High performance/pressure liquid

chromatography (HPLC)

Introduction

High-performance liquid chromatography (HPLC) is a formof liquid chromatography to separate compounds that are dissolvedin solution.

HPLC involves a solid stationary phase, normally packed inside astainless-steel column, and a liquid mobile phase.

The different components in the mixture pass through the column atdifferent rates due to differences in their partitioning brhaviorbetween the mobile liquid phase and the stationary phase

Separation of the components of a solution results from thedifference in the relative distribution ratios of the solutes betweenthe two phases.

Components of HPLC

Components of HPLC

Stationary phase

Mobile phase

Injector

Detectors

Chromatographic column

Pumping system

Components of HPLC

Pumping system

Computer- or microprocessor-controlled pumping systems are

capable of accurately delivering a mobile phase of either constant

(isocratic elution) or varying (gradient elution) composition,

according to a defined programme.

In the case of gradient elution, solvent mixing can be achieved on

either the low- or high-pressure side of the pump(s).

Injector

The sample solution is usually introduced into the flowing mobile phase at

or near the head of the column using an injection system based on an

injection valve design which can operate at high pressure.

Chromatographic column

Columns are usually made of polished stainless steel, are between 50 and

300 mm long

internal diameter of between 2 and 5 mm.

They are commonly filled with a stationary phase with a particle size of 3–

10 μm.

un modified Silica alumina or porous graphite (normal phase)

Modified silica C8 and C18 (reverse phase)

Stationary phase

They are usually solids with polar compounds stick to the

column wall.

HPLC systems consisting of polar stationary phases and non-

polar mobile phases are described as normal-phase

chromatography.

Those with non-polar stationary phases and polar mobile

phases are called reversed-phase chromatography.

Detectors• Detectors provide flow cells that give energy to show result.

Ultraviolet/visible (UV/vis) absorption spectrophotometers

evaporative light-scattering detectors (ELSD)

charged aerosol detectors (CAD)

mass spectrometers (MS) or other special detectors may be used.

Mobile phase

The mobile phase, or solvent, in HPLC is usually a mixture of polarand non-polar liquid components whose respective concentrations arevaried depending on the composition of the sample.

As the solvent is passed through a very narrow column, anycontaminants could at worst plug the column.

Working PrincipleThe sample mixture to be separated and analyzed is

introduced, in a discrete small volume (typically microliters),

into the stream of mobile phase.

The components of sample in the mobile phase moves with

different velocity due to its interaction with the stationary

phase.

The smaller particle size of the stationary phase provides less

binding of the sample in mobile phase with the stationary

phase and also increases the flow rate of the sample to be

eluted out more quickly.

To enhance the fast flow rate pump is

used

Then, outside the column they are sent into a detector, where individual compounds

are detected and recorded in a computer

The detector is wired to the computer data station, that

records the electrical signal to generate the chromatogram to

display and to identify and quantitate the concentration of

the sample constituents.

The recordings (preferably in the form of quantitative peaks)

are compared with those of standard compound's HPLC

values and the individual compounds are identified

Preparatory chromatography

• The time at which a specific analyte elutes (emerges from the column) is called its retention time.

Normal phase HPLC

• The column is filled with tiny silica (polar)particles, and the solvent

is non-polar – hexane.

• Polar compounds in the mixture being passed through the column

will stick longer to the polar silica than non-polar compounds will.

• The non-polar ones will therefore pass more quickly through the

column.

Reverse Phase Chromatography• In this case, the column size is the same, but the silica is modified to make

it non-polar by attaching long hydrocarbon chains to its surface - typicallywith either 8 or 18 carbon atoms in them (Stationary phase).

• A polar solvent is used - for example, a mixture of water and an alcoholsuch as methano (Mobile phase).

• In this case, there will be a strong attraction between the polar solventand polar molecules in the mixture being passed through the column.There won't be as much attraction between the hydrocarbon chainsattached to the silica (the stationary phase) and the polar molecules in thesolution.

• Non-polar compounds in the mixture will tend to form attractions with thehydrocarbon groups because of van der Waals dispersion forces.

