🧬 L10: NMR, ESR (EPR) & Surface Plasmon Resonance (SPR)

Methods in Biology | CSIR-NET Life Sciences | GATE Biotechnology

Detailed theory notes, principles, instrumentation, interpretation, applications and practice MCQs.

NMR Spectroscopy Chemical Shift Peak Splitting H/D Exchange 2D NMR ESR/EPR SPR

Topic at a glance: Nuclear Magnetic Resonance (NMR), Electron Spin Resonance (ESR/EPR), and Surface Plasmon Resonance (SPR) are powerful biophysical and analytical techniques. NMR provides structural and dynamic information about molecules, ESR detects paramagnetic species containing unpaired electrons, while SPR is primarily used to monitor molecular interactions at or near a sensor surface in real time.

1. Introduction to Biophysical Spectroscopic Methods

Biophysical methods are analytical approaches used to study biological molecules, their structure, dynamics, interactions and physical properties. Proteins, nucleic acids, lipids, metabolites and biomolecular complexes can be investigated using different spectroscopic and physical techniques.

NMR spectroscopy provides information about molecular structure, chemical environment, molecular dynamics and interactions.
ESR/EPR spectroscopy detects species containing unpaired electrons, particularly free radicals and many metal-containing proteins.
SPR detects changes in refractive index near a metal surface and is widely used to study biomolecular binding interactions.
These methods are complementary rather than interchangeable.
NMR is especially powerful for atomic-level structural information in solution.
ESR is particularly useful for paramagnetic species.
SPR is highly useful for real-time interaction analysis without requiring fluorescent or radioactive labeling.

Exam concept: Always associate NMR → nuclear spins, ESR/EPR → unpaired electrons, and SPR → surface plasmon resonance and refractive-index changes near a metal surface.

2. NMR Spectroscopy: Principle

Nuclear Magnetic Resonance is based on the magnetic properties of certain atomic nuclei. Nuclei possessing a non-zero nuclear spin can interact with an external magnetic field. Common biologically relevant NMR-active nuclei include ¹H, ¹³C, ¹⁵N and ³¹P.

2.1 Nuclear Spin

Atomic nuclei contain protons and neutrons.
Some nuclei have intrinsic angular momentum called nuclear spin.
A nucleus with spin quantum number I ≠ 0 can generally be observed by NMR.
¹H has I = 1/2 and is therefore highly suitable for NMR.
¹³C has I = 1/2 but occurs naturally at relatively low abundance.
¹²C has I = 0 and is NMR inactive.
¹⁵N has I = 1/2 and is frequently used in protein NMR.
³¹P has I = 1/2 and is useful for studying nucleotides, phospholipids and phosphorylation.

2.2 Effect of an External Magnetic Field

When a spin-active nucleus is placed in a strong external magnetic field, its allowed spin orientations become energetically differentiated. For a spin-1/2 nucleus, two principal energy states are commonly considered: a lower-energy state aligned with the applied field and a higher-energy state opposed to it.

ΔE = hν

The energy difference between the spin states is related to the frequency of electromagnetic radiation required for resonance. The exact resonance frequency depends on the magnetic field and the gyromagnetic properties of the nucleus.

2.3 Resonance

When radiofrequency energy matches the energy gap between allowed spin states, resonance occurs.
The nucleus absorbs energy and changes its spin state.
The detected signal is converted into an NMR spectrum.
The spectrum contains information about the chemical environment of nuclei.

CSIR-NET tip: NMR does not simply identify an element. It provides information about the chemical environment of NMR-active nuclei.

3. NMR Instrumentation

A modern NMR spectrometer consists of several important components that work together to generate and detect nuclear resonance signals.

Strong magnet: produces the static magnetic field.
Probe: holds the sample and contains radiofrequency coils.
RF transmitter: delivers radiofrequency energy to the sample.
RF receiver: detects the emitted NMR signal.
Shim system: improves magnetic-field homogeneity.
Lock system: helps stabilize the magnetic field during acquisition.
Computer/data system: processes the free induction decay and generates the spectrum.

