BASICS OF CHROMATOGRAPHY Prepared by DR: Sherif Abdel- Naby - PowerPoint Presentation

BASICS OF CHROMATOGRAPHY Prepared by DR: Sherif Abdel- Naby Abdel- Gawad PhD Analytical Chemistry-Faculty of Pharmacy - Cairo University

History of Chromatography Mikhail Tswett is credited with the invention of chromatography. Developed a technique that separated various plant pigments (e.g.,chlorophylls and xanthophylls) by passing solutions through glass columns filled with finely ground CaCO3. The separated species appeared as colored bands on the column. Hence Greek chroma = color and graphein = writing M. S. Tswett Russian Scientist (1872-1919)

Tswett’s experiment

CHROMATOGRAPHY Basic PrinciplesTwo phases considered:1) Mobile Phase: solvent moving through the column.2) Stationary Phase: stays in place inside of the column.“Eluent” COLUMN “Eluate”Process is called “elution”

Simple Column Fresh eluentInitial bandof A and BColumnPacking Porous Disk

•Two phases: mobile and stationary •Mobile phase is solvent moving throughcolumn–liquid (Methanol, water, buffer)–gas (He, H2, N2)•Stationary phase fixed inside column–viscous liquid coated on inert support inside of column–solid particles packed inside column•Solutes have different affinities for mobilephase and stationary phase

Classification of chromatographic methods I- Classification according to the nature of mobile phaseA- Liquid chromatography (LC)i-Liquid-solid chromatography (LSC) In this type, stationary phase is solidii- Liquid-liquid chromatography (LLC) In this type the stationary phase is liquid, which may be mechanically held on a solid support in the form of thin coat. It may be packed in a tubular column or spreaded as thin layer on a plate.Bonded phase chromatography (BPC) The liquid stationary phase may be chemically and permanently held to a solid support.

B- Gas Chromatography (GC) The mobile phase used in this type is a gas. This type can be classified according to the nature of stationary phase into:i- Gas-Liquid chromatography (GLC) In this type the stationary phase is a liquid, which may be mechanically held on a solid support in the form of thin coat and packed in a column or it coats the inner wall of capillary column.ii- Gas-Solid Chromatography (GSC) In this type the stationary phase is a solid.

II- Classification according to the mechanism of attachment to the stationary phase.A- Adsorption ChromatographyStationary phase : Solid Mobile phase : Liquid or GasHow it separates–solute adsorbs on tostationary phase surface

B- Partition Chromatography Stationary phase: Liquid on solid support Mobile phase: Liquid or GasHow it separates–solute dissolves intoliquid coating

C- Ion-Exchange Chromatography Stationary phase: Anions or cations covalently bound to solid stationary phaseMobile phase :liquidHow it separatessolute ions of opposite chargeattracted to stationary phase

D- Molecular Exclusion ChromatographyStationary phase: Porous gel Mobile phase: LiquidHow it separates–small moleculestrapped in pores ofstationary phaseAlso calledgel filtration,gel permeation,orsize-exclusionchromatography

E- Affinity ChromatographyStationary phase : Immobilized molecules on liquid or solid stationary phase Mobile phase: LiquidHow it separates–Molecules with specificshape dock with ligands

III- Classification according to the used technique A- Columnar: In this type the stationary phase is packed in a column, e.g. GC, LC and conventional column.B- Planar: In this type the stationary phase is spreaded as a thin layer on glass, plastic or aluminium plates; or it is held in the network structure of paper (paper chromatography).

Summary of types of chromatography Mobile PhaseLiquid Gas Chromatographic method Liquid chromatography (LC) Gas chromatography (GC) Stationary phase Solid liquid Solid liquid Chromatographic method LSC LLC GSC GLC Mechanism of sorption Adsorption chromatography Partition chromatography Adsorption chromatography Partition chromatography Technique Columnar & Planar Columnar & Planar Columnar Columnar

Theory of chromatography The plate theory The plate theory assumes that, a column of stationary phase is regarded as a large number of horizontal, narrow, discrete, consecutive theoretical layers known as theoretical plates (N). At each plate of the stationary phase continuous partitioning and equilibration of solutes (components molecules) occurs as the mobile phase moves down the column.

