1. Define chromatography.

Explain what we mean by stationary and mobile phase in a

chromatography column.
a technique for the separation of a mixture by passing it in solution or suspension through a
medium in which the components move at different rates.

All forms of chromatography work on the same principle. They all have a stationary phase
(a solid, or aliquid supported on a solid) and a mobile phase (aliquid or a gas). The mobile phase
flows through the stationary phase and carries the components of the mixture with it.

2. Explain gel& affinity chromatography techniques used for purification of

biopharmaceuticals.
Gel

Separation based on size, called size/molecular exclusion or gel

permeation chromatography. Stationary phase consists of porous beads with

well‐defined range of pore sizes. Stationary phase for gel filtration is said to have a

fractionation range, meaning that molecules within that molecular weight range can be

separated.

Affinity

Used for mAB capture (Protein A binds to IgG). Relies on highly specific recognition and binding of
target molecules onto ligands attached to the stationary phase such as [Protein A]

Biospecific ligands (Usually very expensive, 1 mg about US$ 10 (protein A))

• Often not stable ,Coupling chemically complex

• Problems with leaching of ligands.

3. Explain the term chromatogram. What types of sensors (or detectors) are used in gas
chromatography for component analysis?
It is a visible record (such as a graph) showing the result of separating the components of a
mixture by chromatography.

Flame ionisation detector, Mass spectrometer & flame photometric detector are examples of
detectors used in gas chromatography.

4. What instrumentation is used in gas chromatography?

Carrier gas, flow controller, injector port, column, column oven, detector, recorder

5. What physical properties of molecules are exploited in HPLC

HPLC works on the principle that some molecules take longer than others to pass through a
chromatography column. This depends on the affinity of the molecule with the mobile phase
(liquid or gas) and the stationary phase (solid or liquid). Size, shame, hydrophobicity and
charge are other examples
 6. One of the concepts used in chromatography is that of HETP (height equivalent to
theoretical plate).
(a) Explain how HETP is related to band spreading.
(b) State the equation that describes the factors that affect HETP and explain these factors.
(c) Describe two strategies in chromatography that can improve the value of HETP.

a. HETP indicates the height of the theoretical plate and usually if the plate length is too think
separation will not be efficient and a smaller HETP is preferred

b. Performance of column determined by HETP (H) , the

height equivalent of a theoretical plate where L=total

length of column (HETP) = L/N

c. To improve HETP, a smaller value is preferred thus smaller plates are smaller number of plates of
smaller sizes.

7a. What is the isoelectric point pI of a protein?

The pH at which the net charge on the protein is zero. For a protein with many basic amino
acids, the pI will be high, while for an acidic protein the pI will be lower.

7b. Draw glycine as positively charged, negatively charged and uncharged amino acid. How can
one form be converted into the other?

8a. Explain the principle of separation by ion exchange with the help of
diagrams.

IEX or IEC

• Very commonly used for protein purification

• High resolution

• High capacity

• Relies on charge‐charge interactions

• Between protein and immobilised charges on resin

• Anion or cation exchange

b. What steps are involved in performing IEX?

Resin (packing material) is bounded to charged molecules by

covalent bonds, Proteins with opposite charge will bind to charged molecule on

resin and remain in column. Later, target protein will be eluted using stronger buffer to

replace proteins.

c. What is the fundamental difference in separation between anion exchange and cation
exchange chromatography in terms of separated proteins?

Anion - Stationary phase is positively charged while counter

ions will be negatively charged.

Cation - Stationary phase will be negatively charged while

counter ions will be positively charged.