Chromatography• Russian scientist Tswett in 1906 used a glass columns

packed with finely divided CaCO3 to separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one.

• Tswett was the first to use the term "chromatography" derived from two Greek words "Chroma" meaning color and "graphein" meaning to write.

Definition of chromatography• Tswett (1906) stated that " chromatography is a method in

which the components of a mixture are separated on adsorbent column in a flowing system”.

• IUPAC definition (International Union of pure and applied Chemistry) (1993):

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction.

The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.

Principles of Chromatography

• Chromatography is a physical process. • Any Chromatography system is composed of

three Components : • Stationary phase• Mobile phase • Mixture to be separated

We can only control stationary and mobile phase as mixtures are the problem we have to deal with.

• Chromatography is a dynamic process in which the mobile phase moves in definite direction.

Classification of Chromatographic methods

• Different methods were attempted for classification of chromatography:

A – According to mechanism of separation:

The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into:

1- Adsorption Chromatography:

It is the oldest and most common type of chromatography. The stationary phase is a solid with adsorption power. Mixture components will be adsorbed on the surface of the stationary phase with different powers and that account for separation. Silica gel is the most common stationary phase in adsorption chromatography.

2- Partition Chromatography: The stationary phase is a liquid forming a thin film on an inert solid acts as support. The stationary liquid is usually more polar than the mobile liquid. The two liquids must be immiscible with each other. Cellulose powder and wet silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. Partition chromatography is preferable over adsorption when dealing with polar compounds.

Solid Support Film of the liqiud stationary Phase

3- Ion Exchange Chromatography:

It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. Both the mixture components and the mobile phase must be changed. Mixture of Alkaloids (compounds with positive charges) can be separated on anionic exchanger, while mixture of organic acids (negative charges) can be separated using cationic exchanger. Both types are used for desalination of water.

4- Molecular Exclusion ( Size Exclusion ) Chromatography:

• Size-exculsion chromatography (SEC), also called gel filtration or gel-permeation chromatography (GPC), molecular sieve chromatography uses porous particles to separate molecules of different sizes.

• It is generally used to separate biological molecules.• It is usually applied to large molecules or macromolecular

complexes such as proteins and industrial polymers.

4- Molecular Exclusion

( Size Exclusion ) Chromatography:

very large molecules eluted first without separation

large molecules can enter some pores

very small molecules enter all pores and eluted at last

5- Affinity Chromatography:

It uses the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose - agarose – dextran.

Or

Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand.

B- ACCORDING TO MOBILE PHASE:In this regard chromatography is classified into:

1- Liquid Chromatography (LC):

The mobile phase is liquid. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography (LSC). If separation occurs through partition the stationary phase is liquid so it is called: Liquid -Liquid Chromatography (LLC).

2- Gas Chromatography (GC)

Where the mobile phase is inert gas nitrogen or helium. Again if the stationary phase is solid it is called: Gas–Solid Chromatography (GSC). When stationary phase is liquid it is called: Gas-Liquid Chromatography (GLC).

C- ACCORDING TO THE TECHNIQUE (methods of holding the stationary phase):

1- Planar or Plane Chromatography:In this type of chromatography the stationary phase is used in the form of layer. Plane chromatography is further classified into:

a- Thin Layer Chromatography (TLC):

A technique used to separate non-volatile mixtures. It is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose.

b- Paper Chromatography (PC):

A specific type of papers is used as stationary phase in the form of sheets.

2- Columnar or Column Chromatography (CC):

The stationary phase is held in to a tube made of glass or metal.

D- ACCORDING TO PURPOSE OF USE:

Chromatography can be used for analytical work and also to obtain pure

materials from mixtures.

1- Analytical Chromatography:

a- Qualitative ChromatographyIn this case Chromatography can be used to:

1- Confirm the absence or probable presence of certain constituent in the sample under investigation

2- Give an idea about the complexity of the mixture and the least number of compounds present.

3- Check purity and identity of any compound.

