Simple separation VS Chromatographic separation Principle of simple separation (exp partitioning between phases): - The separation occurs only one direction.

Simple separation VS Chromatographic separation

Principle of simple separation (exp partitioning between phases):

- The separation occurs only one direction- Increase efficiency by using fresh

extracting phase.The principle of chromatographic

separation: extracting the solutes back and forth between fresh portion of the two phases. The two phases used are called the mobile phase and the stationary phase.

ChromatographyChromatography

Chromatography is the separation of a mixture based on the different degrees to which they interact with two separate material phases:

The two phases are:1) The stationary phase2) The mobile phase

The stationary phase – a phase that is fixed in place either in a column or in a planar surface. The stationary phase is either a porous solid used alone or coated with a stationary liquid phase.

The mobile phase – a phase that moves over or through the stationary phase, carrying with it the analyte mixture. It is also called the eluting fluid. The mobile phase can be a gas, a liquid, or a supercritical fluid.

Column VS Planar Column VS Planar chromatographychromatography

Principle of column chromatography:The stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure.

Principle of planar chromatographyThe stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action.

Exp paper chromatography and Thin layer chromatography.

Column Chromatography Column Chromatography and Planar Chromatographyand Planar Chromatography

Packing material

Separation column

Column Chromatography

Paper or a substrate

coated with particles

Paper ChromatographyThin Layer Chromatography (TLC)

Separation Process and Separation Process and Chromatogram for Column Chromatogram for Column

ChromatographyChromatography

7

Outp

ut

conce

ntr

ati

on

Time

ChromatogramChromatogram

ChromatogramChromatogram

8

Time

tR

t0

Inte

nsi

ty o

f dete

ctor

signal

PeaktR : Retention time

h

A

t0 : Non-retention time

A : Peak area

h : Peak height

1. Retention factor = the degree of the retention of the sample component in column k is defined as the time the solute resides in the stationary phase (ts) relative to the time it resides in the mobile phase (tm).

2. Number of plates (N) is the number of plates of a column toward a particular compound.

3. Height of plate, (H) is the plate height or the length of a column (L) divided by the number of theoretical plates

4. Separation factor of selectivity factor, a for solutes A and B is defined as the ratio of the distribution constant of the more strongly retained solute (B) to the distribution constant of the less strongly held solute (B).

5. Resolution of a chromatographic column is a quantitative measure of its ability to separate analytes A and B

or

Chromatographic Chromatographic TechniquesTechniques Classified according to the type of

equilibration process involved. Equilibration process control by the type of stationary phase.

1. Adsorption Chromatography- The stationary phase is a solid.- Sample components are adsorbed on the

solid stationary phase.- The mobile phase may be a liquid or a gas.- The sample components are distribute

between the two phases through a combination of sorption and desorption process.

- Example: Thin layer chromatography.

Chromatographic Chromatographic TechniquesTechniques2. Partition Chromatography- The stationary phase is a liquid

supported on an inert solid.- The mobile phase may be a liquid or a

gas.- In normal-phase C: a polar SP and non-

polar MP is used for nonpolar solutes.- In reversed-phase C:nonpolar SP and

polar MP is used for polar solutes.- Reversed phase is the most widely used.

Chromatographic TechniquesChromatographic Techniques3. Ion Exchange and 4. Size Exclusion Chromatography

Stationary Phase Mechanism

Ion Exchange Ion exchange resin ion exchange

equilibriaSize Exclusion Chromatography

A sievelike structure penetration of

a certain size

GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY (GC)(GC)A chromatographic technique in

which the mobile phase is a gas.Parts of GC are:

- The mobile phase- The stationary phase- Sample introductions- Temperature control- Detectors

Principle of Gas Principle of Gas ChromatographyChromatographySample should be converted to vapor state (if

it is not already a gas).Separation occurs as the vapor constituents

equilibrate between carrier gas and the SP.The sample is automaticaly detected by

detector.Measuring the retention time and comparing

this time with that of a standard of a pure substances make it possible to identify the peak.

Since the area of the peak is porpotional to the concentration, and so the amount of the substance can be quantitatively determined.

The peak height can be compared with a calibration curved prepared in a same manner.