• That means that now it is the polar molecules that will travel through thecolumn more quickly. Reversed phase HPLC is the most commonly usedform of HPLC .

Applications

Quantification

Purifications

Identifications

Pharmaceutical Environmental

Clinical

Food and Flavor

Pharmaceutical applications

• Identification of active ingredients of dosage forms

• Pharmaceutical quality control

Environmental applications

• Detection of phenolic compounds in Drinking Water

• Identification of diphenhydramine in sedimented samples

• Bio-monitoring of pollutant

Forensics

• Quantification of the drug in biological samples.

• • Determination of cocaine and metabolites in blood

Clinical

• Quantification of ions in human urine Analysis of antibiotics in

blood plasma.

Food and Flavor

• Ensuring the quality of soft drink and drinking water.

• Analysis of beer.

• Sugar analysis in fruit juices.

GAS CHROMATOGRAPHY

• Gas chromatography (GC), also called gas-liquid chromatography

• Common type of chromatography used in analytical chemistry for separating and analyzing volatile organic compounds without decomposition.

USES

• Testing the purity of a particular substance

• separating the different components of a mixture (the relative amounts of such components can also be determined)

• help in identifying a compound.

INTRODUCTION

• Gaseous compounds interact with the walls of the column(narrow tube) which is coated with a stationary phase and located in an oven where the temperature of the gas can be controlled.

• Each compound elutes at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness.

• GC separates the components of a mixture primarily based on boiling point (or vapor pressure) differences.

• Archer John Porter Martin was awarded the Nobel Prize for his

work in:

• developing liquid–liquid (1941) and

• paper (1944) chromatography,

• laid the foundation for the development of gas chromatography

and he later produced liquid-gas chromatography (1950).

HISTORY

• A known volume of gaseous or liquid analyte is injected into

the "entrance" (head) of the column, usually using a

microsyringe .

• As the carrier gas sweeps the analyte molecules through the

column, this motion is inhibited by the adsorption of the

analyte molecules either onto the column walls or onto packing

materials in the column.

• The rate at which the molecules progress along the column

depends on the strength of adsorption, which in turn depends

on the type of molecule and on the stationary phase materials.

• The components of the analyte mixture are separated at the end

of the column at different retention times.

GC ANALYSIS

GAS CHROMATOGRAPH

• Inlet/Sample injector

provides the means to introduce a sample into a continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.

The most common injection method:

A microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column.

The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

INSTRUMENTAL COMPONENTS

The injector can be used in one of two modes; • split or splitless.• The injector contains a

heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode).

• The sample vapourises to form a mixture of carrier gas, vapourised solvent and solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet.

Carrier gas

• Chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide.

• The choice of carrier gas is often dependant upon the type of detector.

• The carrier gas system also contains a molecular sieve to remove water and other impurities.

Detectors

• Non-selective detector responds to all compounds except the carrier gas,

• Selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound.

• Concentration dependant detectors-concentration of solute in the detector.

• Mass flow dependant detectors -rate at which solute molecules enter the detector.

• flame ionization detector (FID)

• thermal conductivity detector (TCD).

Both are sensitive to a wide range of components and concentrations.

TCDs-universal and can be used to detect any component other than the carrier gas.

FIDs are sensitive to hydrocarbons.

Commonly Used Detectors

• The method used depends on the collection of conditions used.

Conditions:

• inlet temperature,

• detector temperature,

• column temperature

• carrier gas

• carrier gas flow rates,

• the column's stationary phase, diameter and length,

• Inlet type and flow rates,

• sample size and injection technique.

METHODS

Carrier gas selection and flow rates• Typical carrier gases include helium, nitrogen,

argon, hydrogen and air.• The purity of the carrier gas• The carrier gas linear velocity.The higher the linear velocity the faster the analysis, but the lower the separation between analytes.Polarity of the stationary phase• Polar compounds interact strongly with a polar

stationary phase, hence have a longer retention time than non-polar columns.

