flashcard 15

Question 1

What is the definition of spectroscopy?

Answer 1

Spectroscopy is the study of the interaction between matter and electromagnetic radiation—basically, how a sample absorbs or emits light, producing a “spectrum” that serves as its fingerprint.

Question 2

From what languages does the term “spectroscopy” derive, and what do they mean?

Answer 2

It derives from Latin “spectrum” (meaning ghost or spirit) and Greek “scopos” (meaning to see), reflecting the idea of observing a “shadow” or “ghost” of a molecule via its interaction with light.

Question 3

What are the three main types of spectroscopy covered in the lecture?

Answer 3

UV/Vis absorbance spectroscopy, photoluminescence (including fluorescence), and bio-/chemi-luminescence.

Question 4

What additional techniques for determining protein structure were mentioned beyond spectroscopy?

Answer 4

X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM).

Question 5

What is the basic physical process underlying absorption spectroscopy?

Answer 5

A photon of a certain energy is absorbed by a molecule, causing a transition from its ground state to an excited state; the decrease in transmitted light intensity is measured as absorbance.

Question 6

Write the Beer–Lambert law and define each term.

Answer 6

A = ε·c·l, where A is absorbance (unitless), ε is the molar extinction coefficient (M⁻¹·cm⁻¹), c is the concentration of the absorbing species (M), and l is the path length of the cuvette (cm).

Question 7

In practice, what additional effects can cause deviation from the Beer–Lambert law?

Answer 7

Light scattering (e.g., from turbidity), reflection, refraction, and matrix effects—all of which reduce or scatter transmitted light beyond simple molecular absorption.

Question 8

How does turbidity in a sample affect absorption measurements?

Answer 8

Turbidity causes light scattering, so photons are diverted out of the beam path, leading to apparent absorption that is not due to the analyte molecules.

Question 9

What is polarization in the context of spectroscopy?

Answer 9

Polarization refers to the orientation of the electric field vector of light; analyzing how a sample rotates or transmits polarized light can reveal information about molecular chirality and structure.

Question 10

Describe how a basic UV/Vis spectrophotometer measures absorbance.

Answer 10

A broadband light source is filtered to a selected wavelength (via interchangeable filters or a monochromator), passed through the sample cuvette, and then detected; the instrument compares incoming and transmitted intensities to calculate absorbance.

Question 11

What types of applications use absorption spectroscopy for kinetic studies?

Answer 11

Enzyme kinetics (e.g., monitoring NADH absorbance at 340 nm), redox reactions, reaction rates, and metabolic enzyme activity assays by tracking characteristic absorbance changes over time.

Question 12

Why is NADH commonly monitored at 340 nm in enzyme assays?

Answer 12

NADH has a strong absorbance peak at 340 nm (ε ≈ 6,220 M⁻¹·cm⁻¹), whereas NAD⁺ does not, allowing easy quantification of its formation or consumption.

Question 13

How can one distinguish oxyhemoglobin from deoxyhemoglobin using absorption spectroscopy?

Answer 13

Oxyhemoglobin and deoxyhemoglobin have different absorbance spectra: oxyhemoglobin shows a Soret band near 415 nm and distinct visible peaks, while deoxyhemoglobin’s peaks are shifted; comparing λ max values allows differentiation.

Question 14

How is DNA quantified and its purity assessed by UV absorbance?

Answer 14

DNA concentration is measured by absorbance at 260 nm (A₂₆₀), and purity is assessed by the A₂₆₀/A₂₈₀ ratio (ideal ~ 1.8 for pure DNA).

Question 15

What defines photoluminescence?

Answer 15

Photoluminescence is the emission of light from a molecule after it absorbs photons, encompassing both fluorescence (prompt emission) and phosphorescence (delayed emission).

Question 16

Compare fluorescence and phosphorescence lifetimes.

Answer 16

Fluorescence typically has lifetimes of 10⁻¹⁰ to 10⁻⁸ s, whereas phosphorescence lifetimes range from 10⁻³ up to 10² s, because phosphorescence involves a spin-forbidden transition (triplet→singlet).

Question 17

What is quantum yield in fluorescence?

Answer 17

The quantum yield is the fraction of excited-state molecules that return to the ground state via fluorescence rather than nonradiative processes; it ranges from 0 (no fluorescence) to 1 (all excited molecules fluoresce).

Question 18

How are excitation and emission spectra obtained in a fluorimeter?

Answer 18

The excitation spectrum is recorded by holding emission wavelength constant and scanning excitation wavelengths; the emission spectrum is recorded by exciting at a fixed wavelength and scanning emission wavelengths, each corrected for source and detector response.

Question 19

What are the key qualitative and quantitative uses of fluorescence spectroscopy?

Answer 19

Qualitatively, one compares excitation/emission peak shapes and positions to standards to identify compounds. Quantitatively, at low concentrations, fluorescence intensity is directly proportional to analyte concentration, allowing precise measurements.

