Chromatographic Techniques

Chromatography is a laboratory technique used to separate mixtures into their individual

components. The term "chromatography" originates from the Greek words "chroma" (color) and
"grapho" (to write), as early methods were used to separate colored substances. Over time,
chromatography has evolved to include a variety of techniques used for separating and analyzing
different types of mixtures.

Definition:

Chromatography is defined as a procedure by which solutes are separated by dynamic

differential migration process in a system consisting of two or more phases, one of which moves
continuously in a given direction and in which the individual substances exhibit different
mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular
size, or ionic charge density.

The sample to be examined is allowed to interact with two physically distinct entities—a mobile
phase and a stationary phase.

The sample most often contains a mixture of several components to be separated.

 The molecules targeted for analysis are called analytes.

 The mobile phase, which may be a liquid or gas, moves the sample components through a
region containing the solid or liquid stationary phase, which is called the sorbent.
 The eluent is the solvent that carries the analyte.
 A chromatogram is the visual output of the chromatograph.

 The eluate is the mobile phase leaving the column.

 The detector refers to the instrument used for qualitative and quantitative detection of
analytes after separation.
 The general process of moving a sample mixture through a chromatographic system is
called development.
 Retention Factor (Rf): In TLC, this is the ratio of the distance traveled by a substance to
the distance traveled by the solvent front.
 Partition Coefficient: Describes the distribution of a compound between the stationary
and mobile phases.
 Resolution: Refers to the ability of the chromatographic system to separate different
components. High resolution is desirable for complex mixtures.

Basic Principle of Chromatography

The basis of all forms of chromatography is the distribution or partition coefficient (Kd), which
describes the way in which a compound (the analyte) distributes between two immiscible phases.
For two such phases A and B, the value for this coefficient is a constant at a given temperature
and is given by the expression:
Chromatography works based on the differential migration of components in a mixture due to
differences in their interactions with a stationary phase (solid or liquid) and a mobile phase (liquid
or gas). Components that interact more strongly with the stationary phase will move slower, while
those that interact more weakly will move faster.

The molecular components in the sample distribute themselves between the mobile phase and
sorbent and thus have the opportunity to interact with the stationary phase.

 If some of the sample molecules (analytes) are preferentially bound by the sorbent, they
spend more time in the sorbent and are retarded in their movement through the
chromatographic system.
 Molecules that show weak affinity for the sorbent spend more time with the mobile
phase and are more easily removed or eluted from the system.

The chromatographic method of separation, in general, involves following steps

• Adsorption or retention of substances on the stationary phase

• Separation of the adsorbed of substances by the mobile phase
• Recovery of the separated substances by a continuous flow of the mobile phase; the
method being called elution.
• Qualitative and Quantitative analysis of the eluted substances.

 Types of Chromatographic Techniques

Adsorption Chromatography
Adsorption Chromatography is a type of chromatography where the separation of components
in a mixture occurs based on their differential adsorption (attachment) to the surface of a solid
stationary phase. This technique is primarily used for separating and analyzing components of
complex mixtures.
1. Principle of Adsorption Chromatography

 Adsorption chromatography is based on the principle that different components in a

mixture have different affinities for the stationary phase, typically a solid surface, which
adsorbs the components.
 Components that are more strongly adsorbed will move more slowly through the
stationary phase, while those that are weakly adsorbed will travel faster.
 The differential adsorption (interaction with the stationary phase) is the key factor that
drives the separation of components.

2. Stationary and Mobile Phases

 Stationary Phase: The stationary phase in adsorption chromatography is a solid material,

often an adsorbent like silica gel, alumina, or activated carbon.
 Mobile Phase: The mobile phase is typically a liquid or a gas that moves over or through
the stationary phase, carrying the components of the mixture with it. The choice of the
mobile phase depends on the nature of the sample and the stationary phase.

3. Mechanism of Separation

 The separation in adsorption chromatography occurs due to the different affinities that the
components of the mixture have for the solid stationary phase.
 Strongly adsorbed components will spend more time interacting with the stationary phase,
slowing down their movement through the system.
 Components that are less strongly adsorbed will move faster and elute from the stationary
phase first.

Adsorbents in Adsorption Chromatography

The efficiency of adsorption chromatography largely depends on the choice of the stationary
phase (adsorbent):

 Silica Gel: Most commonly used adsorbent. It is polar and is ideal for separating polar
compounds in liquid-phase adsorption chromatography.
 Alumina (Al2O3): Also used as an adsorbent for separating less polar compounds.
 Activated Carbon: Often used for separating highly nonpolar compounds.
 Cellulose: Sometimes used for specific applications such as separating larger molecules.

Elution and Detection

 Elution: Elution is the process by which the components are removed from the stationary
phase by the mobile phase.
o In liquid-phase chromatography, solvents of increasing polarity (or strength) are
often used to elute the adsorbed components.
o In gas-phase chromatography, a carrier gas is used to carry the sample through the
stationary phase, and the elution time will depend on the components' adsorption
characteristics.
 Detection: The separated components can be detected using a variety of detectors
depending on the technique used (e.g., UV-VIS detector, flame ionization detector, etc.).
In thin layer chromatography, detection may be done visually or using chemical reactions.

