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Department of QC-QA
Pallavi Rawat
CONTENT
1. Types of Chromatography
A. Preparative Chromatography
B. Semi- Preparative Chromatography
C. Analytical Chromatography
D. Ultra Performance Liquid Chromatography [UPLC]
2. Column Chemistry
A. Reverse Phase Column
B. Normal Phase Column
Chromatography
 Separation of component from a mixture for analysis.
 Develop in 1900 by Mikhail S. Tswett.
 Separate Mixture on:
Stationary Phase (S.P)- solid or liquid
Mobile Phase (M.P)- water or gas (to move over S.P)
 Each component elute from the S.P at specific time called Retention Time
 Once the components are separated, detector detect by various methods depending on
the type of chromatography, such as UV-Vis spectroscopy, mass spectrometry
 Differential partitioning of components between a stationary and a mobile phase
to separate and analyze mixtures.
A. Preparative Chromatography
 Technique used to:
separate and purify larger quantities
of a substance based on its chemical or physical properties.
 Use:
To isolate and collect specific components in larger amounts
 Common techniques include:
column chromatography,
flash chromatography,
TLC
 Methods are widely used in various fields:
pharmaceuticals, chemistry, and biochemistry, where the isolation and
purification of specific compounds are essential for research and
development.
Column Chromatography: Column packed with
stationary phase to separate components based on their
affinity for the stationary phase.
 S.P- solid (silica, aluminum)and M.P- liquid
 Performed Manually or automatically ( AKTA)
 Flash Chromatography: Similar to column
chromatography but faster, by using a pump or by using
a compressed gas (Nitrogen or argon) to push the
solvent through the column.
 Quick separation and less solvent usage.
 It is particularly employed in organic chemistry
laboratories for isolating and purifying target
compounds from complex mixtures.
 Scaled-up version of column chromatography and is
designed to handle larger sample loads with relatively
quick separation times.
 Thin-Layer Chromatography (TLC):
 Preparative Thin-Layer Chromatography (PTLC):Similar to thin-layer
chromatography, but larger quantities can be separated for isolation.
 : Involves the separation of components on a thin layer of adsorbent material. The components
move based on their affinity for the stationary phase.
 Column Chromatography with TLC monitoring: A sample is loaded onto a
column, and fractions are collected based on TLC analysis. This allows for real-
time monitoring of the separation.
 Each method has its strengths and is chosen based on the specific
characteristics of the compounds being separated
B. Semi- Preparative Chromatography
 Intermediate-scale chromatographic technique:
Falls between
analytical chromatography (used for small-scale analysis)and preparative
chromatography (used for large-scale purification)
 Isolation:
few micrograms of analyte for structural elucidation, or isolation of a few
milligrams of analyte
for assessing physiological, toxicological, or pharmacological activity.
 Commonly used:
research laboratories
smaller-scale production settings
where obtaining moderate quantities of purified compounds is sufficient
for the intended purposes, such as biological testing, or limited-scale
synthesis.
 High-Performance Liquid
Chromatography (HPLC):
 Column Type: larger column is used
compared to analytical HPLC.
 S.P is often similar, but the flow rate is
increased to accommodate larger sample
volumes.
 Flash Chromatography:
 Type of liquid chromatography designed for
rapid separation of compounds.
 Typically uses a medium-pressure flow of
liquid through a column containing a
S.P(silica gel or other adsorbents).
 Application: widely used for semi-
preparative purification, especially when
dealing with organic compounds.
 Gas Chromatography (GC): Suitable for volatile
compounds, where a gaseous mobile phase carries the
sample through a column.
 Column Type: Similar to HPLC, semi-preparative GC involves the use of
larger columns to handle larger sample volumes.
 Application: It is often used for the separation of volatile compounds.
 Size-Exclusion Chromatography (SEC):
 Separates molecules based on their size. In semi-preparative larger
columns are employed.
 Application: It is commonly used for the purification of bio molecules
like proteins.
 Ion-Exchange Chromatography:
 Separation based on the charge of the molecules. In semi-preparative
larger columns are used.
 Application: Purification of proteins peptides, or nucleic acids or other
bio molecules with high specificity.
