HPLC

&
GC
 High Performance Liquid
Chromatography

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Outline
 Introduction
 Principle
 Instrumentation
 Applications

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Introduction
 HPLC is one of the most widely used analytical
techniques.

 It is used to separate and analyze compounds through

the mass-transfer of analytes between stationary and
mobile phases.

 The technique is employed in broad range of activities

such as analysis of foods, drugs and agrochemicals.

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Principle
 The process of separating the compounds in a mixture is
carried out between the stationary phase (solid) and the mobile
phase (liquid).
 Modes:
 1. Normal Phase
 2. Reverse Phase

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1.Normal phase

 This method separates analytes based on adsorption to a

stationary surface polarity.
 It uses a polar stationary phase and a non-polar, non-
aqueous mobile phase, and works effectively for separating
analytes readily soluble in non-polar solvents.
 The analyte associates which is retained by the polar
stationary phase.
 Adsorption strengths increase with increased analyte
polarity, and the interaction between the polar analyte and
the polar stationary phase (relative to the mobile phase)
increases the elution time.

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2. Reverse Phase
 It uses a non-polar stationary phase and a polar, aqueous
mobile phase, and works effectively for separating analytes
readily soluble in polar solvents

  Decreasing the mobile phase polarity by using organic

solvents reduces the hydrophobic interaction between the
solute and the solid support resulting in de-sorption. 

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Instrumentation

Gradient
Controller
•
Column
Pump
Detector
Injector
Mobile Phases

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Components

 Mobile Phase
 Pumping system
 Sample Injection System
 Column
 Detector

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Mobile Phase
 Selecting the correct composition and type of mobile phase is
important because it governs the separation.
 The choice is restricted because of the column used, the type
of stationary phase employed.
 The main distinction is between reversed and normal phase
chromatography.
 In normal Phase systems, non-polar solvents such as hexane,
diethyl ether, dichloromethane, isopropyl alcohol, iso-octane
are used.
 In reversed phase, polar systems such as water, acetonitrile,
methanol, tetrahydrofuran are used.

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Choice of Solvent:
 Polarity
 Miscibility with other solvents
 Chemical inertness
 UV cut-off wavelength
HPLC system can be set up either for isocratic or gradient
elution.
 Isocratic solution is where the mobile phase composition
remains constant during the whole analysis.
 Gradient elution is where the mobile phase composition is
steadily changed during the analysis.
 To obtain better resolution
 To decrease analysis time

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Characteristics of the Mobile Phase
 HPLC grade materials should be used.
 Mobile phase should be free of dust and impurities.
 There should be no dissolved gas in mobile phase, this can
cause irregular pumping action and fluctuating signals from
the detector, by performing one or more of the following:
 Degas the mobile phase with helium.
 Place the mobile phase under vacuum.
 Agitate the mobile phase in an ultrasonic bath.
 Sample to be analyzed is soluble in the mobile phase.
 Mobile phase should not react with the stationary phase.
 Important to monitor the levels of the mobile phase and ensure
that they are constantly topped up and the system is never
allowed to run dry.
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Pumping Systems
 Important feature of HPLC
 High Pressures and Pulse free output is required for better
separation.
 The output pressure should be atleast 5000psi.
 Materials in the pump should be chemically resistant to all
solvents.
 Purpose of HPLC pump is to pass a constant flow of mobile
phase through the chromatographic column.
 Types of pump:
 Syringe pump
 Reciprocating pump

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 Syringe Pump:
They operate pulse free. But the total volume of mobile phase
that the pump can deliver is limited by the capacity of the
syringe.
 Reciprocating Pump:
• It is commonly used.
• It is operated by motorized piston and entry of the solvent
and exit of the solvent is regulated by check valves.
• Pulse dampners are incorporated to minimise pulsing
effect.

