Column Chromatography

Column Chromatography (CC)

Stationary phase is held in a narrow tube

through which the mobile phase is forced
under pressure or under the effect of gravity

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 Open Column Chromatography (Traditional column
chromatography)

Traditional column chromatography is characterized by

addition of mobile phase under atmospheric pressure and the
stationary phase is packed in a glass column.

Packing and operating the column

• The selection of the method of packing depends mainly on the density of
the solid.
• Techniques used: wet, dry and slurry methods.
• In all cases avoid inclusion of air bubbles

Sample Application
Apply evenly and in a concentrated solution to the top of the column which is
protected from disturbance (e.g. add glass wool or filter paper).

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 Elution techniques

Technique Procedure

Isocratic elution Addition of solvent mixture of fixed

composition during the whole process.

Gradient elution Continuous or linear elution: in which there is

continuous change in the composition of the
mobile phase over a period of time (e.g. polarity, pH
or ionic strength).
Step wise or fractional elution: in which the change is
not continuous i.e. a sudden change in the
composition of the mobile phase .

Detection
• On-column detection for colored or fluorescent compounds directly after developing the chromatogram.
• Monitoring of eluted fractions (PC or TLC).
• Using special detectors connected to the column such as refractive index, UV detectors, etc…
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 Factors affecting solutes
separation in CC
(Factors affecting
column efficiency)
Factor Effect
Particle size of solid
Decrease of size improves separation (but very small
stationary phase (or
particles need high pressure).
of support)
Column dimensions Efficiency increases as ratio length / width increases.
Non uniform packing results in irregular movement
Uniformity of packing of solutes through column & less uniform zone formation,
(i.e. band broadening or tailing).
Increase in column temperature results in speed of elution
Column temperature
but does not improve separation (tailing).
Solvents should be of low viscosity (to give efficient
Eluting solvent
resolution) and High volatility (to get rapid recovery of the
substances).
Solvent flow rate Uniform and low flow rate gives better resolution.
Continuity of flow Discontinuous flow disturbs resolution
Condition of adsorbent Deactivation of adsorbent decreases separation.
Concentration of solutes Substances of high concentration move
slowly.
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 Adsorption Column Chromatography
Adsorbents:
The most common are Alumina and Silica gel in which the interactions with
solute molecules is due to OH groups present on their surface.

More polar molecules are adsorbed more strongly and thus, will elute more slowly
Strength of adsorption of polar groups (solutes) on polar support is in the following order:

-C=C- < O-CH3 < -COOR < =C = O < -CHO < -NH2 < -OH < -COOH
Olefins < Ethers < Esters < Lactones < Aldehydes < Amines < Phenols < Acids.

Applications in separation of natural products

Alumina: sterols, dyestuffs, vitamins, esters, alkaloids & inorganic compounds.
Not used for compounds containing phenolic or carboxylic groups
Silica gel: sterols & amino acids.
Carbon: peptides, carbohydrates & amino acids.
Calcium carbonate: carotenoids & xanthophylls. 6
 Partition Column Chromatography
In this type, the packing consists of a theoretically inert support
material coated with a film of the liquid stationary phase.

The division into adsorption & partition is only of theoretical

significance as in partition chromatography the adsorption
effects of the support can be felt.

Selection of the solid support

The support material should:
• adsorb & retain the mobile stationary phase.
• expose as large surface as possible to the mobile phase
• be mechanically stable.
• be easy to pack.
• not retard the solvent flow

Examples of solid supports:

Silica gel, diatomaceous earth (as kieselguhr, celite etc.) & cellulose.
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 Selection of the mobile phase

The liquid stationary & mobile phases should have a considerable difference
between their solvent strength parameters.

Pure water > Methanol > Ethanol > Propanol > Acetone > Ethyl acetate> Ether >
Chloroform > Dichloromethane >Benzene > Toluene > Carbon tetrachloride >
Cyclohexane > Hexane > Pentane.

e.g. if the stationary phase is water, pentane would be the eluent of choice.

The mobile phase is usually saturated with the stationary phase to

overcome "stripping" (washing of the stationary phase from the
column by the mobile phase).

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9
• Normal Phase Chromatography:

In normal phase chromatography, the stationary phase is

polar, and so the more polar solutes being separated will adhere
more to the stationary adsorbent phase. When the solvent or
gradient of solvents is passed through the column, the less polar
components will be eluted faster than the more polar ones. The
components can then be collected separately, assuming adequate
separation was achieved, in order of increasing polarity.
• Reverse Phase Chromatography:

In reverse phase chromatography, the polarities of the

mobile and stationary phases are opposite to what they were
when performing normal phase chromatography. Instead of
choosing a non-polar mobile phase solvent, a polar solvent and
non-polar stationary phase will be chosen.
 High Performance Liquid Chromatography
• Liquid chromatography (LC) is a separation technique in which the mobile
phase is a liquid. Liquid chromatography can be carried out either in a column
or a plane. Present day liquid chromatography that generally utilizes very small
packing particles and a relatively high pressure is referred to as high
performance liquid chromatography (HPLC).

