Dr.Nawfal Hussein Aldujaili

Is a technique used to separate and identify thecomponents of a mixture.

Works by allowing the molecules present in the mixture todistribute themselves between a stationary and a mobilemedium.

Molecules that spend most of their time in the mobilephase are carried along faster.

Chromatography is a physical method of separation in which the

components to be separated are distributed between two Phases

one of which is stationary (stationary phase) while the other (the

mobile phase) moves through it in a definite direction.

The chromatographic process occurs due to differences in the

distribution constant of the individual sample components

Components:

• mobile phase: a solvent that flows through the supporting medium

• stationary phase: a layer or coating on the supporting medium that

interacts with the analytes

• supporting medium: a solid surface on which the stationary phase is

bound or coated

.

Chromatography

Classification according to the packing of the stationary phase:

Thin layer chromatography (TLC): the stationaryphase is a thin layer supported on glass, plasticor aluminium plates.

Paper chromatography (PC): the stationaryphase is a thin film of liquid supported on aninert support.

Column chromatography (CC): stationary phaseis packed in a glass column.

Chromatography

This is a form of solid – liquid chromatography. The stationary phase is a

porous, finely divided solid which adsorbs molecules of the test substance

on its surface due to dipole- dipole interactions, hydrogen bonding and / or

van der Waals interactions .

The range of adsorbents is limited e.g. polystyrene-based resins ( for non-

polar molecules .Silica, aluminum oxide and calcium phosphate ( for polar

molecules ).Most adsorbents must be activated by heating to 110-120 ₒC

before use ,since their adsorptive capacity is significantly decreased in the

presence of bound water.

Adsorption chromatography can be carried out in column or thin-layer

form, using a wide range of organic solvents .

o Technique which separates solutes based on their

adsorption to solid

o is a method for identifying substances and testing

the purity of compounds.

o TLC is a useful technique because it is relatively

quick and requires small quantities of material.

This is based on the partitioning of a substance between two liquid

phases. in this instance the stationary and mobile phases.

Substances which are more soluble in the mobile phase will pass rapidly

through the system while those which favor the stationary phase will

retarded .

In normal phase partition chromatography the stationary phase is a polar

solvent , usually water ,supported by solid matrix (e.g. cellulose fibers in

paper chromatography ) and the mobile phase is an immiscible, non-

polar organic solvent.

For reverse-phase partition chromatography ,the stationary phase is a

non-polar solvent (e.g. C18 hydrocarbon, such as octadecylsilane) which

chemically bonded to a porous support matrix (e.g. silica ), while the

mobile phase can be chosen from a wide range of polar solvents .usually

water or an aqueous buffered solution containing one or more organic

solvents .e.g. acetonitrile .

Solutes interact with the stationary phase through non-polar interactions

and so the least polar solutes elute last from the column .

Applications of partition Chromatography

How can ions be separated by using partition Chromatography?

Normal Phase LC and Reversed-phase LC

Stationary phase: polar non-polar

A method of partition chromatography using filter paper strips as

carrier or inert support.

The factor governing separation of mixtures of solutes on filter paper

is the partition between two immiscible phases.

One is usually water adsorbed on cellulose fibres in the paper

(stationary phase).

The second is the organic solvent flows past the sample on the paper

(stationary phase).

o Multiple chromatography includes all procedures in which thedevelopment is repeated after one development is completed.

o A- multiple development: the chromatogram is repeatedly developed inthe same direction and thus the complete resolution of two or moresubstances which have Rf values close together can be obtained.

o As the mobile phase one can use either the same solvent system ordifferent solvent systems.

o B- two-dimensional chromatography: When large numbers ofsubstances are to be separated on a single chromatogram. Developmentin a direction perpendicular to the first, and with a solvent systemdifferent from that used initially is often necessary.

o The sample is applied on one corner of a square piece of paper and afterdevelopment with the first solvent, the paper is dried , rotated 90o anddeveloped in the second direction.

o Usually, different types of solvents systems are used in each direction. Itis essential that the first solvent be completely volatile.

GFC is a separation based on size. It is also called molecular exclusion or

gel permeation chromatography.

The stationary phase consists of porous beads with a well-defined range of

pore sizes.

Proteins that are small enough can fit inside all the pores in the beads and

are said to be included. These small proteins have access to the mobile

phase inside the beads as well as the mobile phase between beads and

elute last in a gel-filtration separation.

