introduction and principle of glc, hplc

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) .

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How can We Analyze the Sample

For example:Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose

1

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4

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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

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High Performance Liquid Chromatograph

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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Detector

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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

introduction and principle of glc, hplc

Education