensitive to slight changes in the composition. The interpretation of these

composite spectra requires sophisticated data analysis, which is still evolving

through the continued development of multivariate methods.

THE COMPLEXITY OF THE FTIR

MICROSPECTROSCOPIC DATA

These approaches are based on the principle that there

exist small, but reproducible changes in the spectra that

can be associated with the variations in sample properties

that are investigated.

There exist several known statistical approaches for

infrared data analysis, but the most frequently used is

principal components analysis (PCA).

It aims at determining if the variance in the spectral pattern

of all the individual entities studied is correlated, or due to

random fluctuations.

Diletta Ami, Paolo Mereghetti and Silvia Maria Doglia, http://dx.doi.org/10.5772/53850

Regression and classification techniques

MULTIVARIATE ANALYSIS FOR FTIR SPECTRA OF

COMPLEX CHEMICAL/BIOLOGICAL SYSTEMS AND PROCESSES

Visual effect of different pre-processing sets on a set of spectra

Some existing FTIR spectroscopy data analysis software

Available at SESAME

Available at SESAME

Available at SESAME

Remote Access authorization for data processing and

analysis for EXPERIENCED USERS

THANK YOU!