3.1 Sample Preparation

Samples are commonly dissolved in an appropriate solvent.
Deuterated solvents are frequently used for proton NMR.
Deuterated solvents reduce interference from solvent proton signals.
They can also provide a signal useful for magnetic-field locking.
Sample concentration depends on the type of experiment and sensitivity required.
Biological NMR may require buffering, controlled pH, suitable ionic strength and stable temperature.

4. Chemical Shift

One of the most important concepts in NMR is the chemical shift. Nuclei in different chemical environments do not experience exactly the same effective magnetic field because surrounding electrons shield them from the external magnetic field.

4.1 Shielding and Deshielding

Shielding: surrounding electrons reduce the effective magnetic field experienced by the nucleus.
Deshielding: reduced electron density causes the nucleus to experience a greater effective magnetic field.
Electron-withdrawing groups often cause proton signals to move downfield.
Electron-donating groups can contribute to increased shielding.
Aromatic ring currents can strongly influence proton chemical shifts.

δ = [(ν_sample − ν_reference) / ν_reference] × 10⁶ ppm

Chemical shifts are usually reported in ppm. Reporting in ppm makes chemical-shift values more comparable between instruments operating at different magnetic field strengths.

4.2 Reference Compound

Tetramethylsilane, commonly abbreviated TMS, is traditionally used as a reference for proton and carbon NMR chemical shifts and is assigned a chemical shift of 0 ppm under standard conventions.

Remember: A larger chemical-shift value in a typical ¹H NMR spectrum generally corresponds to a more downfield position and often indicates a more deshielded proton.

4.3 Factors Affecting Chemical Shift

Electronegativity of neighboring atoms.
Hybridization.
Hydrogen bonding.
Aromatic ring currents.
Solvent.
Temperature.
pH.
Conformation and molecular structure.
Proton exchange.

5. Number of NMR Peaks

The number of signals in an NMR spectrum depends on the number of chemically non-equivalent nuclei.

Chemically equivalent nuclei generally give the same signal.
Chemically non-equivalent nuclei give different signals.
Molecular symmetry can reduce the number of signals.
Equivalent protons usually have the same chemical shift in an achiral environment.
Different environments can produce multiple resonances even within the same type of atom.

Example: Ethanol

Ethanol, CH₃CH₂OH, typically produces separate resonances for the methyl group, methylene group and hydroxyl proton under appropriate conditions.

CH₃ protons → one environment.
CH₂ protons → another environment.
OH proton → another environment.

Therefore, a simple proton NMR spectrum of ethanol can show approximately three major proton environments, although exchange and experimental conditions can affect the appearance of the OH signal.

6. Peak Splitting and Spin-Spin Coupling

NMR signals can be split because magnetic nuclei interact with neighboring non-equivalent nuclei. This phenomenon is called spin-spin coupling.

6.1 The n + 1 Rule

For a simple first-order spectrum, a proton coupled to n equivalent neighboring protons can appear as approximately n + 1 lines.

0 neighboring protons → singlet.
1 neighboring proton → doublet.
2 neighboring protons → triplet.
3 neighboring protons → quartet.
4 neighboring protons → quintet.
5 neighboring protons → sextet.
6 neighboring protons → septet.

6.2 Coupling Constant

The spacing between lines in a multiplet is related to the coupling constant, J, usually expressed in Hz.

J describes the magnitude of spin-spin coupling.
It is measured as a frequency difference.
Coupling constants provide structural information.
Different types of coupling can show characteristic values.

Important: The n + 1 rule is a useful introductory rule, but real biological molecules frequently produce complex multiplets because of multiple coupled nuclei, non-equivalent neighbors and higher-order effects.

7. H/D Exchange

Hydrogen/deuterium exchange is an important concept in NMR, especially for identifying exchangeable protons and studying protein structure.

Exchangeable hydrogen atoms can be replaced by deuterium.
Common exchangeable groups include OH, NH and SH groups.
D₂O can be used to test whether a signal belongs to an exchangeable proton.
After addition of D₂O, an exchangeable proton may disappear from a ¹H NMR spectrum because deuterium is not normally observed in a conventional proton spectrum.
Exchange rates depend on pH, temperature, solvent and molecular environment.

7.1 Protein NMR and H/D Exchange

Hydrogen-deuterium exchange can also provide information about protein dynamics and solvent accessibility. Amide hydrogens involved in stable hydrogen bonding or buried within a protein structure can exchange more slowly than exposed hydrogens.