 Technique depends on:
nature of the sample,
desired purity
specific properties of the compounds being separated.
Each technique has its advantages and limitations, and the selection
should be based on the characteristics of the target molecules
and the overall purification goals.
C. Analytical Chromatography
 Laboratory technique used to:
Separate
Identify
quantify (components in a mixture)
 Analyze sample at a smaller scale:
than preparative or semi-preparative chromatography
 Primary goal :
to understand individual components present in a mixture
and their respective concentrations
 Powerful tool:
for analyze composition of mixtures, providing valuable insights
into the nature and quantity of individual components
within a sample.
 High-Performance Liquid Chromatography
(HPLC):
 Principle: Uses a liquid M.P to move the
sample through a column containing a S.P .
Components are separated based on their
interaction with the stationary phase.
 Uses high pressure to improve separation
efficiency
 Applications: commonly employed for separating
bio-molecules and pharmaceuticals,
environmental samples, and bio-molecules.
 Gas Chromatography (GC):
 Principle: Utilizes a gaseous M.P to carry the
sample through a column with a S.P .
Separation is based on the differential
interaction of components with the S.P.
 Applications: Ideal for volatile and thermally
stable compounds, such as in the analysis of
organic compounds, environmental pollutants,
and forensic samples.
 Liquid Chromatography-Mass Spectrometry
(LC-MS):
 Principle: Combines liquid chromatography with
mass spectrometry for both separation and
identification of compounds. LC separates the
components, and MS provides structural
information.
 Applications: Used in pharmaceuticals, metabolomics,
proteomics, and environmental analysis.
 Thin-Layer Chromatography (TLC):
 Principle: Involves the separation of components on a
thin layer of adsorbent material. The components
move based on their affinity for the S.P.
 Applications: Quick and simple qualitative
analysis, often used in the separation of organic
compounds.
Choice of analytical chromatography Method
depends on:
properties of the sample
specific information required
such as separation efficiency, speed, sensitivity, and specificity.
D. Ultra Performance Liquid
Chromatography [UPLC]
 Advanced chromatographic technique i.e evolution of HPLC
 characterized by :
use of smaller particle size columns
higher pressure systems,
 resulting in:
increased resolution
Sensitivity
faster analysis times (compared to traditional HPLC)
 Key features include:
columns packed with sub-2-micron particles(smaller particle size allows for more
efficient separation of components in a mixture, leading to sharper peaks and
improved resolution
higher operating pressures (allows for faster flow rates, resulting in shorter analysis
times without sacrificing resolution)
 used in various scientific fields, including:
pharmaceuticals, environmental analysis, and biochemistry, where fast and precise
separation of complex mixtures is essential. Its high efficiency and sensitivity
make it a valuable tool for researchers and analysts who need to achieve optimal
results in a shorter time frame.
 :
 Reversed-Phase UPLC (RP-UPLC):
 Principle: Similar to reversed-phase HPLC, RP-UPLC separates
analytes based on their hydrophobicity. It is widely used for the
analysis of non-polar and moderately polar compounds.
 Applications: Pharmaceuticals, metabolomics, environmental
analysis, and natural products.
 Normal-Phase UPLC (NP-UPLC):
 Principle: In NP-UPLC, the stationary phase is polar, and
separation is based on the polarity of analytes. It is suitable for
separating polar compounds.
 Applications: Analysis of polar and hydrophilic compounds, such as
some pharmaceuticals and natural products.
 Ion-Exchange UPLC:
 Principle: Separates analytes based on their charge. Ion-exchange
UPLC uses a stationary phase with charged functional groups to
interact with analytes of opposite charge.
 Applications: Analysis of charged biomolecules like proteins,
peptides, and amino acids.
The choice of the UPLC technique depends on the nature of the analytes
and the specific requirements of the analysis.
Preparative Chromatography Semi- Preparative
Chromatography
Analytical Chromatography Ultra Performance Liquid
Chromatography (UPLC)
Large-scale isolation and
purification of compounds for
further use or analysis.
