High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a powerful analytical technique widely

used in chemistry, biochemistry, and many other fields. It excels at separating, identifying, and
quantifying the individual components within a complex mixture that can be dissolved in a liquid
solvent. Here's a detailed breakdown of HPLC:

Definition:

HPLC is a chromatographic technique that separates a mixture based on the partitioning behavior
of its components between two phases: a mobile phase (liquid) and a stationary phase (solid).
The mobile phase, typically a liquid solvent or mixture of solvents, carries the dissolved sample
through a column packed with the stationary phase. The stationary phase is a porous material
with a high surface area designed to interact with the sample components.

Principles:

Separation in HPLC occurs due to differences in the affinity of sample components for the
mobile and stationary phases. Components with a stronger affinity for the stationary phase spend
more time interacting with it, resulting in a slower travel through the column. Conversely, those
with a weaker attraction for the stationary phase move faster with the mobile phase. This
difference in interaction times translates to distinct retention times, the time it takes for each
component to emerge from the column and be detected. By analyzing these retention times and
comparing them to standards or reference data, chemists can identify and potentially quantify the
separated components.

Components:

An HPLC instrument is an intricate assembly of several key parts:

• Reservoir: This container holds the mobile phase solvent(s).

• Pump: A high-pressure pump continuously delivers the mobile phase through the
system. Unlike Gas Chromatography (GC), HPLC utilizes high pressure to overcome the
resistance of the packed column.
• Injector: This component introduces a small, controlled volume of the dissolved sample
into the mobile phase stream.
• Column: The heart of HPLC, it's a long, narrow tube packed with the stationary phase.
Different column lengths, stationary phase chemistries, and particle sizes can influence
separation.
• Detector: This sensitive device monitors the column effluent and generates a signal
based on the presence of the separated components. Common detectors include
ultraviolet (UV) detectors and mass spectrometers (MS).
• Data Acquisition System: This system records and processes the detector signal,
generating a chromatogram - a graph showing peaks corresponding to the separated
components.
Strengths:

• High Resolution: HPLC offers excellent separation capabilities, allowing complex

mixtures to be resolved into their individual components.
• Versatility: A wide range of samples can be analyzed by HPLC, as long as they are
soluble in a suitable solvent. This makes it applicable to diverse fields like
pharmaceuticals, food science, environmental analysis, and forensics.
• Quantitation: By analyzing the peak area in the chromatogram, HPLC allows for the
quantification of each component in the mixture.
• Compatibility with Non-volatile Analytes: Unlike GC, HPLC can analyze non-volatile
and thermally labile compounds that would decompose at high temperatures.

Limitations:

• Cost: HPLC instruments can be expensive to purchase and maintain due to the high-
pressure pumps and specialized detectors.
• Solvent Usage: HPLC utilizes significant amounts of solvents, which can be hazardous
and require proper disposal.
• Analysis Time: HPLC analyses can take longer than GC, especially for complex
mixtures.
• Sample Preparation: Samples often require pre-treatment to ensure solubility and
remove interferences.
 Difference between High-Performance Liquid Chromatography and
Gas Chromatography
High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) are both
powerful separation techniques used in analytical chemistry, but they differ significantly in the
type of samples they can handle and the information they provide. Here's a breakdown of the key
differences:

Sample Type:

• HPLC: Analyzes soluble samples. It can handle a wide range of compounds, including
polar molecules, ionic species, biomolecules (proteins, peptides), and non-volatile
compounds that would decompose at high temperatures.
• GC: Analyzes volatile samples. It's limited to compounds that can be vaporized without
decomposition. This typically includes low-boiling-point organic molecules like
hydrocarbons, solvents, and many volatile organic compounds (VOCs).

Separation Mechanism:

• HPLC: Separation is based on the partitioning of the sample components between a

liquid mobile phase (solvent) and a solid stationary phase. Components with a stronger
affinity for the stationary phase move slower through the column.
• GC: Separation is based on the volatility and affinity of the sample components for a
gaseous mobile phase (carrier gas) and a stationary phase (liquid or solid coating).
More volatile components travel faster through the column.