7. Retention Factor (Rf)

  Retention factor (Rf): In chromatography, the retention factor is a measure of the relative
affinity of a compound for the stationary phase compared to the mobile phase.
o In adsorption chromatography, Rf is used to quantify how far a particular
component travels relative to the mobile phase.
o It is calculated as:

Rf=Distance travelled by solute/Distance travelled by solvent front

8. Applications of Adsorption Chromatography

 Separation of Organic Compounds: It is commonly used in the separation and

purification of organic compounds, particularly in the analysis of polar and nonpolar
substances.
 Purification of Natural Products: Adsorption chromatography is widely used in the
isolation and purification of natural products from complex mixtures.
 Pharmaceutical Industry: It is used for quality control, including checking the purity of
compounds.
 Food Industry: To identify and separate flavor components, colorants, and preservatives
in food.
 Environmental Analysis: For separating pollutants and other environmental contaminants
from water, soil, and air samples.
 Forensic Science: Used in the analysis of trace evidence, such as drugs and toxins.

9. Advantages of Adsorption Chromatography

 Simplicity: The technique is relatively simple and does not require complex
instrumentation.
 Cost-Effective: It is inexpensive compared to some other chromatographic techniques like
High-Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC).
 High Sensitivity: Adsorption chromatography can separate even small amounts of
compounds efficiently.
 Versatility: It can be used for both qualitative and quantitative analysis of mixtures.

10. Limitations of Adsorption Chromatography

 Resolution: The resolution may not be as high as other techniques like HPLC or GC,
especially for very complex mixtures.
 Slow Process: The separation may take longer than in more advanced chromatographic
methods.
 Limited Capacity: Adsorption chromatography is typically less suitable for large-scale
separations or high-throughput analysis.

11. Applications in Thin Layer Chromatography (TLC)

 In Thin Layer Chromatography (TLC), a thin layer of adsorbent (usually silica gel or
alumina) is spread on a glass or plastic plate.
 A small amount of sample is applied, and the plate is placed in a solvent (mobile phase).
The solvent moves up the plate by capillary action, carrying the components of the sample,
which are separated based on their adsorption affinity.
 TLC is commonly used for the rapid identification of compounds and checking the purity
of substances.

1. THIN LAYER CHROMATOGRAPHY

Thin Layer Chromatography (TLC) is a solid-liquid form of chromatography where the stationary
phase is a polar absorbent and the mobile phase can be a single solvent or combination of
solvents.

In TLC the stationary phase is a polar absorbent, like finely ground alumina (Al2O3) or silica
(SiO2) particles which are coated on a glass slide or plastic sheet to create a thin layer of the
particular stationary phase.

Partition chromatography
Partition Chromatography: Notes

Definition: Partition chromatography is a type of chromatography where separation occurs based

on the differential partitioning (distribution) of analytes between two immiscible phases: a
stationary phase and a mobile phase. In this technique, one phase is usually a liquid and the other
phase is typically either a gas or another liquid.

Key Concepts:

1. Stationary Phase:
o The stationary phase in partition chromatography is typically a liquid that is coated
on a solid support (such as silica or alumina) or a liquid that is immobilized in the
pores of a solid material.
o It is immiscible with the mobile phase, which allows for the partitioning of
compounds between the two phases.
2. Mobile Phase:
o The mobile phase can either be a gas or liquid that moves over the stationary
phase, carrying the sample components.
o The rate at which a compound partitions between the mobile and stationary phases
influences how it moves through the system.
3. Partition Coefficient (K):
o The partition coefficient (K) is a measure of how a compound partitions between
the stationary and mobile phases.
o It is defined as:
K=[Compound]stationary[Compound]mobileK = \frac{[Compound]_{stationary}}
{[Compound]_{mobile}}
o A higher partition coefficient indicates that the compound is more likely to stay in
the stationary phase, resulting in a slower migration through the chromatographic
column.
4. Separation Mechanism:
o Compounds are separated based on their differential solubility in the stationary and
mobile phases.
 o As the mobile phase moves, compounds that have a higher affinity for the
stationary phase (lower partition coefficient) move more slowly, while those with a
greater affinity for the mobile phase move faster.

Procedure:

1. Preparation:
o A sample mixture is applied to the stationary phase in a chromatographic column
or a thin layer of the stationary phase is placed on a surface.
2. Separation Process:
o The mobile phase is passed through or over the stationary phase. As it moves, the
compounds interact differently with the two phases based on their partition
coefficients.
3. Detection:
o As the compounds move at different rates, they are separated and can be detected,
usually through UV light absorption, fluorescence, or mass spectrometry.

Applications:

1. Chemical Analysis:
o Partition chromatography is widely used for the separation and analysis of complex
mixtures of organic compounds, such as amino acids, alcohols, and other small
molecules.
2. Environmental Testing:
o It is used to analyze pollutants in air, water, and soil samples by isolating harmful
compounds.
3. Pharmaceutical Industry:
o Partition chromatography helps isolate and purify active pharmaceutical
ingredients (APIs) and to test for purity.
4. Food Industry:
o It is used to test for additives, preservatives, and contaminants in food products.

Advantages:

 High Resolution: Partition chromatography can provide very high resolution separations,
making it ideal for complex mixtures.
 Flexibility: It allows for the separation of a wide range of compound types (polar, non-
polar, etc.).
 Versatility: It can be applied to liquid and gas phases and is adaptable to various
stationary phases.