Purifying and isolating larger
amounts of individual
components from a mixture
analysis
Primarily used for qualitative
and quantitative analysis of the
components in a sample, often
on a smaller scale than
preparative or semi-preparative
chromatography.
Similar to analytical
chromatography but employs
smaller particle sizes and higher
pressures, resulting in faster
separations and improved
resolution.
Column Characteristics:
larger columns with a higher
loading capacity to handle larger
sample volumes.
Features columns of
intermediate size, suitable for
moderate sample volumes.
Employs smaller columns
designed for analytical purposes,
focusing on high-resolution
separation.
Utilizes columns with very small
particle sizes and operates at
higher pressures, allowing for
rapid separations.
Sample Size:
Processes larger sample
quantities, often in the
milligram to gram range.
Handles moderate sample sizes,
typically in the milligram to sub-
gram range.
Deals with smaller sample sizes,
usually in the microgram to
milligram range.
Analyzes smaller sample
volumes compared to traditional
analytical chromatography.
Resolution and Speed:
Focuses on purification;
resolution may be less critical,
and the process is generally
slower.
Balances resolution and speed to
provide a moderate-scale
purification process.
Emphasizes high resolution for
accurate analysis and
identification of components;
may have longer run times.
Prioritizes both high resolution
and speed, allowing for faster
analyses with improved
separation.
Cost and Efficiency:
Generally more resource-
intensive and expensive due to
the larger quantities involved.
Offers a balance between cost
and efficiency, suitable for
moderate-scale needs.
Often more cost-effective for
routine analyses but may lack
the throughput of preparative or
semi-preparative methods.
May require higher initial
investment but offers enhanced
efficiency and reduced operating
costs over time due to faster
separations.
Column Chemistry
 Plays crucial role in separation of components within a mixture.
 column packed with S.P that interacts with the sample components as they
pass through it.
 The chemistry of the column, include
nature of the S.P, is a key determinant of the separation efficiency and selectivity.
 Stationary Phase:
 key component of column (solid or a liquid).
 In liquid chromatography, S.P coated onto the surface of particles or beads,
while in gas chromatography, it may be immobilized on a solid support.
 The choice of S.P depends on the specific separation requirements. Common
types include
normal phase
reversed phase
ion exchange
size exclusion
Normal Phase Chromatography: Reversed Phase Chromatography:
The stationary phase is polar, and
separation is based on the affinity of
compounds for the polar stationary
phase
The stationary phase is non polar, and
separation is based on the hydrophobic
interactions of compounds with the non
polar stationary phase
Silica gel is a commonly used material
for normal phase columns.
Common reversed phase materials
include C18 (octadecylsilane) and C8
(octylsilane) bonded phases
Normal Phase Column
 The S.P is polar, and the separation is based on the affinity of
compounds for this polar S.P.
 It may include materials such as silica gel or alumina.
 Silica gel is a common choice for normal phase columns. It consists of
porous silica particles with hydroxyl (OH) groups on the surface, making it
polar.
 Basic structure of a siloxane. The R groups can be varied depending on
the type of column and analyte being analyzed.
 Most common R group attached to the Silica gel, cyano type CN,
amino type NH3, diol type OH, dimethylamino.
 Mobile phases
 Non polar Common solvents include hexane, diethyl ether, or chloroform.
 The choice of mobile phase depends on the polarity of the compounds being separated.
 Separation Mechanism:
 Separation is based on the polarity of the compounds. Polar compounds have a stronger
affinity for the polar stationary phase, leading to longer retention times, while less polar
compounds elute more quickly.
 The more polar the compound, the stronger its interaction with the polar stationary phase,
and the longer it remains in the column.
 Applications:
 used for the separation of polar compounds such as alcohols, amines, organic acids, and
other polar substances.
 It is particularly useful for compounds that are difficult to separate by reversed phase
chromatography.
 Elution Order:
 In normal phase, more polar compounds elute first, while less polar compounds elute
later.
 Sample Loading:
 Samples typically dissolved in a less polar solvent to enhance their interaction with the
polar stationary phase.
 widely used in various fields, including organic chemistry, natural product isolation, and
the analysis of polar compounds.