The efficiency of the column is increased as the number of theoretical plates (N) is increased, i.e the efficiency of column is measured by N. Also, the column efficiency is measured by Height Equivalent of Theoretical plates (HETP) which is denoted by (H) N = L/H or H = L/NWhere:N = number of theoretical plates.L = Column length.

Chromatogram: It is a plot of solute concentration as given by detector response versus elution time. The position of peak on the time axis can be used for identification of the sample The area under the peak provides a quantitative measure of the amount of each species. A typical chromatogram for a sample containing two components

Some terms used in chromatography 1- R Value (retardation factor): t m R = _________ t m + t s where t m is the time that the substance spends in the mobile phase and t s is the time that it spends in the stationary phase. R value for a certain substance varies with change of both or either of the mobile and stationary phases , but once we state the conditions of a particular chromatographic method R value will be characteristic for the substance and used for its identification . The differences in migration rates cause the components in a mixture to separate into bands located along the column.

2-R f Value in thin layer chromatography, paper chromatography and in column chromatography where the sample band moves down the column to a definite distance:Rf = the distance traveled by the zone center The distance traveled by solvent (solvent front)  As the solute is more retained as the smaller will be the Rf value.

3- Rt or tR Vaue The Retention Time : (GC and HPLC) it is the time from the injection to the emergence of the peak maximum, (notice that as the solute is more retained the larger will be the Rt value). 4- Rv The Retention Volume : It is the total volume in ml of eluent (mobile phase or solvent ) required to elute the center of the zone of a particular substance. It is used in column, HPLC, and GC, (notice that as the solute is more retained the larger will be the Rv value).5-Capacity Factor K\: It is widely used to describe the migration rates of solutes on columns. K \ = K V s / V m also K \ = t R - t m t m

6-Column resolution (R s)A- Selectivity factor () : for a column separating two components A and B K\B (tR)B – tm  = ________ = ______________ K\A (t R)A – tm K ’ B is the capacity factor of the more retained substance and K ' A is that for the less retained one. The larger the  the more selective the stationary phase and the more effective the separation,  should be more than one.

B- Resolution factor Rs = 2z / WB + WA = 2 [tR(B) – tR(A)]/WB + WA It is clear from the equation that, the greater the separation of the zones from each other and/or the smaller the zone width the larger will be the resolution. Rs should be at least 1 and 1.5 for complete separation

7- The Efficiency of the Column: It is determined by two parameters , the migration of the zone and the zone broadening i.e. the retention time and the zone width Column efficiency is quantitatively determined by calculating the number of theoretical plates N or H (HETP) N =16 tR2 / W2 while H = LW2 / 16 tR2 So as the retention time increases and / or the zone width decreases the more efficient will be the column .

Factors affecting zone broadening . Zone broadening is due to three factors:Eddy diffusion (Multiple path effect)This effect occur in packed column not in open tubular columns. Eddy diffusion is caused by imperfect packing of the column with the stationary phase. Imperfect packing causes the presence of air bubbles or cracks. Imperfect packing is also, due to the use of irregular large particles stationary phase.

Ordinary diffusion. It results from low flow rate of the mobile phase, where molecules of the separated component move from the concentrated zone center to the borders of the zone.Non equilibrium mass transfer It results from high flow rate of the mobile phase. At high flow rate of the mobile phase there is no enough time for molecules of the separated component to distribute themselves between the mobile phase and the stationary phase.

Some of the molecules move ahead with the mobile phase at the front of the zone and some will remain on the stationary phase on the back of the zone. Thus to minimize zone broadening we have to use small regular stationary phase particles, perfect packing (no cracks or air bubbles) and choose optimum flow rate.

Van Deemter EquationPutting the three factors together yields the Van Deemter equation that helps predict how the column flow rate will affect the theoretical plate heightH= A + B/Ux + CUxA (Eddy diffusion) H independent on flow rate.B/Ux (Ordinary diffusion) H inversely proportional to flow rate.CUx (Non equilibrium mass transfer) H proportional to flow rate.