• Since the chromatographic techniques are slow and time consuming, hence
the separation can be greatly improved by using high pressure in the range of
5000-10000 psi(pounds per square inch),hence this technique is also referred
to as high pressure liquid chromatography.

• In HPLC the sample is forced by a liquid at high pressure (the mobile phase)
through a column that is packed with a stationary phase composed of
irregularly or spherically shaped particles.

• The interaction between the mobile and the stationary phase leads to the
separation of the mixture.
HPLC
HPLC Instrumentation
 Rabeprazole

Structure of R-(+)-
Rabeprazole and its
impurities

HPLC method to Separate (and

quantify) R-(+)-Rabeprazole from its
impurities/isomers
Chromatogram of Rabeprazole
HPLC vs
UPLC
• UPLC is Ultra Performance Liquid Chromatography.
• UPLC became the modern standard HPLC platform due to its power for increasing
sample throughput, chromatographic efficiency, sensitivity and decreasing run time.
• UPLC system can handle pressure up to 15,000 psi.

• Both HPLC and UPLC are liquid chromatography techniques used to separate the
different components found in mixtures.
• However, UPLC operates at higher pressures (15,000 psi) and allows for lower particle
sizes in columns, while HPLC operates at lower pressures (max 6000 psi).
• UPLC improves analyte resolution and sensitivity, lower solvent consumption, and
shorten run times.

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 Applications of
HPLC
HPLC is an incredibly useful analytical technique with a broad range of applications.

HPLC is one of the most useful analytical methods in the development and manufacture of pharmaceuticals. Its
applications are not confined to just one area and it is instrumental in a number of critical steps necessary for
robust pharmaceutical analysis.

HPLC is considered a crucial tool in a variety of pharmaceutical applications, including evaluating formulations,
checking purity, and monitoring changes due to process adjustments or during scale-up.

To ensure the consistency of active pharmaceutical ingredients (API)

HPLC can provide quantitative analysis of select molecules, so you can confirm the correct dosage of active
ingredients.

For QA/QC testing, HPLC can be useful in ensuring critical quality attributes such as strength/concentration,
content uniformity, the detection and quantification of impurities, and the quality and identity of raw materials

Impurities can pose a serious safety risk to patients, and their detection and identification is often facilitated by the
use of HPLC. Standard HPLC techniques may be combined with highly efficient detection methods (such as UV
detection) to provide a complete and accurate impurity profile.

HPLC can be vital in evaluating the stability of pharmaceutical products. The composition of formulations can alter
over time due to a variety of environmental factors, such as exposure to humidity, oxygen, heat, and light. HPLC
can assist in the identification of degradation products as well as determine the extent of change over time1 .8
 Gas Chromatography
• Gas chromatography (GC), is a common type of chromatography used
in analytical chemistry for separating and analyzing compounds that can
be vaporized without decomposition.

• Typical uses of GC include testing the purity of a particular substance, or

separating the different components of a mixture (the relative amounts of
such components can also be determined).

• In some situations, GC may help in identifying a compound.

• In preparative chromatography, GC can be used to prepare pure

compounds from a mixture.

• Two types of gas chromatography are encountered

a. Gas-Solid Chromatography (GSC)
b. Gas-Liquid Chromatography (GLC)
 Mobile phase (Carrier gas ) is a chemically inert gas.
• Must be dry, free of oxygen and chemically inert.

• It is most commonly used because it is safer than, but comprable to H2 in

efficiency, has a larger range of flow rates and is compatible with many
detectors.

• Nitrogen, argon, and hydrogen are also used depending upon the desired
performance and the detector being used.

• Both hydrogen and helium are commonly used on most traditional detectors
such as Flame Ionization(FID), thermal conductivity (TCD) and Electron capture
(ECD), provide a shorter analysis time and lower elution temperatures of the
sample due to higher flow rates and low molecular weights.

• MS, uses nitrogen or argon which has a much better advantage than hydrogen or
helium due to their higher molecular weights.

Sample Injection
The vaporization chamber is typically heated 50 °C above the lowest boiling point of the sample
and subsequently mixed with the carrier gas to transport the sample into the column.
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Isothermal method:
• The optimum column temperature for isothermal operation is
about the middle point of the boiling range of the sample.

• Isothermal programming works best only if the boiling point

range of the sample is narrow

Temperature gradient method:

• In the temperature programming method, the column
temperature is either increased continuously or in steps as the
separation progresses. This method is well suited to
separating a mixture with a broad boiling point range.