Exam shortcut: If an NMR proton peak disappears after D₂O treatment, suspect an exchangeable proton, such as OH or NH.

8. 2D NMR

One-dimensional NMR spectra can become very complicated for proteins and other large biological molecules. Two-dimensional NMR techniques provide an additional frequency dimension and allow correlations between nuclei to be analyzed.

8.1 Basic Concept

A 2D NMR spectrum has two frequency axes.
Cross-peaks indicate relationships or correlations between nuclei.
Different 2D experiments provide different types of information.
2D NMR is extensively used for biological macromolecules.

8.2 COSY

COSY stands for Correlation Spectroscopy.
It mainly detects through-bond scalar coupling.
It is useful for identifying coupled proton networks.
Diagonal peaks correspond to the same nuclei along both axes.
Cross-peaks reveal coupled nuclei.

8.3 NOESY

NOESY stands for Nuclear Overhauser Effect Spectroscopy.
It provides information related to spatial proximity between nuclei.
NOE correlations are particularly useful for structural biology.
NOESY can help determine three-dimensional arrangements of atoms in proteins and nucleic acids.

8.4 HSQC

HSQC stands for Heteronuclear Single Quantum Coherence.
It correlates two different types of nuclei.
A common experiment correlates ¹H with ¹⁵N.
Protein backbone amide resonances are often observed in ¹H-¹⁵N HSQC spectra.
It is sometimes described as a protein "fingerprint" spectrum because many residues generate characteristic signals.

8.5 HMBC

HMBC provides longer-range heteronuclear correlations.
It can help connect fragments of a molecule through multiple bonds.

Technique	Main Information
COSY	Through-bond proton-proton coupling
NOESY	Through-space proximity / NOE
HSQC	Heteronuclear one-bond correlations, commonly ¹H–¹⁵N
HMBC	Longer-range heteronuclear correlations

High-yield distinction: COSY → coupling; NOESY → spatial proximity; HSQC → heteronuclear correlation.

9. Applications of NMR in Biology

Determination of three-dimensional structures of proteins.
Analysis of nucleic acid structures.
Investigation of protein folding and unfolding.
Study of protein-ligand interactions.
Metabolomics.
Analysis of metabolic pathways.
Detection and characterization of small molecules.
Study of molecular dynamics.
Measurement of conformational changes.
Analysis of protein-protein interactions.
Study of membrane-associated molecules.
Investigation of enzyme mechanisms.

NMR has an important advantage because samples can often be studied in solution under conditions that preserve biologically relevant conformations. However, sensitivity can be lower than some other analytical methods, and large macromolecules can generate highly complex spectra.

10. ESR/EPR Spectroscopy

Electron Spin Resonance, also called Electron Paramagnetic Resonance (EPR), is a spectroscopic method used to study species containing unpaired electrons.

Definition: ESR/EPR measures transitions between electron spin states in the presence of an external magnetic field.

10.1 What Can ESR Detect?

Free radicals.
Organic radicals.
Reactive oxygen species containing unpaired electrons.
Transition-metal ions with unpaired electrons.
Paramagnetic metal centers in proteins.
Defects in solids and biomaterials.
Spin-labeled biological molecules.

10.2 Paramagnetic vs Diamagnetic

Paramagnetic species: contain one or more unpaired electrons.
Diamagnetic species: have all electrons paired.
ESR/EPR is mainly sensitive to paramagnetic species.

A molecule does not have to be large or complex to be studied by EPR. The key requirement is the presence of an appropriate unpaired electron.

11. ESR Principle and Instrumentation

In ESR, an unpaired electron has spin. When a paramagnetic species is placed in an external magnetic field, electron spin energy levels split. Microwave radiation can induce transitions between these levels when the resonance condition is satisfied.

ΔE = hν = gβB

Here, g is the electron g-factor, β is the Bohr magneton, and B represents the magnetic field under the simplified resonance relationship.

11.1 Main Components

Magnet.
Microwave source.
Resonant cavity.
Sample holder.
Magnetic-field modulation system.
Detector.
Computer/data-processing system.

