SPECTROSCOPY OF
BIOMOLECULES
G0O58a
2025-2026
SCHAKELJAAR BIOCHEMIE EN BIOTECHNOLOGIE
Lowie Boels
,Spectroscopy of biomolecules Lowie Boels
TABLE OF CONTENTS
1. THE ORIGIN OF SPECTROSCOPY ....................................................................................................................... 5
1. THE EM SPECTRA ................................................................................................................................................. 5
2. THE BIRTH OF SPECTROSCOPY.................................................................................................................................. 5
The Photoelectric effect ........................................................................................................................................ 6
3. THE NATURE OF LIGHT ............................................................................................................................................ 6
Wave particle duality ............................................................................................................................................ 6
4. ATOMIC SPECTROSCOPY ........................................................................................................................................ 7
5. ENERGY LEVELS IN THE HYDROGEN ATOM .................................................................................................................... 7
6. WAVE CHARACTER OF MATTER ................................................................................................................................. 9
The wave nature of matter particles ....................................................................................................................... 9
Wave mechanical treatment of the atom: the Schrödinger equation ...................................................................... 10
Description of the energy levels via wave mechanics ............................................................................................ 10
Examples for a hydrogen atom ............................................................................................................................ 10
Relation between orbitals and symmetry ............................................................................................................. 11
Relation between orbitals and line spectra........................................................................................................... 11
Transitions between different wavefunctions by light absorption ........................................................................... 12
2. MOLECULAR ORBITALS AND ENERGY LEVELS IN MOLECULES.......................................................................... 13
1. MOLECULAR ORBITALS ............................................................................................................................................. 13
Molecular spectra .............................................................................................................................................. 13
Energy levels: molecular orbitals ......................................................................................................................... 13
Shape of molecular orbitals ................................................................................................................................ 14
Examples ........................................................................................................................................................... 14
2. CHROMOPHORES ................................................................................................................................................... 16
The energy levels in ethylene ............................................................................................................................... 16
Conjugated dienes (butadiene) ........................................................................................................................... 16
Aldehydes and ketons (formaldehyde) ................................................................................................................. 17
Combination of chemical structure elements: ENONS ......................................................................................... 18
Substitution effects ............................................................................................................................................ 18
3. ELECTRON IN A BOX MODEL ....................................................................................................................................... 19
Particle in a Box .................................................................................................................................................. 19
Application to Organic Molecules ........................................................................................................................ 19
Azacyanines....................................................................................................................................................... 20
Aromatic molecules ........................................................................................................................................... 20
Hetero-aromatics ............................................................................................................................................... 21
Quinones ........................................................................................................................................................... 21
4. STRENGTH OF ABSORPTION BANDS .............................................................................................................................. 21
The Lambert-Beer Law ........................................................................................................................................ 21
3. FLUORESCENCE ..................................................................................................................................... 22
1. HISTORY .............................................................................................................................................................. 22
2. INTRODUCTION TO FLUORESCENCE ............................................................................................................................. 22
Absorption of light .............................................................................................................................................. 22
Emission of light: fluorescence............................................................................................................................ 23
Kasha’s rule ....................................................................................................................................................... 23
Jablonski diagram............................................................................................................................................... 24
Stokes shift ........................................................................................................................................................ 25
3. WHY USE FLUORESCENCE? ...................................................................................................................................... 25
Sensitivity .......................................................................................................................................................... 25
Decreased popularity of radioactivity ................................................................................................................... 25
Appearance of many new fluorescent probes....................................................................................................... 26
1
,Spectroscopy of biomolecules Lowie Boels
Introduction of genetically encoded fluorophores................................................................................................. 26
4. CHROMOPHORES AND FLUOROPHORES ....................................................................................................................... 26
Intrinsic chromophores ...................................................................................................................................... 26
Extrinsic chromophores ...................................................................................................................................... 27
5. HOW DO WE MEASURE ABSORPTION AND FLUORESCENCE................................................................................................. 27
Absorption Measurement (Spectrophotometry).................................................................................................... 27
Fluorescence Measurement (Fluorometry) .......................................................................................................... 28
Emission vs. Excitation Spectra ........................................................................................................................... 28
6. FLUORESCENCE QUANTUM YIELD AND FLUORESCENCE LIFETIME ......................................................................................... 28
Fluorescence quantum yield ............................................................................................................................... 28
