Column

Chromatography
Wali Ullah Shahid
Lecturer
Column Chromatography
• Column chromatography is a critical technique used in
pharmaceutical research and production for separating,
purifying, and analyzing drugs and their components.
• how drugs are isolated from complex mixtures, which is
crucial for both drug development and quality control
Components
• Stationary Phase (Solid):
The material packed inside the column, often silica gel or
alumina. Its properties (e.g., polarity) play a crucial role in
the separation process.
• Mobile Phase (Liquid/Gas):
The solvent or mixture of solvents that carries the sample
through the stationary phase.
• Sample:
The mixture of compounds that need to be separated. In
pharmaceuticals, this could include drugs, impurities, or
metabolites.
Separation Principle
• Adsorption:
The interaction of components with the stationary phase.
Strongly adsorbed compounds travel slowly.
• Partitioning:
Compounds distribute themselves between the stationary
phase and mobile phase, and the distribution coefficient
determines their retention time.
• Elution:
As the mobile phase flows through the column, different
compounds are eluted at different times, based on their
interaction with the stationary phase.
Types of Column Chromatography
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Size exclusion chromatography
• Affinity Chromatography
Adsorption Chromatography
• Principle:
Separation is based on the differential adsorption of
compounds on the stationary phase. Compounds with
higher affinity for the stationary phase will move slower.
• Stationary Phase:
Typically, silica gel or alumina.
• Application in Pharmaceutics:
Purification of small drug molecules
Removal of impurities from drug formulations.
Partition Chromatography
Principle:
Separation is based on the partitioning of compounds
between a liquid stationary phase and the mobile phase.
The mobile phase can be a mixture of solvents that affect
solubility.
• Stationary Phase: A liquid coating on a solid support
(e.g., cellulose or polystyrene).
• Application in Pharmaceutics:
Purification of lipophilic or hydrophilic compounds
Separating metabolites of drugs from biological fluids.
Ion-Exchange Chromatography
• Principle:
Separation of compounds is based on their charge. The
stationary phase is a resin with charged groups that
attract oppositely charged molecules.
• Stationary Phase:
Charged resins such as sulfonated polystyrene or
cellulose.
• Application in Pharmaceutics:
Separation of charged pharmaceutical compounds (e.g.,
amino acids, peptides, or proteins).
Purification of biologic drugs (e.g., monoclonal
antibodies).
Size-Exclusion Chromatography
(SEC)
• Principle:
Separation based on molecular size, with larger
molecules eluting faster as they are excluded from the
pores of the stationary phase.
• Stationary Phase:
Porous gel beads, usually made of crosslinked polymers.
• Application:
Separation of protein aggregates or large drug
formulations
Quality control to verify the molecular weight of biologic
drugs
Practical aspects and consideration
• Column Preparation and Packing
• Sample Preparation and Loading
• Elution Techniques
• Fraction Collection
• Troubleshooting in Column Chromatography
• Scale-up Considerations
• Final Considerations in Pharmaceutical Applications
1. Column Preparation and Packing
a. Stationary Phase Selection and Packing:
Choosing the Right Stationary Phase:
The stationary phase should match the nature of the
compounds being separated. For example, silica gel is
commonly used for normal-phase chromatography (polar
stationary phase), while C18 (octadecylsilane) is widely used for
reverse-phase chromatography (non-polar stationary phase).
Column Packing:
Proper packing is critical for good resolution. If the stationary
phase is not packed uniformly, it can lead to channeling, where
the solvent moves through a particular path in the column,
causing uneven separation.
Procedure:
• First, prepare the stationary phase slurry by mixing the
solid phase with an appropriate solvent (e.g., using a
solvent that matches the polarity of the sample).
• Carefully load the slurry into the column, avoiding air
bubbles, which can disrupt the flow and affect the
resolution.
• Gradually add more solvent to ensure the stationary
phase is well-packed and free from voids or channels.
b. Equilibration
• Before loading the sample, equilibrate the column with
the mobile phase (solvent) to ensure that the stationary
phase is fully saturated with the solvent. This helps in
achieving consistent separation conditions.
2. Sample Preparation and Loading
a. Sample Preparation
• Solvent Choice:
The sample should be dissolved in a minimal amount of
solvent that is compatible with the mobile phase. Using
a solvent that is too polar or non-polar can result in poor
separation or sample loss.