Optimization of column performance To optimize the column performance we have to change  and KB to obtain Rs value of 1.5. This can be achieved by:Change the composition of mobile phase in LC .Change the flow rate of the mobile phase.The column must be perfectly packed with small regular spherical particles, without any cracks, gabs or spaces to minimize zone broadening and to maximize N.If resolution is still less than 1, the stationary phase must be changed.

Types of elution A- Isocratic elution (Simple elution) In this type of elution a mobile phase is either single solvent or mixture of solvents without change of its composition. B- Gradient elution In this type of elution the composition of the mobile phase is changed during chromatographic separation either stepwise or continuously.

Requirements of solvents used as mobile phase It must dissolve the components of the mixture to be separated.It must be of different eluting power.Solvents of weaker elution power can be tried successively by addition of solvent of higher elution power (as in case of gradient elution). The elution power of a solvent is measured by its adsorbability on the stationary phase. Solvents are arranged according to their elution power in what is called the elutropic series.

Example Eluting power of a series of solvents for substances adsorbed on silica gel, arranged in descending series. H2O > methanol > ethanol > propanol > acetone > ethyl acetate > ether > chloroform > dichloromethane > benzene > toluene > trichloroethylene > carbon tetrachioride > cyclohexane > hexane.This order is the order of decreasing dielectric constant of the solvents. The purity of solvents should be as high as possible

Choice of chromatographic separation methods .In case of substances of similar chemical types, partition chromatography is preferable.In case of substances of different chemical types, adsorption chromatography gives better separation.Gaseous and volatile substances need separation adopting gas chromatographic separation.Ionic and inorganic substances are better separated on ion exchange column and zone electrophoresis.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) For conventional column chromatography where the mobile phase is liquid (Liquid Chromatography LC) and the stationary phase is either a solid or a liquid which is coated on solid support.In trials to improve chromatographic separation by increase the stationary phase surface area either we may decrease the particle size in this case the mobile phase flow rate will decrease and stops after few ml or we may increase the column length in this case the elution will take a very long time may be several hours.

However, it was not until the late 1960s, that the technology of producing and using small size columns packed with stationary phase of 10 m particle diameter.To allow the flow of mobile phase within reasonable time it is introduced under Pressure for this reason the method was first referred as High Pressure Liquid Chromatography. In HPLC system, a pressure is applied to the column, forcing the mobile phase through at a much higher rate. The pressure is applied using a pumping system.

Types of Samples For HPLC: The sample can be organic, inorganic (including ionic) compounds, volatile, nonvolatile thermally stable or labile.Molecular weight ranges from ~80-~6000.Advantages of HPLCSpeed the analysis (takes only few minutes).High resolution; very complex mixture can be resolved to its components.High accuracy; Relative error less than 1% for quantitative analysis.

High sensitivity; according to the nature of sample and detector it is possible to measure a concentration of 10 -6 - 10-9 g/ml.Automatic systems; the whole operation starting from injection of the sample to identification of the peaks and determination of the concentration, and then repeat the cycle with next sample.Limitation of HPLCExpensive instrumentation and supplies.The requirement of experience at least 6-12 months of practical experience in methods developments are generally required to become a professionally operator and obtain the maximum benefit, from HPLC.

HPLC - Instrument Schematic diagram showing liquid flow in a typical liquid chromatographic instrument.

Mobile Phases: A good solvent must possess the following requirements:High purity and readily available.Low viscosity, limited flammability and toxicity and low reactivity to avoid chemical interaction with solutes or stationary phase.For LLC, immiscibility with stationary phase.Compatibility with the detector.

PUMPS: The requirements for pumping system are mainly the followinggenerate a pressure up to 500 atmosphereproduce a pulse free output.The flow rates ranging from 0.1 to 10ml/minTo have high resistance to corrosion by a variety of solvents.