Fluorescence lifetime ......................................................................................................................................... 30
Time-Resolved Emission Spectroscopy (TRES) ..................................................................................................... 31
7. EXCIMERS, EXCIPLEX AND AGGREGATES ....................................................................................................................... 32
4. QUENCHING PROCESSES IN FLUORESCENCE: COLLISIONAL QUENCHING AND ENERGY TRANSFER................. 33
1. FLUORESCENCE QUENCHING .................................................................................................................................... 33
2. ENERGY TRANSFER MECHANISMS ............................................................................................................................... 34
Biological Applications of FRET ........................................................................................................................... 35
3. ANISOTROPY ......................................................................................................................................................... 36
Definition and terms ........................................................................................................................................... 36
Experimental determination of anisotropy............................................................................................................ 37
Derivation of equations ....................................................................................................................................... 38
Causes of depolarisation .................................................................................................................................... 39
Time resolved anisotropy .................................................................................................................................... 40
5. CIRCULAR DICHROISM ................................................................................................................................... 42
1. INTRODUCTION TO CIRCULAR DICHROISM .................................................................................................................... 42
2. MODES OF POLARIZATION ......................................................................................................................................... 42
3. MOLECULAR ORIGIN OF CD AND ORD ........................................................................................................................ 43
4. SAMPLE PREPARATION ............................................................................................................................................. 44
6. RAMAN SPECTROSCOPY ................................................................................................................................. 46
1. MOLECULAR SPECTROSCOPY: LIGHT SCATTERING ........................................................................................................... 46
2. VIBRATIONAL MICROSCOPY: PHOTON PICTURE ............................................................................................................... 47
3. RAMAN SCATTERING: CLASSICAL PICTURE .................................................................................................................... 47
4. THE RAMAN EFFECT: INELASTIC SCATTERING ................................................................................................................. 48
Vibrations in polyatomic molecules & Rule of Mutual Exclusion ............................................................................ 48
5. RAMAN VS INFRARED SPECTRUM ................................................................................................................................. 49
6. ADVANTAGES AND DISADVANTAGES OF RAMAN SPECTROSCOPY ......................................................................................... 50
7. SPACE/TIME-RESOLVED RAMAN SPECTRA OF LIVING CELLS ................................................................................................ 50
7. INFRARED SPECTROSCOPY ............................................................................................................................ 51
1. MOLECULAR SPECTROSCOPY BASICS .......................................................................................................................... 51
2. FUNDAMENTALS AND SCALE ...................................................................................................................................... 51
Electric Dipole Moment ...................................................................................................................................... 51
Harmonic vs. Anharmonic Oscillators ................................................................................................................. 52
Characteristic Vibrational Frequencies ................................................................................................................ 53
3. INFRARED MEASUREMENTS AND INSTRUMENTATION......................................................................................................... 54
Fourier Transform IR (FT-IR) Instrumentation ........................................................................................................ 54
ATR: Attenuated Total Internal Reflection ............................................................................................................. 55
Purpose, Advantages, and Disadvantages of IR .................................................................................................... 55
Interpreting Infrared Spectra & Regions................................................................................................................ 56
4. APPLICATIONS OF IR SPECTROSCOPY ........................................................................................................................... 56
Identification of substances ................................................................................................................................ 56
Determination of molecular structure .................................................................................................................. 56
2
, Spectroscopy of biomolecules Lowie Boels
Quantitative Analysis using IR ............................................................................................................................. 58
Biological Applications of IR Spectroscopy .......................................................................................................... 59
LES 1: ................................................................................................................................................................ 60
1. THE ELECTROMAGNETIC SPECTRUM AND DIFFERENT TYPES OF SPECTROSCOPY ...................................................................... 60
2. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY .......................................................................................................... 62
The Evolution and Application of NMR Spectroscopy ............................................................................................ 62
The Physical Origins of NMR: Spin and Magnetic Fields ......................................................................................... 62
Nuclear Spin Groupings and Isotopes .................................................................................................................. 62
Multiplicity and The Zeeman Effect ...................................................................................................................... 63
Energy Transitions and Resonance ...................................................................................................................... 64
The Boltzmann Distribution and The Sensitivity Problem ....................................................................................... 64
Strategies to Overcome Low Sensitivity ................................................................................................................ 65
3. EQUIVALENCE AND SYMMETRY ............................................................................................................................... 67
LES 2: ................................................................................................................................................................ 70
1. SPIN-SPIN COUPLING .............................................................................................................................................. 70
Spin-spin coupling: AX spin system ..................................................................................................................... 70
Spin-spin coupling: A2X and A2X3 systems .......................................................................................................... 71
Pascal's triangle & General Rules ........................................................................................................................ 72
Splitting by two non-equivalent nuclei.................................................................................................................. 72
The Order of a Spectrum ..................................................................................................................................... 73