• Concentration:
The sample should be concentrated enough to produce
detectable peaks, but not too concentrated to overload
the column. Overloading leads to poor resolution and
tailing of peaks.
b. Sample Loading
• Gentle Application: When loading the sample onto the
column, apply it carefully to avoid disturbing the stationary
phase. Ideally, the sample should be applied in a
concentrated but small volume.
• Avoiding Sample Overload: Overloading occurs when too
much sample is applied, causing the components to elute
together, making it hard to separate them.
To prevent overloading:
Use a small sample size.
Ensure the column is of adequate size relative to the sample
amount.
3. Elution Techniques
a. Solvent Choice and Gradient Elution
• Choosing the Right Mobile Phase:
The mobile phase should be chosen based on the properties of the
compounds being separated. For instance, polar solvents are useful for
separating polar compounds, whereas less polar solvents help in separating
non-polar compounds.
• Gradient Elution:
For more complex mixtures, gradient elution is used, where the polarity of the
solvent is gradually changed to enhance separation. This helps elute
compounds that are less soluble in the initial solvent and those with varying
polarities.
Example:
Starting with a non-polar solvent and slowly increasing the polarity with a
more polar solvent (e.g., from hexane to ethyl acetate or methanol) helps
separate both polar and non-polar components.
• b. Flow Rate
The flow rate at which the mobile phase is passed
through the column affects the separation. A faster flow
rate can reduce the resolution but can be useful for
quick separations. A slower flow rate increases the time
for separation, allowing for better resolution.
Optimal Flow Rate:
Find a balance between speed and resolution based on
the complexity of the sample. For a more complex
mixture, use a slower flow rate to ensure proper
separation.
c. Monitoring the Elution
• As compounds are eluted, they should be monitored,
typically using UV-Vis detection, refractive index
detection, or conductivity, depending on the nature of
the compounds
• UV Detection: Commonly used when compounds
absorb light in the UV range (e.g., many
pharmaceuticals have chromophores that absorb UV
light).
• Manual Monitoring: If a detector is unavailable, visual
monitoring (e.g., by spotting fractions on a TLC plate) or
manual collection can help track the progress.
3. Elution Techniques
• Elution involves the separation of compounds from the
stationary phase using a mobile phase.
• Types
Isocratic Elution
Gradient Elution
Normal-Phase Elution
Reverse-Phase Elution
• Isocratic Elution:
The composition of the mobile phase remains constant
throughout the separation. This method is used when
the compounds of interest have similar polarity and
elute at similar rates.
• Gradient Elution:
The mobile phase composition is gradually changed
(e.g., increasing the polarity of the solvent) during the
separation process. Gradient elution is useful when the
compounds vary widely in their polarities and require
different conditions to elute effectively.
• Normal-Phase Elution:
Involves a polar stationary phase (e.g., silica gel) and a
non-polar mobile phase. This is used to separate
compounds based on polarity
• Reverse-Phase Elution:
Involves a non-polar stationary phase (e.g., C18) and a
polar mobile phase. Reverse-phase chromatography is
especially useful for separating hydrophobic compounds
and is widely used in pharmaceutical applications.
• Optimizing Flow Rate:
The flow rate of the mobile phase must be adjusted
according to the column dimensions and the nature of
the sample. Too fast a flow rate may compromise
separation, while too slow a rate may increase the run
time unnecessarily.
4. Fraction Collection
• After separation, the individual components are
collected in fractions for analysis and further
processing.
• Steps
Fractionation
Detection
Fraction Size
• Fractionation:
As the mobile phase carries compounds through the column,
separate fractions can be collected at intervals based on the
expected elution profile.
• Detection:
Depending on the nature of the compound, fractions can be
monitored using detectors like UV-Vis spectrophotometers (for
compounds that absorb light) or other suitable detection methods
(e.g., fluorescence or mass spectrometry).
• Fraction Size:
Collect smaller fractions if the elution times of compounds are
close to each other. This will allow for finer separation and
purification.
• Considerations:
Fraction Analysis:
After collecting the fractions, analyze them using
techniques such as TLC (Thin-Layer Chromatography) or
HPLC to confirm the separation of components.
