Affinity

Chromatography:
Review Of Clinical
Applications
Presented By:
Zyad Nayef 19128
Abdelrahman Ahmed Nawwar 19163
Abdullah Salem Sultan 19169
Mohamed Mamdouh 19212
Mostafa Abdelmoaty Salama 19231
Mostafa Mahmoud Fathy 19232
Youssef Moamn Megally 19309
Mohamed Samir Ahmed 19208
 Chromatography is a technique used to separate and analyze
mixtures of substances based on their differential movement
through a stationary phase. It's widely used in various fields like
Chromatography chemistry, biochemistry, pharmaceuticals, and environmental
science for identifying and quantifying compounds in a mixture.
There are different types of chromatography, including gas
chromatography, liquid chromatography, and thin-layer
chromatography, each with its own applications and principles.
 Liquid Chromatography
Liquid chromatography (LC) is a chromatographic technique where the mobile phase is a liquid. It
involves the separation of components of a mixture based on their interaction with a stationary phase,
which is typically packed into a column. The sample is dissolved in a solvent and passed through the
column, where different components interact differently with the stationary phase, causing them to
elute at different times. LC is widely used in analytical chemistry for the separation and
quantification of compounds in complex mixtures, such as in pharmaceutical analysis, environmental
monitoring, and biochemical research. High-performance liquid chromatography (HPLC) is a
common variant of liquid chromatography known for its high resolution and sensitivity.
 High-Performance Liquid Chromatography
(HPLC)
It's a technique used to separate, identify,
and quantify components in a mixture. It's
widely used in various industries like
pharmaceuticals, food and beverage,
environmental analysis, and more. HPLC
works by pumping a sample mixture
through a column packed with a stationary
phase, where different components in the
sample interact differently with the
stationary phase, causing them to separate.
 Advantages of HPLC
1. High Sensitivity: HPLC can detect and quantify trace amounts of compounds in a sample,
making it suitable for analyzing complex mixtures.

2. Versatility: It can analyze a wide range of compounds, including polar and non-polar
substances, small molecules, proteins, and peptides.

3. Speed: HPLC typically provides faster separations compared to traditional liquid

4. Accuracy and Precision: HPLC systems offer high accuracy and precision in quantitative
analysis, making them reliable for quality control and research applications.

5. Automation: Many HPLC systems are fully automated, reducing the need for manual
intervention and increasing reproducibility.
 Disadvantages of HPLC
1. Cost: HPLC systems can be expensive to purchase, operate, and maintain, especially high-
end instruments with advanced features.

2. Complexity: Operating HPLC systems requires specialized training and expertise,

3. Sample Preparation: Sample preparation can be time-consuming and labor-intensive,

4. Column Lifetime: HPLC columns have a limited lifespan and may need frequent
replacement, adding to operating costs.

5. Solvent Usage: HPLC typically requires large volumes of organic solvents, which can be
costly and generate waste, posing environmental concerns
 Direct Analyte Detection By
Affinity Chromatography
In direct analyte detection by affinity chromatography, the ligand that has high affinity for the
target analyte is immobilized on a chromatography column or surface. When a sample
containing the analyte is passed through the column or over the surface, the target analyte
selectively binds to the immobilized ligand while other components in the sample pass
through or are washed away.

After the sample has been applied and unwanted components have been removed, the bound
analyte can be eluted from the column or surface using a suitable elution buffer or conditions.
The eluted analyte can then be quantified using various detection methods such as UV-vis
spectroscopy, fluorescence spectroscopy, mass spectrometry, or other analytical techniques
depending on the nature of the analyte.
 Direct Analyte Detection By
Affinity Chromatography
separate and detect specific analytes, such as fibrinogen, based on their affinity for a
particular ligand immobilized on the chromatography matrix. In the case of fibrinogen,
which is a blood plasma protein involved in blood clotting, the affinity chromatography
column would be functionalized with a ligand that specifically binds to fibrinogen. As the
sample passes through the column, fibrinogen molecules bind to the immobilized ligand
while other components of the sample flow through or are washed away. After the
separation, the bound fibrinogen can be eluted from the column for further analysis
or quantification.
 Chromatographic
A chromatographic Immunoassays
immunoassay is a technique that combines the principles of
chromatography and immunoassay for the detection and quantification of specific analytes
(such as proteins, hormones, drugs, etc.) in a sample. It involves the use of an antibody or
antigen immobilized on a solid support, which interacts with the target analyte as the sample
passes through a chromatographic matrix. The analyte-antibody or analyte-antigen complex is
then separated based on its affinity for the immobilized molecule, allowing for qualitative or
quantitative analysis. These assays are commonly used in clinical diagnostics, drug testing,
and environmental monitoring.
 Chromatographic
Immunoassays
There are several different methods for performing chromatographic immunoassays, but the most
common method uses a competitive binding format. The easiest approach is to mix the sample with a
labeled analyte analog (i.e., the label) and to inject the mixture simultaneously onto an
immunoaffinity column that contains a relatively small amount of antibody. This format, known as a
“simultaneous injection competitive binding immunoassay”, is the most common approach for
chromatographic immunoassays. A specific example of this method in which theophylline was
measured in serum by a flow-injection competitive binding immunoassay that used
carboxyfluorescein-impregnated liposomes as the label. The injected samples contained 50 m L of
serum diluted 1:100 and combined in a 1:2 ratio with a working solution of the label.
 Affinity Extraction
Affinity extraction of chromatography is a purification technique to isolate target molecules based on a
specific biological interaction, such as that between an antigen and antibody, a lectin and a carbohydrate,
or a chelated metal ion and a histidine peptide (His tag).
now affinity extraction involves combining the
affinity column either off-line or on-line with
some other method for the actual quantification
of analytes. Affinity extraction represents one of
the most common uses of affinity
chromatography in chemical analysis. This
section will examine some applications of
affinity extraction, including both off-line
methods and those that involve the on-line
coupling of affinity columns with techniques
such as HPLC, gas chromatography (GC), or
capillary electro- phoresis (CE).
 Postcolumn affinity detection
Postcolumn affinity detection is a method used in chromatography to detect analytes separated by
chromatographic techniques such as liquid chromatography (LC) or gas chromatography (GC). In this
method, after separation of analytes in the chromatographic column, a postcolumn reaction or interaction is
employed to selectively detect or quantify specific analytes. This can involve a chemical reaction, binding to
a specific ligand or receptor, or other affinity-based interactions.