Reverse Phase Column
 Type of liquid chromatography where the S.P is non polar, and the separation is based on the
hydrophobic interactions between the analytes and the non polar stationary phase. This is in
contrast to normal phase chromatography, where the stationary phase is polar.
 Here are the key features of reversed phase chromatography:
 Stationary Phase:
 Non polar and consists of hydrophobic materials. Common materials include hydrocarbons or
polymers with non polar functional groups.
 Silica gel modified with hydrophobic groups (such as C18 or C8) or any hydocarbon is widely
used in reversed phase columns.
 C18 ODS , C8 Octyl, , C4butyl, Phenyl, cyano type
 Mobile Phase:
 The mobile phase used in reversed phase chromatography is more polar than the stationary phase. It
often includes water (cheap, non toxic , invisible in Uv region) or a mixture of water and an
organic solvent like methanol or acetonitrile.
 The polarity of the mobile phase can be adjusted to control the elution of different compounds.
C8 octylsilane C18 octadecylsilane
OH-Si-C8 STRUCTURE
8 no. of carbon atom
OH-Si-C18 STRUCTURE
18 no. of carbon atom
Short carbon chain less hydrophobic Long carbon chain more hydrophobic
Less dense (less no. of carbon), low
retention time, sample elutes quickly,
retain less component of sample/analyte
More dense (More no. of carbon), high
retention time, sample elutes slowly,
retain less component of sample/analyte
Relatively low separation Relatively high separation
Seprates small organic compound better Separates long chain fatty acid well
used for pharmaceutical analyses,
environmental testing, and the
separation of drug compounds. They are
also suitable for analyzing peptides and
proteins in some cases.
versatile and are widely used in
pharmaceutical, clinical, environmental,
and food industry applications. They are
the standard choice for many HPLC
analyses, including the separation of
pharmaceuticals, natural products, and
biomolecules.
 Separation Mechanism:
 Separation is based on the hydrophobicity of the compounds.
Hydrophobic analytes have a greater affinity for the
hydrophobic stationary phase, resulting in longer retention
times, while more polar analytes elute more quickly.
 Applications:
 widely used for the separation of pharmaceuticals,
natural products, peptides, proteins, and more.
 Elution Order:
 Less polar compounds elute first, while more polar
compounds elute later.
 Sample Loading:
 Samples dissolved in a polar solvent to enhance their
interaction with the non polar stationary phase.

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CHROMATOGRAPHY PALLAVI RAWAT.pptx

  • 2. CONTENT 1. Types of Chromatography A. Preparative Chromatography B. Semi- Preparative Chromatography C. Analytical Chromatography D. Ultra Performance Liquid Chromatography [UPLC] 2. Column Chemistry A. Reverse Phase Column B. Normal Phase Column
  • 3. Chromatography  Separation of component from a mixture for analysis.  Develop in 1900 by Mikhail S. Tswett.  Separate Mixture on: Stationary Phase (S.P)- solid or liquid Mobile Phase (M.P)- water or gas (to move over S.P)  Each component elute from the S.P at specific time called Retention Time  Once the components are separated, detector detect by various methods depending on the type of chromatography, such as UV-Vis spectroscopy, mass spectrometry  Differential partitioning of components between a stationary and a mobile phase to separate and analyze mixtures.
  • 4. A. Preparative Chromatography  Technique used to: separate and purify larger quantities of a substance based on its chemical or physical properties.  Use: To isolate and collect specific components in larger amounts  Common techniques include: column chromatography, flash chromatography, TLC  Methods are widely used in various fields: pharmaceuticals, chemistry, and biochemistry, where the isolation and purification of specific compounds are essential for research and development.
  • 5. Column Chromatography: Column packed with stationary phase to separate components based on their affinity for the stationary phase.  S.P- solid (silica, aluminum)and M.P- liquid  Performed Manually or automatically ( AKTA)
  • 6.  Flash Chromatography: Similar to column chromatography but faster, by using a pump or by using a compressed gas (Nitrogen or argon) to push the solvent through the column.  Quick separation and less solvent usage.  It is particularly employed in organic chemistry laboratories for isolating and purifying target compounds from complex mixtures.  Scaled-up version of column chromatography and is designed to handle larger sample loads with relatively quick separation times.