Concentration:
In pharmaceutical applications, some fractions may need
to be concentrated or further purified.
5. Isolation of Separated compounds
• TLC is done with fractions
• Spots on tlc indicate which fraction contain compound
• Combine same fractions and evaporate the solvent and
pure separated compound will be left behind
• Recrystallization may be used to further purify solid
product
6. Detection of components
• It can be done visually
• Different colored bands moving down
• For colorless compounds, the technique depends upon properties
of components
• Absorption of light – UV VIS detectors
• Fluorescence or light emission characteristics- Fluorescence
Detector
• Using Flame Ionization detectors
• Refractive index detector
• Evaporation of solvent and weighing the residue
• Monitoring the fractions by TLC
7. Recovery of components
• The mobile phase (solvent) moves through the column,
carrying separated components at different rates.
• Fractions are collected at regular intervals and pooled
based on similar composition.
• Solvents are evaporated (e.g., using a rotary
evaporator) to isolate the pure compound.
• After recovery, purity is checked with techniques like
TLC, NMR, or HPLC.
8. Troubleshooting in Column
Chromatography
• Common Problems:
Poor Separation
Slow Flow Rate
Tail or Broad Peaks
• Solutions:
Repack the Column
Adjust Solvent
Sample Dilution
9. Scale-Up Considerations
• When transitioning from laboratory-scale to industrial-
scale chromatography, several factors need careful
adjustment.
• Scaling Parameters needed for
Column Size
Flow Rate
Solvent Volume
Automation
10. Final Considerations in
Pharmaceutical Applications
• Column chromatography is widely used in
pharmaceutical applications, especially for drug
purification and compound isolation.
• Key Considerations:
Purity
Regulatory Compliance
Safety
Cost Efficiency
• Purity:
High purity is critical in pharmaceutical applications.
Ensuring that the separation achieves the desired purity
without contaminating the final product is crucial.
Regulatory Compliance:
Pharmaceutical applications often require adherence to
stringent regulations (e.g., cGMP, FDA) regarding
solvents, equipment, and documentation.
• Safety:
The handling of solvents, chemicals, and compounds
must follow safety protocols to protect lab personnel
and ensure the safety of the final pharmaceutical
products
• Cost Efficiency:
Pharmaceutical companies must balance cost and
efficiency. Scale-up processes, solvent usage, and
equipment costs must be optimized to ensure economic
feasibility while maintaining high-quality results.
Elution Procedure
• Isocratic
• Fractional elution
• Gradient
Isocratic Elution
• Isocratic elution is a type of elution in chromatography
where the mobile phase composition remains constant
throughout the separation process. This method is often
used when the compounds to be separated have similar
polarities and can be effectively separated with a single,
fixed solvent or solvent mixture.
Key Characteristics of Isocratic
Elution:
• Constant Mobile Phase Composition:
The solvent or mixture of solvents used to carry the sample
through the column remains unchanged during the entire
elution process.
• Simple Process:
Since the solvent composition is not changed over time,
isocratic elution is relatively simple to set up and maintain
• Ideal for Similar Compounds:
This method is most effective when the compounds being
separated are of similar polarity or when the stationary phase
and mobile phase provide sufficient separation power without
needing adjustments.
Fractional Elution
• Fractional elution refers to the process in column
chromatography where the elution is conducted in
discrete fractions, typically collected at specific time or
volume intervals, as compounds are gradually
separated from the stationary phase by the mobile
phase. Fractional elution is a critical part of the
separation process because it helps isolate individual
components of a mixture as they elute from the column.
The term "fractional" relates to the collection of the
eluate in discrete portions or fractions..
Key Features of Fractional Elution
• Discrete Collection: The eluate is collected in fractions, with
each fraction ideally containing one or more separated
components of the sample.
• Monitoring and Detection: Fractions are typically
monitored using techniques like UV-Vis detection, thin-layer
chromatography (TLC), or other analytical methods to confirm
the presence of target compounds.
• Progressive Separation: As the mobile phase moves
through the column, different components of the sample elute
at different times based on their interaction with the
stationary phase, and fractions are collected to capture these
separated components.
Gradient Elution
• Gradient elution is a type of elution in
chromatography where the composition of the
mobile phase changes progressively during the
separation process. This is typically used when
separating compounds that have different affinities for
the stationary phase and varying polarities. Gradient
elution allows for better separation of complex
mixtures, especially when the sample contains
components with a wide range of polarities or other
physicochemical properties.