Detection:

• HPLC: Uses various detectors, including ultraviolet (UV) detectors, refractive index (RI)
detectors, and mass spectrometers (MS).
• GC: Primarily uses detectors like flame ionization detectors (FIDs) that ionize and
measure the amount of the eluting components. MS can also be used as a detector for GC.

Strengths:

• HPLC:
o Versatile: Handles a wide range of samples, including non-volatile and polar
compounds.
o Compatible with biological samples: Ideal for analyzing biomolecules like
proteins and peptides.
o Good for quantitation: Can be used to accurately determine the amount of each
component in a mixture.
• GC:
o High resolution: Excellent for separating complex mixtures of volatile
compounds.
 o Sensitive: Can detect very small quantities of a compound.
o Fast analysis time: Analyses are often quicker compared to HPLC.

Limitations:

• HPLC:
o Cost: HPLC instruments can be expensive due to high-pressure pumps and
specialized detectors.
o Solvent usage: Uses significant amounts of solvents, which require proper
disposal.
o Analysis time: Analyses can be slower than GC, especially for complex mixtures.
• GC:
o Limited sample type: Restricted to volatile and thermally stable compounds.
o Less information: Primarily provides identification based on retention times.
Additional techniques might be needed for structural information.
 Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC) is a widely used technique for separating the components of
a mixture. It's a simple, fast, and cost-effective method employed in analytical chemistry for both
qualitative and quantitative analysis.

Principles:

TLC relies on the interplay between two phases:

1. Stationary Phase: A thin layer of adsorbent material like alumina, silica gel, or
cellulose, coated on a rigid plate (glass, plastic, or aluminum).
2. Mobile Phase: A solvent or solvent mixture that moves through the stationary phase by
capillary action.

The separation hinges on the relative affinities of the mixture's components towards these two
phases. Compounds with a stronger attraction to the stationary phase travel slower, while those
with a greater affinity for the mobile phase move faster. This differential movement leads to the
separation of the components into distinct spots on the TLC plate.

Components:

• TLC Plate: The plate serves as the support for the stationary phase.
• Adsorbent: The stationary phase material, typically alumina or silica gel, with a high
surface area that interacts with the components of the mixture.
• Solvent System (Mobile Phase): A solvent or a mixture of solvents chosen based on the
polarity of the sample components.
• Sample Solution: The mixture to be separated, dissolved in a suitable solvent and
applied as a small spot on the TLC plate.
• Developing Chamber: A sealed container saturated with the mobile phase solvent
vapors.

Strengths of TLC:

• Simplicity: The process is easy to set up and requires minimal training.

• Speed: Separations can be achieved in minutes to tens of minutes.
• Low Cost: TLC plates and solvents are relatively inexpensive.
• Sensitivity: TLC can detect very small amounts of sample (in microgram or nanogram
quantities).
• Versatility: The technique can be used for a wide range of samples, including organic
and inorganic compounds.
• Visual Analysis: Separated components often appear as colored spots, allowing for
immediate qualitative analysis.

Limitations of TLC:
 • Limited Resolution: Compared to other chromatographic techniques, TLC offers lower
resolution, meaning it might not be suitable for separating very similar compounds.
• Qualitative Analysis: While visualization techniques can offer clues about the
components, definitive identification often requires additional methods like spectroscopy.
• Quantification: Although some staining techniques allow for rough quantification, TLC
is primarily a qualitative technique.
• Limited Sample Size: The amount of sample loaded needs to be small, as overloading
can lead to poor separation.
• Destructive: The TLC plate is typically destroyed during the analysis process.

Similarities and Differences

Similarities:

• Separation Principle: All three utilize a stationary and a mobile phase to separate
components based on their interactions with these phases. The components with a
stronger affinity for the stationary phase move slower, achieving separation.
• Applications: All find applications in various fields like pharmaceuticals, environmental
analysis, food science, and forensics to separate and analyze complex mixtures.
• Qualitative and Quantitative Analysis: While primarily used for qualitative analysis
(identifying components), all can be adapted for rough quantification with additional
techniques.