Inlet types and flow rates

• The choice of inlet type depends on if the sample is in liquid, gas, adsorbed, or solid form.

Sample size and injection technique

Sample injection

• The amount injected should not overload the column.

• The width of the injected plug should be small.

Column selection

• depends on the sample measured.

• polarity of the mixture must closely match the polarity of the column stationary phase to increase separation while reducing run time.

• The separation and run time also depends on the film thickness (of the stationary phase), the column diameter and the column length.

• Column temperature

• The temperature is precisely controlled electronically.

• The rate at which a sample passes through the column is directly proportional to the temperature of the column

Qualitative analysis

• Data- in the form of graph. detector response (y-axis) is plotted against retention time (x-axis), which is called a chromatogram.

• spectrum of peaks of a sample- analytes eluting at different times.

• Retention time-identify analytes if the method conditions are constant.

DATA REDUCTION AND ANALYSIS

Gas Chromatogram

Quantitative analysis

• area under a peak is proportional to the amount of analyte present in the chromatogram.

• Calculation of the peak area-concentration of an analyte in the original sample can be determined.

•pharmaceuticals, cosmetics, environmental toxins etc.

•Air samples can be analyzed using GC.

APPLICATIONS

MASS SPECTROMETRY

• Mass spectrometry has been described as the smallest scale inthe world, not because of the mass spectrometer’s size butbecause of the size of what it weighs – molecules.

• In a typical MS procedure, a sample, which may be solid, liquid,or gas, is ionized. The ions are separated according to their mass-to-charge ratio. The ions are detected by a mechanism capable ofdetecting charged particles. Signal processing results aredisplayed as spectra of the relative abundance of ions as afunction of the mass-to-charge ratio. The atoms or molecules canbe identified by correlating known masses to the identifiedmasses or through a characteristic fragmentation pattern.

• Due to ionization sources such as electrospray ionization andmatrix-assisted laser desorption/ ionization (MALDI), massspectrometry has become an irreplaceable tool in the biologicalsciences.

Principle

• Four basic components are, for the most part, standard in all

mass spectrometers .

1. Sample inlet

2. Ionization chamber

3. Mass analyzer

4. Ion detector

Sequence of components

Sequence

• Stage 1: Ionisation

The vaporised sample passes into the ionisation chamber.The

electrically heated metal coil gives off electrons which are

attracted to the electron trap which is a positively charged plate.

The particles in the sample (atoms or molecules) are therefore

bombarded with a stream of electrons, and some of the collisions

are energetic enough to knock one or more electrons out of the

sample particles to make positive ions

• Most of the positive ions formed will carry a charge of +1

because it is much more difficult to remove further electrons

from an already positive ion.

Acceleration

• The positive ions are repelled away from the very positive

ionisation chamber and pass through three slits, the final one of

which is at 0 volts. The middle slit carries some intermediate

voltage. All the ions are accelerated into a finely focused beam.

Deflection

Different ions are deflected by the magnetic field by

different amounts.

1.the mass of the ion.

Lighter ions are deflected more than heavier ones.

2. The charge on the ion.

Ions with 2 (or more) positive charges are deflected more

than ones with only 1 positive charge

Mass/charge ratio is given the symbol m/z (or sometimes m/e).

For example, if an ion had a mass of 28 and a charge of 1+, its

mass/charge ratio would be 28. An ion with a mass of 56 and a

charge of 2+ would also have a mass/charge ratio of 28

• In the diagram, ion stream A is most deflected it will contain

ions with the smallest mass/charge ratio. Ion stream C is the least

deflected - it contains ions with the greatest mass/charge ratio.

Detection

• When an ion hits the metal box, its charge is neutralised by an

electron jumping from the metal on to the ion (right hand

diagram). That leaves a space amongst the electrons in the

metal, and the electrons in the wire shuffle along to fill it.

• A flow of electrons in the wire is detected as an electric current

which can be amplified and recorded. The more ions arriving,

the greater the current.

If you vary the magnetic field, you can bring each ion stream in

turn on to the detector to produce a current which is proportional

to the number of ions arriving.