Key Characteristics of Gradient
Elution
• Changing Mobile Phase Composition: The
composition of the mobile phase is gradually altered
(e.g., by changing the polarity or strength of the
solvent) during the chromatographic run.
• Optimized for Complex Mixtures: Gradient elution is
ideal for separating complex mixtures where
compounds vary significantly in their properties (e.g.,
polarity).
• Improved Separation: By altering the mobile phase,
gradient elution can help overcome challenges
associated with separating compounds that are
otherwise too similar in their behavior when using a
Advantages of Column
chromatography
• High Efficiency:
Column chromatography provides excellent separation of
compounds, even in complex mixtures. It can separate
components with similar polarity or molecular size.
• Scalability: It can be used for both small-scale laboratory
work and large-scale industrial applications. The size of the
column can be adjusted depending on the amount of material
to be processed
• Flexibility: You can adjust the mobile phase (solvent) and
stationary phase (e.g., silica gel, alumina) to optimize the
separation depending on the nature of the compounds being
separated.
• Wide Range of Applications: It is used to separate a
variety of substances, including organic compounds,
natural products, and biomolecules like proteins and
nucleic acids.
• Continuous Operation: Unlike batch processes,
column chromatography can be run continuously, which
is useful for large-scale separations.
• Minimal Sample Loss: The sample is typically loaded
onto the column, and the separation occurs as the
sample moves through, which minimizes loss of
material compared to other methods.
• Mild Conditions:
Since column chromatography can operate under
relatively mild conditions (temperature and pressure), it
is less likely to cause degradation of heat-sensitive or
delicate compounds
• High Purity:
The technique often provides high purity of separated
fractions, which is crucial in analytical chemistry,
pharmaceuticals, and biochemistry.
Disadvantages
• Time-Consuming:
The separation process can be slow, especially for complex
mixtures or when a large volume of sample needs to be processed.
It requires patience and careful monitoring.
• Sample Loss:
There can be some sample loss during the process, particularly
when collecting fractions or when the compound is poorly retained
on the column.
• Solvent Usage:
The process often requires a large volume of solvents, which can
be expensive and create disposal challenges. It's also crucial to
select the right solvent to achieve proper separation, which might
require trial and error.
• Requires Expertise:
While the basic principles of column chromatography are straightforward,
successful separation often requires significant expertise in selecting the
right stationary phase, mobile phase, and flow rate. Small adjustments can
have a big impact on the outcome.
• Limited Resolution:
For very similar compounds (e.g., with slight differences in polarity or size),
column chromatography might not provide the best resolution without
optimization of conditions, and separation might not be as effective as
needed.
• Limited Resolution:
For very similar compounds (e.g., with slight differences in polarity or size),
column chromatography might not provide the best resolution without
optimization of conditions, and separation might not be as effective as
needed.
• Size Limitation:
Although it’s scalable, very large-scale chromatography can be
expensive, and the resolution might drop if the column is too large
for the sample size
• Potential Contamination:
If not properly cleaned or maintained, the column can become
contaminated or saturated with previously separated substances,
leading to inconsistent results.
• Labor-Intensive:
Compared to some automated techniques, column chromatography
requires more hands-on time and attention, especially when
isolating multiple fractions or monitoring the separation process.
Factors affecting Column Efficiency
• Particle Size of Stationary Phase
• Column Length
• Flow Rate
• Mobile Phase Composition
• Temperature
• Stationary Phase Quality
• Sample Size
• Compounds’ Chemical Properties
• Particle Size of Stationary Phase:
Smaller particles of the stationary phase (e.g., silica gel) provide a
larger surface area, leading to better separation and improved
efficiency. However, this can increase the backpressure, making the
process slower.
• Column Dimensions:
A longer column provides more time for separation, generally improving
efficiency. However, this also requires more solvent and time.
Length diameter ratio of 20:1 or 30:1 are ideal but for improving the
efficiency 100:1 may be more satisfactory
• Flow Rate:
The flow rate of the mobile phase affects the time compounds spend in
the column. A slower flow rate gives better separation but is more time-
consuming, while a faster flow rate may result in poor separation.