11.2 Why Microwaves?

Electron spin energy separations under typical magnetic fields correspond to microwave frequencies. Therefore ESR/EPR instruments commonly operate in the microwave region.

11.3 Hyperfine Splitting

An unpaired electron can interact magnetically with nearby magnetic nuclei. This interaction can split an ESR signal into multiple lines. This is called hyperfine splitting.

Hyperfine structure provides information about the local environment of the unpaired electron.
The number and spacing of lines can provide information about interacting nuclei.
For simple cases, an electron interacting with equivalent nuclei can generate characteristic multiplets.
Hyperfine coupling is highly useful in identifying radical species and metal centers.

12. Applications of ESR/EPR in Biology

Detection of free radicals.
Study of oxidative stress.
Investigation of reactive oxygen species.
Characterization of metalloproteins.
Study of iron, copper and manganese-containing systems.
Analysis of enzyme active sites containing paramagnetic centers.
Investigation of electron-transfer reactions.
Study of radical intermediates in enzymatic reactions.
Spin-labeling studies of proteins and membranes.
Investigation of protein conformational changes.

CSIR-NET/GATE shortcut: If a question asks which technique is most directly suited to detecting a free radical, think ESR/EPR.

13. Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance is an optical technique used extensively for studying biomolecular interactions. Unlike NMR and EPR, SPR does not rely on nuclear or electron spin resonance.

SPR detects changes in the optical properties near the surface of a thin metal film, commonly involving gold. When molecules bind to an immobilized partner on the sensor surface, the local refractive index changes. This produces a measurable shift in the SPR response.

13.1 Important Terms

Ligand: one binding partner immobilized or presented on the sensor surface.
Analyte: the molecule flowing over the sensor surface and potentially binding to the ligand.
Sensor chip: contains the metal surface and immobilization chemistry.
Response: optical signal generated as binding changes the local refractive environment.
Association: formation of ligand-analyte complex.
Dissociation: release of analyte from the ligand.

14. Principle and Instrumentation of SPR

In a typical SPR instrument, polarized light interacts with a thin metal film under conditions that allow surface plasmons to be excited. The resonance condition is sensitive to the refractive index near the metal surface.

When an analyte binds to a ligand near the surface, the local mass density and refractive index change. This alters the resonance condition and generates a measurable response.

14.1 Label-Free Detection

One major advantage of SPR is that binding can be measured without directly labeling the analyte with a fluorescent or radioactive tag. This allows real-time monitoring of interactions.

14.2 Real-Time Measurement

Sample is passed over the sensor surface.
Binding causes a response increase.
When analyte flow is replaced with buffer, dissociation can be monitored.
The resulting curve is called a sensorgram.
Association and dissociation phases can be analyzed mathematically.

15. SPR Sensorgram

A typical SPR sensorgram plots response against time.

15.1 Association

During association, analyte molecules bind to available ligand molecules on the sensor surface. The response generally increases as complexes form.

15.2 Dissociation

During dissociation, analyte-containing solution is replaced by running buffer. Bound analyte molecules begin to dissociate, and the SPR response decreases.

15.3 Kinetic Constants

For a simple 1:1 interaction:

Ligand + Analyte ⇌ Ligand–Analyte

k_a = association rate constant.
k_d = dissociation rate constant.
K_D = equilibrium dissociation constant.

K_D = k_d / k_a

A lower K_D generally indicates higher apparent binding affinity for a simple binding model.

Very important: Lower K_D → generally stronger affinity. Higher K_D → generally weaker affinity.

16. Applications of SPR

Protein-protein interaction studies.
Protein-DNA interaction analysis.
Protein-RNA interaction studies.
Antibody-antigen binding.
Receptor-ligand interactions.
Small-molecule drug screening.
Measurement of binding affinity.
Measurement of association and dissociation kinetics.
Characterization of biosensors.
Analysis of biomolecular recognition.
Investigation of therapeutic antibodies.
Comparative screening of ligands.

16.1 Advantages of SPR

Label-free detection.
Real-time measurement.
Quantitative kinetic information.
Can determine association and dissociation behavior.
Can estimate affinity constants.
Useful for many classes of biomolecular interactions.