Yet another way in which affinity columns can be used is for monitoring the elution of specific solutes from
other chromatographic columns. This involves the use of a postcolumn reactor and an affinity column
attached to the exit of an analytical HPLC column. A large number of affinity ligands can be used for this
purpose. One specific example is the reported use of anion-exchange chromatography followed by an HPLC
boronate column for the determination of glycated albumin in serum samples
 Affinity-based Chiral Separations
Another important application of affinity ligands has been in the analysis of chiral compounds.
Because of pressure from regulatory agencies such as the US Food and Drug Administration, there
has been increasing interest in the pharmaceutical field for methods capable of discriminating
between the individual chiral forms of drugs. various naturally occurring proteins and
carbohydrates have been used as ligands for chiral separations of clinical analytes.

One class of natural carbohydrates that can be used as stereoselective ligands in HPLC are the
cyclodextrins. The cone-shaped structure and hydrophobic interior cavity of cyclodextrins give
them the ability to form inclusion complexes with numerous small, aromatic solutes. Furthermore,
the well-defined arrangement of hydroxyl groups about the upper and lower faces of the
cyclodextrins provide these agents with the ability to discriminate between various chiral
compounds. Examples of some clinical applications for cyclodextrins in HPLC are
chlorpheniramine and citalopram
 Affinity-based Chiral Separations
Although all proteins are chiral, only one [a1-acid glycoprotein (AGP)] has seen any significant use in the
analysis of chiral drugs in clinical samples. The low isoelectric point the AGP has makes it useful in
binding to cationic compounds. There are many drugs and related solutes that have been separated by
AGP in human urine, serum, or plasma. Examples of clinical interest include ibuprofen, ketoprofen,
ketamin.
 Characterization of Drug- and
Hormone-Protein Interactions
In addition to its applications as a method for quantifying or isolating specific solutes, affinity
chromatography can also be used in studying the interactions that take place between biomolecules.
Such an approach is known as “analytical” or “quantitative affinity chromatography”. This area has
been the subject of several past reviews and has been used to examine a variety of biological systems,
including lectin/sugar, enzyme/inhibitor, protein/protein, and DNA/protein interactions. For example,
it has been shown that association constants measured by equilibrium dialysis for soluble HSA with
Rand S-warfarin or l-tryptophan (i.e., solutes that interact with one of the two major binding regions of
HSA) are in close agreement with values determined using immobilized HSA columns (206–208). It
has also been found that displacement phenomena and allosteric interactions observed for HSA
columns are representative of behavior noted for HSA in solution.
 Future Trends and Developments
Future trends and development may involve enhanced selectivity and improved methodologies,
addressing current limitations and expanding applications. Affinity Chromatography is essential in
protein purification due to its specificity. Biomedical applications, like antibody purification,
highlight its importance.

Conclusion
Affinity chromatography has found extensive applications in clinical settings due to its ability to isolate
and purify biomolecules with high specificity and efficiency. It plays a crucial role in protein purification
for therapeutic use, diagnostic assays, and drug development. Its precision in targeting specific
biomolecules makes it invaluable in areas such as antibody purification, enzyme immobilization, and
isolation of nucleic acids. Overall, affinity chromatography is a powerful tool that significantly contributes
to advancements in clinical research and healthcare.
Thank
you