Liquid Chromatography (GC)

Hashim BaharinDepartment of Chemistry, Faculty of Science

Universiti Teknologi Malaysia81310 Skudai, Johor, Malaysia

THIN LAYER CHROMATOGRAPHY (TLC)THIN LAYER CHROMATOGRAPHY (TLC)

•Simple and inexpensive technique.Simple and inexpensive technique.

•Often used to judge the purity of a synthesized compound or to Often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction (qualitative indicate the extent of progress of a chemical reaction (qualitative analysis)analysis)

•Small quantity of a solution of the mixture to be analyzed is Small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate.deposited as a small spot on a TLC plate.

•TLC plate consists of a thin layer of silica gel (SiOTLC plate consists of a thin layer of silica gel (SiO22) or alumina ) or alumina

(Al(Al22OO33) coated on a glass or plastic (stationary phase). ) coated on a glass or plastic (stationary phase).

•The sheet is then placed in a chamber containing a small amount The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase.of solvent, which is the mobile phase.

The solvent gradually moves up the plate via capillary action, The solvent gradually moves up the plate via capillary action, and it carries the deposited substances along with it at different and it carries the deposited substances along with it at different rates. rates.

The desired result is that each component of the deposited The desired result is that each component of the deposited mixture is moved a different distance up the plate by the mixture is moved a different distance up the plate by the solvent. solvent.

The components then appear as a series of spots at different The components then appear as a series of spots at different locations up the plate. locations up the plate.

The RThe Rff Value Value

Rf is the retardation/retention factor, or how far up a plate the Rf is the retardation/retention factor, or how far up a plate the compound travels.compound travels.

The Rf value for a substance is the ratio of the distance that the The Rf value for a substance is the ratio of the distance that the substance travels to the distance that the solvent travels up the substance travels to the distance that the solvent travels up the plate. plate.

frontsolvent by travelledDistance

substanceby travelledDistance R Factor,n Retardatio f

Visualizing the Spots (Qualitative Analysis)

•If the compounds are colored, they are easy to see with the naked eye.

•If not, a UV lamp is used or spray with iodine vapour, ninhydrin, flourescamine.

Applications

• To determine the number of components in a To determine the number of components in a mixture. mixture.

• To determine the identity of two substances. To determine the identity of two substances.

• To monitor the progress of a reaction. To monitor the progress of a reaction.

• To determine the effectiveness of a purification. To determine the effectiveness of a purification.

• To determine the appropriate conditions for a To determine the appropriate conditions for a column chromatographic separation. column chromatographic separation.

• To monitor column chromatographyTo monitor column chromatography.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

•Evolved from preparative column chromatography.

•Performance (efficiency and resolution) enhanced by use of stationary phase particles with small diameter (2-5 m).

•IN ORDER TO OBTAIN SATISFACTORY FLOW RATES, THE LIQUID MUST BE PRESSURIZED TO SEVERAL HUNDRED PSI OR MORE.

Solvent

•Common solvents used are water, methanol, acetonitrile, and tetrahydrofuran.

•Solvents are filtered (0.45 m membrane filter) and degas.

•Inlet filter at the line tubing.

ADVANTAGES OF HPLC

Greater sensitivity - various detectors.

Ideal for ionic species and large molecules.

Analytical or preparative (highly purified compound in small or large quantities).

Applications include analysis of sugars, pesticide residues, organic acids, lipids, amino acids, toxins, and vitamins.

COMPONENTS OF AN HPLC SYSTEM

•Consist of several components with defined functions.

•Components in modular form or as an integrated unit.

•Modular system allows users to tailor the instruments to their needs and budget.

•Components interconnected by transfer tubing with small diameter (0.1 mm) e. g. stainless steel or flexible polymer tubing (polyetheretherketone - PEEK)

Solvents(Mobile Phase)

Pump

Injector

Thermostated Column

Detector

Computer

Pumping system

•The function is to deliver mobile phase through the system at flow rates ranging from 0.1 to 10 mL/min.

•Needs to be controlled, accurate and precise.

•System and connecting lines are made from stainless steel (can handle pressure of up to 6000 psi).

•Pumps are sensitive to particles in the mobile phase and air. Filter and degas mobile phase.

•Types: reciprocating pumps, screw-driven syringe type, pneumatic pump.

Injector

The function is to put sample in the mobile phase.

Valve Injector

•Trouble free, good precision and can change loop for different volumes.

•Place in load position and load sample into an external fixed volume loop at atmospheric pressure.

•Rotate to inject position. Loop becomes part of the eluent flow stream and the sample is carried to the column.

Auto Samplers

•Can inject a large number of samples.

•Small vials with a septum placed in a tray.

•Needle penetrates septum and withdraws the sample.

•Valve introduces the sample into the column.

COLUMNS•Straight, stainless steel column, typically 3-25 cm length and internal diameter 0.5-5 mm.•Stationary phase held in column by one porous disc at each extremity.•Dead volumes within column kept as small as possible.•Normal columns allow mobile phase through at a few mL/min.•Usually constructed from stainless steel tubing (some glass tubing but <600 psi).•Packing materials silica, alumina, porous polymer, ion exchange resins.

To increase lifetime of column, a precolumn or guard column is often used.

Precolumn - auxillary column that preceed the analytical column.

Guard column - protect analytical column by removing particulate matter and contaminants.

Analytical columnSize: 10, 15, or 25 cm long with internal diameter of 4.6 or 5 mm.Packing material particle size - 3, 5, or 10 m particles.Shorter columns (3 cm) packed with <3 m particles are becoming popular because of faster separation.