16.2 Limitations

Immobilization may sometimes affect biological activity.
Non-specific binding can complicate interpretation.
Mass transport limitations can influence observed kinetics.
Surface chemistry must be carefully controlled.
Very small molecules may produce relatively small signals.
Appropriate reference surfaces and controls are important.

17. NMR vs ESR/EPR vs SPR

Feature	NMR	ESR/EPR	SPR
Primary principle	Nuclear spin resonance	Electron spin resonance	Surface plasmon resonance
Main target	NMR-active nuclei	Paramagnetic species	Surface biomolecular interactions
Key requirement	Non-zero nuclear spin	Unpaired electron	Optically active metal sensor surface and binding event
Typical radiation	Radiofrequency	Microwave	Optical light
Major information	Structure, environment, dynamics	Radicals, metal centers, electron environment	Binding, kinetics and affinity
Label-free?	Generally yes	Yes for naturally paramagnetic samples	Yes
Important parameter	Chemical shift, J coupling	g-value, hyperfine coupling	k_a, k_d, K_D

18. Important CSIR-NET/GATE Examination Points

NMR-active nucleus: generally has non-zero nuclear spin.
¹²C: NMR inactive because I = 0.
¹H: highly sensitive and widely used in NMR.
TMS: conventional reference at 0 ppm.
Chemical shift: reported in ppm.
Downfield: generally higher δ value.
Spin-spin coupling: causes signal splitting.
n + 1 rule: simple first-order approximation.
D₂O exchange: useful for identifying exchangeable H.
COSY: through-bond coupling correlations.
NOESY: through-space proximity information.
HSQC: heteronuclear correlations, commonly H-N.
ESR/EPR: detects unpaired electrons.
Hyperfine splitting: electron-nuclear magnetic interaction.
SPR: detects changes in refractive index near a metal surface.
SPR sensorgram: response versus time.
Association: binding phase.
Dissociation: release phase.
K_D = k_d/k_a for a simple binding model.
Lower K_D: generally higher affinity.

19. Conceptual Questions for Self-Study

Why is NMR called a magnetic resonance technique?

Because the experiment involves transitions between magnetic spin states of nuclei in an applied magnetic field when electromagnetic radiation of the appropriate frequency is supplied.

Why is ESR different from NMR?

NMR detects transitions involving nuclear spin states, whereas ESR/EPR detects transitions involving electron spin states. Electron spin transitions are associated with much larger magnetic moments and therefore typically occur at microwave frequencies under conventional EPR conditions.

Why is SPR useful for drug discovery?

SPR can rapidly characterize interactions between candidate compounds and target molecules. It can provide binding responses and, under suitable experimental conditions and models, association and dissociation kinetics and affinity estimates.

Why does D₂O help identify exchangeable protons?

Exchangeable protons can exchange with deuterium from D₂O. Because deuterium is not normally detected in a standard proton NMR experiment, the corresponding proton resonance can decrease or disappear.

20. CSIR-NET / GATE Practice MCQs

Instructions: Select one option for each question and click Submit Quiz. The correct answers and explanations are intentionally hidden until submission.

Q1. Which nucleus is normally NMR inactive because its nuclear spin quantum number is zero?

A. ¹H B. ¹³C C. ¹²C D. ³¹P

Correct answer: C. ¹²C
¹²C has nuclear spin I = 0 and therefore does not produce a conventional NMR resonance.

Q2. In a simple first-order ¹H NMR spectrum, a proton coupled to two equivalent neighboring protons is expected to appear as:

A. Singlet B. Doublet C. Triplet D. Quartet

Correct answer: C. Triplet
Using the simple n + 1 rule, two equivalent neighboring protons give 2 + 1 = 3 lines, producing a triplet.

Q3. Which technique is most directly associated with detection of free radicals?

A. ESR/EPR B. SDS-PAGE C. Agarose electrophoresis D. Southern blotting

Correct answer: A. ESR/EPR
Free radicals contain unpaired electrons and are therefore suitable targets for electron spin resonance spectroscopy.

Q4. Which statement about chemical shift is correct?

A. It is normally reported in ppm B. It is always reported in volts C. It measures protein molecular weight directly D. It is independent of chemical environment

Correct answer: A. It is normally reported in ppm
Chemical shift is a dimensionless normalized frequency difference and is conventionally expressed in parts per million.

