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analytic techniques Flashcards

1
Q

Describe the principle behind chromatography.

A

Chromatography separates components of a mixture based on size, charge, hydrophobicity, composition, and specificity.

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2
Q

List some types of chromatography techniques.

A

Techniques include Gel filtration, Ion exchange, Affinity, High Performance Liquid Chromatography (HPLC), Reversed Phase, and Gas chromatography (GC).

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3
Q

Explain the purpose of electrophoresis.

A

Electrophoresis separates macromolecules such as DNA, RNA, and proteins according to their size and/or charge.

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4
Q

Identify the types of electrophoresis mentioned.

A

Types include SDS PAGE, Isoelectric focusing, and 2D gel electrophoresis.

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5
Q

What is the role of spectroscopy in analytical techniques?

A

Spectroscopy is used for structure determination by measuring the absorption and transmission of electromagnetic radiation as the wavelength varies.

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6
Q

Define mass spectrometry and its function.

A

Mass spectrometry is used for structural characterization by fragmenting molecules and measuring the resulting masses.

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7
Q

How does column chromatography work?

A

Column chromatography relies on the interaction between the mobile and stationary phases to separate target molecules from the mobile phase.

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8
Q

Describe the process of elution in column chromatography.

A

Elution in column chromatography involves collecting fractions that elute from the column at specified time or volume points while monitoring the protein content.

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9
Q

How does ion exchange chromatography work for protein purification?

A

Ion exchange chromatography purifies proteins by adjusting the mobile phase’s salt concentration or pH, allowing charged molecules to interact with the stationary phase.

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10
Q

Define gel filtration chromatography and its separation mechanism.

A

Gel filtration chromatography separates proteins, peptides, and oligonucleotides based on size, where large proteins exit the column first as they cannot enter the beads.

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11
Q

Explain the principle behind affinity chromatography.

A

Affinity chromatography utilizes the specific affinity of certain proteins for particular chemicals or groups, allowing only those proteins to be retained on the column.

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12
Q

What role does centrifugation play in salt fractionation?

A

Centrifugation in salt fractionation separates components based on size and shape after adding a dilute salt solution and a gradient to the sample in a centrifuge tube.

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13
Q

How are proteins separated using chromatography techniques?

A

Proteins are separated using various chromatography techniques such as ion exchange, gel filtration, and affinity chromatography, each based on different properties like charge, size, or affinity.

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14
Q

Do large proteins behave differently in gel filtration chromatography compared to small proteins?

A

Yes, large proteins cannot enter the beads in gel filtration chromatography and exit the column first, while small proteins can enter the beads and exit later.

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15
Q

Describe the relationship between resolving power and interaction sites in chromatographic techniques.

A

The resolving power of any chromatographic technique is related to the number of potential sites of interaction between the mobile phase and the stationary phase. More fine beads allow for greater interactions, thus increasing resolving power.

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16
Q

How does High Performance Liquid Chromatography (HPLC) enhance resolving power?

A

HPLC uses very fine beads in metal columns and high pressure pumps, which increases the number of interaction sites between the mobile phase and stationary phase, resulting in greater resolving power compared to normal columns.

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17
Q

Define the types of methods that can be used in HPLC.

A

The methods used in HPLC can include Gel filtration, Ion exchange, Affinity, or Reversed phase.

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18
Q

Explain the role of the mobile phase in Gas Chromatography (GC).

A

In Gas Chromatography, the mobile phase is a stream of inert gas that carries vaporized molecules to be analyzed, affecting their retention times based on interactions with the stationary phase.

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19
Q

What is the purpose of the column in Gas Chromatography?

A

The column in Gas Chromatography is coated with a microscopic layer of liquid or polymer on an inert solid support, which facilitates the separation of components based on their interaction with the mobile phase.

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20
Q

How are proteins separated using Gel Electrophoresis?

A

Proteins and other molecules are separated by Gel Electrophoresis according to their size and/or charge, utilizing techniques such as SDS-PAGE and isoelectric focusing.

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21
Q

Describe the significance of Gas Chromatograms in forensic analysis.

A

Gas Chromatograms are significant in forensic analysis as they can be used to identify substances, such as in the analysis of seized drugs or blood alcohol content.

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22
Q

Describe the principle behind SDS-PAGE.

A

SDS-PAGE separates proteins based on mass by denaturing them with sodium dodecyl sulfate (SDS), which binds to proteins, ensuring they have the same charge to mass ratio, allowing migration through a gel under an electric field.

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23
Q

How do proteins migrate in a polyacrylamide gel during electrophoresis?

A

Proteins migrate in the gel due to an electric field, with their velocity being directly proportional to the electric field strength and the charge on the protein, and inversely proportional to their mass, shape, radius, and the density of the medium.

