Bring real, hands-on EPR experiments and teaching into your … · 2016-03-03 · Bring real, hands-on EPR experiments and teaching into your classroom! Dr. Kalina Ranguelova Application

Bring real, hands-on EPR experiments and teaching into your classroom!

Dr. Kalina Ranguelova Application Scientist, Bruker BioSpin

Dr. Ralph T. Weber Application Scientist, Bruker BioSpin

Webinar - November 5, 2014

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The Next Generation – EPR LabExperiments in the Curriculum

2Innovation with Integrity

What is EPR?

• EPR stands for Electron Paramagnetic Resonance

• It is also called Electron Spin Resonance (ESR)

• EPR is a spectroscopic technique that detects unpaired electrons in paramagnetic substances

• Unpaired electrons occur in free radicals and many transition metals

• EPR is the only technique that unambiguously detects free radicals

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Ascorbic Acid AscorbateRadical

Why are Free Radicals and EPR so Important?

• Free radicals are very common in everyday life

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Why are Free Radicals and EPR so Important?

• Beer: an important EPR application

• EPR is used to study the shelf-life of beer at breweries throughout the world

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Why are Free Radicals and EPR so Important?

• Electrons’ spins are usually paired in orbitals (Pauli Exclusion Principle)

• An unpaired electron results from the electron transfer

• The radical is paramagnetic because the unpaired electron no longer has its magnetic moment canceled by the other electron

• Free radicals can be very reactive and short-lived

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Who uses EPR?Chemistry

• Polymers

• Catalysis

• Redox chemistry

• Electrochemistry

• Photochemistry

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Who uses EPR?Material Science

• Polymers

• Magnetic properties of materials

• Semiconductors

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Who uses EPR?Biology and Medicine

• Free radicals such as ROS and NO in living tissues and fluids

• Antioxidants, radicals scavengers

• Structural biology

• Preclinical EPR imaging such as oximetry

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Hemoglobin-NO

Who uses EPR?Industrial Research

• Food and beverages

• Pet food analysis

• Diamond grading

• Optical fiber defects

• Semiconductors

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Who uses EPR?Ionizing Radiation

• Alanine dosimetry

• Food irradiation

• Archaeology

• Retrospective dosimetry

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Irradiated mango

Alanine dosimeter EPR spectrum of Alanine radical

Why is it Important to Develop an Undergraduate EPR Curriculum?

• EPR offers a wealth of solutions for many scientific problems and questions

• Most undergraduates are not exposed to EPR during their education

• EPR is quite often seen as difficult to understand

and difficult to teach

• Bruker has developed an ideal EPR teaching package

• It offers the students an exciting

and interactive learning experience

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The Bruker EPR Educational Package

• E-scanE bench-top spectrometer

• Educational Powerpointlecture presentation

• EPR Primer and Practicum

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• Two standard samples

• Collection of sample tubes and capillaries

Bruker e-scanEbench-top EPR system

• Sensitivity: ease-of-use

• Rapid automated tuning and measurement

• Software automates data acquisition, processing, and storage

• Dedicated application support

• Easy site-planning, compact footprint (71 50 cm)

• X-band

• Accommodates multiple sample dimensions and types

• Ease of use makes it ideal for undergraduate level analytical labs

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A Brief Powerpoint Lecture

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An EPR Primer

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An EPR PrimerIntroduction to Spectroscopy

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An EPR PrimerBasic Principles of EPR

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An EPR PrimerLineshapes and Linewidths

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An EPR PrimerSpectrometer Parameters

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A Six Section EPR Lab Practicum

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Sample preparation

Spectrum acquisition

Questions for students

A Six Section EPR Lab Practicum

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1. EPR Signals from Real Life Samples

• Detection of stable C-centered free radicals in coffee, cocoa, cinnamon, etc. and e’ center in sand

• How antioxidants work: detection of ascorbate (Vitamin C) free radical

AA AA●

Coffee Sand

Cocoa Cinnamon

Correct CF and SW Incorrect CF

Incorrect SW

1 G 5 G

10 G

1 scan 36 scans

MW Power

2. Instrumental Parameters – How to Choose Them and What is Their Effect on the EPR Spectra?

• Center Field and Sweep Width

• Modulation Amplitude

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• Averaging

• Microwave Power

g = 2.00254

CF = 3477 G

1,4-Benzosemiquinone anion radical

Structure information: • the unpaired electron is

distributed over the whole ring and the coupling with the four protons is the same.

What to expect from the EPRspectrum? Experiment

CF = 3515 G

g = 1.9848

3. Spin Hamiltonian Parameters

• g – factor

• Hyperfine interactions – examples of semiquinone radicals

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3. Spin Hamiltonian Parameters

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3. Spin Hamiltonian Parameters

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0% Glycerol

50% Glycerol

70% Glycerol

1 mM

8 mM

17 mM

4. Lineshape of EPR Spectra

• Effects from molecular motion

• (sample: TEMPOL with glycerol)

• Effects from spin-spin relaxation

• (sample: TEMPOL in H2O)

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Full 3-line spectrum

Calibration curve Triplicate experiments

5. Quantitative EPR – Creating a Calibration Curve with TEMPOL

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6. Kinetics and Quantitative Analysis of Oil Oxidation

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FAH + Initiating Oxidant FA●

(Fatty acid)

Fe,Cuor heat

Initiation

Propagation

Termination

Rancidity(Aldehydes, ketones and alcohols)

(Fatty acid free radical)

AH(Reducedantioxidant)

FAH + A●

O2

FA−OO●

(Fatty acid peroxyl radical)

FAH

FA● + FA−OOH

(Oxidized antioxidant)

(Fatty acid hydroperoxide)

FA−O●

The EPR method detects free radical intermediates during the oxidation process

(Heat, light, Fe, Cu)

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6. Kinetics and Quantitative Analysis of Oil Oxidation

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The middle lines of PBN-radical adducts in oils

• Step 1: Building a calibration curve with Tempol dissolved in oil

• Step 2: Measuring two different types of edible oils (spin-trapping EPR at 70C, 1 hr)

• Step 3: Calculating the free radical concentration

• Step 4: Calculating the rate of free radical formation

2.35 x 106

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y = Aekt

6. Kinetics and Quantitative Analysis of Oil Oxidation

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Bruker e-scanEBench-top EPR System

• We are trying to keep the target price down to make it affordable for an undergraduate lab

• Only accredited educational institutions are eligible

• At the moment the curriculum is only available with the purchase of the instrument

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Materials and ConsumablesSupplied by Bruker

• BDPA sample

• Alanine marker sample

• 4 mm EPR quartz tubes

• 50 and 100 L capillaries

• A list of consumables that the instructor is responsible for providing (with catalog numbers from possible suppliers is provided with the package

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Q & A

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