The mass of each ion being detected is related to the size of the

magnetic field used to bring it on to the detector

The machine can be calibrated to record current against m/z

directly.

OutputMolybdenum 7 isotopes92,94,95,96,97,98,100

Sample introduction techniques• Sample must be introduced so that the vacuum inside instrument

remains unchanged.

• Sample placed on probe/plate

• Inserted into the ionization chamber

Direct insertion

• Capillary or capillary column. Sample – gas or solution

• Small quantities of sample

Direct infusion

Ionization methods

Ionization methods

Protonation

Deprotonation

Cationization

Transfer into gas phase

Electron ejection

Electron Capture

Protonation

M + H MH+

H2N-RGASSRR-OH + H+ MH+

Protonation is a method of ionization by

which a proton is added to a molecule,

producing a net positive charge of 1+ for every

proton added. Positive charges tend to reside

on the more basic residues of the molecule,

such as amines, to form stable cations.

Peptides are often ionized via protonation.

Protonation can be achieved via matrix-

assisted laser desorption/-ionization (MALDI),

electrospray ionization (ESI) and atmospheric

pressure chemical ionization (APCI)

Deprotonation

M - H+ [M – H] --

Deprotonation is an ionization method by

which the net negative charge of 1- is achieved

through the removal of a proton from a

molecule. This mechanism of ionization is

very useful for acidic species including

phenols, carboxylic acids, and sulfonic acids.

Cationization

M + Cation+ → MCation+

Cationization is a method of ionization thatproduces a charged complex by non-covalently adding a positively charged ion toa neutral molecule. While protonation couldfall under this same definition, cationization isdistinct for its addition of a cation adductother than a proton (e.g. alkali, ammonium).Moreover, it is known to be useful withmolecules unstable to protonation. Thebinding of cations other than protons to amolecule is naturally less covalent, therefore,the charge remains localized on the cation.This minimizes delocalization of the chargeand fragmentation of the molecule.Cationization is commonly achieved viaMALDI, ESI, and APCI. Carbohydrates areexcellent candidates for this ionizationmechanism, with Na+a common cationadduct.

Electron ejection

-e--

M M+

electron ejection achieves ionization

through the ejection of an electron to

produce a 1+ net positive charge,

often forming radical cations.

Observed most commonly with

electron ionization (EI) sources,

electron ejection is usually performed

on relatively nonpolar compounds

with low molecular weights and it is

also known to generate significant

fragment ions.

Electron capture

+e--

M M--

With the electron captureionizationmethod, a net negative charge of 1- is achieved with the absorption or capture of an electron. It is a mechanism of ion-ization primarily observed for molecules with a high electron affinity, such as halogenated compounds.

Transfer of charged molecule to gas phase

M+ solution M+ gas

The transfer of compounds already charged in solution is normally achieved through the desorption or ejection of the charged species from the condensed phase into the gas phase. This transfer is commonly achieved via MALDI or ESI.

Ionization sources

• Electron ionization (EI) limited chemists and biochemists to small molecules well below the mass range of common bio-organic compounds. This limitation motivated scientists to develop the new generation of ionization techniques

• These techniques have revolutionized biomolecular analyses, especially for large molecules. Among them, ESI and MALDI have clearly evolved to be the methods of choice when it comes to biomolecular analysis.

Ionization Source Acronym Event

Electrospray ionization ESI Evaporation of charged droplets

Nanoelectrospray ionization nanoESI Evaporation of charged droplets

Atmospheric pressure chemical ionization

APCI Corona discharge and proton transfer

Matrix-assisted laser desorption ionziation

MALDI Photon absorption/proton transfer

Desorption/ ionization on silicon DIOS Photon absorption/proton transfer

Fast atom/ion bombardment FAB Ion desorption/ proton transfer

Electron ionization EI Electron beam/electron

Chemical ionization CI Proton transfer

Ionization

MethodAdvantages Disadvantages

Protonation

(positive

ions)

•many compounds will

accept a proton to become

ionized

•many ionization sources

such as ESI, APCI, FAB, CI

and MALDI will generate

these species

•some compounds are not

stable to protonation (i.e.