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24
Q

Define the role of SDS in SDS-PAGE.

A

Sodium dodecyl sulfate (SDS) denatures proteins and binds to them, ensuring a consistent charge to mass ratio, which allows for separation based solely on mass during electrophoresis.

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25
Explain the sieving effect of the polyacrylamide gel.
The porous structure of the polyacrylamide gel acts as a sieve, allowing smaller proteins to move more quickly through the gel compared to larger proteins, which migrate more slowly.
26
What is the purpose of staining the gel after SDS-PAGE?
Staining the gel with dyes like Coomassie blue allows for visualization of the separated proteins, making it possible to analyze the bands and estimate their sizes using a standard protein mixture.
27
How is the electrophoretic mobility of proteins related to their mass in SDS-PAGE?
The electrophoretic mobility of proteins in SDS-PAGE is inversely proportional to the logarithm of their mass, meaning that lighter proteins move faster than heavier ones.
28
Do multiple samples undergo electrophoresis simultaneously in SDS-PAGE?
Yes, several samples are typically loaded into wells on a single flat polyacrylamide gel and undergo electrophoresis simultaneously.
29
Describe the role of β-mercaptoethanol in Reducing SDS-PAGE.
β-mercaptoethanol is used to break (reduce) disulphide bridges in proteins, which are covalent bonds formed by oxidation, allowing for proper separation of proteins during electrophoresis.
30
How does glycosylation affect protein movement in SDS-PAGE?
Heavily glycosylated proteins, which have extended sugar chains, may move anomalously on the gel, leading to misleading weight indications during analysis.
31
Describe the purpose of spectroscopy in analytical techniques.
Spectroscopy is used for structure determination by measuring the absorption and transmission of electromagnetic radiation as the wavelength is varied.
32
How does chromatography separate components of a mixture?
Chromatography separates components based on size, charge, hydrophobicity, composition, and specificity.
33
Define mass spectrometry and its function in analysis.
Mass spectrometry is a technique for structural characterization by fragmenting molecules and measuring the resulting masses.
34
Explain the role of infrared spectroscopy in molecular analysis.
Infrared spectroscopy uses specific frequencies matched to vibrating bonds, causing the absorption of specific wavelengths of infrared light.
35
What types of macromolecules can be separated using electrophoresis?
Electrophoresis can separate macromolecules such as DNA, RNA, and proteins according to their size and/or charge.
36
Describe the significance of energy levels in spectroscopy.
Energy levels within a molecule are discrete (quantized), and transitions between these levels occur only at energies that match the difference between them.
37
How does the electromagnetic spectrum relate to spectroscopy?
The electromagnetic spectrum is continuous and covers a wide range of wavelengths, which are crucial for determining molecular structure through spectroscopy.
38
Describe the purpose of Infrared Spectroscopy in molecular analysis.
Infrared Spectroscopy measures the bond vibration frequencies in a molecule and is used to determine functional groups.
39
How does UV-visible spectroscopy utilize wavelengths for molecular analysis?
UV-visible spectroscopy uses wavelengths of 100–700nm to facilitate electron transitions from outer occupied electron shells to higher unoccupied orbitals.
40
Define the role of Nuclear Magnetic Resonance (NMR) in studying molecules.
NMR spectroscopy provides information about the structure, dynamics, reaction state, and surrounding environment of molecules by measuring how nuclei absorb and re-emit electromagnetic radiation in a magnetic field.
41
Explain the significance of the fingerprint region in Infrared Spectroscopy.
The fingerprint region, which ranges from 400-1500 cm-1, contains complex vibrations that are unique to each molecule, allowing for identification.
42
Identify the relationship between light absorption and concentration in UV-visible spectroscopy.
In UV-visible spectroscopy, the amount of light absorbed is directly related to the concentration of the absorbing molecule in the sample.
43
How are characteristic absorbance maxima used in UV-visible spectroscopy?
Characteristic absorbance maxima (λmax) for functional groups allow for their identification in the UV-vis spectrum, such as the carbonyl group (C=O) at 186nm and 280nm.
44
What is the typical wavelength range for Infrared Spectroscopy?
Infrared Spectroscopy typically operates within the wavelength range of 2.5-25 mm, corresponding to the energies of molecular vibrations.
45
Describe the significance of absorbance peaks in the UV-vis spectrum of propanone.
The absorbance peaks at 280 and 186 nm correspond to the characteristic λmax for the carbonyl (C=O) group, indicating the presence of this functional group in propanone.
46
How does Nuclear Magnetic Resonance (NMR) spectroscopy determine protein structure?
NMR spectroscopy determines protein structure by bombarding the sample with radio frequency pulses and monitoring the absorption of radio waves, which occurs when protons change spin states at resonance.
47
Define 'Chemical shift' in the context of NMR spectroscopy.