Structural Information from Coupling Constants (J) .............................................................................................. 73
Long Range Coupling & Exceptional Cases .......................................................................................................... 74
2. 13C NMR SPECTROSCOPY....................................................................................................................................... 75
Correlating Chemical Shift with Molecular Structure ............................................................................................ 75
Calculating Chemical Shifts ................................................................................................................................ 76
Key Differences: 1H vs. 13C NMR ........................................................................................................................ 77
3. FACTORS THAT INFLUENCE THE APPEARANCE OF 13C NMR SPECTRA ................................................................................... 78
RELAXATION ...................................................................................................................................................... 78
Decoupling ........................................................................................................................................................ 80
LES 3: ................................................................................................................................................................ 82
1. NUCLEAR OVERHAUSER EFFECT (NOE) ....................................................................................................................... 82
2. INTERPRETING 13C SPECTRA (DEPT & APT) ................................................................................................................. 83
3. 2D NMR SPECTROSCOPY: AN IN-DEPTH OVERVIEW ....................................................................................................... 84
COSY ................................................................................................................................................................. 84
TOCSY ............................................................................................................................................................... 86
2D Heteronuclear Correlation Spectroscopy (HETCOR) ........................................................................................ 86
HSQC (Heteronuclear Single Quantum Correlation) ............................................................................................. 87
2D-NOESY ......................................................................................................................................................... 88
4. STUDYING DYNAMIC PROCESSES BY NMR ................................................................................................................. 88
The Time Scale of Spectroscopic Measurements.................................................................................................. 88
Dynamic Chemical Processes ............................................................................................................................ 89
LES 4: ADVANCED APPLICATION OF NMR ........................................................................................................... 93
1. HISTORY OF PROTEIN NMR ....................................................................................................................................... 93
2. THE "WATER PROBLEM" ........................................................................................................................................... 93
3. SOURCES OF PROTEIN FOR NMR STUDIES ..................................................................................................................... 94
4. FROM SPECTRUM TO STRUCTURE ................................................................................................................................ 94
The Assignment Problem and Magnetization Transfer ........................................................................................... 94
Multidimensional NMR ....................................................................................................................................... 94
To know before starting the experiments .............................................................................................................. 95
The 3 phases ...................................................................................................................................................... 96
5. THE LIMITS OF PROTEIN NMR .................................................................................................................................. 102
3
BIOMOLECULES
G0O58a
2025-2026
SCHAKELJAAR BIOCHEMIE EN BIOTECHNOLOGIE
Lowie Boels
,Spectroscopy of biomolecules Lowie Boels
TABLE OF CONTENTS
1. THE ORIGIN OF SPECTROSCOPY ....................................................................................................................... 5
1. THE EM SPECTRA ................................................................................................................................................. 5
2. THE BIRTH OF SPECTROSCOPY.................................................................................................................................. 5
The Photoelectric effect ........................................................................................................................................ 6
3. THE NATURE OF LIGHT ............................................................................................................................................ 6
Wave particle duality ............................................................................................................................................ 6
4. ATOMIC SPECTROSCOPY ........................................................................................................................................ 7
5. ENERGY LEVELS IN THE HYDROGEN ATOM .................................................................................................................... 7
6. WAVE CHARACTER OF MATTER ................................................................................................................................. 9
The wave nature of matter particles ....................................................................................................................... 9
Wave mechanical treatment of the atom: the Schrödinger equation ...................................................................... 10
Description of the energy levels via wave mechanics ............................................................................................ 10
Examples for a hydrogen atom ............................................................................................................................ 10
Relation between orbitals and symmetry ............................................................................................................. 11
Relation between orbitals and line spectra........................................................................................................... 11
Transitions between different wavefunctions by light absorption ........................................................................... 12
2. MOLECULAR ORBITALS AND ENERGY LEVELS IN MOLECULES.......................................................................... 13
1. MOLECULAR ORBITALS ............................................................................................................................................. 13
Molecular spectra .............................................................................................................................................. 13
Energy levels: molecular orbitals ......................................................................................................................... 13
Shape of molecular orbitals ................................................................................................................................ 14
Examples ........................................................................................................................................................... 14
2. CHROMOPHORES ................................................................................................................................................... 16
The energy levels in ethylene ............................................................................................................................... 16
Conjugated dienes (butadiene) ........................................................................................................................... 16
Aldehydes and ketons (formaldehyde) ................................................................................................................. 17
Combination of chemical structure elements: ENONS ......................................................................................... 18
Substitution effects ............................................................................................................................................ 18
3. ELECTRON IN A BOX MODEL ....................................................................................................................................... 19
Particle in a Box .................................................................................................................................................. 19