Differences:

High-Performance Thin Layer

Gas Chromatography
Feature Liquid Chromatography Chromatography
(GC)
(HPLC) (TLC)
Liquid (solvent or solvent Liquid (solvent or
Mobile Phase Inert Gas (Helium)
mixture) solvent mixture)
Liquid or soluble in Liquid or soluble in
Sample State Volatile
mobile phase mobile phase
Separation Partitioning between Differential interaction Differential interaction
Mechanism gas and stationary liquid with stationary phase with stationary phase
Instrumentation Complex and expensive Complex and expensive Simple and inexpensive
Minutes to tens of
Analysis Time Minutes Minutes to hours
minutes
Resolution High High to very high Low to moderate
Microliters to tens of
Sample Size Microliters Microliters to Milliliters
microliters
Destructive or Non-destructive (can be
Destructive Destructive
Non-destructive recovered)
 Volatile and thermally Wide range of compounds Most suitable for
Suitability
stable compounds (polar, non-polar, ionic) preliminary analysis

Key Differentiators:

• Mobile Phase: GC uses an inert gas (helium) for faster separation, while HPLC and TLC
utilize liquids.
• Sample State: GC requires volatile samples, while HPLC and TLC can handle liquids or
compounds soluble in the mobile phase.
• Separation Mechanism: GC relies on partitioning between the gas and stationary liquid
phase, while HPLC and TLC separate based on interactions with the stationary phase
itself.
• Instrumentation: GC and HPLC are complex and expensive instruments, whereas TLC
is a simple and inexpensive technique.
• Resolution and Analysis Time: GC and HPLC offer superior resolution for complex
mixtures, but with longer analysis times compared to TLC's faster separations and lower
resolution.
 Gas Chromatography
Gas Chromatography (GC) is a workhorse technique in analytical chemistry. It excels at
separating and analyzing the individual components within a complex mixture. Here's a deep
dive into its definition, working principles, the components that make it tick, its strengths as an
analytical tool, and the limitations to consider.

Definition:

GC is a separation technique that relies on the different partitioning behaviors of compounds

between two phases: a mobile phase and a stationary phase. The mobile phase, typically an inert
gas like helium, carries the vaporized sample through a long, narrow column containing the
stationary phase. The stationary phase can be a solid coated with a high-boiling-point liquid or a
special solid material.

Principles:

The separation magic happens due to interactions between the sample components and the two
phases. Compounds with a stronger affinity for the stationary phase spend more time interacting
with it, leading to a slower travel through the column. Conversely, those with a weaker attraction
move faster with the mobile phase. This difference in interaction times translates to distinct
retention times, the time each component takes to emerge from the column and be detected. By
analyzing these retention times, chemists can identify and potentially quantify the separated
components.

Components:

A GC instrument is an assembly of several key parts:

• Injector: This port vaporizes the sample for introduction into the mobile phase.
• Column: The heart of GC, it's a long, narrow tube packed with the stationary phase.
Different column lengths and stationary phase types can influence separation.
• Carrier Gas: An inert gas like helium continuously flows through the column, carrying
the vaporized sample along.
• Detector: This sensitive device monitors the column effluent (the exiting stream) and
generates a signal based on the presence of the separated components. Common detectors
include flame ionization detectors (FIDs) and mass spectrometers (MS).
• Data Acquisition System: This system records and processes the detector signal,
generating a chromatogram - a graph showing peaks corresponding to the separated
components.

Strengths:

• High Resolution: GC excels at separating complex mixtures into their individual

components.
• Sensitivity: Modern detectors can detect incredibly small quantities of a compound.
 • Quantitation: By analyzing the peak area in the chromatogram, GC allows for the
quantification of each component in the mixture.
• Speed: Compared to other separation techniques, GC analyses are often relatively fast.
• Versatility: A wide range of samples can be analyzed using GC, provided they are
volatile and thermally stable.

