Advanced analytical Chemistry

ADVANCED ANALYTICAL
CHEMISTRY
Assignment No. 1

Name:
▪ Iram Javed
▪ Azra Batool
▪ Misbah Shaheen
▪ Balqees
▪ Javeria Zahid
▪ Muhammad Arslan

Department: BS Chemistry

Submitted to: Mam Arooba

University of Education Lahore, Jauharabad campus

1 Advanced analytical Chemistry

Question No. 1

How will you purify the mixture of enzyme and protein by using
chromatography?

Answer:
Chromatography is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for qualitative and quantitative
analysis.
Column chromatography is commonly used for protein purification. Column chromatography
can separate protein on based on their size, charge, hydrophobicity, or specific binding
interactions. There are several types of column chromatography commonly in protein purification,
including:
➢ Ion exchange chromatography: Separates proteins based on their net charge.
➢ Size exclusion chromatography: Separates proteins based on size.
➢ Affinity chromatography: Utilizes specific interactions between a ligand and the target
protein/enzyme.
Ion Exchange Chromatography
Ion exchange chromatography is broken in to two types - anion & cation exchangers. There
are many different types of moieties that are used from weakly to very strongly charged thus
allowing a huge range of molecules the ability to interact.
Unlike gel filtration chromatography, here proteins directly interact with the resin. So
generally, the column is equilibrated in a buffer solution to establish a constant pH in the column,
then the protein mixture is loaded where all or some of the proteins interact with the resin
depending upon their own charge. Buffer is continued to be applied until all proteins not interacting
with the resin have been washed off. At that point usually, a gradient of increasing salt
concentration (disrupts ionic and hydrogen binding) in the buffer is applied to column allowing
the weakest interacting proteins to release first followed by the more strongly and finally the most
strongly interacting. This can also be accomplished by changing the pH of the buffer being applied
to the column.
Anion Exchanger
Anion exchanger means that it removes anions from protein mixture so that means the resin
must be decorated with positively charged moieties. Before elution begins all positively and
uncharged proteins will fall through the column. When you start eluting, first you will knock off
the weakly negative proteins (e.g. -1 charge), followed by those with a stronger negative charge (-
2), and finally the most negatively charged proteins (-3).
Cation Exchange
It is exactly opposed with a cation exchanger -- here cations are removed from the protein
solution so the resin must be negatively charged. Again, before elution begins all negatively and
uncharged proteins will fall through the column. When you start eluting, first you will knock off
the weakly positive proteins (e.g. +1 charge), followed by those with a stronger positive charge.
Affinity Chromatography Steps
In affinity chromatography, proteins are loaded on the column under conditions that
influence binding between the protein (or tag) and its ligand. The bound protein is washed under
conditions that do not disrupt the specific interaction, but that can disrupt any non-specific

1|Page
2 Advanced analytical Chemistry

interactions between contaminating proteins and the stationary phase. The bound protein is then
eluted with a buffer containing a competing molecule or conditions that disrupt all protein/protein
interactions. Competing molecules bind to the ligand, displacing the protein of interest. This
competing molecule is typically removed from the protein of interest either through another
chromatographic procedure or dialysis.
Gel Filtration/Size Exclusion
In gel filtration, or as it is sometimes referred to as size exclusion, chromatography the
resin are porous (see figure to the left). Some molecules (blue here) can enter the resin and as the
lines try to indicate it is not a straight path through; thus, it takes longer for small molecules to
traverse the column than large molecules which travel around the outside of the resin. This is
highlighted in the figure to the right where big molecules (blue) come off first and smaller
molecules (red) later.

Affinity Ion exchange Size-exclusion Hydrophobic

chromatography chromatography chromatography interaction
chromatography
Protein Biorecognition Charge Size Hydrophobicity
property
Applications Receptor and Charged Large Proteins and
ligand, enzyme molecules molecules, peptides with
and substrate, macromolecular hydrophobic
antigen and complexes amino acid side
antibody chains on
surfaces
Advantages • Able to isolate • High accuracy • High recovery • High selectivity
one specific and precision yield • Mild, non-
protein at a time • High matrix • Well defined denaturing
• High recovery tolerance separation time conditions
yield • High • Narrow bands
• Rapid selectivity available
separation
Disadvantages Demand ligand Inconsistency Demand Too strong
with high from column to differences in interactions
selectivity column MW
Question No.2
Write down the applications of Ion-Exchange chromatography?
Answer:
Ion exchange chromatography for purification of protein
Significant cations and anions separation are possible by the use of ion-exchange methods.
It facilitated the study of environmental speciation. Toxic metal pollutants present in
aquatic and environmental can be analyzed by ion-exchange methods.

2|Page
3 Advanced analytical Chemistry

It is used for the separation of purification blood components. It is also used to detect
different kinds of renal diseases.
It is used for the determination of sulfur compounds and analysis of inorganic anions in the
petrochemical and mining industries.
To analysis micronutrients in soils, we used ion exchange chromatography methods.
Therefore, ion exchange technique has very wide and interesting applications. It provides
a sensitive method of analysis. It separated or purification many ions present on molecules,
biomolecules in very low time intervals.

3|Page