Importance parts of GCImportance parts of GC

There are three parts most important for GC

1. The columns2. The detectors3. The mobile phase gas supply

The ColumnsThe ColumnsCommonly used columns are

packed columns and capillary columns.

Packed ColumnAbout 1-10 m long and 0.2-0.6

cm in diameter.Short columns made of glass and

longer columns made of stainless steel or can also made of Teflon.

Functions of GC ColumnsFunctions of GC ColumnsTo contain the stationary phase

and the passing way of the mobile phase.

The site where the separation of analyte occurs.

To provide analysis in terms of resolution, sensitivity and retention time.

The DetectorsThe Detectors Over 40 detectors have been

developed since the introduction of GC.

Commonly used detectors1. Thermal Conductivity Detector (TDC)- The original detector2. Flame Ionization Detector (FID) - The most sensitive and widely used

detector for organic compounds.3. Flame Photometric Detectors (FPD)

Functions of DetectorsFunctions of DetectorsTo respond to compounds

analysed.To automatically detect the

sample as it emerges from the column.

The mobile phase gas supplyThe mobile phase gas supplyUsually an inert gas that available in pure

form such as argon, helium or nitrogen.A highly dense gas is more effective.The choice of gas determine by the type of

detector.Functions:- To bring along gas and injected

compounds throughout the column up to detector.

- To provide equilibration between the carrier gas and the stationary phase for compounds separation.

Sample Introduction- Must consider three rules:1. All constituents injected into GC

must be volatile.2. The analyte must be present at an

appropriate concentration.3. Injecting the sample must not

degrade the separation (thermally stable).

Volatile SampleA volatile compound is a compound

that easily evaporated because of their low molecular weight.

In GC, the sample constituents need to be volatiled in order to move through the column.

Nonvolatile solutes will condense on the column, degrading the column’s performance.

Exp. of volatile compounds are from the monoterpenoids group (limonene, linalool,

champor, menthol etc.)Applications of GCWidely used for the analysis of

diverse array of samples in environmental, clinical, pharmaceutical, biochemical, forensic, food science and petrochemical laboratories.

GC – Mass Spectrometry (GC-MS)GC – Mass Spectrometry (GC-MS)

GC-MS is a sophisticated instrumental technique that produces, separates, and detects ion in the gas phase.

Today, relatively inexpensive compact benchtop system are available and widely used in laboratories.

• Mass spectroscopy is used to determine the molecular formula of the unknown compound.

• Mass spectroscopy data that provides structural information tends to be unreliable and thus will only be used to verify a possible structure or in the event that the other spectral techniques are unsuccessful.

GC – Mass Spectrometry (GC-MS)GC – Mass Spectrometry (GC-MS)

Principle of GC-MSPrinciple of GC-MS

Block diagram of mass spectroscopy

• The inlet transfers the sample into the vacuum of the mass spectrometer. In the source region, neutral sample molecules are ionized and then accelerated into the mass analyzer.

• The mass analyzer is the heart of the mass spectrometer. This section separates ions, either in space or in time, according to their mass to charge ratio.

• After the ions are separated, they are detected and the signal is transferred to a data system for analysis.

• All mass spectrometers also have a vacuum system to maintain the low pressure, which is also called high vacuum, required for operation.

• High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions.

• In some experiments, the pressure in the source region or a part of the mass spectrometer is intentionally increased to study these ion-molecule reactions. Under normal operation, however, any collisions will interfere with the analysis.

Ionization SourceIonization Source

Electron Impact (EI)Electron Impact (EI)

Electron Ionization (EI) is the most common ionization technique used for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations.

Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

Electron Ionization SourceElectron Ionization Source

A) Ionizing electron approaches the electron cloud of a molecule

B)Electron cloud distorted by ionizing electron

C) Electron cloud further distorted by ionizing electron

Electron Ionization Process

E) Electron cloud of molecule ejecting an electron

F) Molecular ion and ejected electron.

D) Ionizing electron passes by the molecule

Chemical IonizationChemical Ionization

Chemical Ionization (CI) is a “soft” ionization technique that produces ions with little excess energy. As a result, less fragmentation is observed in the mass spectrum.