Proteins that are too large to fit inside any of the pores are said to be

excluded. They have access only to the mobile phase between the beads

and, therefore, elute first.

Proteins of intermediate size are partially included—meaning they can fit

inside some but not all of the pores in the beads. These proteins will then

elute between the large (“excluded”) and small (“totally included”) proteins.

o Separate proteins by molecular weight

o Determination of molecular weight

o Separation of salts and other small molecules from a protein sample.

o Buffer exchange

o Ion-exchange chromatography separates protein molecules based on their

electrical charge.

o Applicable to the separation of almost any type of charged molecule, from

large proteins to small nucleotides and amino acids.

o consisting of a mobile phase and a stationary phase and exchanging ions

between the phases depending on their charges.

o Anion-exchange chromatography is used to separate negatively charged

proteins (anions). The column has positively charged groups on the inert

phase, which bind negative sites on the protein. Example: DEAE groups.

o Cation-exchange chromatography is used to separate positively charged

proteins (cations). Example :carboxymethyl (CM) groups covalently linked to

a cellulose or agarose matrix.

o Water softening: Removal of Ca2+, Mg2+ & other multivalent ionscausing hardness of water by filtration through a layer of strong cationresin.

o Water demineralization: Removal of cations & anions dissolved inwater. Usually carried by the two step technique in which two columns ofstrongly acid cation exchanger in [H+] form & strongly basic anionexchanger in [OH-] form are used in sequence.

o Neutralization: Cationic exchanger in [H+] can be used to neutralizealkali hydroxide & anionic exchanger in [OH+] form to neutralize theacidity.

o Separation of electrolytes from non-electrolytes.

o Separation of carbohydrates & their derivatives:

o Chromatofocusing (isoelectric focusing by ion-exchange chromatography)

is a procedure in which the biomolecules separate according to their

isoelectric points in an ion-exchange column.

o The proteins are allowed to bind to the ion-exchange bed in an

equilibrating buffer of low ionic strength and eluted with a polybuffer at a

pH lower than that of the starting buffer. The polybuffer, containing

cationic and amphoteric buffering species, makes a pH gradient, which is

used to elute bound proteins from the ion-exchange resin in order of their

isoelectric points.

o Proteins with pI differences as small as 0.05 pH unit can be resolved in

chromatofocusing. Chromatofocusing is useful to separate isoforms of

closely spaced pIs after affinity chromatography.

The most powerful procedure for the purification of proteins is

affinity chromatography.Under ideal conditions the target protein can

be purified in a single step.

The purified protein is obtained in a biologically active form, as the

purification is based on its biospecific interaction with an

immobilized ligand. The procedure involves the adsorption of crude

protein extract onto a ligand conjugated solid support (commonly

called a matrix).

Proteins in the extract having a binding site complementary to the

ligand remain bound to the matrix, while the unbound components

are removed by washing the matrix with a buffer. The bound protein

is eluted with an elution buffer.

Affinity chromatography

Insert matrix

Antibody

Enantiomer with

Low affinity to the

antibody

Enantiomer with

high affinity to the

antibody

Antibody-antigen

For biomolecule

separation

Molecules are separated according to their hydrophobic interaction

between the stationary phase and mobile phase.

Proteins and peptides interact with the hydrophobic surfaces of the matrix

by adsorption in an aqueous buffer.

Salt solutions are often used to mediate the binding of sample molecules

to a hydrophilic matrix substituted with a hydrophobic ligand.

Ammonium sulfate is often used in the equilibrating buffer to increase the

hydrophobic interaction of the proteins.

Protein molecules differ in their surface hydrophobicity according to the

distribution of hydrophobic or non-polar amino acid residues.

The matrix is silica that has been substituted with n-alkyl chains, usually

C4, C8, and C18. The mobile phase is usually a mixture of water and a less

polar organic solvent.

The name “reversed-phase” is introduced to distinguish it from “normal

phase” chromatography, in which the matrix is silica and the mobile phase

is a non-polar solvent such as hexane.

In RPC ,water present in the mobile phase is more polar than the stationary

phase, C8 or C18-derivatized silica.

RPC is rarely used for the purification of biologically active protein

molecules.

RPC used to separate peptides obtained from chemically or enzymatically

digested purified protein and other applications where loss of the protein’s

biological activity is not a concern. Separation of peptides is commonly

achieved on reversed-phase columns using a high-performance liquid

chromatography system rather than a conventional chromatography

system.

.

