Introduction and principle of GLC, HPLC
Vishnu Vardhan Reddy.PTVM/2015-029
Department of Animal nutritionCollege of Veterinary Science, TirupatiSri Venkateswara Veterinary University
Chromatography• Chromatography is an analytical technique where in a sample mixture
under test is separated into different components based on difference
in their affinity for a stationary phase and mobile phase.
General classification of chromatographic methods1. Column chromatography
2. Paper chromatography
3. Thin layer chromatography
4. Gas chromatography
5. High pressure liquid chromatography
6. Ion exchange chromatography
7. Gel filtration chromatography
8. Super critical fluid chromatography
Gas Chromatography
• It is a technique where by the components of a mixture (sample) in
the gaseous state are separated as the sample passes over a
stationary liquid or solid phase and a gaseous mobile phase.
• Based on stationary phase G.C classified into two types.
Gas Solid Chromatography (G.S.C)
Gas Liquid Chromatography (G.L.C)
GLCGas Liquid Chromatography
In gas-liquid chromatography the
mobile phase is an unreactive gas,
such as nitrogen (the carrier gas), and
the stationary phase comprises of a
small amount of non volatile liquid
held on a finely-divided inert solid
support.
Gas Liquid Chromatography (G.L.C)
Gas liquid Chromatography Principle of Operation
• Gas liquid chromatography runs on the principle of partition.
• In GLC the components of vaporize samples are fractionated due to
partition between a gaseous mobile phase and a liquid stationary
phase held in column.
Instrumentation
• Tank of carrier gas
• Flow regulator and flow meter
• Injection port
• Column
• Temperature controlled device
• Detector
• Microprocessor/recorder
The Mobile Phase (Carrier Gas)
• An inert gas such as He or N2
Function is to transport sample vapors through column
No chemical interaction with sample
• Typical parameters
Column inlet pressure: 10-50 psi (above ambient)
Flow rate: 25-50 mL/min (packed column)
• Precise control of carrier gas flow rate is critical to obtaining reproducible retention
times.
Sample Injection Sample is injected using a syringe into a flowing stream of hot
mobile phase High temperature (at least 50 oC above boiling point of
sample) causes vaporization of sample Introduces a narrow plug of sample vapor onto the column
Various designs For packed columns, inject 1 to 5 L of sample For capillary columns, a split valve is used to introduce a
small fraction of sample onto column
Columns
• Column is heart of GC, which decides the separation efficiency.
• It is made up of glass or copper.
• Columns are two types based on it’s use:
Analytical column:
Length 1-2 mts, outer diameter 3-6 mm.
Preparative column:
Length 3-6 mts, outer diameter 6-9 mm.
Columns are two types based on nature:
• Packed column:
• Capillary columns:(Golay column)
wall coated open tubular (WCOT) column
porous layer open tubular (PLOT) column
Support coated open tubular (SCOT) columns
• Wall Coated Open Tubular (WCOT) column
Internal wall of capillary is coated with a very fine film of liquid stationary phase.
• Surface Coated Open Tubular (SCOT) column
Capillary tube wall is lined with a thin layer of solid support on to which liquid
phase is adsorbed. The separation efficiency of SCOT columns is more than WCOT
columns because of increased surface area of the stationary phase coating.
• Fused Silica Open Tubular (FSOT) column
Walls of capillary fused silica tubes are strengthened by a polyimide coating.
These are flexible and can be wound into coils.
Columns for Gas Chromatography
Selection of appropriate column geometry and dimensions may be critical to a successful separation
Column Oven
Precise control of column temperature.
Column temperature should be slightly below the boiling points of the
solutes (but above the dew point; i.e., no condensation)
For complex mixtures with a broad range of boiling points, use
programmed temperature
Precise control of oven temperature is critical to obtaining reproducible
retention times.
DetectorsGenerate an electrical signal proportional to solute concentration or mass flow rate
Ideal characteristics High sensitivity
Rapid response time
Non destructive technique
Applicable to wide range of samples
Easy to use
Stable ,predictable response
Detectors for GC•
May be universal or selective
Universal (responds to wide range of solutes)
Thermal conductivity detector (TCD)
Simple, inexpensive and modest sensitivity.
For greater sensitivity or selectivity
Flame Ionization detector (FID)
Responds to most organic compounds
High sensitivity and wide dynamic range (9 orders of magnitude).