DETECTORS

Choice is determined by solute type and concentration, detector sensitivity, linear range, compatibility with solvent and elution mode.

Most common - UV/VIS, fluorescence, refractive index, diode-array (DAD).

In UV/VIS absorption detectors: absorption proportional to analyte concentration.

Coupled technique: LC-MS, LC-NMR.

HPLC NORMAL PHASE

Stationary Phase: Polar•Chemically attached polar nonionic functional group (alcoholic hydroxy, nitrile, or amino).

Mobile Phase: Nonpolar•Nonpolar solvent (e.g. hexane) with a more polar modifier (e.g. methylene chloride).•Solvent strength and selectivity is controlled which influences solute retention (weak solvents increase retention and strong solvents decrease retention).

Applications•Best for compounds highly soluble in organic solvent (fat soluble vitamins) or that have low solubility in an aqueous mobile phase.•Also can be used for compound classes, isomers, and highly hydrophilic species like carbohydrates.

HPLC REVERSE PHASEStationary Phase: Non-Polar

SilicaChemically bound to silica via surface silanol.Usually an octadecyl (C18).Bonded phase (non polar octadecylsilyl). Chromatographic behavior is influenced by the type of organic group bound to silica, chain length of the organic group (C4 - C18), support particle size and shape.

PolymericHighly cross-linked polystyrene-divinylbenzene (PS-DVB).

Mobile Phase: Polar•Usually water mixed with methanol, acetonitrile, or tetrahydrofuran.•Solute retained due to hydrophobic interactions with the non polar stationary phase.•Solute eluted in order of increasing hydrophobicity (least hydrophobic first, most hydrophobic last).•Increasing polarity of the mobile phase increases solute retention.•Increasing organic solvent content decreases solute retention.•Additives: an amine to deactivate silanols, aqueous buffers to suppress ionization of sample components, ion-pair reagents to neutralize charged solutes and make them more lipophilic

Applications of HPLC Reverse Phase

•Proteins,- including cereal proteins.

•Water soluble and fat soluble vitamins.

•Carbohydrates using ion-pairing reagents.

•Antioxidants, dyes, pigments, and phenolic flavor compounds.

Elution Mode

Isocratic Elution

•The composition of the solvent remains constant.

Gradient Elution

•The composition of the solvent is changed continuously or in a series of steps. This technique can overcome the general elution problem.

TYPES OF HPLC METHOD

•Liquid/liquid (partition) chromatography

•Liquid/solid (adsorption) chromatography

•Ion-exchange chromatography

•Size-exclusion (gel-permeation, gel-filtration) chromatography

ION EXCHANGE CHROMATOGRAPHY

•An ion exchange technique: low concentrations of anions or cations are separated via column materials that carry an opposite charge.

•The ionic groups of exchanger columns are covalently bound to the gel matrix and are compensated by small concentrations of counter ions, which are present in the buffer.

•When a sample is added to the column, an exchange with the weakly bound counter ions takes place.

sampleresin

ligandCounter ion

Functional Group Functional Group

Condition

Type

SO3H+ Sulfonic acid

Strong acid

Cation

COOH+ Carboxylic acid

Weak acid

Cation

CH2N+(CH3)3 Cl Quartenar

y amino group

Strong base

Anion

CH2N(CH3)2H+OH

3 amino group

Weak base

Anion

Stationary Phase•Ion exchanger functional groups

When a cation exchanger is immersed in an aqueous solution containing the M+ cations, an exchange equilibrium is establised:

xRSO3H+ + Mx+ (RSO3)xMx+ + xH+

(solid) (solution) (solid) (solution)

where, M: cation, R: part of the resin molecule containing one sulfonic acid group.

For an anion exchange resin:xRN(CH3)3

+OH + Ax- [RN(CH3)3+]xAx- + xOH

(solid) (solution) (solid)(solution)

where Ax- is an anion.

Mobile PhaseSolute retention is controlled by changing ionic strength and pH of the mobile phase.

Aqueous solution (sometime mix with the organic solvent) which contain ionic solute.

ApplicationsSimple inorganic compounds (cation and anion).Amino acids, carbohydrates, organic acids.

Preparative purification of proteins, fractionation of peptides.

InstrumentationIEC is practiced in two forms. Suppressed ICA second column (suppressor column) is used to remove the buffer ions so that sample ions can be more easily detected. Nonsuppressed ICWeakly conducting buffers at low concentration are carefully selected, and the entire effluent is passed through the detector; ions are detected above the background signal.

Supressed column

SIZE EXCLUSION CHROMATOGRAPHY •Size-exculsion chromatography (SEC), also called gel-filtration or gel-permeation chromatography (GPC).•Separation of mixtures based on the molecular size of the components. Uses porous particles to separate molecules of different sizes. •Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transist time than larger molecules that cannot enter the particles.•Molecules larger than the pore size can not enter the pores and elute together as the first peak in the chromatogram.

•Molecules that can enter the pores will have an average residence time in the particles that depends on the molecules size and shape. •Different molecules therefore have different total transit times through the column. •Molecules that are smaller than the pore size can enter all pores, and have the longest residence time on the column and elute together as the last peak in the chromatogram.

Stationary and Mobile Phase•Select stationary phase for a matrix pore size that matches the molecular weight range of the species to be resolved.•Hydrophilic packing (surface modified silica or methacrylate resins): Use with water soluble samples and an aqueous mobile phase.•Hydrophobic packing (polystyrenedivinylbenzene): Use with non-aqueous mobile phase that is chosen based on sample solubility.

Applications•To separate biological molecules (amino acid, virus, carbohydrate).•To determine molecular weights and molecular weight distributions of polymers.