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Considerations of the pump:
 It must be able to deliver the mobile phase at high pressures to
overcome the flow resistance associated with HPLC columns.
 The components of the pump must be resistant to corrosive
chemicals and solvents.
 Flow rates should be between ~0.1 and 10mL/min.
 Should be able function routinely with only minimum
requirement for maintenance and servicing.
 Flow should be pulse free and stable.
 The pump should be a ‘constant flow’ device.
 Pump should be never operated without a solvent reservoir.
 Pumps should be checked for leaks before and after the
analysis. (Important when the system is unattended for long
periods of time)

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Injecting Systems
Load Inject
Pump
Pump

Column
Column
Needle Port
Vents Needle Port Vents

Loop
Loop

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Columns
 Made of stainless steel, can withstand pressures upto 8000psi.
 Crucial in determining the performance and resolution of the
system.
 Choice of the column depends on the type of chromatography
used.
 Straight columns with internal mirror finish are generally used
for better separation.
 Porous plugs of S.S or teflon are used in the end of the column
to retain column material.
 Plugs must be homogenous to ensure flow of the solvents through
the column.

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 Stationary Type Application Mobile Typical Analytes
Phase Phase
Silica Normal Hexane, Pesticides,
Phase(NP) alcohols Natural Products
Octadecyl C-18 Reversed Water, Peptides, amino
silyl Phase(RP) Methanol, acids
buffers
(pH2-8)
C-8 C-8 RP Water, Drugs,
Hydrocarbon Methanol, Pharmaceuticals
Chain buffers
(pH2-8)
Cyanopropyl Cyanopropyl RP and NP RP- Foods, Fatty
bonded to water,alcohol acids
silica support NP-
hexane,ether
Amino propyl Aminopropyl RP and NP RP- Surfactants
bonded to water,alcohol
silica support NP-
hexane,ether

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Common type of columns(Increasing Polarity)
Docosyl Octadecyl Octyl Hexyl Trimetyl Silyl
-(CH2)12CH3 -(CH2)17CH3 -(CH2)7CH3 -(CH2)5CH3 -(CH3)3
(C-22) (C-18) (C-8) (C-6) (C-3)

Column Specification
Nucleosil ODS 5µm 25cm x 4.6mm

Type of silica Particle

size Column internal
material diameter

C(18) functional
group Column length

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 Detectors
 UV
 Single wavelength (filter) -254nm
 Variable wavelength (monochromator)190-600nm.
 Multiple wavelengths (PDA)
 Fluorescence
 Electrochemical
 Mass Spectrometric
 Refractive Index Indicator

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Absorption detectors:
 – UV-Vis: Most widely used
• Based on the light absorption characteristics of the sample.
• Z-shape, flow-through cell (V, 1 ~ 10 μL and b, 2 ~ 10 mm)
• Photometer: Hg 254 nm and 280 nm line

• D2 or W filament + interference filter

• versatile

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Applications
 Separation process has been applied to variety of
natural products such as Nucleic acids, biological
fluids, carbohydrates, amino acids, bile acids and
manufactured products such as pharmaceuticals,
pesticides, herbicides, surfactants and antioxidants.
 Determination of purity of compounds, presence of
related compounds and Assay of drugs.
 Reverse Phase HPLC is particularly useful for
separating polar compounds such as drugs and their
metabolites, peptides, vitamins, polyphenols, steroids,
etc.
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 Resolution of the numerous aminoacids formed in
the hydrolysis of a protein.
 The separation and analysis of closely related
aliphatic alcohols and separation of sugar derivatives.
 Biopharmaceutic and Pharmacokinetic studies.
 Stability studies.
 Gas Chromatography
Gas Chromatography

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Outline
 Principle
 Instrumentation
 Applications

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Principle
 The process of separating the compounds in a mixture is carried out
between a liquid stationary phase and a gas phase based on the partition
coefficient between the two phases.

 The column through which the gas phase passes is located in an oven
where the temperature of the gas can be controlled.

 The concentration of a compound in the gas phase is solely a function of

the vapor pressure of the gas.

 Volatility and thermostability of the samples are the important criteria in

gas chromatography.