4- Establish a (finger print ) pattern for extracts , volatile oils or pharmaceutical preparations. These finger prints can be then used to check the identity and purity in the future.

5- Monitor both column chromatography and organic chemical reactions.

b- Quantities Chromatography:

The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications.

2- Preparative application:

This was the first and is the main application of chromatography. The technique was developed primarily for this purpose.

Chromatography is used to obtain reasonable quantities of pure compounds from mixtures.

HPLC• High performance liquid chromatography is basically a

highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

• It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture

Types of HPLC• Normal phase HPLC• separation mode in which the retention material is

polar and mobile phase is nonpolar. Retained sample components are eluted in ascending order of polarity

• Although it is described as "normal", it isn't the most commonly used form of HPLC.

• The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.

• 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.

• Reversed phase HPLC• separation mode in which the stationary phase is

nonpolar and mobile phase is polar. Elution order of components is in decreasing order of polarity. It is the most commonly used mode of HPLC separations.

• Silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.

• In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.

• Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.

• That means that now it is the polar molecules that will travel through the column more quickly.

• Size-exclusion chromatography (SEC), • also known as gel permeation chromatography or gel

filtration chromatography, separates particles on the basis of molecular size (actually by a particle's Stokes radius). It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification.

• SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.

• This technique is widely used for the molecular weight determination of polysaccharides

. BASED ON ELUTION TECHNIQUE

• 1.Isocratic elution

• A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic elution

• Best for simple separations

• Often used in quality control applications that support and are in close proximity to a manufacturing process

• Gradient elution

• A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution

• Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower peaks . This also improves the peak shape and the peak height

• Best for the analysis of complex samples

• Often used in method development for unknown mixtures

• Linear gradients are most popular

Instrumentation and working

Instrumentation and working

• A . Solvent delivery system(mobile phase reservoir)

• The mobile phase in HPLC refers to the solvent being continuously applied to the column or stationary phase

• The mobile phase acts as a carrier to the sample solution

• A sample solution is injected into the mobile phase of an assay through the injector port

• As a sample solution flows through a column with the mobile phase, The components of that solution migrate according to the non-covalent interaction of the compound with the column

• The chemical interaction of the mobile phase and sample , with the column , determine the degree of migration and separation of components contained in the sample

• The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. this is because as the particle size of stationary phase is around 5-10µ, so the resistance to the flow of solvent is high.

• B. Pumps

• •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).

• C. Injector:

• The injector serves to introduce the liquid sample into the flow stream of the mobile phase for analysis.

• It is equipped with six port valves so that a sample can be injected into the flow path at continuous pressure

• For a manual injector, the knob is manually operated to deliver the sample to the column

• The knob is set to LOAD position for sample injection using a syringe , the sample is injected into the sample loop , which is separated from the flow path

• The knob is turned to INJECT position and the eluent travels through the loop from the pump and delivers the sample to the column

• •Typical sample volumes for manual injector are 5-to 20-microliters (μL).

• •The injector must also be able to withstand the high pressures of the liquid system.

• •An autos ampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical. It can continuously Inject variable volume a of 1 μL – 1 mL

• D. Column

• Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters.

• It is usually made of stainless steel to withstand high pressure caused by the pump to move the mobile phase through the column packing other material include PEEK and glass

• •The small particles inside the column are called the “packing” what cause the high back pressure at normal flow rates.

• Column packing is usually silica gel because of its particle shape , surface properties , and pore structure give us a good separation

• Other material used include alumina, a polystyrene-divinyl benzene

• synthetic or an ion-exchange resin

• – Pellicular particle: original, Spherical, nonporous beads,

• proteins and large biomolecules separation (dp: 5 μm)

• – Porous particle: common used, dp: 3 ~ 10 μm. Narrow size

• distribution, porous microparticle coated with thin organic film

• The dimensions of the analytical column are usually

• -straight, Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm), diameter of particle(35 μm). Number (40 k ~ 70 k plates/m)

Guard column is used to remove particular matter and contamination, it protect the analytical column and contains similar packing its temperature is controlled at < 150 °C, 0.1 °C

As mention before , columns are divided into different types according to their functions.