Limitations:

• Sample Volatility: Compounds must be vaporizable without decomposing at the

injection temperature to be analyzed by GC.
• Non-volatile Analytes: If a compound isn't volatile, derivatization techniques may be
required to modify it for GC analysis.
• Limited Information: While GC provides excellent separation, it may not always
definitively identify unknown compounds. Coupling GC with other techniques like MS
can offer more structural information.
• Destructive Detectors: Many detectors destroy the sample during analysis, making
further characterization challenging.

A Gas Chromatography (GC) instrument is a workhorse technique in analytical chemistry. It

excels at separating and analyzing the individual components within a complex mixture. Here's a
deep dive into its components:

• Injector: This port vaporizes the sample for introduction into the mobile phase. It is a
heated chamber that rapidly converts the liquid sample into a gaseous state.

Gas Chromatography Injector

• Column: The heart of GC, it's a long, narrow tube packed with the stationary phase.
Different column lengths and stationary phase types can influence separation. The
stationary phase is a thin layer of high-boiling-point liquid or a special solid material
coated on an inert solid support within the column.

Gas Chromatography Column

• Carrier Gas: An inert gas like helium continuously flows through the column, carrying
the vaporized sample along. The carrier gas plays a crucial role in transporting the sample
through the column and maintains a constant linear velocity.
• Detector: This sensitive device monitors the column effluent (the exiting stream) and
generates a signal based on the presence of the separated components. Common detectors
include flame ionization detectors (FIDs) and mass spectrometers (MS). Some detectors
like FID uses a flame to ionize the eluting molecules, while others like MS use high
energy to ionize the molecules and separate them based on their mass-to-charge ratio.

Gas Chromatography Detector

 • Data Acquisition System: This system records and processes the detector signal,
generating a chromatogram - a graph showing peaks corresponding to the separated
components. The data acquisition system is a computer program that controls the GC
instrument, collects data from the detector, and generates a chromatogram. The
chromatogram is a visual representation of the separation process, with each peak
corresponding to a different component in the sample.

Mass Spectrometry-Gas Chromatography (MS-GC)

Mass Spectrometry-Gas Chromatography (MS-GC) is a powerful analytical technique that
combines the strengths of two individual methods: Gas Chromatography (GC) and Mass
Spectrometry (MS). This marriage of technologies allows for the separation, identification, and
structural characterization of a vast array of compounds within a complex mixture.

Definition:

MS-GC is a hyphenated technique where GC acts as a separation tool and MS acts as a detector.
GC separates the mixture's components based on their interaction with a stationary phase, while
MS identifies and characterizes them by measuring their mass-to-charge ratio (m/z).

Principles:

1. Separation by GC: The sample is first injected into the GC. Here, the components are
separated based on their differing affinities for the mobile and stationary phases in the
GC column. Components with a stronger affinity for the stationary phase travel slower,
emerging from the column at distinct retention times.
2. Detection and Identification by MS: As each separated component exits the GC
column, it enters the MS. Here, the molecules are ionized (given an electric charge) and
fragmented into smaller pieces. The MS then measures the mass-to-charge ratio (m/z) of
these ions. Each compound has a unique fragmentation pattern and m/z signature,
allowing the MS to identify the compound by comparing it to a spectral library database.

Components:

An MS-GC instrument combines the key components of both GC and MS:

• GC Components:
o Injector: Vaporizes the sample for introduction into the GC.
o Column: Separates the mixture's components based on their interactions with the
stationary phase.
o Carrier Gas: Carries the vaporized sample through the GC column.
• MS Components:
o Ion Source: Ionizes the sample molecules.
o Mass Analyzer: Separates the ions based on their mass-to-charge ratio.
 oDetector: Measures the abundance of each ion.
• Data Acquisition System: Controls the instrument, collects data from the MS detector,
and processes it to generate a mass spectrum for each separated component.