Disadvantages:

 Limited Speed: Compared to other forms of chromatography, partition chromatography

can be slower due to the time it takes for compounds to partition between phases.
 Sensitivity to Temperature: The distribution of compounds can be affected by
temperature, which can limit its precision under varying conditions.

Conclusion:

Partition chromatography is a widely used and effective separation technique based on the
differential distribution of substances between two immiscible phases. Its versatility allows it to
be applied in numerous industries and scientific fields, from environmental testing to
pharmaceutical analysis.

Feature Adsorption Chromatography Partition Chromatography

Definition Separation based on differential Separation based on differential
adsorption of solutes onto a solid partitioning of solutes between two
stationary phase. immiscible liquid phases.
Stationary Solid (e.g., silica gel, alumina) Liquid coated on a solid support (e.g.,
Phase water or organic solvent coated on
inert material).
Mobile Phase Liquid or gas Liquid or gas
Separation Components adsorb to the Components dissolve in the stationary
Mechanism stationary phase with varying and mobile phases to different extents,
strengths, leading to different leading to separation.
retention times.
Interaction Physical adsorption or chemical Solute distribution between the
Type bonding between solutes and the stationary and mobile liquid phases
stationary phase. based on solubility.
Examples Thin Layer Chromatography Paper Chromatography, Gas-Liquid
(TLC), Column Chromatography, Chromatography (GLC), High-
Gas-Solid Chromatography (GSC). Performance Liquid Chromatography
(HPLC - reverse phase).
Key Factors Nature of adsorbent, polarity of Partition coefficient, solubility of
Affecting solutes, and strength of interactions solutes in both phases, polarity of
Separation (Van der Waals forces, hydrogen solvents.
bonding).
Usage Used for separation of compounds Used for separation of compounds
with significant differences in based on their relative solubilities in
adsorption properties. two liquid phases.
Size Exclusion Chromatography (SEC) - Notes

Definition: Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography
(GFC), is a chromatographic technique used to separate molecules based on their size. In SEC, the
stationary phase consists of porous beads, and the separation occurs as molecules pass through
these pores, with smaller molecules entering the pores and taking longer to travel through the
column, while larger molecules bypass the pores and elute more quickly.

Key Concepts:

1. Principle of Separation:
o SEC separates molecules based on their hydrodynamic size (also referred to as
molecular size or Stokes radius).
o The stationary phase in SEC consists of porous gel beads. As the sample is loaded
onto the column, the molecules interact differently with the pores:
 Large molecules: These molecules are excluded from entering the pores of
the stationary phase and move through the column faster.
 Small molecules: These molecules can enter the pores and spend more time
inside, causing them to elute slower than large molecules.
o The separation is based on the difference in the time spent by the molecules inside
the pores, with larger molecules being excluded from the pores and thus eluting
first.
2. Stationary Phase:
o The stationary phase in SEC is typically made of porous materials such as
agarose, dextran, or polyacrylamide, which form a gel-like structure with varying
pore sizes.
o The pore size distribution of the stationary phase determines the range of molecular
sizes that can be separated.
o Common stationary phases have pore sizes tailored for specific molecular weight
ranges (e.g., low-molecular-weight compounds, proteins, or nucleic acids).
 3. Mobile Phase:
o The mobile phase in SEC is typically an aqueous buffer or organic solvent,
depending on the nature of the sample being separated.
o The mobile phase does not interact with the sample molecules in terms of chemical
affinity but serves to transport the sample through the column.
4. Elution Profile:
o As the sample is eluted through the column, molecules of different sizes will
appear as separate peaks in the elution profile.
o Larger molecules will elute earlier, while smaller molecules will elute later.
o The elution is typically monitored using detectors such as UV absorption,
refractive index, or light scattering.
5. Void Volume (V₀) and Total Volume (Vt):
o Void Volume (V₀): The volume in the column that is outside the stationary phase
pores; this is where the largest molecules elute.
o Total Volume (Vt): The total volume of the column, including both the mobile
and stationary phases.
o The molecules elute in a range of times that correlates with their size relative to the
pore size distribution of the stationary phase.
6. Molecular Weight Estimation:
o By comparing the elution times or volumes of a sample with known molecular
weight standards, the molecular weight of the sample can be estimated.
o SEC is commonly used for the determination of the molecular weight distribution
in complex mixtures, especially for proteins and polymers.

Procedure:

1. Preparation:
o Select an appropriate column with the correct pore size range based on the size of
the molecules being separated.
o Choose a suitable mobile phase (e.g., buffer or solvent) for the sample.
o Calibrate the system with molecular weight standards to establish a calibration
curve for molecular size estimation.
2. Loading the Sample:
o A small volume of the sample is injected onto the column. The sample should
ideally be dissolved in the mobile phase to avoid interference with the separation
process.
3. Separation:
o The sample is carried through the column by the mobile phase. Larger molecules
elute first, followed by smaller molecules. The separation occurs as molecules
either enter or bypass the pores of the stationary phase.
4. Detection:
o The separated components are monitored and detected as they elute from the
column, often using UV absorbance, refractive index, or multi-angle light
scattering detectors.
5. Analysis:
o The elution profile is analyzed, and the size or molecular weight distribution of the
components is determined using the calibration curve.