• Mobile Phase Composition:
The choice of solvent or solvent mixture (polarity,
viscosity) affects how well components interact with the
stationary phase, influencing separation. Proper solvent
selection is crucial for efficient separation.
• Temperature:
Higher temperatures can increase the solubility of
compounds in the mobile phase, speeding up the
process but potentially reducing resolution. Temperature
must be carefully controlled for optimal efficiency.
• Stationary Phase Quality:
The purity and consistency of the stationary phase (e.g.,
silica gel, alumina) affect the uniformity of interactions
between compounds and the column, influencing the
separation efficiency.
• Compounds’ Chemical Properties: The polarity, size,
and shape of the components being separated affect
their interactions with the stationary and mobile phases,
influencing how well they separate.
• Pressure :
Pressure affects column chromatography by influencing
the flow rate of the mobile phase. Higher pressure
speeds up the process but can reduce resolution, while
lower pressure improves separation but takes longer.
Excessive pressure can also damage the column or
cause cloggings
By applying a vacuum, the solvent is drawn through the
column faster, reducing separation time. However, if the
vacuum is too strong, it may lead to poor separation or
affect the integrity of the column packing. It is
important to control the vacuum to maintain good
resolution and prevent column damage.
Applications
• Purification of Active Pharmaceutical Ingredients
(APIs):
Column chromatography is used to purify APIs by separating
them from impurities, side products, or residual solvents,
ensuring the drug’s purity and potency.
Separation of Components in Drug Formulations:
It helps in separating and analyzing different components in
pharmaceutical formulations (e.g., tablets, injections) to
ensure consistent quality and efficacy.
• Detection of Contaminants:
It is used to identify and quantify contaminants such as
solvents, heavy metals, and other impurities in drug samples.
• Analysis of Metabolites:
Column chromatography helps in studying the metabolic products
of drugs by separating parent compounds from metabolites in
biological samples.
• Stereoisomer Separation:
For chiral drugs, column chromatography can be used to separate
stereoisomers, as the biological activity of each isomer may differ
significantly
Quality Control:
In pharmaceutical quality control (QC), column chromatography is
used to ensure that drug substances meet required specifications
for identity, purity, and potency, in accordance with
pharmacopoeial standards
Examples
• Ibuprofen:
Column chromatography is used to purify ibuprofen from synthetic by-
products or impurities during its production process. It helps ensure the drug
is of high purity for therapeutic use.
• Caffeine:
In drug formulations containing caffeine, column chromatography can be
used to separate caffeine from other components, ensuring its correct
dosage and purity in the final product
• Penicillin:
Used to purify penicillin from fermentation mixtures, column
chromatography helps isolate the active antibiotic and remove contaminants.
• Paracetamol:
Column chromatography can separate paracetamol from other reaction by-
products during its synthesis, ensuring the final product’s purity.
• Ginseng:
Column chromatography is used to isolate active compounds like
ginsenosides from Panax ginseng, which are responsible for its
medicinal properties. It helps in quality control and standardization of
ginseng-based products
• Echinacea:
Active components like echinacoside and cichoric acid are isolated
from Echinacea species using column chromatography to ensure
their effectiveness in immune-boosting preparations.
• St. John’s Wort:
Hypericin and hyperforin are key active compounds in St. John’s
Wort, used in antidepressant formulations. Column chromatography
is employed to isolate and purify these compounds for quality control.
Flash Chromatography
• Flash chromatography is a faster, more efficient
version of traditional column chromatography.
• It uses the same principle of separating compounds
based on their interaction with the stationary phase, but
with a few key differences that make it faster and more
convenient
Features
• High Pressure:
Flash chromatography applies higher air pressure or gas
pressure to push the solvent through the column
quickly, resulting in faster separation compared to
gravity-based column chromatography.
• Shorter Columns:
Flash chromatography uses shorter columns with
smaller particle sizes in the stationary phase, which
increases the surface area and speeds up the
separation.
• Pre-packed Columns:
Often, flash chromatography is done using pre-packed
columns that are optimized for fast separations. These
columns are convenient, easy to use, and consistent in
performance.
• Elution and Solvent Gradient:
A gradient of solvents (from non-polar to polar) is often
used to enhance separation. This allows for better
resolution and faster analysis of the sample.