  • 7.  Thin-Layer Chromatography (TLC):  Preparative Thin-Layer Chromatography (PTLC):Similar to thin-layer chromatography, but larger quantities can be separated for isolation.  : Involves the separation of components on a thin layer of adsorbent material. The components move based on their affinity for the stationary phase.  Column Chromatography with TLC monitoring: A sample is loaded onto a column, and fractions are collected based on TLC analysis. This allows for real- time monitoring of the separation.  Each method has its strengths and is chosen based on the specific characteristics of the compounds being separated
  • 8. B. Semi- Preparative Chromatography  Intermediate-scale chromatographic technique: Falls between analytical chromatography (used for small-scale analysis)and preparative chromatography (used for large-scale purification)  Isolation: few micrograms of analyte for structural elucidation, or isolation of a few milligrams of analyte for assessing physiological, toxicological, or pharmacological activity.  Commonly used: research laboratories smaller-scale production settings where obtaining moderate quantities of purified compounds is sufficient for the intended purposes, such as biological testing, or limited-scale synthesis.
  • 9.  High-Performance Liquid Chromatography (HPLC):  Column Type: larger column is used compared to analytical HPLC.  S.P is often similar, but the flow rate is increased to accommodate larger sample volumes.  Flash Chromatography:  Type of liquid chromatography designed for rapid separation of compounds.  Typically uses a medium-pressure flow of liquid through a column containing a S.P(silica gel or other adsorbents).  Application: widely used for semi- preparative purification, especially when dealing with organic compounds.
  • 10.  Gas Chromatography (GC): Suitable for volatile compounds, where a gaseous mobile phase carries the sample through a column.  Column Type: Similar to HPLC, semi-preparative GC involves the use of larger columns to handle larger sample volumes.  Application: It is often used for the separation of volatile compounds.
  • 11.  Size-Exclusion Chromatography (SEC):  Separates molecules based on their size. In semi-preparative larger columns are employed.  Application: It is commonly used for the purification of bio molecules like proteins.  Ion-Exchange Chromatography:  Separation based on the charge of the molecules. In semi-preparative larger columns are used.  Application: Purification of proteins peptides, or nucleic acids or other bio molecules with high specificity.  Technique depends on: nature of the sample, desired purity specific properties of the compounds being separated. Each technique has its advantages and limitations, and the selection should be based on the characteristics of the target molecules and the overall purification goals.
  • 12. C. Analytical Chromatography  Laboratory technique used to: Separate Identify quantify (components in a mixture)  Analyze sample at a smaller scale: than preparative or semi-preparative chromatography  Primary goal : to understand individual components present in a mixture and their respective concentrations  Powerful tool: for analyze composition of mixtures, providing valuable insights into the nature and quantity of individual components within a sample.
  • 13.  High-Performance Liquid Chromatography (HPLC):  Principle: Uses a liquid M.P to move the sample through a column containing a S.P . Components are separated based on their interaction with the stationary phase.  Uses high pressure to improve separation efficiency  Applications: commonly employed for separating bio-molecules and pharmaceuticals, environmental samples, and bio-molecules.  Gas Chromatography (GC):  Principle: Utilizes a gaseous M.P to carry the sample through a column with a S.P . Separation is based on the differential interaction of components with the S.P.  Applications: Ideal for volatile and thermally stable compounds, such as in the analysis of organic compounds, environmental pollutants, and forensic samples.
  • 14.  Liquid Chromatography-Mass Spectrometry (LC-MS):  Principle: Combines liquid chromatography with mass spectrometry for both separation and identification of compounds. LC separates the components, and MS provides structural information.  Applications: Used in pharmaceuticals, metabolomics, proteomics, and environmental analysis.  Thin-Layer Chromatography (TLC):  Principle: Involves the separation of components on a thin layer of adsorbent material. The components move based on their affinity for the S.P.  Applications: Quick and simple qualitative analysis, often used in the separation of organic compounds. Choice of analytical chromatography Method depends on: properties of the sample specific information required such as separation efficiency, speed, sensitivity, and specificity.