HIC and RPC are based on the interactions between the hydrophobic

moieties of a sample and an insoluble immobilized hydrophobic group. In

HIC, the immobilized matrix is hydrophilic (e.g., Sepharose) substituted

with short chain phenyl or octyl non-polar groups.

In RPC, the matrix is silica substituted with longer n-alkyl chains such as

C8 and C18. In HIC, the mobile phase is an aqueous salt solution,

whereas in RPC, the mobile phase is usually a mixture of water and a less

polar organic modifier. Separation on HIC occurs in non-denaturing

conditions, but separation on RPC is achieved in a mixture of aqueous

and organic solvents, which often denature proteins

High-performance Liquid Chromatography:

All types of liquid chromatography both adsorption and partition

chromatography can be performed through HPLC as in conventional

chromatography

HPLC is more widely used for the separation of small molecules but can

be applied to the separation of proteins in some applications.

HPLC uses high pressure to force the mobile phase through a closed

column packed with micromeres sized particles. This allows rapid

separation of complex mixtures. Several operating modes OF HPLC are

possible. These are:

Normal phase (NPHPLC); the sample should be soluble in a hydrophobic

solvent, e.g. hexane. and should be non-ionic. The mobile phase is non-

polar while the stationary phase is polar, e.g. silica, cyano, amino.

Reversed phase (RPHPLC): the sample should be soluble in water or a

poIar organic solvent, e.g. methanol, and should be non-ionic. The mobile

phase is polar while the stationary phase is non-polar, e.g. C18 (ODS),

C8 (octyI), phenyl.

• Reverse phase HPLC can be used to separate a wide range of polar,

non-polar, and ionic molecules such as proteins, sugars, and

oligosaccharides, vitamins, peptides , amino acids, and lipids, etc.

• The speed, sensitivity and versality of HPLC make this method of choice

for the separation of many small molecules of biological interest. normally

using reverse phase partition chromatography .Separation of

Macromolecules (especially proteins and nucleic acids )usually requires

biocompatible systems in which stainless steel components are replaced

by titanium, glass, or fluoroplastics, using lower pressures to avoid

denaturation

HPLC (high-performance liquid chromatography)

Yellow is more polar compound , Red less polar compound

,Blue is nonpolar compound

Solvent

Refractiv

e Index

Viscosity

(cP)

Boiling

Point (oC)

Polarity

Index (P)

Eluent

Strength (eo)

Fluoroalkanes 1.27-1.29 0.4-2.6 50-174 <-2 -0.25

cyclohexane 1.423 0.90 81 0.04 -0.2

N-hexane 1.327 0.30 69 0.1 0.01

1-chlorobutane 1.400 0.42 78 1.0 0.26

Carbon tetrachloride 1.457 0.90 77 1.6 0.18

i-propyl ether 1.365 0.38 68 2.4 0.28

toluene 1.494 0.55 110 2.4 0.29

Diethyl ether 1.350 0.24 35 2.8 0.38

tetrahydrofuran 1.405 0.46 66 4.0 0.57

chloroform 1.443 0.53 61 4.1 0.40

ethanol 1.359 1.08 78 4.3 0.88

Ethyl acetate 1.370 0.43 77 4.4 0.58

dioxane 1.420 1.2 101 4.8 0.56

methanol 1.326 0.54 65 5.1 0.95

acetonitrile 1.341 0.34 82 5.8 0.65

nitromethane 1.380 0.61 101 6.0 0.64

Ethylene glycol 1.431 16.5 182 6.9 1.11

water 1.333 0.89 100 10.2 large

Selection of a mobile phase for a particular LC application can be done by using

various tables that summarize properties for common LC solvents:

LIKE DISSOLVES LIKE!

• Fast protein liquid chromatography (FPLC) is a similar concept as

HPLC, but specifically designed for protein separations. FPLC also uses

special columns and pumps to achieve high flow rates and therefore faster

separations. In general the flow rates obtained with FPLC are not as great

as those achieved with HPLC.

• e.g. the Pharmacia FPLC system .such separations are carried out using

ion –exchange ,gel permeation and /or hydrophobic interaction

chromatography

Fast protein liquid chromatography (FPLC)

o A gaseous solute (or the vapor from a volatile liquid) is carried by the

gaseous mobile phase.

o In gas-liquid partition chromatography, the stationary phase is a non-volatile

liquid coated on the inside of the column or on a fine support.

o In gas solid adsorption chromatography, solid particles hat adsorb the solute

act as the stationary phase.

o A volatile liquid is injected through a septum into a heated port. Which

volatilizes the sample.

o A gaseous mobile phase carries the sample through the heated column, and

the separated components are detected and recorded.

o GLC is used to separate volatile. non-polar compounds ,substances with

polar groups must be converted to less polar derivatives prior to analysis, in

order to prevent adsorption on the column, resulting in poor resolution and

peak tailing.

