Common asked Exam Q

Question 1

A negatively charged protein is bound to a positively charged column. The
protein has a pI of 6.2. What buffer pH could you use to elute the protein from the
column?

Answer 1

The positively charged column is an anion exchanger, which binds negatively charged proteins
The binding buffer pH must be above 6.2. To elute the protein, you need to change the buffer pH

Question 2

Suggest 2 modes of chromatography that could be used in the early stages of
a protein purification. [5 Marks]

Answer 2

Ion Exchange Chromatography (IEC): Separates based on net surface charge
Hydrophobic Interaction Chromatography (HIC): Separates based on differences in protein surface hydrophobicity

Question 3

Differentiate between the sorption mechanisms of adsorption and partition in relation
to chromatography.

Answer 3

Adsorption chromatography: The stationary phase is a solid material.
Separation is based on the adsorption of sample compounds onto the surface of this active.

Partition chromatography: The stationary phase is a thin film of liquid coated on a solid support. Solutes separate by equilibrating between the mobile phase (liquid or gas) and the stationary liquid phase

Question 4

Explain the difference between isothermal and temperature programming which is
commonly used with Gas Chromatography and discuss the effect of using both
programmes on the separation of a mixture (containing compounds of varying
volatility).

Answer 4

Isothermal GC: The oven temperature is held uniform and constant throughout the analysis

Temperature Programming GC: The oven temperature is changed over time during the analysis It is suitable for separating mixtures with a wide range of volatilities or similar polarities.

Effect on separation Isothermal separation leads to poor resolution of peaks. Temperature programming improves the separation quality in shorter times

Question 5

Briefly explain the following four chromatographic terms:
Elution, Resolution, Efficiency and void time

Answer 5

Elution: The process where solutes are washed through the stationary phase by the mobile phase

(ii) Resolution: A measure of the separation quality between two peaks

(iii) Efficiency: A measure of how well a column minimises peak broadening,

(iv) Void time (to): The time taken for a component that is not retained by the stationary phase to reach the detector

Question 6

In Mass Spectrometry (MS), a soft ionisation method is typically used to ionise large
biomolecules such as proteins. Briefly explain the mode of operation of one soft
ionisation method w0hich could be used for the ionisation of proteins.

Answer 6

Electrospray Ionization (ESI)
In ESI, the liquid sample containing the protein is forced through a capillary at a high voltage.
This process electrostatically disperses the analyte into a fine spray
The charge is imparted to the protein molecules.
ESI typically produces multiply charged ions.
Soft ionization methods keep the molecule of interest intact

Question 7

Which amino acids bind to divalent cations such as Nickel?

Answer 7

Histidine (His), Cysteine (Cys), and Tryptophan (Trp)

Question 8

Define the term “ligand” in relation to affinity chromatography. Provide 3 examples of ligand-protein interactions that reflect the variety of affinity
chromatography types. [5 Marks]

Answer 8

A ligand is the molecule that is immobilised on the stationary phase that binds specifically and reversibly to the target molecule

Recombinant His-tag on a protein binding to immobilised Nickel ions

An antibody binding to a specific protein of interest

Protein A or Protein G binding to the Fc region of antibodies

Question 9

Differentiate between normal phase HPLC and reverse phase HPLC. In each case,
include an example of a suitable stationary phase and mobile phase, and explain how
polar and non polar sample analytes will interact with both the stationary and mobile
phases.

Answer 9

Normal Phase HPLC: Polar Stationary Phase, non Polar Mobile Phase .
Analyte Interaction: Less polar compounds interact more with the non-polar MP and move faster

Reverse Phase HPLC: Non polar Stationary Phase hydrocarbons, Polar Mobile Phase typically aqueous,
Analyte Interaction: Less non-polar compounds interact more with the polar MP and move faster

Question 10

Briefly explain column bleed in relation to Gas Liquid Chromatography (GLC)
stationary phases. In your answer, explain what effect column bleed has on the
separation of compounds and what type of columns can reduce/eliminate column
bleed.

Answer 10

Column bleed refers to the gradual release of stationary phase components from the column.
To reduce column bleed, chemically bonded stationary phases are used. In these columns, the liquid stationary phase is covalently bonded to the support particle which lessens the chance of losing the sp

Question 11

Identify 2 techniques that are used to support the interpretation of chromatographic
data when separating proteins. State the purpose of these techniques. [5 Marks]

Answer 11

SDS-PAGE: Its a denaturing technique that separates proteins by their mass.
It can be used to determine if the protein is pure after chromatography .
The fractions that contain the target protein and its purity can be visualized.

Activity Assay : used to determine if the protein is still active after the separation process. This is crucial in purification to ensure that the protein retains its function.