carbohydrates) or cannot

accept a proton easily (i.e.

hydrocarbons)

Cationization

(positive

ions)

•many compounds will

accept a cation, such as

Na+ or K+ to become

ionized

•many ionization sources

such as ESI, APCI, FAB and

MALDI will generate these

species

•tandem mass spectrometry

experiments on cationized

molecules will often

generate limited or no

fragmentation information

Ionization methods, advantages and disadvantagesods, advantages and

disadvantages

Deprotonation

(negative ions)

•most useful for compounds that

are somewhat acidic

•many ionization sources such as

ESI, APCI, FAB and MALDI will

generate these species

•compound specific

Transfer of

charged

molecule to gas

phase

(positive or

negative ions)

•useful when the compound is

already charged

•many ionization sources such as

ESI, APCI, FAB and MALDI will

generate these species

•only for precharged ions

Electron

ejection

(positive ions)

•observed with electron ionization

and can provide molecular mass as

well as fragmentation information

•often generates too much

fragmentation

•it can be unclear whether the

highest mass ion is the molecular

ion or a fragment

Electron

capture

(negative ions)

•observed with electron ionization

and can provide molecular mass as

well as fragmentation information

•often generates too much

fragmentation

•it can be unclear whether the

highest mass ion is the molecular

ion or a fragment

Electrospray Ionization MS

• Most commonly used for biomolecular MS

• Strong electric field is applied to a liquid passing through a capillary

tube with a weak flux under atmospheric pressure. Multiply charged

ions are obtained from proteins.

• Research of non-covalent complexes with the advantages of speed,

sensitivity, specificity and low sample consumption.

• Conjugates retain their structural integrity upon the transition from

solution state into gas phase

• Evidence for complex formation and accurate determination of their

binding stoichiometries

Matrix-Assisted Laser Desorption/Ionization

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)

was first introduced in 1988 by Tanaka, Karas, and Hillenkamp. It has since

become a widespread analytical tool for peptides, proteins, and most other

biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).

The efficient and directed energy transfer during a matrix-assisted laser-

induced desorption event provides high ion yields of the intact analyte, and

allows for the measurement of compounds with sub-picomole sensitivity.

In addition, the utility of MALDI for the analysis of heterogeneous samples

makes it very attractive for the mass analysis of complex biological samples

such as proteolytic digests.

In MALDI analysis, the analyte is first co-crystallized with a large molar

excess of a matrix compound, usually a UV-absorbing weak organic

acid. Irradiation of this analyte-matrix mixture by a laser results in the

vaporization of the matrix, which carries the analyte with it. The matrix

plays a key role in this technique. The co-crystallized sample molecules

also vaporize, but without having to directly absorb energy from the

laser. Molecules sensitive to the laser light are therefore protected from

direct UV laser excitation.

Once in the gas phase, the desorbed charged molecules are then

directed electrostatically from the MALDI ionization source into the

mass analyzer. Time-of-flight (TOF) mass analyzers are often used to

separate the ions according to their mass-to-charge ratio (m/z). The

pulsed nature of MALDI is directly applicable to TOF analyzers since

the ion’s initial time-of-flight can be started with each pulse of the laser

and completed when the ion reaches the detector

The matrix consists of crystallized molecules, of

which the three most commonly used are 3,5-

dimethoxy-4-hydroxycinnamic acid (sinapinic acid

), α-cyano-4-hydroxycinnamic acid (alpha-cyano

or alpha-matrix) and 2,5-dihydroxybenzoic acid

(DHB). A solution of one of these molecules is

made, often in a mixture of highly purified water

and an organic solvent (normally acetonitrile

(ACN) or ethanol ).Trifluoroacetic acid (TFA) may

also be added. A good example of a matrix-solution

would be 20 mg/mL sinapinic acid in

ACN:water:TFA (50:50:0.1).

MATRIX

p = m/z

p1 = (Mr + z1)/z1

p is a peak in the mass spectrum

m is the total mass of an ion

z is the total charge

Mr is the average mass of protein