Gas chromatography

o A centrifuge is a device for separating particles from a solution

according to their sedimentation rate, which depends on factors like

size, shape, density, viscosity of the medium, and centrifugal force

(rotor speed).

o This process of separation of particles based on its sedimentation rate

is called centrifugation.

o In biology, the particles are usually cells, sub-cellular organelles,

viruses, and large molecules such as proteins and nucleic acids. The

rate of sedimentation will be directly proportional to the molecular

weight or size, if all other factors are constant.

o Ultracentrifugation is carried out at speed faster than 30,000 rpm.

Highspeed centrifugation is at speeds between 10,000 and 30,000

rpm. Low-speed centrifugation is at speeds below 10,000 rpm

(mostly between 3,000 to 9,000 rpm).

Centrifugation

o The acceleration of a centrifuge is usually expressed as relative

centrifugal field (g) or RCF. RCF depends on the speed of the rotor

represented as Revolution per minute or RPM (n) and radius of the rotor

(r). The relation between RPM and RCF is given by the following equation.

Centrifugation

o Native PAGE is generally carried out to determine the molecular

weight of a protein at its active state in tertiary or quaternary structure.

o To determine the number of subunits and the nature of the Three-

dimensional structure (whether having one or more subunits) and the

molecular weight of each subunit, the native gel experiment should be

followed by a denaturing gel electrophoresis.

o SDS-PAGE or denaturing PAGE for separating protein subunits after

they have been denatured by heating under reducing conditions and

bound with the non-ionic detergent SDS.

o The SDS binds to the unfolded proteins giving all proteins a similar

shape and a uniform charge-to-mass ratio.

o The polypeptides coated with SDS separate by size.

o Used for estimation of molecular size

o Used for monitoring the purification process

Proteins can be separated electrophoretically on the basis of their relative

contents of acidic and basic residues.

This method of separating proteins according to their isoelectric point

The isoelectric point (pl) of a protein is the pH at which its net charge is

zero. At this pH, its electrophoretic mobility is zero .

Suppose that a mixture of proteins undergoes electrophoresis in a pH

gradient in a gel. Each protein will move until it reaches a position in the

gel at which the pH is equal to the pI of the protein.

The pH gradient in the gel is formed first by subjecting a mixture of

polyampholytes (small multicharged polymers) having many pI values to

electrophoresis.

Isoelectric focusing can readily resolve proteins that differ in one net

charge and can be separated .

Isoelectric focusing can be combined with SDS–PAGE to obtain very

high resolution separations.

A single sample is first subjected to isoelectric focusing. This single-lane

gel is then placed horizontally on top of an SDS–polyacrylamide slab.

The proteins are thus spread across the top of the polyacrylamide gel

according to how far they migrated during isoelectric focusing.

They then undergo electrophoresis again in a perpendicular direction

(vertically) to yield a two dimensional pattern of spots. In such a gel,

proteins have been separated in the horizontal direction on the basis of

isoelectric point and in the vertical direction on the basis of mass.

It is remarkable that more than a thousand different proteins in the

bacterium Escherichia coli can be resolved in a single experiment by

two-dimensional electrophoresis

Two-dimensional gel electrophoresis. (A) A protein sample is initially

fractionated in one dimension by isoelectric focusing .The isoelectric focusing

gel is then attached to an SDS–polyacrylamide gel, and electrophoresis is

performed in the second dimension, perpendicular to the original separation.

Proteins with the same pI are now separated on the basis of mass. (B) Proteins

from E. coli were separated by two-dimensional gel electrophoresis, resolving

more than a thousand different proteins.

Spectroscopic

Techniques

o Used for the qualitative and quantitative estimation of biomolecules

such as proteins, sugars, carbohydrates, amino acids, nucleic acids,

vitamins, flavonoids, isoquinoline alkaloids, and coumarins etc

o technique for measuring the absorption of radiation in the visible

and UV regions of the spectrum. A spectrophotometer is an

instrument designed to allow precise measurement at a particular

wavelength, while a colorimeter is a simpler instrument, using filters

to measure broader wavebands (e.g. light in the green, d or blue

regions of the visible spectrum).