Question 12

List 3 major considerations when carrying out gel filtration chromatography.

Answer 12

Usually used late in purification as a “polishing step”: A low amount of proteins can typically be resolved in a single gel filtration run, making it best suited for samples that are already partially purified.

Sample volume: The volume of protein injected onto the column should not be more than 2% of the total column volume (Vt)

Only one buffer should be used, and it should be the optimal pH for the protein of interest

Question 13

List 3 modes of adsorption chromatography and the basis for their ability to separate
proteins.

Answer 13

Ion Exchange Chromatography: Separation is based on charge. It exploits the reversible interactions between proteins and oppositely charged ion groups bonded to the stationary phase.

Hydrophobic Interaction Chromatography (HIC): Separation is based on protein surface hydrophobicity. Proteins bind to a hydrophobic stationary phase through hydrophobic interactions, which are enhanced at high salt concentrations Elution is achieved by decreasing the salt concentration,

Affinity Chromatography: Separation is based on the specific affinity of a protein for an immobilised ligand on the stationary phase. The protein of interest specifically binds to this ligand, while impurities flow through.

Question 14

Briefly explain why a wide range of stationary phases are needed in GC, while a
smaller number are required for HPLC.

Answer 14

In Gas Chromatography (GC), the mobile phase is typically a gas. Separation relies heavily on the interaction between the analyte and the stationary phase, since it’s a gas a wide range of stationary phases with varying polarities are needed to provide different interactions.

In High Performance Liquid Chromatography (HPLC), the mobile phase is a liquid, Separation can be significantly influenced and controlled by changing the composition of the mobile phase.

Question 15

Define the term IMAC and identify 4 metals that could be used in this type of
chromatography. [5 Marks]

Answer 15

IMAC stands for Immobilised Metal Affinity Chromatography.
It is a type of affinity chromatography where metals are immobilised on a solid stationary phase.
Proteins can then bind to these immobilised metals, typically via residues like Histidine.
Four metals that could be used in IMAC are: Cu, Zn, Co and Ni

Question 16

Define the term void volume (Vo) and identify a molecule that could be used to
determine the void volume of a gel filtration column.

Answer 16

Void volume (Vo) is the volume of liquid outside the resin beads in a gel filtration column, It is not retained by the stationary phase.
Blue dextran (2000 kDa) Its large and elutes in the void volume.

Question 17

Explain the role of ammonium sulphate in hydrophobic interaction chromatography.

Answer 17

High salt concentrations, increase the hydrophobic interactions between the protein and the hydrophobic stationary phase.
Salts can increase the ordered structure of water around hydrophobic regions.
Proteins are loaded onto the column at high salt concentration to bind to the hydrophobic stationary phase decreasing the salt allows proteins to detach

Question 18

What are the advantages and disadvantages of Gas Chromatography (GC) over
High Performance Liquid Chromatography (HPLC)?

Answer 18

GC is quicker, easier and cheaper to use.
Capillary GC often offers higher resolution.
Detection limits are often lower.

Question 19

Explain what is meant by the term “solvation shell” as it relates to proteins.

Answer 19

When a protein is in water, water molecules form a highly ordered network or “solvation shell” around it .
This shell forms around the hydrophobic and hydrophilic patches on the surface.

This needs to be disrupted for hydrophobic parts of the protein surface to interact with the sp

As the salt conc is decreased, the solvation shell is restored, weakening hydrophobic interactions.
This causes the protein to elute from the column.

Question 20

Explain the chromatographic term “adjusted retention time (t’r)”. Include in your
answer details of how t’r is calculated (defining each of the terms) and explain what
does t’r actually measure.

Answer 20

Adjusted retention time (t’r) is a measure of the actual amount of time a sample component spends on the stationary phase
The formula is: t’r = tr - t₀

Question 21

Explain the difference between a weak and a strong ion exchanger, outline one advantage/ disadvantage

Answer 21

Strong exchanger can resist pH change over a broad range,
Weak exchanger cannot resist change over broad pH range,

Strong is methyl sulphonate and Weak is DEAE .
Adv of strong is wide ph stability. Disad of strong is will bind wide range of proteins including unwanted.

Adv of weak is more selective binding and dis of weak is lower binding capacity.

Question 22

List 4 major differences between HPLC and FPLC

Answer 22

HPLC small molecule analysis. FPLC biomolecule purification.

HPLC smaller column and smaller particle size. FPLC larger column and larger particle size.

HPLC high pressure FPLC low pressure.

HPLC high flow rate and fast separation. FPLC low flow rate and slower separation

Question 23

A biochemist wanted to isolate a recombinant enzyme without the use of an affinity tag and they chose antibody mediated affinity chromatography instead. An antibody that typically binds to the target protein was prepared.