• The analysis begins at a low temperature to resolve the low

boiling components and increases during the separation to
resolve the less volatile, high boiling components of the
sample. Rates of 5-7 °C/minute are typical for temperature
programming separations. 21
 GC

GC Instrumentation

22
 Hyphenated techniques of GC

• Gas chromatography techniques:

• Gas Chromatography [GC]
• Gas Chromatography Mass Spectrometry Analysis [GCMS]
• High Resolution GCMS [GC/MS/SIM/HR]
• Gas Chromatography Tandem Mass Spectroscopy [GC-MS-MS]
• Gas Chromatography Inductively Coupled Plasma Mass Spectroscopy [GC-ICP-MS]

Gas chromatography detectors:

• Flame Ionization Detection, (GC/FID)
• Thermal Conductivity Detection, (GC/TCD)
• Flame Photometric Detection, (GC/FPD)
• Electron Capture Detection, (GC/ECD)
• Photo-ionization Detection, (GC/PID)
• Sulfur Chemiluminescence Detection (GC/SCD)
• Gas Chromatography Mass Spectrometry (GC/MS)
• Two-Dimensional Gas Chromatography (GCxGC, i.e., 2D GC)
• PIONA Gas Chromatography (GC/PIONA)
• Atomic Emission Detection, (GC/AED) 23
• Pyrolysis Gas Chromatography (Py/GC)
• Pulsed Discharge Helium Ionization Detector (PD/HID)
 Gas chromatography mass spectrometry analytical
techniques

GCMS Headspace Analysis

Headspace is often used when clients are faced with odor issues. During this process a sample is heated in a sealed
vial to an elevated temperature for a period of time. A proportion of the headspace is then introduced into the GCMS in
order to identify volatiles produced. Headspace essentially replicates volatiles being released during the production
stage of materials or whilst a product is in use.
GC-MS Selected ion monitoring (SIM)
HR/GC/MS (High Resolution Gas Chromatography Mass Spectrometry)
GC/MS/MS (GC Tandem MS)
Tandem GC Mass Spectrometry, GC/MS/MS
Thermal desorption GCMS:Thermal desorption is similar to headspace analysis but provides a wider temperature
range. A useful technique for identifying volatile compounds released from systems without the need to introduce the
whole sample in to the GCMS. Thermal desorption has many uses including NIAS, regulatory, air sampling, residual
monomer, additives and solvent identification.
GC-QTOF MS: QTOF MS combines both a Quadrupole and Time of Flight mass spectrometer. This provides high
resolution and high mass accuracy. Where it is not possible to find a match for compounds using conventional MS
using QTOF mass accuracy will be able to provide a definitive chemical formula. This can be combined with
headspace or thermal desorption.
Pyrolysis GCMS: Pyrolysis Gas Chromatography Mass Spectrometry analysis combines three analytical tools:
Pyrolysis, Gas Chromatography and Mass Spectrometry. Adding pyrolysis to the traditional GC-MS provides the ability
to heat samples to 1200°C in either an inert atmosphere or under air, breaking down large, complex molecules into
smaller and more analysable fragments. In industry, pyrolysis provides the ability to identify, characterise and
fingerprint complex polymer systems such as plastics, rubber and paints. The process allows analysis of high
molecular weight, insoluble, crosslinked and thermally stable polymers. Additionally, pyrolysis, when harnessed with
GC-MS, does not require the use of solvents which means that it is possible to identify low level monomers, residual
solvents, additives and crosslinkers without adding further contaminants.
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 Hyphenated techniques of GC

Gas chromatography mass spectrometry techniques including GCMS,

Pyrolysis, GC-QTOF MS measure components in compounds to low ppm
and ultra-trace levels

Gas chromatography mass spectrometry (GC/MS) has long been used in industry to identify and
quantify unknown components in compounds down to ultra-trace levels.

GCMS is ideal for impurity analysis and as a QC method.

GCMS provides trace-level molecular characterization of many sample types and is a powerful
analytical tool for trace and ultra-trace analysis, unknowns identification, and other applications.
It is the technique of choice for a large proportion of regulatory work where it is essential to identify
components present down to very low levels, and is also one of the foremost techniques used for
organic analysis.

GCMS provides molecular characterization of many sample types and is a powerful analytical tool
for trace analysis, unknowns identification, and other applications.

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 Gas Chromatography - Inductively Coupled Plasma (GC/ICP/MS) for separation and
measurement of ultra-trace levels of metals and organo- metallic compounds

• Gas Chromatography - Inductively Coupled Plasma - Mass Spectrometry

analysis combines three analytical tools: Gas Chromatography, Inductively
Coupled Plasma and Mass Spectrometry, into one integrated system.

• It allows lower limits of detection for metals, sulphur and mercury speciation in
crude oils and
• oil products.

• GC/ICP/MS provides a powerful laboratory technique for analyses, with the

capability to separate and quantitate ultra-trace levels of metals and organo-
metallic compounds.