Electron capture detector (ECD)
Selective and very sensitive for halogenated organics.
Thermal conductivity detector
• Element is electrically heated at constant
power.
• Temperature depends on thermal
conductivity of surrounding gas.
• Measure conductivity with respect to a
reference.
• When analyte comes off, filament
temperature goes up, resistance goes down.
Thermal Conductivity Detector
• Mechanism: A detector cell contains a heated filament with an applied
current. As carrier gas containing solutes passes through the cell, change
in the filament current occurs. The current change is compared against
current in reference cell. The difference is measured and a signal is
generated.
• Sensitivity: 5-20 ng.
• Selectivity: All compounds.
• Gases: Hydrogen, Helium.
• Temperature: 150-2500C
Flame Ionization Detector
• Column effluent is passed through a H2-air
flame produces ions and electrons.
• Charged particles are accelerated by voltage
applied between jet and collector-results in
current
• Less sensitive to non hydrocarbon groups.
• Insensitive to H2o,Co2,So2
Flame Ionization DetectorFor most organic compounds
• Mechanism: Compounds are burned in a hydrogen-air flame. carbon
containing compounds produce ions that are attracted to the collector. The
number of ions hitting the collector is measured and a signal is generated.
• Sensitivity: 0.1-10 ng.
• Selectivity: compounds with C-H bonds.
• Gases: combustion –hydrogen and air, makeup-He,N2.
• Temperature: 250-3000C.
Electron capture detector
• Carrier gas (and analyte) passes over β-
emitter, resulting in ionization and electron
production.
• Produce current between electrodes.
• Most commonly used for halogenated
organics.
• Mechanism: Electrons are supplied from a 63Ni foil lining the detector
cell. A current is generated in the cell. Electronegative compounds
capture electrons resulting in a reduction in the current. The amount of
current loss is indirectly measured and a signal is generated.
• Sensitivity: 0.1-10 ng.
• Selectivity: Halogens, nitrates.
• Gases: Nitrogen or argon.
• Temperature: 300-4000C
Nitrogen phosphorous detector
• Mechanism: compounds are burned in a plasma surrounding rubidium bead
supplied with hydrogen and air. Nitrogen and phosphorous containing
compounds produce ions that are attracted to the collector. The number of
ions hitting the collector is measured and a signal is generated.
• Sensitivity: 1-10 pg.
• Selectivity: Nitrogen phosphorous containing compounds.
• Gases: combustion- hydrogen ,make up – helium.
• Temperature: 250 -3000C.
Recorder
• Recorder is a device that draws the chromatogram that results from a
chromatographic process onto chart paper.
• The device can have a full scale deflection (FSD) voltage that commonly
ranges from 1 mv to 10 v.
• The time scale of the chart movement normally ranges from about 1 cm
per second to 1 cm per hour.
Advantages of GLC
• Both qualitative and quantitative analysis are possible.
• Instrument is simple ,time of analysis is short.
• High sensitivity.
• The method is applicable to about 60% of organic compounds.
• Very small samples sizes can be used.
• Analysis can be highly accurate and precise.
Factors affecting separation
• Particle size and surface area.
• Carrier gas flow rate.
• Type and amount of stationary phase.
• Column length.
• Column diameter.
• Column temperature.
Applications Of GLC In animal feed industry
• Quantitative and/or qualitative analysis of feed composition that is
estimation of:
Amino acids, hydroxyl (poly)carboxylic acids, fatty acids, phenolic
compounds, sugars, vitamins, and many veterinary drugs, herbicides,
and ‘‘natural’’ chemical toxins present in feed.
• Quantitative and/or qualitative analysis of feed additives.
• Estimation of flavor and aroma components in feed.
• Estimation of spoilage components, such as histamine and carbonyls,
that cause rancidity.
• Identification of contaminants, such as pesticides, fumigants,
environmental pollutants, natural toxins, veterinary drugs, and
packaging materials in animal feeds.
• Variety of transformation products like polycyclic aromatic
hydrocarbons, heterocyclic amines, urethane, nitrosamines,
chloropropanols, cholesterol oxides, irradiation products, microbial
marker chemicals
• Only the non-volatile compounds, such as inorganic salts, proteins,
polysaccharides, nucleic acids, and other large molecular weight
organics, are outside the realm of GLC.