Describe how the biochemist should immobilise the antibody on an agarose medium.

Answer 23

Protein A agarose or protein G agarose can be used for antibody immobilization, as these come pre-activated for antibody binding.

Streptavidin can be coupled to pre-activated agarose beads.
Biotin binds to a number of different reactive groups found on proteins (including antibodies)

Biotinylated antibodies passed over the column will bind very strongly to the streptavidin

Question 24

3 reasons agarose is a suitable matrix for affinity chromatography

Answer 24

Extremely low non-specific adsorption, OH groups for easy derivatisation, Open pore structure to allow binding of large molecules

Question 25

An antibody expressed by CHO cells have a mw of 150 kDa. Protein x has a mw of 42 KDA and protein Y has a mw of 210 kDa

Answer 25

In gel filtration, protein y elutes first as it is largest at 2120kda then recombinant antibody as it is 150kda, then protein x as it is the smallest. In protein a affinity chromatography, the two contaminants elute together as they have no affinity to ligand and then the antibody is eluted by a change in ph.

Question 26

Outline the principle of gel filtration

Answer 26

Gel filtration chromatography separates molecules by size using a column packed with porous beads. Larger molecules elute first because they cannot enter the pores, while smaller ones are delayed as they travel through the pores. Protein 3 is largest so will elute first followed by protein 2 because is can enter the larger pores so will take longer to elute than protein 3 and protein 1 is smallest so enters all the pores and takes longest to elute.

Question 27

Why is it beneficial to add 100-200 mM or KCL to any buffer used for gel filtration.

Answer 27

Minimizes nonspecific ionic interactions between proteins and the matrix. Reduces protein aggregation or sticking to the column wall or resin.

Question 28

What happens to proteins that are loaded onto a column that have a mw outside the fractionation range

Answer 28

Above range (too large): Cannot enter any pores → elutes at Vo (very early). Below range (too small): Enters all pores → elutes at or near Vt (very late).

Question 29

Outline 2 options to elute a protein from a hydrophobic interaction column and explain its principle Identify 2 hydrophobic ligands

Answer 29

b. Lower salt conc reforms solvation shell and protein detatches from column. Salt “steals” water from proteins. Add a small amount of organic solvent like isopropanol to disrupt solvation shell butyl, phenyl, octyl in order of increasing hydrophobicity

Question 30

State the Ligand thats used in immuno-affinity chromatography and explain in detail one way the ligand is coupled to an agarose matrix

Answer 30

Immobilise Streptavidin on a pre-activated agarose bead matrix . Generate an antibody against the protein of interest and biotinylate it. Pass the biotinylated antibody over the column; it will bind very strongly to the immobilised streptavidin

Question 31

Name 3 ways to elute a protein bound to an IMAC column

Answer 31

Imidazole competes with histidine residues for binding to the metal ions. * Lowering the pH protonates the histidine residues, preventing them from coordinating with the metal ion. Add a chelating agent that strips the metal ion from the resin.

Question 32

Provide 3 examples of amino acids that facilitate the binding of proteins to HIC column

Answer 32

Phenylalanine, leucine and isoleucine

Question 33

Explain the mode of operation of the thermal conductivity detector. Include which carrier gas should be used for maximum response

Answer 33

TCD is a universal detector, meaning it can detect both organic and inorganic compounds. It works by measuring the difference in thermal conductivity between the mobile phase alone which is mixed with eluting analyte molecules It uses a dual-channel system with a reference channel through which the carrier gas flows, and a sample channel through where the column effluent passes. Helium should be the carrier gas used.

Question 34

Differentiate between a fixed wavelength UV detector and diode array detector which can be used in HPLC. which is better

Answer 34

Fixed Wavelength UV Detector: measures the absorbance of the eluent at a single, specific wavelength, typically 254 nm. If an analyte absorbs at this wavelength, a signal is generated. Diode Array Detector (DAD): uses a photodiode array to simultaneously measure the absorbance across a range of wavelengths for each eluting component. It can capture the full UV-Vis spectrum of each peak as it passes through the detector cell. The Diode Array Detector (DAD) is generally more advantageous than a fixed wavelength detectors

Question 35

Outline the main differences in packed and capillary GC columns

Answer 35

Packed columns are shorter and wider with solid support, good for large samples but lower resolution. Capillary columns are narrow and long, with a stationary phase on the inner wall, providing high efficiency, resolution, and speed, ideal for detailed analytical work.

Question 36

If you increase the % methanol in the mobile phase what effect would this have on the retention time hydrocortisone

Answer 36

methanol competes more effectively with hydrocortisone for interactions with the non-polar stationary phase. As a result, hydrocortisone elutes faster, leading to a shorter retention time.