Applications:

1. Protein Purification:
o SEC is commonly used to purify proteins based on size. It is an excellent method
for separating proteins or peptides without denaturing them.
 o It is also used to remove aggregates or impurities from purified proteins.
2. Polymer Characterization:
o SEC is used for determining the molecular weight distribution of synthetic
polymers, such as plastics and resins.
o It can provide insights into the degree of polymerization and polydispersity (the
distribution of polymer sizes).
3. Nucleic Acid Analysis:
o SEC is used in the separation of nucleic acids, like DNA or RNA, by size, allowing
for analysis of fragmentation or purification of oligonucleotides.
4. Carbohydrates and Small Molecule Separation:
o SEC can be used for the separation and analysis of sugars, polysaccharides, and
other small molecules based on their size.

Advantages:

 Gentle Separation: SEC separates molecules based purely on size, making it a mild
technique that doesn't rely on chemical interactions, which helps preserve the integrity of
sensitive molecules like proteins and nucleic acids.
 No Sample Degradation: The technique does not cause degradation or modification of
the analytes, unlike other chromatographic methods that may involve chemical
interactions.
 High Resolution for Size-Based Separation: It can effectively resolve different size
species in a mixture, particularly useful for proteins, polymers, and other biopolymers.

Disadvantages:

 Limited to Size-Based Separation: SEC is only effective for separating molecules with
significant differences in size. It is not suitable for separating molecules with similar sizes
or those that do not interact significantly with the stationary phase.
 Broad Elution Peaks for Large Molecules: For larger molecules, SEC may not provide
highly resolved peaks, as they may elute in a broad range.
 Slow: The separation can be slower compared to other chromatographic techniques such
as ion exchange chromatography or affinity chromatography, especially for large sample
volumes.

Conclusion:

Size Exclusion Chromatography is a powerful and non-destructive technique for separating

molecules based on size, widely used in biochemistry, molecular biology, and polymer science. It
is particularly beneficial for purifying proteins, determining molecular weight distributions, and
analyzing large biomolecules, all without the need for complex chemical interactions. Its primary
limitation is that it cannot be used to separate molecules that are of similar sizes or for complex
mixtures where size differences are not pronounced.
Size Exclusion Chromatography (SEC) - Notes

Definition: Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography
(GFC), is a chromatographic technique used to separate molecules based on their size. In SEC, the
stationary phase consists of porous beads, and the separation occurs as molecules pass through
these pores, with smaller molecules entering the pores and taking longer to travel through the
column, while larger molecules bypass the pores and elute more quickly.

Key Concepts:

1. Principle of Separation:
o SEC separates molecules based on their hydrodynamic size (also referred to as
molecular size or Stokes radius).
o The stationary phase in SEC consists of porous gel beads. As the sample is loaded
onto the column, the molecules interact differently with the pores:
 Large molecules: These molecules are excluded from entering the pores of
the stationary phase and move through the column faster.
 Small molecules: These molecules can enter the pores and spend more time
inside, causing them to elute slower than large molecules.
o The separation is based on the difference in the time spent by the molecules inside
the pores, with larger molecules being excluded from the pores and thus eluting
first.
2. Stationary Phase:
o The stationary phase in SEC is typically made of porous materials such as
agarose, dextran, or polyacrylamide, which form a gel-like structure with varying
pore sizes.
o The pore size distribution of the stationary phase determines the range of molecular
sizes that can be separated.
o Common stationary phases have pore sizes tailored for specific molecular weight
ranges (e.g., low-molecular-weight compounds, proteins, or nucleic acids).
3. Mobile Phase:
o The mobile phase in SEC is typically an aqueous buffer or organic solvent,
depending on the nature of the sample being separated.
o The mobile phase does not interact with the sample molecules in terms of chemical
affinity but serves to transport the sample through the column.
4. Elution Profile:
o As the sample is eluted through the column, molecules of different sizes will
appear as separate peaks in the elution profile.
o Larger molecules will elute earlier, while smaller molecules will elute later.
 o The elution is typically monitored using detectors such as UV absorption,
refractive index, or light scattering.
5. Void Volume (V₀) and Total Volume (Vt):
o Void Volume (V₀): The volume in the column that is outside the stationary phase
pores; this is where the largest molecules elute.
o Total Volume (Vt): The total volume of the column, including both the mobile
and stationary phases.
o The molecules elute in a range of times that correlates with their size relative to the
pore size distribution of the stationary phase.
 6. Molecular Weight Estimation:
o By comparing the elution times or volumes of a sample with known molecular
weight standards, the molecular weight of the sample can be estimated.
o SEC is commonly used for the determination of the molecular weight distribution
in complex mixtures, especially for proteins and polymers.

Procedure:

1. Preparation:
o Select an appropriate column with the correct pore size range based on the size of
the molecules being separated.
o Choose a suitable mobile phase (e.g., buffer or solvent) for the sample.
o Calibrate the system with molecular weight standards to establish a calibration
curve for molecular size estimation.
2. Loading the Sample:
o A small volume of the sample is injected onto the column. The sample should
ideally be dissolved in the mobile phase to avoid interference with the separation
process.
3. Separation:
o The sample is carried through the column by the mobile phase. Larger molecules
elute first, followed by smaller molecules. The separation occurs as molecules
either enter or bypass the pores of the stationary phase.
4. Detection:
o The separated components are monitored and detected as they elute from the
column, often using UV absorbance, refractive index, or multi-angle light
scattering detectors.
5. Analysis:
o The elution profile is analyzed, and the size or molecular weight distribution of the
components is determined using the calibration curve.