HPLCHigh Performance Liquid Chromatography
High Performance Liquid Chromatography
• HPLC is a form of liquid chromatography used to separate compounds that
are dissolved in solution.
• HPLC is characterized by the use of high pressure to push a mobile phase
solution through a column of stationary phase allowing separation of
complex mixtures with high resolution.
• Mobile phase is Liquid
• Stationary phase is Solid or Liquid
Principle• The process involves the interaction of the compounds in the analyte or sample across an
immobile surface (stationary phase).
• The compounds bind at specific regions of stationary phase based on certain physical and
chemical properties. These bound molecules are then eluted with a suitable buffer and
the same are collected with time.
The properties are –
• Polarity
• Charge
• Molecular weight
• Present of functional group
Types of HPLC
• There are many ways to classify liquid column chromatography based on
the nature of the stationary phase and the separation process, three
modes can be specified.
• Adsorption chromatography
Here stationary phase is an adsorbent (like silica gel) and the separation is
based on repeated adsorption-desorption steps.
• Ion-exchange chromatography
Here the stationary bed has an ionically charged surface of opposite charge
to the sample ions. This technique is used almost exclusively with ionic or
ionizable samples.
The stronger the charge on the sample, the stronger it will be attracted to
the ionic surface and thus, the longer it will take to elute.
The mobile phase is an aqueous buffer, where both pH and ionic strength are
used to control elution time.
• Size exclusion chromatography
Here the column is filled with material having precisely controlled pore sizes,
and the sample is simply screened or filtered according to its solvated
molecular size.
Larger molecules are rapidly washed through the column; smaller molecules
penetrate inside the porous of the packing particles and elute later.
This technique is also called gel filtration or gel permeation
chromatography.
• Concerning the Adsorption chromatography two modes are defined
depending on the relative polarity of the two phases:
Normal-phase chromatography
• The stationary bed is strongly polar in nature (e.g., silica gel)
• The mobile phase is nonpolar (such as n-hexane or tetrahydrofuran).
• Polar samples are thus retained on the polar surface of the column packing
longer than less polar materials.
Reversed-phase chromatography
• The stationary bed is nonpolar (hydrophobic) in nature.
• The mobile phase is a polar liquid, such as mixtures of water and
methanol or acetonitrile.
• Here the more nonpolar the material is, the longer it will be retained.
Flow chart of HPLC mechanism
Instrumentation of HPLC
• Solvent (mobile phase)
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column (stationary phase)
• Detectors (Diode Array)
• Waste Collector
• Recorder (Data Collection)
Solvent (mobile phase)
• In normal phase typically non polar solvents such as hexane, heptane, iso-
octane are used in combination with slightly more polar solvents such as
isopropanol, ethyl-acetate or chloroform.
• In reverse phase applications water is usually the base solvent. Other polar
solvents such as Methanol, Acetonitrile or Tetrahydrofuran are added in
fixed or varying proportions.
• pH is adjusted by buffers to modify separations of ionizable solutes.
Pump•The role of the pump is to force a liquid (called the mobile phase) through the
liquid chromatograph at a specific flow rate, expressed in milliliters per min
(mL /min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-
bar).
•During the chromatographic experiment, a pump can deliver a constant mobile
phase composition (isocratic) or an increasing mobile phase composition
(gradient).
Injector
•The injector serves to introduce the liquid sample into the flow stream of the
mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of the
liquid system.
•An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
Column• Considered the “heart of the chromatograph” the column’s stationary phase
separates the sample components of interest using various physical and
chemical parameters.
• The small particles inside the column are what cause the high back pressure at
normal flow rates.
• The pump must push hard to move the mobile phase through the column and
this resistance causes a high pressure within the chromatograph.
Modes of High Performance Liquid Chromatography
Types of Compounds Mode Stationary
PhaseMobile Phase
NeutralsWeak AcidsWeak Bases
ReversedPhase
C18, C8, C4cyano, amino
Water/Organic Modifiers
Ionics, Bases, Acids Ion Pair
C-18, C-8 Water/Organic Ion-Pair Reagent
Compounds notsoluble in water
NormalPhase
Silica, Amino,Cyano, Diol
Organics
Ionics Inorganic Ions Ion Exchange
Anion or CationExchange Resin
Aqueous/Buffer Counter Ion
High Molecular WeightCompoundsPolymers
Size Exclusion
Polystyrene Silica
Gel Filtration- AqueousGel Permeation-Organic
Types of columns in HPLC• Guard Column
• Fast Column
• Preparative (i.d. > 4.6 mm; lengths 50 –250 mm)
• Capillary (i.d. 0.1 -1.0 mm; various lengths)
• Nano (i.d. < 0.1 mm, or sometimes stated as < 100 μm)
• Analytical (internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm)
Guard Column
These are placed anterior to the separating column. This serves as protective
factor.
