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Assignment on Chromatography

Chromatography is a separation technique in which a mixture of components (to be separated) is

applied into the surface or solid stationery phase with partitioning effected by mobile phase. The
separation is accomplished by utilising main molecular characteristics which relate to adsorption
(liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights
(Cuatrecasas et al., 1968; Porath, 1997). Due to these discrepancies in characteristics, separation
occurs as some components are retained in the in the stationery phase and some are eluted through
the mobile phase (Harris, 2004).

My research project will employ two chromatographic techniques. My project is based on purification
of H22(scFv)-ETA’, a recombinant immunotherapeutic construct which is a fusion protein made form
the humanised (hence H22) single chain variable fragment (scFv) of antibody and truncated
Pseudomonas exotoxin A’. The first chromatographic technique that I will employ is the immobilized
metal affinity chromatography (IMAC), which was originally proposed by Porath et al. (1975). This
technique is based on the affinity of transition metal ions like Zn(II), Cu(II), Ni (II), and Co(II) ions toward
cysteine, histidine, and tryptophan in aqueous solutions. The protein of interest, H22(scFv)-ETA’ will
be tagged with histidine.

The chromatographic sorbent utilized in IMAC consist of a support and between is a linker that
connects the chelating ligand to a support. When a metal is added (Nickel (ii) will be used in my project)
it forms a complex with the chelating ligands. As the metal is chelated, some coordinate sites of the
metal ions are not complexed and therefore they remain unoccupied until they bind histidine which
is attached to the protein (Kagedal 2011). Elution of the target protein can be carried by using (1) a
low-pH buffer, (2) a competitive displacement agent, or (3) chelating agents which would occupy the
binding sites of the metal. Elution buffers with a low pH value (for example, pH 4.0) are normally used.
The pKa of the imidazole ring of the histidine residue is about 6.0. Because the pKa is greater than pH
of the solution of the buffer therefore the imidazole (structure in the histidine) will be protonated.
Due to this condition, the protonated imidazole ring can no longer bind to the nickel (ii) and is thus
eluted. For the protein that are sensitive to pH, employing a competitive displacement with imidazole
at approximately neutral pH is more appropriate for proteins sensitive to low pH.

The second chromatographic technique that I will use in my project is size exclusion chromatography
(SEC). The understanding of the principle behind the size-based separations by chromatography was
initially suggested by Synge and Tiselius (Synge, 1950), following the observation that small molecules
can be separated through the small pores of zeolites based on their molecular size (Barrer, 1944). The
chromatography column is packed with fine, porous beads which are composed of dextran polymers
(Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore sizes of these
porous beads are used to estimate the dimensions of macromecules. H22(scFv)-ETA is 70 kDa in size
and by using a size range of the column that retains 70 kDa, H22(scFv)-ETA can be separated with
some proteins having a similar size. Small proteins including H22(scFv)-ETA diffuse into the pores of
the gel beads and their flow through the column is retarded according to their size, whereas those
molecules larger than 70 kDa large do not enter through the pores and are eluted first in the column's
void volume. I will use Bio-Prep SE-100/17 column (because of the molecular size range that
corresponds with the target protein) and will conduct the setup according to the manufacturer’s

instructions. H22(scFv)-ETA which diffused through the porous beads will be will be eluted with
Phosphate-buffered saline (PBS) (pH 7.4) and 1 m NaCl.


Barrer, R.M., 1944. General and Physical Chemistry. III. Zeolites as adsorbents and molecular sieves.
Annu. Rep. Prog. Chem, 41, pp.31-46.

Cuatrecasas, P., Wilchek, M. and Anfinsen, C.B., 1968. Selective enzyme purification by affinity
chromatography. Proceedings of the National Academy of Sciences, 61(2), pp.636-643.

Garrett, Reginald H.; Grisham, Charles M., 2013. Biochemistry (5th ed.). Belmont, CA: Brooks/Cole,
Cengage Learning. p. 108.

Harris, D.C., Exploring chemical analysis. 3rd ed. WH. Freeman&Co; 2004.

Kagedal, L., 2011. Immobilized metal ion affinity chromatography. In: Janson JC, Ryden L (eds) Protein
purification: principles, highresolution methods, and applications, 3rd edn. Wiley, New York, pp 183–

Porath, J., 1997. From gel filtration to adsorptive size exclusion. Journal of protein chemistry, 16(5),

Synge, R.L., 1950. Fractionation of hydrolysis products of amylose by electrokinetic ultrafiltration in

an agaragar jelly. The Biochemical journal, 2, p.41.