  • 15. D. Ultra Performance Liquid Chromatography [UPLC]  Advanced chromatographic technique i.e evolution of HPLC  characterized by : use of smaller particle size columns higher pressure systems,  resulting in: increased resolution Sensitivity faster analysis times (compared to traditional HPLC)  Key features include: columns packed with sub-2-micron particles(smaller particle size allows for more efficient separation of components in a mixture, leading to sharper peaks and improved resolution higher operating pressures (allows for faster flow rates, resulting in shorter analysis times without sacrificing resolution)  used in various scientific fields, including: pharmaceuticals, environmental analysis, and biochemistry, where fast and precise separation of complex mixtures is essential. Its high efficiency and sensitivity make it a valuable tool for researchers and analysts who need to achieve optimal results in a shorter time frame.
  • 16.  :  Reversed-Phase UPLC (RP-UPLC):  Principle: Similar to reversed-phase HPLC, RP-UPLC separates analytes based on their hydrophobicity. It is widely used for the analysis of non-polar and moderately polar compounds.  Applications: Pharmaceuticals, metabolomics, environmental analysis, and natural products.  Normal-Phase UPLC (NP-UPLC):  Principle: In NP-UPLC, the stationary phase is polar, and separation is based on the polarity of analytes. It is suitable for separating polar compounds.  Applications: Analysis of polar and hydrophilic compounds, such as some pharmaceuticals and natural products.  Ion-Exchange UPLC:  Principle: Separates analytes based on their charge. Ion-exchange UPLC uses a stationary phase with charged functional groups to interact with analytes of opposite charge.  Applications: Analysis of charged biomolecules like proteins, peptides, and amino acids. The choice of the UPLC technique depends on the nature of the analytes and the specific requirements of the analysis.
  • 17. Preparative Chromatography Semi- Preparative Chromatography Analytical Chromatography Ultra Performance Liquid Chromatography (UPLC) Large-scale isolation and purification of compounds for further use or analysis. Purifying and isolating larger amounts of individual components from a mixture analysis Primarily used for qualitative and quantitative analysis of the components in a sample, often on a smaller scale than preparative or semi-preparative chromatography. Similar to analytical chromatography but employs smaller particle sizes and higher pressures, resulting in faster separations and improved resolution. Column Characteristics: larger columns with a higher loading capacity to handle larger sample volumes. Features columns of intermediate size, suitable for moderate sample volumes. Employs smaller columns designed for analytical purposes, focusing on high-resolution separation. Utilizes columns with very small particle sizes and operates at higher pressures, allowing for rapid separations. Sample Size: Processes larger sample quantities, often in the milligram to gram range. Handles moderate sample sizes, typically in the milligram to sub- gram range. Deals with smaller sample sizes, usually in the microgram to milligram range. Analyzes smaller sample volumes compared to traditional analytical chromatography. Resolution and Speed: Focuses on purification; resolution may be less critical, and the process is generally slower. Balances resolution and speed to provide a moderate-scale purification process. Emphasizes high resolution for accurate analysis and identification of components; may have longer run times. Prioritizes both high resolution and speed, allowing for faster analyses with improved separation. Cost and Efficiency: Generally more resource- intensive and expensive due to the larger quantities involved. Offers a balance between cost and efficiency, suitable for moderate-scale needs. Often more cost-effective for routine analyses but may lack the throughput of preparative or semi-preparative methods. May require higher initial investment but offers enhanced efficiency and reduced operating costs over time due to faster separations.