The chemical shift is the resonant frequency of a nucleus relative to a standard in a magnetic field, influenced by the electron distribution around the nucleus.
48
Explain the relationship between chemical shifts and protein structure.
Chemical shifts depend on the environment of the nuclei, which is influenced by the protein structure, leading to variations in resonant frequencies for the same type of nucleus.
49
What is the role of mass spectrometry in analyzing proteins?
Mass spectrometry fragments molecules and measures their masses, allowing for the accurate detection of mass and composition, including the identification of peptides and proteins.
50
How does one-dimensional NMR differ from two-dimensional NMR?
One-dimensional NMR reveals changes to a particular chemical group under different conditions, while two-dimensional NMR (such as NOESY) displays groups that are in close proximity.
51
Identify a characteristic of nuclei that have a nuclear spin.
Any nucleus with an odd atomic number or odd mass has a nuclear spin, such as 13C, which contains 6 protons and 7 neutrons.
52
Describe the process of converting a sample into gas-phase ions in mass spectrometry.
A sample, whether solid, liquid, or gas, is converted into gas-phase ions by methods such as laser irradiation or electron bombardment.
53
How are gas-phase ions separated in mass spectrometry?
Gas-phase ions are separated by accelerating them and subjecting them to an electric or magnetic field, causing ions of the same mass-to-charge ratio (m/z) to undergo the same deflection.
54
Define the MALDI technique in mass spectrometry.
MALDI, or Matrix-Assisted Laser Desorption/Ionization, is an ionization technique that uses a laser energy-absorbing matrix to create ions from large molecules with minimal fragmentation.
55
What is the significance of the time of flight (TOF) in MALDI-TOF mass spectrometry?
The time of flight (TOF) measures the time it takes for ions to travel through the mass spectrometer, which helps in determining their mass-to-charge ratio.
56
Explain the role of tandem mass spectrometry (MS-MS) in protein analysis.
Tandem mass spectrometry facilitates protein sequence determination by cleaving proteins, separating the resulting peptides based on mass/charge ratio, and then fragmenting them for identification.
57
How does peptide mass fingerprinting work in tandem mass spectrometry?
Peptide mass fingerprinting involves cleaving proteins into peptides, separating them by mass/charge ratio, and identifying individual proteins based on their unique mass patterns.
58
Describe the process of peptide sequencing using tandem mass spectrometry.
In tandem mass spectrometry, selected peptides are fragmented to generate product ions, which are then detected to determine the sequence of amino acids in the precursor peptide.
59
Describe the role of mass spectrometry in gas chromatography.
Mass spectrometry is used with gas chromatography (GC-MS) for the rapid identification of compounds that have been separated by gas chromatography. It allows for the analysis of specific ions of interest by using a quadrupole analyser.
60
How does 1H-NMR spectrum of ethanol differ from that of a protein fragment?
The 1H-NMR spectrum of ethanol shows clearly resolved chemical shifts for hydrogen, while the spectrum of a 55 amino acid protein fragment displays greater complexity with many overlapping peaks.
61
Describe the role of X-ray crystallography in protein structure determination.
X-ray crystallography is used to determine the atomic structure of proteins by interpreting the scattering pattern produced by a crystalline molecular array.
62
How does chromatography separate components of a mixture?
Chromatography separates components based on size, charge, hydrophobicity, composition, and specificity.
63
Define the purpose of mass spectrometry in structural characterization.
Mass spectrometry characterizes structures by fragmenting molecules and measuring the resulting masses.
64
Explain the significance of Nuclear Magnetic Resonance (NMR) in protein analysis.
NMR provides high-resolution details at the atomic level, primarily for small proteins, aiding in understanding protein structure and dynamics.
65
What advancements have been made in electron microscopy for protein structure determination?
High-resolution electron microscopy can now provide atomic-level detail for large proteins and viruses, a capability that is rapidly evolving.
66
List the main experimental methods used for determining protein structures and their total contributions as of October 2024.
The main methods include X-ray crystallography (188,747 structures), Electron Microscopy (23,203), NMR (14,400), and others, totaling 226,707 structures.
67
Describe the process of X-ray crystallography.
X-ray crystallography involves irradiating protein crystals with X-rays, which scatter off the electrons of the atoms. The scattered waves recombine and are collected on a detector, revealing the atomic arrangement of the protein.
68
How does the scattering pattern provide information about atomic arrangement?
The scattering pattern, known as the diffraction pattern, contains information about the nature and location of the scatterers (atoms) in the crystal, allowing for the determination of atomic arrangement.
69
Define the role of electrons in X-ray scattering.
Electrons of the atoms in the crystal scatter the X-rays, and the resulting scattering pattern is used to interpret the structure of the protein.
70