Applications:

1. Protein Purification:
o SEC is commonly used to purify proteins based on size. It is an excellent method
for separating proteins or peptides without denaturing them.
o It is also used to remove aggregates or impurities from purified proteins.
2. Polymer Characterization:
o SEC is used for determining the molecular weight distribution of synthetic
polymers, such as plastics and resins.
o It can provide insights into the degree of polymerization and polydispersity (the
distribution of polymer sizes).
3. Nucleic Acid Analysis:
o SEC is used in the separation of nucleic acids, like DNA or RNA, by size, allowing
for analysis of fragmentation or purification of oligonucleotides.
4. Carbohydrates and Small Molecule Separation:
o SEC can be used for the separation and analysis of sugars, polysaccharides, and
other small molecules based on their size.

Advantages:
  Gentle Separation: SEC separates molecules based purely on size, making it a mild
technique that doesn't rely on chemical interactions, which helps preserve the integrity of
sensitive molecules like proteins and nucleic acids.
 No Sample Degradation: The technique does not cause degradation or modification of
the analytes, unlike other chromatographic methods that may involve chemical
interactions.
 High Resolution for Size-Based Separation: It can effectively resolve different size
species in a mixture, particularly useful for proteins, polymers, and other biopolymers.

Disadvantages:

 Limited to Size-Based Separation: SEC is only effective for separating molecules with
significant differences in size. It is not suitable for separating molecules with similar sizes
or those that do not interact significantly with the stationary phase.
 Broad Elution Peaks for Large Molecules: For larger molecules, SEC may not provide
highly resolved peaks, as they may elute in a broad range.
 Slow: The separation can be slower compared to other chromatographic techniques such
as ion exchange chromatography or affinity chromatography, especially for large sample
volumes.

Conclusion:

Size Exclusion Chromatography is a powerful and non-destructive technique for separating

molecules based on size, widely used in biochemistry, molecular biology, and polymer science. It
is particularly beneficial for purifying proteins, determining molecular weight distributions, and
analyzing large biomolecules, all without the need for complex chemical interactions. Its primary
limitation is that it cannot be used to separate molecules that are of similar sizes or for complex
mixtures where size differences are not pronounced.

Affinity Chromatography: Notes

Definition: Affinity chromatography is a type of liquid chromatography that separates molecules

based on their specific binding affinity to a ligand that is attached to the stationary phase. This
technique takes advantage of the highly selective interactions between a target molecule (ligand)
and its specific binding partner (receptor, enzyme, antibody, etc.).

Key Concepts:

1. Principle of Separation:
o Affinity chromatography is based on the specific and reversible interactions
between the target molecule (usually called the analyte) and a ligand that is
immobilized on the stationary phase.
o The stationary phase typically consists of beads (such as agarose or Sepharose) that
are functionalized with a ligand (such as an antibody, enzyme, or small molecule).
o Molecules that have a high affinity for the ligand will bind to the stationary phase
and be retained, while molecules that do not bind will be washed away.
o The bound analyte can be eluted by changing the conditions (e.g., altering pH, salt
concentration, or using a competing molecule) to disrupt the interaction between
the analyte and the ligand.
Purification of a macromolecule, A, by affinity chromatography. Ligand B, which has a
specific affinity for A, is immobilized on the gel. Y represents an eluting agent that causes
dissociation of A

Stationary Phase:

oThe stationary phase in affinity chromatography is a solid support, often consisting

of porous beads made of materials like agarose, polyacrylamide, or silica.
o The ligand (the molecule that specifically binds the target analyte) is covalently
attached to the solid support. Common ligands include:
 Antibodies (for immunoaffinity chromatography),
 Amino acids or peptides (for enzyme or protein affinity),
 Metals (e.g., nickel for histidine-tagged proteins),
 Lectins (for carbohydrate binding).
2. Mobile Phase:
o The mobile phase is typically a buffer or solvent that moves the sample through the
column.
 o The mobile phase does not interact with the analyte but helps to separate free
components from those bound to the stationary phase.
3. Ligand-Analyte Interaction:
o The ligand on the stationary phase binds the target molecule (analyte) based on a
highly specific interaction, such as:
 Antigen-antibody interactions (e.g., immunoaffinity chromatography),
 Enzyme-substrate binding (e.g., enzyme affinity chromatography),
 Receptor-ligand binding (e.g., receptor affinity),
 Metal-ion coordination (e.g., histidine-tagged protein affinity).
o This selective binding allows for the specific capture of the analyte from a complex
mixture of compounds.
4. Elution:
o Once the target molecule is bound to the stationary phase, it can be eluted
(separated) from the column by altering conditions to weaken the ligand-analyte
interaction.
o Common methods for elution include:
 Changing pH or ionic strength to disrupt electrostatic interactions.
 Adding a competing molecule (e.g., excess ligand or a structurally similar
molecule) to displace the analyte.
 Using a denaturant (such as urea or guanidine) to break the binding
interaction.
5. Purification:
o The key advantage of affinity chromatography is its ability to purify a target
molecule from a complex mixture. This makes it ideal for isolating specific
proteins, nucleic acids, enzymes, or other biomolecules.
o The specificity of the ligand ensures that only the desired analyte binds, while all
other substances are washed away, resulting in high purity.