• In conjunction with Liquid Chromatography - Inductively Coupled Plasma - Mass

Spectrometry (LC/ICP/MS) both techniques facilitate enhanced water analysis26
allowing comprehensive information to be obtained.
 Advantages of GC/ICP/MS analysis:

Detection limit: ~50 ng/g, 50 ppb is several orders of magnitude lower than rival detectors.

Speciation of Sulphur.

Stability and Robustness

Sensitivity towards a specific element, such as sulphur, is virtually unaffected by large quantities
of other components present in the matrix

Testing applications for GC/ICP/MS:

• Metals Speciation Analysis and Testing
• Sulphur Speciation
• Mercury Speciation
• Determination of Organometallic Species
• Mercury Speciation in Refinery Products and Feedstocks
• Trace Metal Analysis in Water
• Trace Metals in Petroleum
• Determination of Phosphorus containing Pesticides
• Determination of Sulphur containing Pesticides

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 Applications of GC
Qualitative Analysis: by comparing the retention time or volume of the sample to the
standard / by collecting the individual components as they emerge from the chromatograph
and identifying these compounds by other methods like UV, IR, NMR.

Quantitative Analysis: area under a single component elution peak is proportional to the
quantity of the detected component/response factor of the detectors.

General Applications:
Environmental monitoring : pollutants of environment analysis of various classes of
persistent organic contaminants in air, water, soils
Criminal forensics : Analyze the particles (Fibre) from a human body in order to help link
a criminal to a crime.
Forensic toxicology : Find drugs and/or poisons in biological specimens of suspects,
victims, or the deceased.
Sports anti-doping analysis : Test athletes' urine samples
Law enforcement : Detection of illegal narcotics.
Security : Explosive detection as in airports.
Food, beverage and perfume : from spoilage or Adulteration - aromatic compounds,
esters, fatty acids, alcohols, aldehydes, terpenes. 28
 Pharmaceutical Applications:

Impurity profiling:
• Process of acquiring and evaluating data that establishes biological safety of a
drug.
• Impurity levels directly correlated with the safety profile of the drug.
• Common impurities are (Organic impurities, Inorganic impurities, Residual
solvents.)
• Hyphenated techniques (GC-IR) and (GC-MS) are commonly used for this
purpose.

Functional Group Isolation/Identification:

• GC coupled with other identification techniques such as Infrared spectroscopy
(IR)
and fourier Transform Infrared spectroscopy (FTIR).
• Useful in identification of novel compounds especially in phytoextracts.

Residual Solvent Analysis 29

• Organic volatile chemicals that are used during manufacturing of certain drugs.
• These chemicals should not be present in the final product since they are
harmful to the body.
 Advantages of GC:

• The main advantage of gas chromatography is its high sensitivity, resolution and
separation capacity, which allow it to separate a wide range of volatile compounds.
• Gas chromatography can analyze a sample much faster than other chromatographic
techniques.
• Gas chromatography is simple, automated, and allows rapid data analysis that offers
relatively high accuracy, precision, and reproducible results.
• It comes with a variety of detectors and injectors that can be used for various
pharmaceutical and other applications.
Disadvantages of GC:

• The main disadvantage of GC is that only volatile and thermally stable compounds can be
separated by gas [Link] main disadvantage of GC is that only volatile and
thermally stable compounds can be separated by gas chromatography.
• The selectivity in HPLC or TLC is also better, because a mobile phase can be easily changed.
In GC you can only change the temperature of the column and the oven, but you cannot
change the mobile phase because you have a constant flow of carrier gas (He, N2).
• Since hydrogen gas is used for the flame (highly flammable), care should be taken when
using it.
• It is not possible to recover the individual components of the sample. 30
Super Critical Fluid Chromatography
 SCFC

Mobile Phase
Line A: CO2
Line B: Pure MeOH or MeOH
(+0.1% basic modifier)

32
SCF Chromatography Instrumentation
 Applications of SCFC

Basic needs of the pharmaceutical industry

The basic requirements of clinical laboratory/pharmaceutical field for chromatography are as follows:
• Minimum requirements of sample and sample preparation
• High sensitivity, specificity, reproducibility, and throughput
• Quantitative and accurate
• Automated, rapid, and easy-to-operate instrumentation
• Simple and easy to use on a daily basis
• Economical
• Easy interpretation of results

SFC has met the chromatography needs of the pharmaceutical industry by providing efficient and
selective testing capabilities on the analytical, semi-preparative, and preparative scale.
It has been helpful in all stages of pharmaceutical drug preparation:
• Chiral separation of the enantiomers of a molecule
• Purification of each of the enantiomers in sufficient quantities to permit a study of the enantiomer’s
pharmacokinetic and metabolic properties
• Identification of the enantiomer of choice as a possible therapeutic agent
• Purification on higher (production) scales