Application to Organic Molecules ........................................................................................................................ 19
Azacyanines....................................................................................................................................................... 20
Aromatic molecules ........................................................................................................................................... 20
Hetero-aromatics ............................................................................................................................................... 21
Quinones ........................................................................................................................................................... 21
4. STRENGTH OF ABSORPTION BANDS .............................................................................................................................. 21
The Lambert-Beer Law ........................................................................................................................................ 21
3. FLUORESCENCE ..................................................................................................................................... 22
1. HISTORY .............................................................................................................................................................. 22
2. INTRODUCTION TO FLUORESCENCE ............................................................................................................................. 22
Absorption of light .............................................................................................................................................. 22
Emission of light: fluorescence............................................................................................................................ 23
Kasha’s rule ....................................................................................................................................................... 23
Jablonski diagram............................................................................................................................................... 24
Stokes shift ........................................................................................................................................................ 25
3. WHY USE FLUORESCENCE? ...................................................................................................................................... 25
Sensitivity .......................................................................................................................................................... 25
Decreased popularity of radioactivity ................................................................................................................... 25
Appearance of many new fluorescent probes....................................................................................................... 26
1
,Spectroscopy of biomolecules Lowie Boels
Introduction of genetically encoded fluorophores................................................................................................. 26
4. CHROMOPHORES AND FLUOROPHORES ....................................................................................................................... 26
Intrinsic chromophores ...................................................................................................................................... 26
Extrinsic chromophores ...................................................................................................................................... 27
5. HOW DO WE MEASURE ABSORPTION AND FLUORESCENCE................................................................................................. 27
Absorption Measurement (Spectrophotometry).................................................................................................... 27
Fluorescence Measurement (Fluorometry) .......................................................................................................... 28
Emission vs. Excitation Spectra ........................................................................................................................... 28
6. FLUORESCENCE QUANTUM YIELD AND FLUORESCENCE LIFETIME ......................................................................................... 28
Fluorescence quantum yield ............................................................................................................................... 28
Fluorescence lifetime ......................................................................................................................................... 30
Time-Resolved Emission Spectroscopy (TRES) ..................................................................................................... 31
7. EXCIMERS, EXCIPLEX AND AGGREGATES ....................................................................................................................... 32
4. QUENCHING PROCESSES IN FLUORESCENCE: COLLISIONAL QUENCHING AND ENERGY TRANSFER................. 33
1. FLUORESCENCE QUENCHING .................................................................................................................................... 33
2. ENERGY TRANSFER MECHANISMS ............................................................................................................................... 34
Biological Applications of FRET ........................................................................................................................... 35
3. ANISOTROPY ......................................................................................................................................................... 36
Definition and terms ........................................................................................................................................... 36
Experimental determination of anisotropy............................................................................................................ 37
Derivation of equations ....................................................................................................................................... 38
Causes of depolarisation .................................................................................................................................... 39
Time resolved anisotropy .................................................................................................................................... 40
5. CIRCULAR DICHROISM ................................................................................................................................... 42
1. INTRODUCTION TO CIRCULAR DICHROISM .................................................................................................................... 42
2. MODES OF POLARIZATION ......................................................................................................................................... 42
3. MOLECULAR ORIGIN OF CD AND ORD ........................................................................................................................ 43
4. SAMPLE PREPARATION ............................................................................................................................................. 44
6. RAMAN SPECTROSCOPY ................................................................................................................................. 46
1. MOLECULAR SPECTROSCOPY: LIGHT SCATTERING ........................................................................................................... 46
2. VIBRATIONAL MICROSCOPY: PHOTON PICTURE ............................................................................................................... 47
3. RAMAN SCATTERING: CLASSICAL PICTURE .................................................................................................................... 47
4. THE RAMAN EFFECT: INELASTIC SCATTERING ................................................................................................................. 48
Vibrations in polyatomic molecules & Rule of Mutual Exclusion ............................................................................ 48
5. RAMAN VS INFRARED SPECTRUM ................................................................................................................................. 49
6. ADVANTAGES AND DISADVANTAGES OF RAMAN SPECTROSCOPY ......................................................................................... 50
7. SPACE/TIME-RESOLVED RAMAN SPECTRA OF LIVING CELLS ................................................................................................ 50
7. INFRARED SPECTROSCOPY ............................................................................................................................ 51
1. MOLECULAR SPECTROSCOPY BASICS .......................................................................................................................... 51
2. FUNDAMENTALS AND SCALE ...................................................................................................................................... 51
Electric Dipole Moment ...................................................................................................................................... 51
