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
Pump
Injector
Column
Detector
Gradient
Controller
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
Inject
Vents
Pump
Column
Needle Port
Loop
Vents
Pump
Column
Needle Port
Loop
Load
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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
Phase
Type Application Mobile
Phase
Typical Analytes
Silica Normal
Phase(NP)
Hexane,
alcohols
Pesticides,
Natural Products
Octadecyl
silyl
C-18 Reversed
Phase(RP)
Water,
Methanol,
buffers
(pH2-8)
Peptides, amino
acids
C-8 C-8
Hydrocarbon
Chain
RP Water,
Methanol,
buffers
(pH2-8)
Drugs,
Pharmaceuticals
Cyanopropyl Cyanopropyl
bonded to
silica support
RP and NP RP-water,
alcohol
NP-hexane,
ether
Foods, Fatty
acids
Amino propyl Aminopropyl
bonded to
silica support
RP and NP RP-water,
alcohol
NP-hexane,
ether
Surfactants
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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
material
C(18) functional
group
Particle
size
Column length
Column internal
diameter
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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 CGhars oCmhraomtoatgorgarapphhyy
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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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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 09/29/14 fire or explosion hazard 30

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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Hplc & GC

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