They are dependable columns designed to filter or remove :
Particles that clog the separation column
Compounds and ions that could ultimately cause “ Baseline drift ”,
decreased resolution, decreased sensitivity and create false peaks.
These columns must be changed on a regular basis in order to optimize their
protective function.
Fast Column
• One of the primary reasons for using these column is to obtain improved sample output
( amount of compound per unit time).
• Fast column are designed to decrease the time of chromatographic analysis
• Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm
diameter.
• Advantages-
Increased sensitivity
Decreased analysis time
Decreased mobile phase usage
Increase reproducibility
Capillary Column
• It is also known as micro columns
• It has a diameter much less than a millimeter and there 3 types:
Open tubular
Partially packed
Tightly packed
They allow the user to work with nanoliter sample volume , decreased
flow rate and decreased solvent usage volume , led to cost effectiveness
Preparatory Column
• Used when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.
• It has usually a large column diameter , which is designed to facilitate large
volume injections into the HPLC system
Detector
The detector can see (detect) the individual molecules that come out (elute)
from the column.
•A detector serves to measure the amount of those molecules
so that the chemist can quantitatively analyze the sample
components.
•The detector provides an output to a recorder or computer
that results in the liquid chromatogram(i.e., the graph of the
detector response).
HPLC Detectors
Common HPLC Detectors• UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
• Mass Spectrometers
• Refractive Index
• Fluorescence
• Light Scattering
• Electrochemical
• Radioactivity
• Conductivity
UV-Vis Detectors
Characteristics: Specific, Concentration Sensitive, good stability, gradient
capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).
b
c
Detector Flow Cell
I0 I
Log I0 = A = abcI
Fluorescence Detection
Emission Monochromatorsignal & spectra mode
PMT detector
Reference Diode
8 µl Flow Cell, auto-recognition
Trigger pack
Exitation Monochromator,signal & spectra mode
Mirror
Lens(condensor EX)
Lens (condensor EM)
Slit EM Slit PMTSlit EX
Diffuser
Xenonflash Lamp,15 W
Electrochemical Detectors
• Gold for carbohydrates.
• Platinum for chlorite, sulfate, hydrazine, etc.
• Carbon for phenols, amines.
• Silver for chloride, bromide, cyanide.
Computer
• Frequently called the data system,
The computer not only controls all the modules of the HPLC instrument
but it takes the signal from the detector and uses it to:
1. Determine the time of elution (retention time) of the sample
components (qualitative analysis)
2. Determine amount of sample ( quantitative analysis) .
61
How can We Analyze the Sample
For example:Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose
1
23
4
5
mAU
time
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Separations
Separation in based upon differential migration between the stationary and mobile phases.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
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Separations Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Separations Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
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Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
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Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
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Separations Injector
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Column
Solvents
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Separations Injector
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Separations Injector
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Separations Injector
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Separations Injector
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Solvents
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Separations Injector
Detector
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Separations Injector
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Solvents
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Separations Injector
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Separations Injector
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Separations Injector
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Separations Injector
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Separations Injector
Detector
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Solvents
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Start Injection
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The Chromatogram
Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportionalto the quantity of analyte.
HPLC uses in Feed industry
• Fat soluble vitamins (A,D,E and K)
• Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic
acid, Pantothenic acid, B12, VitaminC)
• Residual pesticides such as 2, 4-D and Monochrotophos.
• Antioxidants such as TBHQ, BHA and BHT.
• Sugars: Glucose, Fructose, Maltose and other saccharides.
• Cholesterol and sterols
• Dyes and synthetic colours.
• Mycotoxins such as Aflatoxins B1,B2,G1,G2,M1,M2and ochratoxin
• Amino acids
• Residual antibiotics
• Steroids and flavonoids
• Aspartame and other artificial sweeteners.
• Active ingredients of farm produce such as allin in garlic and catachin in tea
extracts.
THANK YOU
Vishnu Vardhan Reddy.PTVM/2015-029
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