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

ColumnDetector

Mobile Phases

Gradient Controller

•

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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

Column

Needle Port

Pump

Loop

Vents

Column

Needle Port

Pump

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)

Column Specification

Nucleosil ODS 5µm 25cm x 4.6mm

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)

Type of silica material

C(18) functional group

Particle size

Column length

Column internal diameter

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Detectors UV

Single wavelength (filter) -254nmVariable 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 ChromatographyGas 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 hazard09/29/14 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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