Chromatographic techniques and mass spectrometry
1. What is chromatography?
separation method used to remove other compounds that could interfere with compound of
interest.
2. Give examples of chromatography methods?
o Ion exchange chromatography
o Size-exclusion chromatography
o Affinity
o Chromatography
o HPLC: normal and reverse phase
3. What are the types of chromatography beds?
Planer or column
4. What separation mechanism is used and the type of mobile phases?
Separated either by size, polarity and 2 mobile phases include gas or liquid.
5. What is the equation for equilibrium constant and K value?
6. What is retention time?
Time for sample injection to extract from the column and each analyte has there their own
Rt.
7. What happens if 2 or more analytes have the same Rt/or K value?
Chromatography system is modified to increase a difference in value, e.g. different mobile
phase solvent.
8. Explain the theory of chromatography (separation and elution)?
Analytes will travel down the column separating out, but not all as this needs readjustment
of the system mainly done in mobile phase.
Ion exchange chroma
9. What do analytes split based on?
Split based on electrical charge?
10. What do bound analytes remove?
Separate amino acids, peptides, proteins and small nucleotides.
11. What does affinity CHROM techniques separate analytes base on?
Based on interactions between antibody & antigen, enzyme and substrates.
12. What happens to unwanted analytes after ligand selectivity?
Unwanted analytes pass through column.
13. What does affinity CHROM separate?
Nucleic acid, proteins (very specific)
14. What is SP?
Gel matrix which ligands is covalently bound to.
15. Explain HPLC method?
•Column filled with porous, inert beads with SP compound chemically bound to them.
•MP containing sample mixture pumped through column at high pressure.
•Analytes bind with SP via polar-polar interaction or non-polar – non-polar interaction.
•Analytes also interact with SP via any method e.g. ion-exchange, size-exclusion, affinity
chromatography.
, 16. What is an advantage of HPLC?
Faster extraction times due to high pressure and better resolution due to smaller stationary
phase particles.
17. What does normal phase HPLC separate and what does it result in?
Separates analytes based on polar interaction with polar SP.
Results in more polar molecules extracted from last column.
18. What does HPLC reverse phase separate and what does it result in?
Separates analytes based on non-polar hydrophobic interaction with non-polar SP. Results in
more hydrophobic molecules extracted from last column.
19. What are the uses of HPLC?
Fraction collectors, manufacturing (pharmaceutical), legal/ forensic
20. What Is mass spectrometry?
An analytical technique that ionises gaseous molecules producing fragment ions detected &
classified according to their mass-to-charge ratio (Used to measure the mass of molecular
ions)
21. What are the essential features for mass spectrometry?
• Production of ions in gas phase.
• Acceleration of ions to specific velocity in electric field.
• Separation of ions in magnetic field.
• Detection of each ion → a mass spectrum
22. What are the uses of mass spectrometry?
Forensic biochemistry, detection of illegal drugs, clinical biochemical, biochemical/ medical
research etc.
Electrophoretic techniques
23. Define electrophoresis?
The movement of a charged particle in an electrical field.
24. Why do molecules have different velocities?
Due to electrical charge or same charge but different molecule sizes
25. What is Potential deference?
Difference in electrical potential energy between the electrodes. Measured in Volts (V)
26. What does Potential difference allow?
Allows a current of charged molecules to follow.
cathode → anode AND anode → cathode
27. What does frictional coefficient depend on?
Hydrodynamic size & shape of molecule.
Pore size of electrophoresis medium (the gel).
Buffer viscosity
28. what do molecules running through the gel generate?
Heat from gel resistance.
29. How do we decrease heating effects?
By controlling voltage.
30. What happens when we have uneven temperature in the gel?
Molecules move faster causing uneven bands.
31. what does loading dye contain?
Contains ionizable tracking dye and density reagent.
32. How are proteins separated?
Separated according to size and native charge (Ph- dependant)
, 33. What are Advantages of isoelectric focusing gel electrophoresis?
Forensic science: detects different isoenzymes in biological fluid
Immunological methods
34. Where is B-cells derived from?
Derived from stem cells in bone marrow and produce antibodies in response to foreign
substances.
35. What is the definition of antibody/Immunoglobin?
protein synthesised by an animal in response to an antigen
36. Define antigen?
A substance producing immune response that produces specific antibodies to fit.
37. Give example of a part of immune system that will destroy antibody-labelled antigens?
T lymphocytes destroy antibody-labelled cells
38. Describe the structure of an antibody?
o consist of four chains:
o Two heavy chains (blue) & two light chains (red)
o Heavy & light chains linked by disulphide bonds.
o Heavy and light chains combine = Fab domains.
o Antigen-binding sites at ends of Fab domains.
o Two heavy chains form the Fc domain.
o Fab domains linked to Fc domain by flexible linkers
39. How do antigens bind to fab domain?
Antibodies and antigens have complementary shapes which allows large surface interaction.
40. What is affinity and specificity and what are the bonding types?
Affinity: strength of interaction between antibody & antigen.
Specificity: degree of compellability between antibody &antigen
• Electrostatic interactions.
• Hydrogen bonds.
• Van der Waals forces
• Hydrophobic interactions
41. In what other way can the affinity of an antibody be shown?
Ag + Ab AgAb
Antibody production
42. How is antibody produced?
Antigen injected into host animal (rabbit, mouse, goat, horse sheep).
Increased production of B lymphocytes in lymph nodes & spleen.