Types of Affinity Chromatography:

1. Metal Chelate Affinity Chromatography:

o A metal (such as nickel or cobalt) is immobilized on the stationary phase, and the
analyte must have a specific metal-binding tag (e.g., a polyhistidine tag) to interact
with the stationary phase.
o Commonly used for purifying recombinant proteins with histidine tags.
2. Enzyme Affinity Chromatography:
o Involves the use of specific enzyme substrates as ligands to purify enzymes or
proteins that interact with these substrates.
o Can be used for purifying enzymes, cofactors, and other small molecule-binding
proteins.
3. DNA/RNA Affinity Chromatography:
o Involves the use of nucleic acid sequences (e.g., DNA or RNA) as ligands to
capture interacting proteins, transcription factors, or other nucleic acid-binding
molecules.
o Useful in genomics and proteomics to study DNA-protein interactions.

Procedure:

1. Column Preparation:
o Select an appropriate stationary phase with the appropriate ligand attached.
o The ligand must be immobilized in such a way that it does not leach from the
column during the separation process.
2. Sample Loading:
 o The sample containing the analyte of interest is loaded onto the column.
o The sample is usually applied in a buffer solution to maintain stable conditions for
binding.
3. Binding:
o The target analyte binds to the ligand on the stationary phase based on its specific
interaction (e.g., antigen-antibody, enzyme-substrate).
o Non-binding molecules are washed away using the mobile phase.
4. Elution:
o The analyte is eluted from the column by changing the conditions (e.g., adding
excess ligand, changing pH or ionic strength).
o Elution is typically monitored by collecting fractions and analyzing them for the
presence of the target molecule.
5. Analysis:
o The eluted fractions are analyzed to verify the purity and identity of the target
molecule. This can be done using methods like SDS-PAGE, Western blotting, or
mass spectrometry.

Applications:

1. Protein Purification:
o Affinity chromatography is widely used in the purification of recombinant proteins,
antibodies, and enzymes.
o It allows for the isolation of a single target protein from complex mixtures (e.g.,
cell lysates) with high specificity.
2. Antibody Isolation:
o Immunoaffinity chromatography is used to isolate antibodies for research,
diagnostics, and therapeutic purposes.
3. Enzyme Studies:
o Used for the purification of enzymes or cofactors for biochemical studies.
4. Biotechnology and Drug Development:
o In biotechnology, affinity chromatography is often used to purify therapeutic
proteins, such as monoclonal antibodies or insulin.
o It is also applied in the development of biosensors or diagnostic assays.
Advantages:

 High Specificity: The high affinity and specificity of ligand-analyte interactions lead to
very efficient separation and purification of target molecules.
 High Purity: It can provide high-purity isolates due to the selective binding nature of the
technique.
 Mild Conditions: The technique often requires mild conditions (e.g., neutral pH,
physiological salt concentrations), preserving the structure and function of biological
molecules.

Disadvantages:

 Limited to Specific Interactions: The success of affinity chromatography depends on

having a known, high-affinity ligand for the target molecule. If such a ligand does not
exist, the technique cannot be used.
 Cost: The ligands used in affinity chromatography (e.g., antibodies) can be expensive to
produce or purchase.
 Regeneration of Columns: The stationary phase may lose its binding capacity over time,
and regenerating or reusing the column can be challenging in some cases.

Conclusion:

Affinity chromatography is a powerful and highly specific technique for isolating and purifying a
wide range of biomolecules, particularly proteins, nucleic acids, and small molecules. Its high
selectivity makes it an invaluable tool in many areas of research, diagnostics, and biotechnology.
However, the need for specific ligands and the potential cost of reagents can limit its broader
application.

Terminologies Used in Chromatography:

Chromatography is a versatile technique used to separate and analyze mixtures of compounds.

The following are key terms commonly used in chromatography:

1. Chromatogram:

 A graphical representation of the detector response (e.g., absorbance, fluorescence) versus

time or elution volume.
 Peaks in the chromatogram correspond to the separated components of the mixture.

2. Stationary Phase:

 The phase that remains fixed within the chromatographic system, providing the surface or
material for interaction with the sample components.
 It can be solid, liquid, or gel-like, depending on the type of chromatography (e.g., silica gel
in thin-layer chromatography (TLC), or a polymer in size exclusion chromatography).

3. Mobile Phase:

 The phase that moves through or along the stationary phase, carrying the sample
components.
 It can be a gas (in gas chromatography) or a liquid (in liquid chromatography).
  The mobile phase transports the analyte through the stationary phase, and its properties
(polarity, viscosity, etc.) influence the separation process.

4. Elution:

 The process of washing the sample through the stationary phase using the mobile phase.
 In liquid chromatography, elution involves the separation of the analytes as they travel
through the column, with different compounds eluting (leaving the column) at different
times.