Strengths:

• High Resolution and Specificity: MS-GC offers exceptional separation power from GC
combined with the high specificity of MS for identifying unknown compounds. This
makes it ideal for complex mixtures.
• Structural Information: MS provides structural information about the identified
compounds due to the characteristic fragmentation patterns.
• Sensitivity: Modern MS detectors can detect incredibly small quantities of a compound,
making MS-GC valuable for trace analysis.
• Quantitation: By combining peak area from the GC chromatogram with the MS signal
intensity, MS-GC allows for both qualitative (identification) and quantitative (amount)
analysis.

Limitations:

• Cost: MS-GC instruments are complex and expensive, requiring significant investment
for purchase and maintenance.
• Sample Volatility: Like GC, MS-GC is limited to volatile and thermally stable
compounds. Non-volatile or thermally labile samples may require derivatization for
analysis.
• Expertise: Operating and interpreting MS-GC data requires specialized training and
knowledge due to the complexity of the combined techniques.
Difference between Mass Spectrometry-Gas Chromatography (MS-
GC) and only Gas Chromatography
Both MS-GC and GC are analytical techniques, but they differ significantly in their capabilities:

Focus:

• GC: Separation. GC excels at separating the components within a complex mixture. It

identifies them based on their retention times within the column, which can be a good
initial indication of the compound's identity.
• MS-GC: Identification and characterization. MS-GC builds upon GC's separation power
by adding the identification capabilities of Mass Spectrometry (MS). MS provides a
fingerprint of the compound based on its mass-to-charge ratio (m/z) and fragmentation
patterns, allowing for definitive identification and structural information.

Information Provided:

• GC: Retention time. GC provides retention times for each separated component. While
retention times can be used for tentative identification by comparing them to standards,
they can be misleading for similar compounds.
• MS-GC: m/z ratio and fragmentation pattern. MS-GC offers a much richer dataset. It
provides the m/z ratio of the ionized molecule and its fragments, creating a unique
"fingerprint" for each compound. This fingerprint can be compared to spectral libraries
for definitive identification and even provides some structural information about the
molecule.

Applications:

• GC: GC is a workhorse technique for routine analysis where the components are already
known or can be identified by retention times and comparison to standards. It's widely
used in quality control, environmental monitoring, and volatile organic compound (VOC)
analysis.
• MS-GC: MS-GC is ideal for analyzing complex mixtures with unknown components,
pinpointing trace amounts of a specific compound, and obtaining structural information
about the identified molecules. It's valuable in forensic science, drug discovery,
metabolite identification, and analyzing complex biological samples.

Cost and Complexity:

• GC: GC instruments are relatively inexpensive and easy to operate.

• MS-GC: MS-GC instruments are significantly more expensive and complex to operate
due to the combination of GC and MS technologies. They require specialized training and
expertise for data interpretation.
In essence:

• GC separates the mixture's "who" based on their travel times.

• MS-GC identifies the "who" and provides some clues about "what" they are made
of through the mass spectrum.

When choosing between GC and MS-GC, consider the complexity of your sample and the level
of detail required for analysis. For routine analysis of known components, GC might suffice.
However, if you need definitive identification, structural information, or are dealing with
unknown compounds in a complex mixture, MS-GC is the more powerful tool.
 PHARM 207
College of
Nursing
(Pharmaceutical Analysis 2
Pharmacy &(Instrumental Methods of Analysis)
Allied
Health
Sciences
STEPHANIE MEGHAN N. CRISTUTA, RPh
Department of Pharmacy
CHROMATOGRAPHY
Department of Pharmacy

Chromatography is a chemical analysis or purification

technique used to separate complex mixtures (in either a solid
or liquid sample matrix) into their different components.

There are a number of different chromatography separation

techniques, but they all share the same basic principles
entailing a solvent called the mobile phase for its ability to
move through a system on a fixed stationary phase.
Department of Pharmacy

The mobile phase may be either a liquid, supercritical fluid

(usually carbon dioxide) or inert gas, while the stationary phase
is a solid phase, or a liquid phase coated on a solid phase.