Procedure
1. Prepare the Column and Stationary Phase
2. Condition the Column
3. Sample preparation
4. Select the Mobile Phase (Solvent)
5. Run the Chromatography
6. Evaluate the Results
7. Post-Chromatography Steps
8. Cleanup and Maintenance
1. Prepare the Column and
Stationary Phase
• Select a Column:
Choose a column with appropriate dimensions. Typically, a column with a
diameter of 2.5–4 cm and a length of 10–30 cm is used, depending on the
sample size.
• Packing the Column:
The column is packed with a stationary phase (usually silica gel or alumina).
The stationary phase is packed into the column using a slurry method:
Mix the stationary phase with a suitable solvent (usually hexane or a
mixture of solvents) to create a slurry.
Slowly pour the slurry into the column while allowing the solvent to flow
through to ensure even packing.
Tap the column gently to settle the packing material and remove air
bubbles.
Make sure the stationary phase is evenly packed and the solvent level is
maintained just above the column bed.
2. Condition the Column
• Condition the Column:
Run a small volume of the elution solvent (or a mixture
of solvents, depending on the polarity of the sample)
through the column to ensure that the stationary phase
is equilibrated and that the column is properly wetted.
Set up the Flash System:
If using a flash chromatography system, ensure the
system’s pressure is set correctly. Flash
chromatography is typically run under positive pressure
to speed up elution.
3. Prepare the Sample
• Dissolve the Sample:
Dissolve the mixture that you want to separate in a
small volume of the appropriate solvent. Typically, use a
solvent that has a similar polarity to the mobile phase.
• Load the Sample:
Once the column is conditioned, carefully load the
sample onto the column. This is done by applying the
dissolved sample directly to the top of the column.
Ensure that the sample is applied evenly to prevent
disruption of the stationary phase.
4. Select the Mobile Phase (Solvent)
• Choose Solvent(s):
The mobile phase (the solvent or mixture of solvents)
should be chosen based on the polarity of the
compounds in the mixture. Generally, a gradient of
increasing polarity is used to gradually elute more polar
compounds.
• Set up Solvent Reservoir:
If using a flash chromatography system, prepare the
solvent reservoir and ensure that the solvent is flowing
through the column under appropriate pressure.
5. Run the Chromatography
• Start Elution:
Begin elution by applying pressure (if using a flash
chromatography system) or by gravity if not. The solvent will
start to move down the column, and different components in
the mixture will interact with the stationary phase differently,
causing them to separate as they move.
• Monitor Elution:
As the solvent passes through the column, fractions are
collected. The time or volume of solvent passed can be used
to divide the collected fractions. Monitor the elution process
visually, usually by spotting fractions on a TLC plate, or by
using a UV detector for more precise detection.
• Collect Fractions:
Collect fractions in separate containers based on the
elution profile. Typically, the more polar compounds will
elute later if using a non-polar mobile phase, while non-
polar compounds will elute earlier.
6. Evaluate the Results
• Monitor TLC (Thin Layer Chromatography):
Thin layer chromatography is often used to check the
progress of separation. Spot fractions on a TLC plate
and compare the Rf values (distance traveled by the
compound relative to the solvent front) to determine if
separation has occurred.
• Analyze Fractions:
Combine fractions that contain pure compounds based
on their TLC or other analytical data (such as UV
absorbance).
7. Post-Chromatography Steps
• Concentration (if needed):
If the solvent used in the elution is volatile, the solvent
can be removed under reduced pressure (rotary
evaporation, for example).
• Purification:
The individual fractions can now be concentrated, or
additional purification steps (like re-chromatography)
can be performed if necessary to achieve higher purity
8. Cleanup and Maintenance
• Clean the Column:
After the chromatography run, clean the column by
flushing it with an appropriate solvent to remove any
residual compounds and prepare it for future use.
• Store the Stationary Phase:
If reusing the stationary phase, dry and store it properly
to prevent contamination.
Important Considerations
• Gradient Elution:
For complex mixtures, using a gradient of solvents (e.g.,
from non-polar to polar) helps achieve better
separation.
• Column Pressure:
Flash chromatography typically uses a positive pressure
system to accelerate the elution process, allowing for
faster separations.
• Flow Rate:
The flow rate must be optimized to balance the speed of
separation and the efficiency of separation.