Since this increases the abundance of the molecular ion, the technique is complimentary to 70 eV EI.

CI is often used to verify the molecular mass of an unknown. Only slight modifications of an EI source region are required for CI experiments.

In Chemical Ionization the source is enclosed in a small cell with openings for the electron beam, the reagent gas and the sample.

The reagent gas is added to this cell at approximately 10 Pa (0.1 torr) pressure.

This is higher than the 10-3 Pa (10-5 torr) pressure typical for a mass spectrometer source. At 10-3 Pa the mean free path between collisions is approximately 2 meters and ion-molecule reactions are unlikely.

In the CI source, however, the mean free path between collisions is only 10-4 meters and analyte molecules undergo many collisions with the reagent gas.

The reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions. The reagent gas ions in this cloud react and produce adduct ions like CH5

+ ,which are excellent proton donors.

When analyte molecules (M) are introduced to a source region with this cloud of ions, the reagent gas ions donate a proton to the analyte molecule and produce MH+ ions.

The energetic of the proton transfer is controlled by using different reagent gases.

The most common reagent gases are methane, isobutane and ammonia. Methane is the strongest proton donor commonly used with a proton affinity (PA) of 5.7 eV. For softer ionization, isobutane (PA 8.5 eV) and ammonia (PA 9.0 eV) are frequently used.

Acid base chemistry is frequently used to describe the chemical ionization reactions. The reagent gas must be a strong enough Brønsted acid to transfer a proton to the analyte.

Fragmentation is minimized in CI by reducing the amount of excess energy produced by the reaction. Because the adduct ions have little excess energy and are relatively stable, CI is very useful for molecular mass determination.

Mass AnalyzerMass Analyzer

After ions are formed in the source region they are accelerated into the mass analyzer by an electric field. The mass analyzer separates these ions according to their m/z value.

The selection of a mass analyzer depends upon the resolution, mass range, scan rate and detection limits required for an application.

Each analyzer has very different operating characteristics and the selection of an instrument involves important tradeoffs.

Quadrapole Mass FilterQuadrapole Mass Filter

The quadrupole mass spectrometer is the most common mass analyzer.

Its compact size, fast scan rate, high transmission efficiency, and modest vacuum requirements are ideal for small inexpensive instruments.

It ‘filter’ and only allow specific ions to pass.

Most quadrupole instruments are limited to unit m/z resolution and have a mass range of m/z 1000.

Many benchtop instruments have a mass range of m/z 500 but research instruments are available with mass range up to m/z 4000.

Quadrapole Mass AnalyzerQuadrapole Mass Analyzer

•Only compound with specific m/z ratio will resonate along the field (stable path)•Achieved by rapidly varying the voltage

Time of Flight AnalyzerTime of Flight AnalyzerThe time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight tube.

This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less than 500.

These instruments have high transmission efficiency, no upper m/z limit, very low detection limits, and fast scan rates.

For some applications these advantages outweigh the low resolution.

Recent developments in pulsed ionization techniques and new instrument designs with improved resolution have renewed interest in TOF-MS.

This picture shows the working principle of a linear time of flight mass spectrometer.

To allow the ions to fly through the flight path without hitting anything else, all the air molecules have been pumped out to create an ultra high vacuum.

Ions of different m/z ratio travel at different velocities.

The difference in arrival times separate two (or more) ions.

Graph of ion intensity versus mass-to-charge ratio (m/z) (units daltons, Da)

Liquid ChromatographyLiquid Chromatography

Chromatography in which the mobile phase is a liquid.◦The liquid used as the mobile phase

is called the “eluent”.The stationary phase is usually a solid

or a liquid.In general, it is possible to analyze any

substance that can be stably dissolved in the mobile phase.

43

HIGH PERFORMANCE LIQUID HIGH PERFORMANCE LIQUID CHROMATOGRAPHYCHROMATOGRAPHYAnalyze sample in liquid form.The sample carried through a

chromatographic column by a liquid mobile phase.