Explain the significance of maintaining protein integrity in crystals.
Maintaining protein integrity in crystals is crucial because the structures obtained must truly represent the biologically active molecules, ensuring accurate interpretation of their function.
71
What is the time scale for protein crystallography projects?
The time scale for protein crystallography can range from 1 man-week to 50 man-years, depending on the complexity of the protein and the crystallization process.
72
How do X-rays compare to atomic distances in protein structures?
X-rays are used for structure determination because their wavelength is about the same size as the separation between atoms in a protein structure, typically around 1 to 2 Ångstroms.
73
Describe the characteristics of protein crystals.
Protein crystals are typically small (0.1-0.2mm), fragile, and contain around 50-70% solvent. They must retain the biological activity of the protein and consist of an ordered three-dimensional arrangement of protein molecules.
74
How are protein crystals obtained?
To obtain protein crystals, an aqueous environment is needed to maintain integrity, along with suitable pH and temperature. The solubility of the proteins is gradually reduced to encourage the molecules to come together.
75
Define the role of solvent in protein crystal formation.
Solvent, typically comprising 50-75% of the crystal, is crucial as it helps maintain the integrity of the protein molecules while allowing them to aggregate into a regular three-dimensional array.
76
Explain the importance of favorable interactions in protein crystallization.
Favorable interactions, such as charge and shape compatibility, are essential for protein crystallization. They determine how protein molecules interact and aggregate, leading to the formation of stable crystals.
77
What is the significance of reducing solubility in protein crystal growth?
Reducing solubility is significant because it encourages protein molecules to come together and form favorable intermolecular contacts, which is necessary for the orderly arrangement required for crystal growth.
78
How does the environment affect protein crystallization?
The environment, including factors like pH, salinity, and protein solubility, influences which interactions dominate among protein molecules, ultimately affecting the stability and formation of protein crystals.
79
Describe the role of ionic strength in protein crystallization.
Ionic strength affects the interactions between proteins and solvents, influencing solubility and precipitation. Adjusting ionic strength can help in achieving the desired conditions for crystallization.
80
How does pH influence protein solubility during crystallization?
The net charge on a protein varies with pH, and proteins are least soluble at their isoelectric point (PI), where they have no net charge, making pH a critical factor in crystallization.
81
Define the hanging drop method in crystallization.
The hanging drop method involves placing a drop of protein solution above a reservoir solution, allowing vapor diffusion to occur, which gradually removes water and promotes crystallization.
82
What is the significance of the isoelectric point in protein crystallization?
At the isoelectric point, proteins have no net charge, leading to reduced solubility and increased likelihood of precipitation, which is crucial for crystallization.
83
Explain the challenges faced in predicting protein crystallization outcomes.
It is difficult to predict the rate of equilibrium, whether proteins will reach supersaturation, or if precipitation will lead to crystal formation, necessitating the testing of various conditions.
83
How do sparse matrix screens assist in protein crystallization?
Sparse matrix screens provide a variety of pre-formulated solutions that represent major crystallization conditions, streamlining the process of finding suitable conditions for protein crystallization.
84
Describe the role of amphiphiles in membrane protein crystallization.
Amphiphiles are used to displace lipids in membrane proteins, making the whole membrane spanning protein soluble, which is essential for crystallization.
85
How is the integrity of protein and lattice maintained in cryocrystallography?
In cryocrystallography, the integrity is maintained by preventing radiation damage and using a single crystal, often by diffusing cryoprotectants and flash-freezing the crystal.
86
Define the purpose of using detergents in the crystallization of membrane proteins.
Detergents are used to create micelles or lipid bilayer nanodiscs that help solubilize membrane proteins, allowing them to be crystallized.
87
Explain the significance of the Nobel Prize in Chemistry 1988 related to membrane proteins.
The Nobel Prize in Chemistry 1988 was awarded for the determination of the three-dimensional structure of the photosynthetic reaction center of the bacterium Rhodopseudomonas viridis.
88
What is the challenge associated with crystal mounting in protein crystallization?
Crystal mounting is challenging because it requires maintaining an aqueous environment, and the crystals are very fragile, often leading to damage during the process.
89
How does cryopreservation contribute to the process of X-ray crystallography?
Cryopreservation allows the crystal to be stored at low temperatures, preventing radiation damage and preserving the crystal's integrity for X-ray exposure.

biochem (9 decks)

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