5. Eluent:

 The mobile phase used to elute or carry the sample components through the stationary
phase.
 In liquid chromatography, this can be a solvent or a mixture of solvents.

6. Retention Time (tₓ):

 The time it takes for a particular analyte to travel from the point of injection to the
detector.
 It is used as a characteristic parameter to identify compounds (under consistent
experimental conditions).

7. Retention Factor (Rf):

 A dimensionless value used primarily in thin-layer chromatography (TLC) to describe the

migration of a compound.
 It is the ratio of the distance travelled by the analyte to the distance travelled by the solvent
front.
 Rf=distance travelled by compound/distance travelled by solvent front

8. Elution Volume (Ve):

 The volume of mobile phase required to elute a component from the column.
 It is often used in size exclusion chromatography (SEC), where larger molecules elute
earlier with a smaller volume.

9. Column:

 The primary separation medium in chromatographic techniques, particularly in liquid and

gas chromatography.
 It contains the stationary phase and provides the environment for the separation process.
 Columns can be packed (with solid particles) or open (e.g., capillary columns in gas
chromatography).

10. Peak:

 A graphical feature in the chromatogram, representing a compound that has been separated
and detected.
 The area under the peak often corresponds to the amount of the analyte present in the
sample.
11. Resolution (R):

 A measure of how well two analyte peaks are separated from each other in a
chromatogram.
 It is calculated based on the difference in their retention times and the width of the peaks.
 Higher resolution results in better separation.

12. Selectivity (α):

 A measure of the ability of the chromatographic system to differentiate between two

analytes.
 It is the ratio of the retention factors (or times) of two compounds.
 Higher selectivity means better differentiation between the analytes.

13. Dead Time (t₀):

 The time it takes for an unretained compound (one that does not interact with the
stationary phase) to pass through the chromatographic system.
 This is typically the time it takes for the mobile phase to travel through the system.

14. Band Broadening:

 The spreading of analyte bands (or peaks) as they move through the stationary phase,
which leads to less sharp peaks in the chromatogram.
 Band broadening can result in decreased resolution, often caused by factors such as
diffusion, column packing issues, or high flow rates.

15. Void Volume (V₀):

 The volume of the mobile phase in the column that is outside the stationary phase.
 It represents the volume of solvent required for the earliest-eluting, unretained compounds
to pass through the column.

 Pressure influences the speed of analysis and the separation efficiency, especially in
packed columns.

16. Partition Coefficient (K):

 A ratio that describes the distribution of a compound between two phases (stationary and
mobile).
  In partition chromatography, it is the ratio of the concentration of a substance in the
stationary phase to the concentration in the mobile phase.

17. Adsorption (in Adsorption Chromatography):

 The process by which analytes adhere to the surface of the stationary phase (e.g., silica
gel) through physical or chemical interactions.
 In adsorption chromatography, separation occurs based on the different affinities of
analytes for the stationary phase.

18. Solvent Front:

 The leading edge of the mobile phase (or solvent) as it moves through a chromatographic
medium (such as in thin-layer chromatography).
 It marks the furthest point reached by the solvent during the chromatographic process.

Conclusion:

Chromatography is a complex yet powerful technique with a variety of terms that describe
different components and processes involved in separating, analyzing, and purifying mixtures of
compounds. Understanding these terminologies is crucial for interpreting chromatograms and
optimizing separation conditions in various chromatographic techniques.

High-Performance Liquid Chromatography (HPLC): Notes

Definition: High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique

used to separate, identify, and quantify components in a liquid sample. It relies on the interaction
of the sample with a stationary phase and a mobile phase to achieve separation based on the
differing affinities of the components for these phases.
Key Components of HPLC System:

1. Solvent Reservoir:
o Contains the mobile phase, which is a liquid or a mixture of liquids (solvents).
o The mobile phase carries the sample through the column.
2. Pump:
o Delivers the mobile phase at a consistent flow rate through the system.
o Typically, the flow rate ranges from 0.1 to 10 mL/min, depending on the column
size and type of separation.
o Pumps are capable of applying high pressure, which is necessary to force the
mobile phase through the packed column.
3. Injector:
o A device used to introduce the sample into the mobile phase.
o The sample is injected into the mobile phase stream before it enters the column.
o Common injectors include manual syringes, automatic injection systems, or loop
injectors (where a defined volume of sample is loaded into a loop and introduced
into the mobile phase).
4. Column:
o The heart of the HPLC system where the separation takes place.
o It is packed with the stationary phase (usually silica-based particles or polymer
materials) that interacts with the sample components.
o Columns vary in length (typically 30-250 mm) and diameter (typically 2-4 mm),
and the stationary phase may be selected based on the chemical nature of the
analytes.
o The choice of column material and the size of the particles within it will affect
resolution and analysis speed.
5. Detector:
o The detector monitors the eluting compounds as they pass through the column.
o Common types of detectors include:
  UV/Vis Detector: Detects analytes that absorb UV or visible light.
 Refractive Index Detector (RI): Detects changes in the refractive index of
the mobile phase caused by the analyte.
 Fluorescence Detector: Measures the emission of light from compounds
that can fluoresce.
 Mass Spectrometer (MS): Can be coupled to HPLC to provide molecular
weight and structural information (LC-MS).
 Evaporative Light Scattering Detector (ELSD): Sensitive to compounds
that are not UV-active or do not fluoresce.
6. Data System:
o The software and hardware that controls the HPLC system, records detector
responses, and analyzes the chromatogram.
o It provides information about the retention time, peak area, and concentration of
components in the sample.