There are two source lamps, one a tungsten filament that provides

wavelengths of visible range (400-700nm) and a hydrogen or

deuterium lamp that forms the source of light in the UV range (200-

400 nm).

Glass and pIastic absorb UV light, so quartz cells must be used at

wavelengths below 300nm.

o Atoms of certain metals will absorb and emit radiation of specific

wavelengths when heated in a flame, in direct proportion to the

number of atoms present.

o Atomic spectrophotometric techniques measure the absorption or

emission of particular wavelengths of UV and visible light, to identify

and quantify such metals.

Measuring of frequencies produced by the vibration of chemical

bonds (stretching and bending).

For identification of groups or atoms in simple compound but

inappropriate for quantitative measurement.

Determines different functional groups, e.g.,—C¼O, —OH, —NH2,

aromaticity, and so on, present in a molecule.

FT(Fourier transformation ) (FTIR) for interconverting frequency

functions and time or distance function.

Computer use FT to convert the pattern into spectrum in under

minutes

Applications (Identification of drugs, small peptides ,pollutants,food

contamitants

Involves the absorption of energy by specific atomic nuclei in magnetic

fields and is probably the most powerful tool available for the structural

determination of molecules

NMR: Reveals information on the number and types of protons and

carbons (and other elements like nitrogen, fluorine, etc.) present in the

molecule, and the relationships among these atoms .

NMR and x-ray crystallography have greatly enriched understanding of

how proteins fold, recognize other molecules, and catalyze chemical

reactions.

NMR reveals the structure and dynamics of proteins in solution.

The technique is also known as the x-ray diffraction technique.

powerful technique used to study the three-dimensional structure of

crystals including macromolecules such as protein and nucleic acids.

As the x-ray passes through the crystal, it is diffracted by the electrons in

each atom present in the molecule. The numbers of electrons in the

atom determine the intensity of the scattering of x-ray. The intensity of x-

rays scattered by carbon will be six times greater than that of a hydrogen

atom

A series of patterns taken from different angles contains the information

needed to determine the three-dimensional structure.

Finally, what we see as the result of a crystallographic experiment is not

really a picture of the atoms, but a map of the distribution of electrons in

the molecule

Based on the fragmentation of compounds into smaller units. The resulting

positive ions are then separated according to their mass-to charge ratio .

A substance is bombarded with an electron beam having sufficient energy

to fragment the molecule. The positive fragments, which are produced

(cations and radical cations) are accelerated in a vacuum through a

magnetic field and are sorted on the basis of mass-to-charge ratio.

The value m/e is equivalent to the molecular weight of the fragment. The

analysis of mass spectroscopy information involves the reassembling of

fragments, working backward to generate the original molecule

A mass spectrometer creates charged particles (ions) from molecules. It

then analyzes those ions to provide information about the molecular weight

of the compound and its chemical structure.

Determine the sequence of proteins and peptides.

Analysis of nucleic acids.

Identification and molecular weight determination of comparatively small

organic molecules.

Identify the structure of other biomolecules such as lipids, carbohydrates,

oligonucleotides, etc.

Perform forensic analysis to detect the presence of certain substances.

Detect the presence of banned substances in athletes.

Determine the composition of rocks and thus determine their age and

origin.

Identify isotopes of various elements.

It is possible to do two-dimensional gel electrophoresis of proteins of a cell

(to separate the few thousand different proteins) and identify them using

mass spectrometer. This approach of protein analysis and identification is

one of the important techniques of what is called proteomics—the study of

the complete protein complement of a cell.

Mass Spectrometry used in conjunction with chromatographic methods can

provide a powerful tool for identifying the components of complex mixture, e.g

pharmaceuticals

ELISA widely used to detect and estimate the concentration of a protein

in a sample.

It is based on covalently linking an enzyme to a known antigen or

antibody, reacting the enzyme-linked material with the patient's

specimen, and then assaying for enzyme activity by adding the substrate

of the enzyme

Involves measurement of a color change, which results from addition of

substrate that is specific for the enzyme; the intensity of the color is

proportional to the amount of antigen detected in the sample.

o For analysis of antigen: antibody is attached to plate and captures

antigen, and an enzyme-linked antibody is used to detect and quantitate

the antigen.

o For analysis of antibody: antigen is attached to plate, antibody binds, and

an enzyme-linked anti-antibody is used to detect and quantitate the

antigen.