Advantages
• Speed:
The separation process is much faster than traditional column
chromatography due to higher pressure and optimized columns.
• Efficiency:
Improved resolution, especially with smaller particle sizes in the
stationary phase.
• Ease of Use:
Pre-packed columns and simple operation make it more user-friendly
for rapid separations.
• Scalability:
Flash chromatography can be scaled up or down depending on the
sample size, making it suitable for both small- and large-scale
separations.
Applications
• Organic Chemistry:
For the purification of small molecules, such as organic
compounds, synthetic intermediates, and natural
products.
• Pharmaceutical Industry:
For the isolation and purification of drug compounds,
especially in the research and development stages.
• Environmental Analysis:
In the purification of pollutants or environmental
samples for further analysis.
Reversed-phase column
chromatography
• Reversed-phase column chromatography (RP
chromatography) is a type of liquid chromatography
where the stationary phase is nonpolar (hydrophobic),
and the mobile phase is polar (hydrophilic).
• This is the reverse of normal-phase chromatography,
where the stationary phase is polar, and the mobile
phase is nonpolar.
Stationary phases
• In reversed-phase chromatography, typically, the stationary
phase consists of hydrophobic materials, such as silica particles
that are coated with long-chain alkyl groups (e.g., C18, C8).
• The mobile phase usually consists of a polar solvent, often a
mixture of water and organic solvents (e.g., acetonitrile,
methanol), and sometimes with varying pH or ionic strength.
• Examples
C18 (Octadecylsilane)
C8 (Octylsilane)
C4 (Butylsilane)
Phenyl (Phenylsilyl)
C30 (Triacontylsilane)
• Silica-based materials with mixed functional groups
In some cases, the stationary phase may contain a
mixture of functional groups, such as a blend of C18
with polar groups like amino or cyano groups. This can
lead to hybrid interactions and is useful for very specific
separation challenges.
Modes of Chromatography
• Adsorption Chromatography
• Partition chromatography
• Ion Exchange Chromatography
• Size Exclusion Chromatography
• Affinity Chromatography
Adsorption chromatography
• separation of components occurs based on their
differential adsorption (affinity) to a stationary phase.
• one of the most commonly used methods in column
chromatography
• stationary phase is typically a solid (adsorbent), and the
components of the mixture interact differently with this
stationary phase
Solvents
• solvents (also called elution solvents or mobile
phases) are used to carry the components through the
column
• choice of solvent plays a key role in determining the
efficiency of separation and depends on the polarity of
the compounds in the mixture
• Non Polar Solvents
Hexane, petroleum ether, chloroform.
These solvents are low in polarity and are used to elute
non-polar compounds. They don't interact strongly with
the stationary phase (which is often polar), so the non-
polar compounds elute faster
• Polar Solvents:
Methanol, ethanol, acetonitrile, acetone, ethyl acetate
Polar solvents have a higher affinity for polar compounds. When
used in column chromatography, they can disrupt interactions
between the stationary phase and the compounds, aiding in the
separation of polar molecules.
• Mixed Solvent Systems:
Mixtures of chloroform and methanol, hexane and ethyl acetate
These mixtures can vary in polarity depending on the ratio of the
solvents used. By adjusting the ratio, you can control the elution
behavior, allowing for better separation of components with varying
polarities
for complex mixtures, where fine control over polarity is necessary
Adsorbants
• In column chromatography is typically a solid that
interacts with the components of the mixture through
adsorption
• selection of adsorbent depends on the nature of the
compounds being separated
• Silica Gel:
one of the most commonly used adsorbents in column
chromatography
polar and provides strong interactions with polar
compounds
Sterols, Amino Acids
• Alumina(Al₂O₃):
a polar adsorbent, similar to silica gel but with different polarity
characteristics
used for separating non-polar to moderately polar compounds
Commonly used for separating larger non-polar molecules
Sterols, Dyestuff, Vitamin, esters, Alkaloids, Inorganic compounds
• Activated Charcoal:
non-polar adsorbent that is used for separating non-polar compounds
or decolorizing liquids
high surface area and is generally used for purifying mixtures
• Magnesium Silicate
less common adsorbent used in column chromatography
Sterols, esters, glycerides, alkaloids
Partition Chromatography