The mobile phase moves over a stationary phase (commonly

silica gel) resulting in a separation of the compounds into its
different components, based on the affinity of each
component’s molecules for the mobile and stationary phases.
Department of Pharmacy

When should you use chromatography?

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What are the types of chromatography?

Department of Pharmacy
Gas Chromatography
Department of Pharmacy

GC is a separation technique that relies on the different

partitioning behaviors of compounds between two phases: a
mobile phase and a stationary phase.
The mobile phase, typically an inert gas like helium, carries
the vaporized sample through a long, narrow column containing
the stationary phase.
The stationary phase can be a solid coated with a high-boiling-
point liquid or a special solid material.
Department of Pharmacy
PRINCIPLE
Gas Chromatography offers a powerful tool for analyzing
mixtures by separating and identifying their volatile
components based on their interactions with a stationary phase
and their movement through a column with a carrier gas.
Department of Pharmacy
The Separation Process:
Sample Preparation
Injection
Mobile Phase
Stationary Phase
Separation
Retention Time
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Detection and Analysis:
Detector: At the end of the column, a detector measures the
presence and amount of each component as it exits.

Chromatogram: The detector signal is plotted as a graph

called a chromatogram. It shows a series of peaks, where each
peak represents a component in the mixture.
Department of Pharmacy
Department of Pharmacy
Components:
• Injector: This port vaporizes the sample for introduction into
the mobile phase.
• Column: The heart of GC, it's a long, narrow tube packed
with the stationary phase. Different column lengths and
stationary phase types can influence separation.
• Carrier Gas: An inert gas like helium continuously flows
through the column, carrying the vaporized sample along.
Department of Pharmacy

• Detector: This sensitive device monitors the column effluent

(the exiting stream) and generates a signal based on the
presence of the separated components. Common detectors
include flame ionization detectors (FIDs) and mass
spectrometers (MS).
• Data Acquisition System: This system records and processes
the detector signal, generating a chromatogram - a graph
showing peaks corresponding to the separated components.
Department of Pharmacy
Department of Pharmacy
Department of Pharmacy
Strengths:
High Resolution: GC excels at separating complex mixtures into
their individual components.
Sensitivity: Modern detectors can detect incredibly small
quantities of a compound.
Quantitation: By analyzing the peak area in the chromatogram, GC
allows for the quantification of each component in the mixture.
Speed: Compared to other separation techniques, GC analyses are
often relatively fast.
Versatility: A wide range of samples can be analyzed using GC,
provided they are volatile and thermally stable.
Department of Pharmacy
Limitations:
Sample Volatility: Compounds must be vaporizable without
decomposing at the injection temperature to be analyzed by GC.
Non-volatile Analytes: If a compound isn't volatile, derivatization
techniques may be required to modify it for GC analysis.
Limited Information: While GC provides excellent separation, it
may not always definitively identify unknown compounds. Coupling
GC with other techniques like MS can offer more structural
information.
Destructive Detectors: Many detectors destroy the sample during
analysis, making further characterization challenging.
Department of Pharmacy Mass Spectrometry-Gas
Chromatography (MS-GC)

MS-GC is a hyphenated technique where GC acts as a

separation tool and MS acts as a detector. GC separates the
mixture's components based on their interaction with a
stationary phase, while MS identifies and characterizes them by
measuring their mass-to-charge ratio (m/z).
Department of Pharmacy
Department of Pharmacy

Difference between Mass Spectrometry-

Gas Chromatography (MS-GC) and only
Gas Chromatography
Department of Pharmacy

GC: Separation. GC excels at separating the components

within a complex mixture. It identifies them based on their
retention times within the column, which can be a good initial
indication of the compound's identity.