The ELISA is used in many different fields. Diagnostic kits that rely on the

ELISA are produced for clinical diagnosis of human disease, dairy and

poultry diseases, and even for plant diseases.

ELISA kits can detect the presence of minute amounts of pathogenic

viruses or bacteria.

Clinical ELISA kits detect various disease markers.

Microscopy

o The fluorescence microscope is based on the phenomenon that certain

material emits energy detectable as visible light when irradiated with the

light of a specific wavelength. The sample can either be fluorescing in its

natural form such as chlorophyll and some minerals, or treated with

fluorescing chemicals.

o In fluorescence microscopy, the sample you want to study is itself the light

source. The technique is used to study specimens, which can be made to

fluoresce.

o used to locate and quantify specific molecules or organelles in a cell. It

can be applied to study the various activities of living cells.

o Fluorescence microscopy is a rapidly expanding technique, both in the

medical and biological sciences. The technique has made it possible to

identify cells and cellular components with a high degree of specificity. For

example, certain antibodies and disease conditions or impurities in organic

material can be studied with the fluorescence microscope.

o A phase-contrast microscope uses the fact that the light passing through a

transparent part of the specimen travels slower and, due to this its phase,

is shifted compared to the uninfluenced light. This makes the transparent

object shine out in contrast to its surroundings.

o widely used for examining such specimens as biological tissues without

staining. It is a type of light microscopy that enhances contrasts of

transparent and colorless objects by influencing the optical path of light.

o A phase-contrast microscope is able to show components in a cell or

bacteria.

o is a vital instrument in biological and medical research. When dealing with

transparent and colorless components in a cell, dyeing is an alternative

but at the same time stops all processes in it. The phase-contrast

microscope has made it possible to study living cells, and cell division is

an example of a process that has been examined in detail with it.

o These microscopes have a dark-field condenser instead of the normal

one. The dark-field condenser focuses light obliquely on the object, so

that only the deflected light enters the objective.

o The entire background field appears black and only the object is bright

against the dark background.

o The contrast between specimen and the background permits the

visualization of even the smallest of objects such as bacteria and virus

particles.

o The is a type of electron microscope that shows the surface images of a

sample. In the SEM, the structure of a surface is studied using a stylus that

scans the surface at a fixed distance from it.

o The study of surfaces is an important part of physics, with particular

applications in semiconductor physics and microelectronics. In chemistry,

surface reactions also play an important part, for example, in catalysis.

o The SEM works best with conducting materials, but it is also possible to fix

organic molecules on a surface and study their structures. For example,

this technique has been used in the study of DNA molecules.

o SEM offer higher magnification, resolution and depth of field

o SEM apply information about

Topography (The surface features of an object and its texture)

Morphology (The shape and size of the particles making up the object )

Composition (The elements and compounds that the object is composed of

and the relative amounts of them)

Depth of field

Depth of field: The range of heights (distances) on the specimen

surface for which the image is in focus

Optical

microscopeSEM

o TEMs use electrons as the “light source,” and their much lower

wavelength makes it possible to get a resolution a thousand times better

than with a light microscope. You can see objects to the order of a few

angstrom (10-10 m).

o You can study small details in the cell or different materials down to near

atomic levels. The possibility of high magnifications has made the TEM

a valuable tool in medical, biological, and materials research.

o A “light source” (electron source) at the top of the microscope emits the

electrons that travel through the vacuum in the column of the

microscope. Instead of glass lenses focusing the light in the light

microscope, the TEM uses electromagnetic lenses to focus the

electrons into a very thin beam. The electron beam then travels through

the specimen studied.

o The image can be studied directly by the operator or photographed with

a camera.

o AFM is suitable for imaging surfaces coated with biological entities such

as DNA or proteins. The AFM operates in air and not under a vacuum.

Some versions of the instrument also allow operation in liquid, which is

very advantageous when imaging biological samples that often need

buffers to remain biologically active.

o The AFM measures the interaction force (attractive or repulsive) between

the probe and the surface. The solid probe is located at the end of a very

flexible cantilever; an optical system detects the deflection of a laser beam

that bounces off the reflective back of the cantilever, thus reporting

cantilever fluctuations, which are proportional to the applied force.

o The probe is continuously moved along the surface and the cantilever

deflection is constantly monitored. A feedback loop continuously changes

the height of the probe on the surface in order to keep the applied force

constant. The vertical movement of the probe is recorded to create

a topographic map of the surface under study.

THANK YOU