There are THREE types of pumps: Reciprocating pumpsThis is the most commonly used.It consists of a small cylindrical chamber that is filled and then emptied by the back and forth motion of a piston. The pumping motion produces a pulsed flow that must be damped.

2-Syringe type: single stroke type it has the advantage of introducing the solvent at pulse free constant flow rate, but it suffers from impossibility of solvent gradient and the limited solvent volume. 3-The pneumatic or constant pressure type, it is cheap but it has the same disadvantages as the syringe type.

PRECOLUMN: It contains a packing chemically identical to that in the analytical column. The purpose of the precolumn is to remove impurities from the solvent, and thus prevent contamination of the analytical column. In addition the precolumn saturates the mobile phase with the stationary phase thus eliminate stripping (washing) of the stationary phase from the analytical column during elution with the solvent.

SAMPLE INJECTION SYSTEM It is used for introduction of the sample. By changing the position of the valve, the loop can be switched in or out the stream of solvent as it passes from the pump to the column. While the loop in the position out of the stream of the solvent they can be filled with syringe till overflow occurs, this insures that the loop is completely filled, then the position of the loop is changed by rotating or sliding the valve, and allow it to pass with the solvent to the column. Loops of different volumes are used (10-50l).

COLUMNS USED FOR HPLC Columns for HPLC are manufactured from heavy walled glass tubing or stainless steel.The typical range for column length is from 15-25cm with an inside diameter from 5-10mm. Column packings typically have particle sizes of 5 to 10m diameter. Recently high speed and high performance microcolumns with an inside diameter of 1 to 4 mm and length of 3 to 7.5 cm are also available. They have the advantages of speed and minimal solvent consumption.

Column Packing Materials: There are TWO main types of column packing:1- Pellicular Particles: This consists of spherical, nonporous glass or polymer beads 30-40 m diameter, deposited on its surface a thin porous layer or crust of silica, alumina, porous polymer or an ion exchange resin. Also liquid stationary phase or chemically bonded phase can be attached to the surface. 2- Porous Micro Particles: This particles have diameter ranging from 3 to 10m, they are composed of silica, alumina, porous polymer or ion exchange resin. Silica particles may be coated with thin organic film, which is physically or chemically bonded to the surface.

Si OH HO - R Hydroxyl Si OH HOOC - R Carboxyllc acid Si OH HN-R 1 o and 2 o Amino Si OH HS-R Thiols Silanol Group Giass Surface

Detectors Questions to ask when evaluating an LC detector:–Is the detection universal?–Is the detector response linear?–What is the limit of detection (LOD)?–Is the detector useful with gradient elution?

UV Absorbance Detector: The detector consists of source of UV radiation, the beam of which is split and pass through two flow cells 10 to 14l.

One through which pure solvent flows and the other through which solvent containing the sample flows. Light from both cells will then pass to wavelength selector, then to two phototubes for detection of absorbed radiation. When the same solvent flows through the cell no signal is obtained, while if sample passes through one cell difference in current occurs and a signal is obtained. LOD ~ 0.1 ng Good detection method with gradient elution Use solvents that do not absorb UV radiation

Fluorescence Detector Similar idea to UV detectorFew molecules fluoresceDerivatize solutes with fluorescent tagDerivatize solutes as they elute from columnbefore detection(Post-column Derivatization)Essentially linearLOD ~ 0.001 ngFine with gradient elutionUse solvents that do not fluoresce

Refractive Index Detector Compare refractive indices of eluate & referenceCannot use with gradient elutionEssentially universalLOD ~ 100 ngEssentially non-linear (Linear range very small)Disaster with gradient elution

Comments AdvantageSampleTypeOf intermediate sensitivity.Detect wide range of samples.Common detector. For light absorbing compounds UV-VIS Most samples need derivatization (except native fluorescent molecules) Highly sensitive detector. For Fluorescent compounds Fluorescence Of intermediate sensitivity. Can be used for both separation and structure elucidation. Universal detector MS Specific for ions Specific detector; for all ions Electrochemical conductometric Specific for electroactive elements. Specific detector; Electroactive compounds Electrochemical amperometric Comparison of LC Detectors