Parts of HPLC are:- HPLC column- The mobile phases- The stationary phases- Sample introductions- HPLC plumbing- Detectors

High Performance Liquid High Performance Liquid ChromatographyChromatography

http://www.waters.com/WatersDivision/ContentD.asp?watersit=JDRS-6UXGYA&WT.svl=1

From Liquid Chromatography to High From Liquid Chromatography to High Performance Liquid ChromatographyPerformance Liquid Chromatography

Higher degree of separation! Refinement of packing material (3 to 10 µm)

Reduction of analysis time! Delivery of eluent by pump Demand for special equipment that can withstand high pressures

The arrival of high performance liquid chromatography!

47

Pump

Sample injection unit

(injector)

Column

Column oven(thermostatic

column chamber)

Detector

Eluent (mobile phase)

Drain

Data processorDegasser

Flow Channel Diagram for High Flow Channel Diagram for High Performance Liquid ChromatographPerformance Liquid Chromatograph

http://www.lcresources.com/resources/getstart/2c01.htm

What’s the different between What’s the different between HPLC and GC?HPLC and GC?

Solvent Delivery PumpA solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required.

Sample Injection UnitThere is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required.

ColumnThe technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing.

DetectorHigher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.

Detection in HPLCDetection in HPLC

*There are six major HPLC detectors:Refractive Index (RI) DetectorEvaporative Light Scattering Detector (ELSD)UV/VIS Absorption DetectorsThe Fluorescence DetectorElectrochemical Detectors (ECDs)Conductivity Detector

* The type of detector utilized depends on the characteristics of the analyte of interest.

http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4

Refractive Index DetectorRefractive Index Detector

Based on the principle that every transparent substance will slow the speed of light passing through it.◦ Results in the bending of light

as it passes to another material of different density.

◦ Refractive index = how much the light is bent

The presence of analyte molecules in the mobile phase will generally change its RI by an amount almost

linearly proportional to its concentrations.

http://farside.ph.utexas.edu/teaching/302l/lectures/img1154.png

Refractive Index DetectorRefractive Index Detector

Affected by slight changes in mobile phase composition and temperature.

Universal-based on a property of the mobile phase

It is used for analytes which give no response with other more sensitive and selective detectors.◦ RI = general

responds to the presence of all solutes in the mobile phase.

Reference= mobile phaseSample= column effluent Detector measures the

differences between the RI of the reference and the sample.

http://hplc.chem.shu.edu/HPLC/index.html

Evaporative Light Scattering Evaporative Light Scattering Detector (ELSD)Detector (ELSD)

Analyte particles don’t scatter light when dissolved in a liquid mobile phase.

Three steps:1) Nebulize the mobile phase effluent

into droplets. Passes through a needle and

mixes with hydrogen gas. 2) Evaporate each of these droplets.

Leaves behind a small particle of nonvolatile analyte

3) Light scattering Sample particles pass through a

cell and scatter light from a laser beam which is detected and generates a signal.

http://www.sedere.com/WLD/whatis.html

HPLC ChromatogramHPLC Chromatogram

Types of HPLCTypes of HPLC

There are numerous types of HPLC which vary in their separation chemistry.◦All chromatographic modes are

possible: Ion-exchange Size exclusion

Also can vary the stationary & mobile phases:◦Normal phase HPLC◦Reverse phase HPLC

Chromatographic Modes of HPLCChromatographic Modes of HPLC

Ion exchange: ◦ Used with ionic or ionizable samples.◦ Stationary phase has a charged surface.

opposite charge to the sample ions◦ The mobile phase = aqueous buffer ◦ The stronger the charge on the analyte, the more it will

be attracted to the stationary phase, the slower it will elute.

Size exclusion: ◦ Sample separated based on size.◦ Stationary phase has specific pore sizes.◦ Larger molecules elute quickly.◦ Smaller molecules penetrate inside the pores of the

stationary phase and elute later.