MS-GC: Identification and characterization. MS-GC builds

upon GC's separation power by adding the identification
capabilities of Mass Spectrometry (MS).
Department of Pharmacy
Applications:
GC: GC is a workhorse technique for routine analysis where
the components are already known or can be identified by
retention times and comparison to standards. It's widely used in
quality control, environmental monitoring, and volatile organic
compound (VOC) analysis.
MS-GC: MS-GC is ideal for analyzing complex mixtures with
unknown components, pinpointing trace amounts of a specific
compound, and obtaining structural information about the
identified molecules. It's valuable in forensic science, drug
discovery, metabolite identification, and analyzing complex
biological samples.
Department of Pharmacy
High-Performance Liquid Chromatography
Department of Pharmacy
HPLC is a chromatographic technique that separates a
mixture based on the partitioning behavior of its
components between two phases: a mobile phase (liquid)
and a stationary phase (solid).
The mobile phase, typically a liquid solvent or mixture of
solvents, carries the dissolved sample through a column
packed with the stationary phase.
The stationary phase is a porous material with a high
surface area designed to interact with the sample
components.
Department of Pharmacy
Principles:
Separation in HPLC occurs due to differences in the affinity of
sample components for the mobile and stationary phases.
Components with a stronger affinity for the stationary phase spend
more time interacting with it, resulting in a slower travel through the
column.
Conversely, those with a weaker attraction for the stationary phase
move faster with the mobile phase. This difference in interaction
times translates to distinct retention times, the time it takes for each
component to emerge from the column and be detected.
By analyzing these retention times and comparing them to
standards or reference data, chemists can identify and potentially
quantify the separated components.
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COMPONENTS:
Department of Pharmacy
Strengths:
High Resolution
Versatility
Quantitation
Compatibility with Non-volatile Analytes
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Limitations:
Cost
Solvent Usage
Analysis Time
Sample Preparation
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Department of Pharmacy
Department of Pharmacy
Thin Layer Chromatography (TLC)
TLC is a chromatographic separation technique where a thin
layer of adsorbent material (like silica gel or alumina) is coated
on a plate (usually glass, plastic, or aluminum).

The mixture you want to analyze is spotted onto the plate,

and a solvent system then travels up the plate by capillary
action.
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The Separation Principle:
Adsorption
The Race
Separation Achieved
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Components of a TLC System:
Stationary Phase: This is the thin layer of adsorbent material
(usually silica gel or alumina) coated on the plate. These
materials have a large surface area that interacts with the
components of the mixture.

Mobile Phase: This is the solvent system that travels up the

plate, carrying the mixture along. The choice of solvent is
crucial and affects the separation of components.
Department of Pharmacy
Sample: The mixture you want to analyze is typically dissolved
in a volatile solvent and then spotted as a small dot onto the
starting point of the TLC plate.

Developing Chamber: This is a sealed container used to hold

the solvent during the run. It allows the solvent to travel up the
plate by capillary action.

Detection: After the development, the separated spots on the

plate might not be visible directly. Visualization techniques like
UV light, staining with specific reagents, or charring are often
used to reveal the spots.
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Strengths
Simplicity
Speed
Low Cost
Sensitivity
Versatility
Visual Analysis
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Limitations
Limited Resolution
Qualitative Analysis
Quantification
Limited Sample Size
Destructive
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Department of Pharmacy
Paper Chromatography
Paper chromatography is a separation technique that utilizes
specially designed paper as a stationary phase.

A solvent, acting as the mobile phase, carries the components

of a mixture through the paper, separating them based on their
differing interactions with the paper and the solvent.
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The Separation Principle:
Partitioning: The paper acts as a porous matrix, holding water
within its fibers. The components in the mixture partition
between the water (stationary phase) and the flowing solvent
(mobile phase).
Department of Pharmacy

Affinity's Role: Molecules with a stronger affinity for the

water will spend more time interacting with the paper, traveling
slower. Conversely, those with a greater affinity for the solvent
will move faster with the solvent flow.

The Separation Dance: This differential partitioning creates a

separation dance on the paper. Each component travels a
specific distance based on its unique interaction with the
system, resulting in distinct bands or spots after development.
Department of Pharmacy
Department of Pharmacy

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