E . Detector:

•The detector can detect the individual molecules that elute from the column and convert the data into an electrical signal

•A detector serves to measure the amount of those molecules

•The detector provides an output to a recorder or computer that results in the liquid chromatogram

•Detector is selected based on the analyte or the sample under detection

Commonly used detectors in HPLC

Ultraviolet (UV)•This type of detector responds to substances that absorb light.

•The UV detector is mainly to separate and identify the principal active components of a mixture.

•UV detectors are the most versatile, having the best sensitivity and linearity.

•UV detectors cannot be used for testing substances that are low in chromophores (colorless or virtually colorless) as they cannot absorb light at low range.

•They are cost-effective and popular and are widely used in industry

Fluorescence

•This is a specific detector that senses only those substances that emit light. This detector is popular for trace analysis in environmental science.

•As it is very sensitive, its response is only linear over a relatively limited concentration range. As there are not many elements that fluoresce , samples must be syntesized to make them detectable.Mass Spectrometry

•The mass spectrometry detector coupled with HPLC is called HPLC-MS. HPLC-MS is the most powerful detector, widely used in pharmaceutical laboratories and research and development.

•The principal benefit of HPLC-MS is that it is capable of analyzing and providing molecular identity of a wide range of components.

Refractive Index (RI) DetectionThe refractive index (RI) detector uses a monochromator and is one of the least sensitive LC detectors.

•This detector is extremely useful for detecting those compounds that are non-ionic, do not absorb ultraviolet light and do not fluoresce.

•e.g. sugar, alcohol, fatty acid and polymers.

F . Data processing unit (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 determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).

•The concentration of each detected component is calculated from the area or height of the corresponding peak and reported.

• Ultra High Performance Liquid Chromatography (uHPLC): Where standard HPLC typically uses column particles with sizes from 3 to 5µm and pressures of around 400 bar, uHPLC use specially designed columns with particles down to 1.7µm in size, at pressures in excess of 1000 bar.

ApplicAtions

HPLC is one of the most widely applied analytical separation techniques.

Pharmaceutical:

• Tablet dissolution of pharmaceutical dosages.

• Shelf life determinations of pharmaceutical products.

• Identification of counterfeit drug products.

• Pharmaceutical quality control.

Environmental

•Phenols in Drinking Water.

•Identification of diphenhydramine in sediment samples.

•Biomonitering of PAH pollution in high-altitude mountain lakes through the analysis of fish bile.

•Estrogens in coastal waters - The sewage source.

•Toxicity of tetracyclines and tetracycline degradation products to

•environmentally relevant bacteria.

•Assessment of TNT toxicity in sediment..

Forensics

•A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy.

•Identification of anabolic steroids in serum, urine, sweat and hair.

•Forensic analysis of textile dyes.

•Determination of cocaine and metabolites in meconium.

•Simultaneous quantification of psychotherapeutic drugs in human plasma.

Clinical

•Quantification of DEET in Human Urine.

•Analysis of antibiotics.

•Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.

•Detection of endogenous neuropeptides in brain extracellular fluids.

Food and Flavor

•Ensuring soft drink consistency and quality.

•Analysis of vicinal diketones in beer.

•Sugar analysis in fruit juices.

•Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.

•Trace analysis of military high explosives in agricultural crops.Stability of aspartame in the presence of glucose and vanillin

ADVANTAGES OF HPLC:

1. Separations fast and efficient (high resolution power)

2. Continuous monitoring of the column effluent

3. It can be applied to the separation and analysis of very complex mixtures

4. Accurate quantitative measurements.

5. Repetitive and reproducible analysis using the same column.

6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

7. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC

8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment

9. A variety of solvents and column packing are available, providing a high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple components in a single analysis.