Normal Phase HPLCNormal Phase HPLC

Stationary phase: polar, silica particlesMobile phase: non-polar solvent or

mixture of solventsPolar compounds:

◦Will have a higher affinity for the polar, stationary phase

◦Will elute slower Non-polar compounds:

◦Will have a higher affinity for the non-polar, mobile phase

◦Will elute faster

Reverse Phase HPLCReverse Phase HPLC

Stationary phase: non-polar◦ Non-polar organic groups are covalently attached to

the silica stationary particles. Most common attachment is a long-chain

n-C18 hydrocarbon Octadecyl silyl group, ODS

Mobile phase: polar liquid or mixture of liquids

Polar analytes will spend more time in the polar mobile phase.◦ Will elute quicker than non-polar analytes

Most common type of HPLC used today.

http://www.lcresources.com/ resources/getstart/3a01.htm

Advantages of High Advantages of High Performance Liquid Performance Liquid ChromatographyChromatography

High separation capacity, enabling the batch analysis of multiple components

Superior quantitative capability and reproducibilityModerate analytical conditions

◦ Unlike GC, the sample does not need to be vaporized.

Generally high sensitivityLow sample consumptionEasy preparative separation and purification of

samples

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Fields in Which High Fields in Which High Performance Liquid Performance Liquid Chromatography Is UsedChromatography Is Used

Biogenic substances◦ Sugars, lipids, nucleic

acids, amino acids, proteins, peptides, steroids, amines, etc.

Medical products◦ Drugs, antibiotics, etc.

Food products◦ Vitamins, food

additives, sugars, organic acids, amino acids, etc.

Environmental samples◦ Inorganic ions◦ Hazardous organic

substances, etc. Organic industrial

products◦ Synthetic polymers,

additives, surfactants, etc.

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HPLC ApplicationsHPLC Applications

Can be used to isolated and purify compounds for further use.

Can be used to identify the presence of specific compounds in a sample.

Can be used to determine the concentration of a specific compound in a sample.

Can be used to perform chemical separations◦ Enantiomers (mirror image molecular

structure)◦ Biomolecules

HPLC ApplicationsHPLC Applications*HPLC has an vast amount of current & future applications*

Some uses include:◦ Forensics: analysis of explosives, drugs, fibers, etc.◦ Proteomics: can be used to separate and purify protein

samples Can separate & purify other biomolecules such as:

carbohydrates, lipids, nucleic acids, pigments, proteins, steroids

◦ Study of disease: can be used to measure the presence & abundance of specific biomolecules correlating to disease manifestation.

◦ Pharmaceutical Research: all areas including early identification of clinically relevant molecules to large-scale processing and purification.

Ion Exchange Ion Exchange ChromatographyChromatographyThe principle- To separate inorganic ions, both

cations and anions.- Separate based on exchange of

ions in the stationary phase.- The stationary phase consists of

beads made of polystyrene polymer crosslinked with divinylbenzene.

ElectrophoresisElectrophoresis

ElectrophoresisElectrophoresis

• Electrophoresis is a separations technique that is based on the mobility of ions in an electric field.

• Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.

• For safety reasons one electrode is usually at ground and the other is biased positively or negatively.

• Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

ElectrophoresisElectrophoresis• An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube.

• Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments.

• After a separation is completed the support is stained to visualize the separated components.

• Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At this pH the protein is natural and no longer migrates, i.e, it is focused into a sharp band on the gel.

Capillary ElectrophoresisCapillary Electrophoresis

Schematic of capillary electrophoresis

How Does CE Work?How Does CE Work?

• Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length. The applied potential is 20 to 30 kV.

•Due to electro osmotic flow, all sample components migrate towards the negative electrode. A small volume of sample (10 nL) is injected at the positive end of the capillary and the separated components are detected near the negative end of the capillary.

•CE detection is similar to detectors in HPLC and include absorbance, fluorescence, electrochemical, and mass spectrometry.

•The capillary can also be filled with a gel, which eliminates the electro osmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection.

well

Sample(blue)

Gel Electrophoresis(sodium dodecyl sulfate

polyacrylamide gel electrophoresis)

Is a vertical gel electropheresisTo separate proteins according to their size

How Does GE Work?How Does GE Work?

• Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.• •This phenomenon is called sieving.

•Electrophoresis refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix.

•By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass

•Molecules move toward the (negatively charged) cathode if positively charged or toward the (positively charged) anode if negatively charged.

Gel stainingGel staining

immersed in solution e.g ethidium bromide

ResultResult

marker

reference

Sample 1Sample 2 Sample 3

Bigger protein

Smaller protein