Question 20

List examples of analytes measurable by fluorescence spectroscopy.

Answer 20

DNA (via intercalating dyes), antibodies/antigens (via fluorescent tags), drugs and metabolites (e.g., quinine), vitamins, minerals, amino acids, and contaminants in food or environmental samples—provided they fluoresce or can be derivatized.

Question 21

What are the advantages of fluorescence over absorbance spectroscopy?

Answer 21

Higher sensitivity (lower detection limits), enhanced selectivity (via distinct excitation/emission wavelengths), ability to probe chemical environment (e.g., pH, polarity), and use of spatially resolved techniques (e.g., fluorescence microscopy).

Question 22

What limitations prevent fluorescence spectroscopy from diagnosing diseases directly?

Answer 22

It generally requires clear, non-turbid samples and analytes that fluoresce—or must be labeled. Many biomarkers do not fluoresce natively, and complex matrices can quench signals or cause interference, reducing clinical utility for direct diagnosis.

Question 23

What is quenching, and how does it affect fluorescence analysis?

Answer 23

Quenching is the reduction of fluorescence intensity due to interactions such as energy transfer, collisions, or binding of quenchers (e.g., halide ions). It forms the basis of Förster resonance energy transfer (FRET) assays and can also be used to measure molecular proximity or concentration changes.

Question 24

Define Förster resonance energy transfer (FRET).

Answer 24

FRET is a distance-dependent energy transfer from an excited donor fluorophore to an acceptor molecule (fluorophore or quencher). It requires donor-acceptor separation of 1–10 nm, overlap of donor emission and acceptor absorption spectra, and results in either acceptor emission or nonradiative dissipation.

Question 25

How is fluorescence recovery after photobleaching (FRAP) used?

Answer 25

In FRAP, a region of a fluorescently labeled cell membrane (or other structure) is photobleached with intense light; recovery of fluorescence via diffusion of unbleached fluorophores into the area is monitored to determine membrane fluidity or molecular mobility.

Question 26

What is bioluminescence, and which enzyme is commonly used in assays?

Answer 26

Bioluminescence is light emission resulting from a biochemical reaction (no external light excitation). Luciferase is commonly used, catalyzing oxidation of luciferin (with ATP and O₂) to produce light, often employed as a reporter in gene expression assays.

Question 27

Describe how a luciferase reporter assay measures transcriptional activation.

Answer 27

The promoter of interest is cloned upstream of the luciferase gene. When transcription factors activate that promoter, luciferase is expressed; adding luciferin substrate generates light proportional to promoter activity, quantifiable with a luminometer.

Question 28

What is chemiluminescence, and how is luminol used in forensics?

Answer 28

Chemiluminescence is light emission from a chemical reaction. Luminol reacts with hydrogen peroxide (and a catalyst such as iron in hemoglobin) to form a high-energy intermediate that emits blue light when returning to the ground state, revealing trace blood at crime scenes.

Question 29

In luminol chemiluminescence, what role does hydrogen peroxide play?

Answer 29

H₂O₂ oxidizes luminol in alkaline solution, generating a dianion that reacts with O₂ to form an unstable peroxide intermediate; when this breaks down (releasing N₂), the molecule returns to its ground state by emitting blue photons.

Question 30

What is epi-fluorescence microscopy?

Answer 30

Epi-fluorescence microscopy illuminates the sample from above with excitation light, collecting emitted fluorescence through the same objective. It enables multicolor imaging of labeled structures (e.g., nuclei with DAPI, actin with phalloidin-AlexaFluor).

Question 31

How do fluorescent proteins enable in vivo imaging?

Answer 31

Genes encoding fluorescent proteins (e.g., GFP and its variants) are fused to genes of interest. When expressed in cells or organisms, they produce intrinsically fluorescent fusions that allow real-time localization and tracking without exogenous dyes.

Question 32

What is confocal microscopy, and why is it advantageous?

Answer 32

Confocal microscopy uses point illumination (typically lasers) and a pinhole to reject out-of-focus light, collecting sharp optical sections. By scanning in x–y and z planes, one can reconstruct 3D images with high resolution and contrast.

Question 33

Why must protein chromatography often be accompanied by electrophoresis or structural methods?

Answer 33

Because chromatography alone doesn’t reveal precise molecular structure or confirm protein identity/purity at the subunit level. Electrophoresis (e.g., SDS-PAGE), X-ray crystallography, or NMR are used to validate size, conformation, or atomic resolution structure.

Question 34

What wavelength range characterizes near-infrared (NIR) absorption spectroscopy?

Answer 34

NIR absorption spans roughly 800–2,500 nm (wavenumbers 12,500–4,000 cm⁻¹), capturing overtone and combination vibrational bands useful for compositional analysis of liquids, solids, or biological samples.