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Components
 Carrier Gas
 Sample injection system
 Separation column
 Detector
 Thermostat
 Recorder

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INSTRUMENTATION

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Carrier Gas
 Most common gases N2, H2, He.
 The lighter gases He and H2 require faster analysis flow rates 20-
50 cm/min.
 Helium is generally used because of excellent thermal
conductivity, low density and it greater flow rates.
 Hydrogen has better thermal conductivity but it may react with
unsaturated compounds.
 Properties
 Should be inert
 Suitable for the detector employed
 Should be readily available in high purity
 Should give best column performance
 Should be cheap
 Should not cause the risk of fire or explosion hazard
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Sample Injection system
 It is important to rapidly vaporize the sample.
 Slow vaporization increases band broadening, by increasing
the sample“plug”.
 Injection port temperature is usually held 50 C higher than the
BP of the least volatile compound.
 Sample should be introduced in a reproducible manner and
must vapourize instantly so that sample enters the column as a
single slug.

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Column

 They are constructed of glass or metal tubing.

 It can be coiled, bent or straight.
 Types:
 Wall coated open tubular
 Support coated open tubular
 Porous layer open tubular

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 Wall coated open tubular (Capillary columns)
 The inside wall of the capillary tubing is coated with a
liquid phase in the form of a thin and uniform film.
 The carrier gas flow faces least resistance because ther is n
packing in the column
 Support coated open tubular
 They are made by depositing a micron size porous layer of
support material on the inside wall of a capillary column
and the coating with a thin film of liquid phase.
 They have more sample capacity and inlet splitter is not
required.
 Used for trace analysis.

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 Porous layer open tubular
 Prepared by packing metal or glass tubings with granular
stationary phase.
 Advantages:
 No column bleed. Stationary phase is stable upto 250˚C
and uses highly sensitive detector.
 No adsorption of polar compounds and are eluted as sharp
peaks
 Porous polymer beads are mechanically strong and can be
easily packed on column.
 Separations are unique.

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Stationary Phase
 The most common stationary phases in gas-chromatography columns
are polysiloxanes, which contain various substituent groups to change
the polarity of the phase.
 A wide variety of stationary phases like polyethylene glycols, high
molecular weight esters, amides, hydrocarbons, microporous cross-
linked polyaromatic compounds.
 For very polar analytes, polyethylene glycol (carbowax) is commonly
used as the stationary phase.
 After the polymer coats the column wall or packing material, it is often
cross-linked to increase the thermal stability of the stationary phase and
prevent it from gradually bleeding out of the column.
 Small gaseous species can be separated by gas-solid chromatography.

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DETECTORS

 Thermal conductivity detector

 Electro chemical detector
 Flame ionization detector
 Electron Capture detector

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Thermal conductivity Detector

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 Carrier gas has a thermal conductivity.
 The presence of analyte molecules in the carrier gas
alter (lowers) the thermal conductivity of the gas
 Second filament to act as a reference (the carrier gas
is split)
 Increased sensitivity with decreasing temperature,
flow rate and applied current.
 Universal detector

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Flame Ionization Detector

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 The ionization detector is based upon the electrical
conductivity of gases.
 At normal temperature and pressures, gases acts as
insulators but will become conductive of ions if
electrons are present.
 The detector responds to all organic compounds
except formic acid and the response greatest for
organic componds.

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Temperature controller
 It facilitates controlled increase of even temperature
during an analysis
 Components with wide boiling range can be evolved
efficiently.

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Applications
 Qualitative
 Qualitative analysis of individual components of a mixture
may be obtained by either
 By comparing the retention times or volumes of the

unknown to the retention time or volumes of a series of

standards
 By collecting the individual components as they emerge

from chromatography and subsequently identifying the

components by other methods.

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  Quantitative
 Depends upon the area under a single component
elution peak is proportional to the quantity of the
detected component.
 Area = (½ W)/H

 W= width of the peak

 H = height of the peak

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Miscellaneous
 Detection of steroid drugs in athletes.
 Hazardous pollutants such as HCHO, benzene,CO.
 Analysis of foods, separation and identification of lipids, proteins,
carbohydrates, flavors, colorants.
 GC finds valid applications in drug analysis, like commercial drug
preparations, illicit drug samples, blood, urine samples and stomach
contents
 Separation and identification of polycyclic hydrocarbons,
chlorinated pesticides, organophosphorous and sulphur compounds,
phenols, amines etc.
 Determination of purity of compounds, presence of related
compounds and Assay of drugs.

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References
 Instrumental Analysis by Skoog.
 Instrumental Analysis by Gurdeep R
Chatwal.

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