Lymphocytes produce antibody to antigen.
1. What is chromatography?
separation method used to remove other compounds that could interfere with compound of
interest.
2. Give examples of chromatography methods?
o Ion exchange chromatography
o Size-exclusion chromatography
o Affinity
o Chromatography
o HPLC: normal and reverse phase
3. What are the types of chromatography beds?
Planer or column
4. What separation mechanism is used and the type of mobile phases?
Separated either by size, polarity and 2 mobile phases include gas or liquid.
5. What is the equation for equilibrium constant and K value?
6. What is retention time?
Time for sample injection to extract from the column and each analyte has there their own
Rt.
7. What happens if 2 or more analytes have the same Rt/or K value?
Chromatography system is modified to increase a difference in value, e.g. different mobile
phase solvent.
8. Explain the theory of chromatography (separation and elution)?
Analytes will travel down the column separating out, but not all as this needs readjustment
of the system mainly done in mobile phase.
Ion exchange chroma
9. What do analytes split based on?
Split based on electrical charge?
10. What do bound analytes remove?
Separate amino acids, peptides, proteins and small nucleotides.
11. What does affinity CHROM techniques separate analytes base on?
Based on interactions between antibody & antigen, enzyme and substrates.
12. What happens to unwanted analytes after ligand selectivity?
Unwanted analytes pass through column.
13. What does affinity CHROM separate?
Nucleic acid, proteins (very specific)
14. What is SP?
Gel matrix which ligands is covalently bound to.
15. Explain HPLC method?
•Column filled with porous, inert beads with SP compound chemically bound to them.
•MP containing sample mixture pumped through column at high pressure.
•Analytes bind with SP via polar-polar interaction or non-polar – non-polar interaction.
•Analytes also interact with SP via any method e.g. ion-exchange, size-exclusion, affinity
chromatography.
, 16. What is an advantage of HPLC?
Faster extraction times due to high pressure and better resolution due to smaller stationary
phase particles.
17. What does normal phase HPLC separate and what does it result in?
Separates analytes based on polar interaction with polar SP.
Results in more polar molecules extracted from last column.
18. What does HPLC reverse phase separate and what does it result in?
Separates analytes based on non-polar hydrophobic interaction with non-polar SP. Results in
more hydrophobic molecules extracted from last column.
19. What are the uses of HPLC?
Fraction collectors, manufacturing (pharmaceutical), legal/ forensic
20. What Is mass spectrometry?
An analytical technique that ionises gaseous molecules producing fragment ions detected &
classified according to their mass-to-charge ratio (Used to measure the mass of molecular
ions)
21. What are the essential features for mass spectrometry?
• Production of ions in gas phase.
• Acceleration of ions to specific velocity in electric field.
• Separation of ions in magnetic field.
• Detection of each ion → a mass spectrum
22. What are the uses of mass spectrometry?
Forensic biochemistry, detection of illegal drugs, clinical biochemical, biochemical/ medical
research etc.
Electrophoretic techniques
23. Define electrophoresis?
The movement of a charged particle in an electrical field.
24. Why do molecules have different velocities?
Due to electrical charge or same charge but different molecule sizes
25. What is Potential deference?
Difference in electrical potential energy between the electrodes. Measured in Volts (V)
26. What does Potential difference allow?
Allows a current of charged molecules to follow.
cathode → anode AND anode → cathode
27. What does frictional coefficient depend on?
Hydrodynamic size & shape of molecule.
Pore size of electrophoresis medium (the gel).
Buffer viscosity
28. what do molecules running through the gel generate?
Heat from gel resistance.
29. How do we decrease heating effects?
By controlling voltage.
30. What happens when we have uneven temperature in the gel?
Molecules move faster causing uneven bands.
31. what does loading dye contain?
Contains ionizable tracking dye and density reagent.
32. How are proteins separated?
Separated according to size and native charge (Ph- dependant)
, 33. What are Advantages of isoelectric focusing gel electrophoresis?
Forensic science: detects different isoenzymes in biological fluid
Immunological methods
34. Where is B-cells derived from?
Derived from stem cells in bone marrow and produce antibodies in response to foreign
substances.
35. What is the definition of antibody/Immunoglobin?
protein synthesised by an animal in response to an antigen
36. Define antigen?
A substance producing immune response that produces specific antibodies to fit.
37. Give example of a part of immune system that will destroy antibody-labelled antigens?
T lymphocytes destroy antibody-labelled cells
38. Describe the structure of an antibody?
o consist of four chains:
o Two heavy chains (blue) & two light chains (red)
o Heavy & light chains linked by disulphide bonds.
o Heavy and light chains combine = Fab domains.
o Antigen-binding sites at ends of Fab domains.
o Two heavy chains form the Fc domain.
o Fab domains linked to Fc domain by flexible linkers
39. How do antigens bind to fab domain?
Antibodies and antigens have complementary shapes which allows large surface interaction.
40. What is affinity and specificity and what are the bonding types?
Affinity: strength of interaction between antibody & antigen.
Specificity: degree of compellability between antibody &antigen
• Electrostatic interactions.
• Hydrogen bonds.
• Van der Waals forces
• Hydrophobic interactions
41. In what other way can the affinity of an antibody be shown?
Ag + Ab AgAb
Antibody production
42. How is antibody produced?
Antigen injected into host animal (rabbit, mouse, goat, horse sheep).
Increased production of B lymphocytes in lymph nodes & spleen.
Lymphocytes produce antibody to antigen.