Working Principle of HPLC:

1. Separation Process:
o In HPLC, the sample is injected into the column, where it interacts with the
stationary phase.
o The different components in the sample will have varying affinities for the
stationary phase.
 Stronger interaction with the stationary phase causes a slower migration
through the column.
 Weaker interaction with the stationary phase causes faster migration
through the column.
o Components with stronger interactions are retained longer, while those with
weaker interactions elute (leave the column) more quickly.
2. Elution:
o As the sample moves through the column, it is separated into its individual
components.
 o The mobile phase carries these components to the detector, where they are detected
and quantified.
3. Retention Time (tₓ):
o The time it takes for a specific compound to travel from the injection point to the
detector is called the retention time.
o It is a characteristic property of each compound under a given set of conditions
(stationary phase, mobile phase, flow rate, etc.).
4. Chromatogram:
o A chromatogram is a graphical representation of the detector response versus time
(or elution volume).
o Each peak in the chromatogram corresponds to a separated component of the
sample.
o The area under each peak is proportional to the amount of that component in the
sample.

Dead time (often represented as t0) is the time it takes for an unretained compound (a
compound that does not interact with the stationary phase) to travel through the
chromatographic column from the point of injection to the detector. It represents the time
it takes for the mobile phase to pass through the system without any interaction with the
stationary phase.

Types of HPLC:

1. Normal-Phase HPLC (NP-HPLC):

o Uses a polar stationary phase (e.g., silica) and a non-polar mobile phase (e.g.,
hexane).
o In normal-phase HPLC, polar compounds are retained longer due to their stronger
interaction with the stationary phase, while non-polar compounds elute faster.
2. Reverse-Phase HPLC (RP-HPLC):
o The most common form of HPLC.
o Uses a non-polar stationary phase (e.g., C18, C8) and a polar mobile phase (e.g.,
water mixed with organic solvents like methanol or acetonitrile).
 o In reverse-phase chromatography, non-polar compounds are retained longer
because of their interaction with the non-polar stationary phase, while polar
compounds elute more quickly.
3. Ion-Exchange HPLC:
o Uses a stationary phase with charged groups (e.g., sulfonic acid or quaternary
amines).
o This type of HPLC separates ions or polar compounds based on their charge and
the strength of their interaction with the stationary phase.
4. Size-Exclusion HPLC (SEC-HPLC):
o Also known as gel filtration chromatography.
o Separates molecules based on size using a stationary phase consisting of porous
beads.
o Larger molecules pass through the column more quickly because they do not enter
the pores of the beads, while smaller molecules take longer as they penetrate the
pores.
5. Affinity HPLC:
o Uses a stationary phase with a ligand that specifically interacts with a target
molecule (e.g., antibodies or enzymes).
o This method is used for purifying or separating biomolecules based on specific
binding interactions.

Applications of HPLC:

1. Pharmaceuticals:
o Used for quality control, method validation, and purity testing of pharmaceutical
products.
o It is also used for the identification and quantification of drugs and their
metabolites.
2. Environmental Testing:
o Used for analyzing environmental samples (e.g., water, air, and soil) for pollutants
such as pesticides, herbicides, and industrial chemicals.
3. Food and Beverage Industry:
o Used to analyze food additives, preservatives, vitamins, flavor compounds, and
contaminants.
o HPLC is employed to determine the composition and safety of food products.
4. Biotechnology:
o HPLC is used for protein and peptide purification, analysis of nucleic acids, and
separation of bioactive compounds.
5. Forensic Science:
o Used in forensic toxicology to analyze biological samples for drugs, poisons, and
other toxic substances.
6. Clinical Diagnostics:
o HPLC is used in clinical labs for the analysis of biomarkers, metabolic profiles,
and drugs in blood or urine samples.

Advantages of HPLC:
  High Resolution: HPLC can resolve complex mixtures into individual components with
high sensitivity and precision.
 Quantitative Analysis: Provides reliable and reproducible quantitative analysis of
components in the sample.
 Flexibility: Can be used for a wide range of analytes, including small organic molecules,
peptides, proteins, and inorganic compounds.
 Speed: Modern HPLC systems can perform rapid separations, with analysis times often
ranging from minutes to an hour.

Disadvantages of HPLC:

 Cost: HPLC systems can be expensive to purchase and maintain, particularly those
equipped with detectors like mass spectrometers.
 Sample Preparation: Requires appropriate sample preparation to ensure that the analytes
are soluble and do not interfere with the column or detector.
 Mobile Phase Selection: The choice of mobile phase (solvents and additives) must be
optimized for each analysis, and in some cases, can lead to issues with solvent
compatibility and waste disposal.

Conclusion:

High-Performance Liquid Chromatography (HPLC) is a versatile, highly efficient, and widely

used analytical technique for separating, identifying, and quantifying chemical compounds in a
variety of industries, including pharmaceuticals, food, environmental analysis, and biotechnology.
Its high resolution, sensitivity, and flexibility make it one of the most important tools in analytical
chemistry.