Harmonic vs. Anharmonic Oscillators ................................................................................................................. 52
Characteristic Vibrational Frequencies ................................................................................................................ 53
3. INFRARED MEASUREMENTS AND INSTRUMENTATION......................................................................................................... 54
Fourier Transform IR (FT-IR) Instrumentation ........................................................................................................ 54
ATR: Attenuated Total Internal Reflection ............................................................................................................. 55
Purpose, Advantages, and Disadvantages of IR .................................................................................................... 55
Interpreting Infrared Spectra & Regions................................................................................................................ 56
4. APPLICATIONS OF IR SPECTROSCOPY ........................................................................................................................... 56
Identification of substances ................................................................................................................................ 56
Determination of molecular structure .................................................................................................................. 56
2
, Spectroscopy of biomolecules Lowie Boels
Quantitative Analysis using IR ............................................................................................................................. 58
Biological Applications of IR Spectroscopy .......................................................................................................... 59
LES 1: ................................................................................................................................................................ 60
1. THE ELECTROMAGNETIC SPECTRUM AND DIFFERENT TYPES OF SPECTROSCOPY ...................................................................... 60
2. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY .......................................................................................................... 62
The Evolution and Application of NMR Spectroscopy ............................................................................................ 62
The Physical Origins of NMR: Spin and Magnetic Fields ......................................................................................... 62
Nuclear Spin Groupings and Isotopes .................................................................................................................. 62
Multiplicity and The Zeeman Effect ...................................................................................................................... 63
Energy Transitions and Resonance ...................................................................................................................... 64
The Boltzmann Distribution and The Sensitivity Problem ....................................................................................... 64
Strategies to Overcome Low Sensitivity ................................................................................................................ 65
3. EQUIVALENCE AND SYMMETRY ............................................................................................................................... 67
LES 2: ................................................................................................................................................................ 70
1. SPIN-SPIN COUPLING .............................................................................................................................................. 70
Spin-spin coupling: AX spin system ..................................................................................................................... 70
Spin-spin coupling: A2X and A2X3 systems .......................................................................................................... 71
Pascal's triangle & General Rules ........................................................................................................................ 72
Splitting by two non-equivalent nuclei.................................................................................................................. 72
The Order of a Spectrum ..................................................................................................................................... 73
Structural Information from Coupling Constants (J) .............................................................................................. 73
Long Range Coupling & Exceptional Cases .......................................................................................................... 74
2. 13C NMR SPECTROSCOPY....................................................................................................................................... 75
Correlating Chemical Shift with Molecular Structure ............................................................................................ 75
Calculating Chemical Shifts ................................................................................................................................ 76
Key Differences: 1H vs. 13C NMR ........................................................................................................................ 77
3. FACTORS THAT INFLUENCE THE APPEARANCE OF 13C NMR SPECTRA ................................................................................... 78
RELAXATION ...................................................................................................................................................... 78
Decoupling ........................................................................................................................................................ 80
LES 3: ................................................................................................................................................................ 82
1. NUCLEAR OVERHAUSER EFFECT (NOE) ....................................................................................................................... 82
2. INTERPRETING 13C SPECTRA (DEPT & APT) ................................................................................................................. 83
3. 2D NMR SPECTROSCOPY: AN IN-DEPTH OVERVIEW ....................................................................................................... 84
COSY ................................................................................................................................................................. 84
TOCSY ............................................................................................................................................................... 86
2D Heteronuclear Correlation Spectroscopy (HETCOR) ........................................................................................ 86
HSQC (Heteronuclear Single Quantum Correlation) ............................................................................................. 87
2D-NOESY ......................................................................................................................................................... 88
4. STUDYING DYNAMIC PROCESSES BY NMR ................................................................................................................. 88
The Time Scale of Spectroscopic Measurements.................................................................................................. 88
Dynamic Chemical Processes ............................................................................................................................ 89
LES 4: ADVANCED APPLICATION OF NMR ........................................................................................................... 93
1. HISTORY OF PROTEIN NMR ....................................................................................................................................... 93
2. THE "WATER PROBLEM" ........................................................................................................................................... 93
3. SOURCES OF PROTEIN FOR NMR STUDIES ..................................................................................................................... 94
4. FROM SPECTRUM TO STRUCTURE ................................................................................................................................ 94
The Assignment Problem and Magnetization Transfer ........................................................................................... 94
Multidimensional NMR ....................................................................................................................................... 94
To know before starting the experiments .............................................................................................................. 95
The 3 phases ...................................................................................................................................................... 96
5. THE LIMITS OF PROTEIN NMR .................................................................................................................................. 102
3