  • 18. Column Chemistry  Plays crucial role in separation of components within a mixture.  column packed with S.P that interacts with the sample components as they pass through it.  The chemistry of the column, include nature of the S.P, is a key determinant of the separation efficiency and selectivity.  Stationary Phase:  key component of column (solid or a liquid).  In liquid chromatography, S.P coated onto the surface of particles or beads, while in gas chromatography, it may be immobilized on a solid support.  The choice of S.P depends on the specific separation requirements. Common types include normal phase reversed phase ion exchange size exclusion
  • 19. Normal Phase Chromatography: Reversed Phase Chromatography: The stationary phase is polar, and separation is based on the affinity of compounds for the polar stationary phase The stationary phase is non polar, and separation is based on the hydrophobic interactions of compounds with the non polar stationary phase Silica gel is a commonly used material for normal phase columns. Common reversed phase materials include C18 (octadecylsilane) and C8 (octylsilane) bonded phases
  • 20. Normal Phase Column  The S.P is polar, and the separation is based on the affinity of compounds for this polar S.P.  It may include materials such as silica gel or alumina.  Silica gel is a common choice for normal phase columns. It consists of porous silica particles with hydroxyl (OH) groups on the surface, making it polar.  Basic structure of a siloxane. The R groups can be varied depending on the type of column and analyte being analyzed.  Most common R group attached to the Silica gel, cyano type CN, amino type NH3, diol type OH, dimethylamino.
  • 21.  Mobile phases  Non polar Common solvents include hexane, diethyl ether, or chloroform.  The choice of mobile phase depends on the polarity of the compounds being separated.  Separation Mechanism:  Separation is based on the polarity of the compounds. Polar compounds have a stronger affinity for the polar stationary phase, leading to longer retention times, while less polar compounds elute more quickly.  The more polar the compound, the stronger its interaction with the polar stationary phase, and the longer it remains in the column.  Applications:  used for the separation of polar compounds such as alcohols, amines, organic acids, and other polar substances.  It is particularly useful for compounds that are difficult to separate by reversed phase chromatography.  Elution Order:  In normal phase, more polar compounds elute first, while less polar compounds elute later.  Sample Loading:  Samples typically dissolved in a less polar solvent to enhance their interaction with the polar stationary phase.  widely used in various fields, including organic chemistry, natural product isolation, and the analysis of polar compounds.
  • 22. Reverse Phase Column  Type of liquid chromatography where the S.P is non polar, and the separation is based on the hydrophobic interactions between the analytes and the non polar stationary phase. This is in contrast to normal phase chromatography, where the stationary phase is polar.  Here are the key features of reversed phase chromatography:  Stationary Phase:  Non polar and consists of hydrophobic materials. Common materials include hydrocarbons or polymers with non polar functional groups.  Silica gel modified with hydrophobic groups (such as C18 or C8) or any hydocarbon is widely used in reversed phase columns.  C18 ODS , C8 Octyl, , C4butyl, Phenyl, cyano type  Mobile Phase:  The mobile phase used in reversed phase chromatography is more polar than the stationary phase. It often includes water (cheap, non toxic , invisible in Uv region) or a mixture of water and an organic solvent like methanol or acetonitrile.  The polarity of the mobile phase can be adjusted to control the elution of different compounds.
  • 23. C8 octylsilane C18 octadecylsilane OH-Si-C8 STRUCTURE 8 no. of carbon atom OH-Si-C18 STRUCTURE 18 no. of carbon atom Short carbon chain less hydrophobic Long carbon chain more hydrophobic Less dense (less no. of carbon), low retention time, sample elutes quickly, retain less component of sample/analyte More dense (More no. of carbon), high retention time, sample elutes slowly, retain less component of sample/analyte Relatively low separation Relatively high separation Seprates small organic compound better Separates long chain fatty acid well used for pharmaceutical analyses, environmental testing, and the separation of drug compounds. They are also suitable for analyzing peptides and proteins in some cases. versatile and are widely used in pharmaceutical, clinical, environmental, and food industry applications. They are the standard choice for many HPLC analyses, including the separation of pharmaceuticals, natural products, and biomolecules.
  • 24.  Separation Mechanism:  Separation is based on the hydrophobicity of the compounds. Hydrophobic analytes have a greater affinity for the hydrophobic stationary phase, resulting in longer retention times, while more polar analytes elute more quickly.  Applications:  widely used for the separation of pharmaceuticals, natural products, peptides, proteins, and more.  Elution Order:  Less polar compounds elute first, while more polar compounds elute later.  Sample Loading:  Samples dissolved in a polar solvent to enhance their interaction with the non polar stationary phase.