Question 35

How does NIR absorption spectroscopy explain why water appears blue?

Answer 35

Water strongly absorbs in the red portion of the visible spectrum (> 600 nm). Since longer red wavelengths are attenuated, only shorter (blue) wavelengths transmit, making bodies of water appear blue.

Question 36

How can “heavy water” be distinguished from normal water by NIR spectroscopy?

Answer 36

Heavy water (D₂O) has vibrational absorption bands similar to H₂O but shifted to higher wavelengths (lower wavenumbers). Measuring outside the visible range (e.g., near 2,000 nm) can differentiate D₂O from H₂O.

Question 37

What is X-ray crystallography, and what data does it produce?

Answer 37

X-ray crystallography directs an X-ray beam at a protein crystal; electrons in the crystal diffract X-rays into spots whose positions and intensities map the electron density, allowing reconstruction of atomic structures.

Question 38

What is a major limitation of X-ray crystallography?

Answer 38

It requires proteins (or other molecules) to form well-ordered crystals. Many proteins are difficult or impossible to crystallize, preventing structural determination by X-ray methods.

Question 39

How does nuclear magnetic resonance (NMR) measure distances between protein atoms?

Answer 39

NMR uses the quantum mechanical property of nuclear spin (e.g., ¹H, ¹³C, ¹⁵N) in a strong magnetic field. Radiofrequency pulses perturb spin states; the resulting NMR spectrum provides information about chemical environments and internuclear distances (via NOE cross-peaks).

Question 40

Which isotopes are NMR-active and most commonly used for macromolecular studies?

Answer 40

¹H, ¹³C, ¹⁵N, ¹⁹F, and ³¹P are NMR-active nuclei commonly used because they have spin angular momentum, generating detectable magnetic dipoles.

Question 41

Describe the basic NMR process for a protein in solution.

Answer 41

The protein’s NMR-active nuclei align with a strong static magnetic field. A short RF pulse at each nucleus’s resonant frequency flips spins to higher energy states. As nuclei relax back, they emit RF signals; Fourier transform yields a spectrum revealing chemical shifts and coupling patterns.

Question 42

What extra information can NMR provide beyond static structure?

Answer 42

NMR can monitor conformational changes, protein folding/unfolding, dynamics, and interactions with ligands or other biomolecules in solution—capabilities not accessible by crystallography alone.

Question 43

Why is spectroscopy critical in forensics and clinical diagnostics?

Answer 43

In forensics, chemiluminescence (e.g., luminol) reveals trace blood; fluorescence can detect drugs or toxins. In clinical settings, absorbance and fluorescence assays measure biomarkers (enzymes, metabolites) sensitively, aiding diagnosis.

Question 44

How does an absorbance spectrum reveal the “shadow” of a molecule?

Answer 44

Each molecule has characteristic electronic transitions that absorb specific wavelengths; plotting absorbance vs. wavelength produces peaks (the molecule’s “shadow” or fingerprint), enabling identification and quantification.

Question 45

What information does polarised spectroscopy (e.g., circular dichroism) provide about proteins?

Answer 45

Polarized absorption (circular dichroism) measures differential absorption of left vs. right circularly polarized light, revealing protein secondary structure content (α-helix vs. β-sheet) because chiral peptide bonds interact differently with polarized light.

Question 46

In photoluminescence spectroscopy, why must samples be clear and non-turbid?

Answer 46

Turbidity scatters excitation and emission light, reducing intensity and distorting spectra. Clear samples ensure that measured fluorescence reflects only molecular emission, not scattering artifacts.

Question 47

How do fluorescent dyes allow quantification of low-concentration analytes (e.g., DNA)?

Answer 47

Fluorescent dyes (e.g., SYBR Green) bind specifically to nucleic acids; their fluorescence intensity at a given excitation correlates with DNA concentration, enabling quantification down to picomolar levels—far below absorbance-based limits.

Question 48

What is a Stokes shift in fluorescence?

Answer 48

The Stokes shift is the difference between the peak wavelength of excitation and the peak wavelength of emission. It arises because excitation energy is partially lost via nonradiative processes before emission, causing emission to appear at longer wavelengths.

Question 49

Why is confocal microscopy preferred over widefield fluorescence for 3D imaging?

Answer 49

Confocal microscopy uses a pinhole to reject out-of-focus light, enhancing axial resolution. By collecting optical sections at different focal planes, one can reconstruct a 3D image with minimal background blur—unlike widefield imaging.

Question 50

Summarize the key steps in a fluorescence-based gene expression assay using luciferase.

Answer 50

1) Clone the promoter of interest upstream of luciferase reporter. 2) Transfect cells with this construct. 3) Treat cells (e.g., with an inducer or inhibitor). 4) Add luciferin substrate. 5) Measure emitted light, which is proportional to promoter activity.