Q5. Which 2D NMR technique is particularly useful for observing spatial proximity between nuclei through the nuclear Overhauser effect?

A. COSY B. NOESY C. SDS-PAGE D. ELISA

Correct answer: B. NOESY
NOESY detects nuclear Overhauser effect correlations and can provide information about nuclei that are close in space.

Q6. In a simple SPR experiment, what usually causes an increase in response during the association phase?

A. Binding of analyte to immobilized ligand B. Destruction of the sensor surface C. Removal of all molecules from the surface D. Loss of the metal film

Correct answer: A. Binding of analyte to immobilized ligand
During association, analyte binds to ligand on the sensor surface, changing the local refractive environment and increasing the response.

Q7. For a simple 1:1 binding interaction, which relationship is correct?

A. K_D = k_a/k_d B. K_D = k_d/k_a C. K_D = k_a × k_d D. K_D = k_a − k_d

Correct answer: B. K_D = k_d/k_a
For the simple 1:1 model, equilibrium dissociation constant is related to the dissociation and association rate constants by K_D = k_d/k_a.

Q8. Addition of D₂O to a proton NMR sample causes a signal to disappear. Which group is most likely responsible?

A. A non-exchangeable alkyl proton B. An exchangeable OH proton C. A carbon atom D. A methyl carbon

Correct answer: B. An exchangeable OH proton
OH, NH and SH protons can exchange with deuterium. The resulting deuterated species generally does not appear in a standard proton spectrum.

Q9. Which technique primarily detects changes in refractive index close to a metal surface?

A. NMR B. ESR C. SPR D. PCR

Correct answer: C. SPR
SPR monitors optical changes associated with the local refractive environment near a suitable metal surface.

Q10. Which statement is most appropriate regarding K_D in a simple binding system?

A. Lower K_D generally indicates stronger affinity B. Higher K_D always means stronger affinity C. K_D is unrelated to affinity D. K_D is always equal to zero

Correct answer: A. Lower K_D generally indicates stronger affinity
For comparable simple binding models, a lower equilibrium dissociation constant corresponds to a greater tendency of the interacting partners to remain associated.

21. Quick Revision Sheet

🧬 NMR

NMR studies nuclei with non-zero nuclear spin.
¹H, ¹³C, ¹⁵N and ³¹P are important biological NMR nuclei.
¹²C is NMR inactive because I = 0.
External magnetic field separates spin states.
RF radiation produces resonance.
Chemical shift is reported in ppm.
TMS is conventionally assigned 0 ppm.
Shielding and deshielding influence chemical shift.
Spin-spin coupling causes splitting.
Simple n + 1 rule predicts multiplicity.
D₂O exchange identifies exchangeable protons.
COSY → through-bond coupling.
NOESY → spatial proximity.
HSQC → heteronuclear correlations.

⚡ ESR/EPR

ESR = Electron Spin Resonance.
EPR = Electron Paramagnetic Resonance.
Detects species with unpaired electrons.
Useful for free radicals.
Useful for paramagnetic metal centers.
Microwave radiation is used.
Hyperfine coupling gives information about electron-nuclear interactions.
g-value provides information about the electronic environment.

🔬 SPR

SPR = Surface Plasmon Resonance.
It is an optical technique.
Commonly uses a thin metal sensor surface.
Detects refractive-index changes near the surface.
Useful for real-time interaction analysis.
Can be label-free.
Sensorgram plots response versus time.
Association = binding phase.
Dissociation = release phase.
k_a = association rate constant.
k_d = dissociation rate constant.
K_D = k_d/k_a for a simple 1:1 model.
Lower K_D generally indicates stronger affinity.

Final CSIR-NET/GATE memory trick:

🧬 NMR → Nuclear spin → RF → Chemical shift → J coupling → Molecular structure
⚡ ESR/EPR → Electron spin → Microwave → Unpaired electrons → Radicals/metal centers
🔬 SPR → Surface plasmon → Optical signal → Binding → Kinetics/affinity

End of L10: NMR, ESR (EPR) & Surface Plasmon Resonance Notes
Prepared for CSIR-NET Life Sciences & GATE Biotechnology preparation.

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NMR, ESR (EPR) & SPR SPECTROSCOPY