Gas chromatography (GC) separates volatile compounds using an inert gas as the mobile phase. The sample is injected into a heated port to volatilize it. The gas mobile phase carries the volatilized sample through a heated column coated with a stationary phase that interacts with analytes. Components are separated based on differences in volatility and affinity for the stationary phase, then detected and recorded. GC is useful for separating volatile, non-polar compounds.

1. Dr.Nawfal Hussein Aldujaili

3.  Is a technique used to separate and identify the
components of a mixture.
 Works by allowing the molecules present in the mixture to
distribute themselves between a stationary and a mobile
medium.
 Molecules that spend most of their time in the mobile
phase are carried along faster.

4.  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

5. Classification according to the packing of the
stationary phase:
 Thin layer chromatography (TLC): the stationary
phase is a thin layer supported on glass, plastic
or aluminium plates.
 Paper chromatography (PC): the stationary
phase is a thin film of liquid supported on an
inert support.
 Column chromatography (CC): stationary phase
is packed in a glass column.
Chromatography

9.  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 .

10. 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.

13.  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 .

14. 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

15.  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).

18. o Multiple chromatography includes all procedures in which the
development is repeated after one development is completed.
o A- multiple development: the chromatogram is repeatedly developed in
the same direction and thus the complete resolution of two or more
substances which have Rf values close together can be obtained.
o As the mobile phase one can use either the same solvent system or
different solvent systems.
o B- two-dimensional chromatography: When large numbers of
substances are to be separated on a single chromatogram. Development
in a direction perpendicular to the first, and with a solvent system
different from that used initially is often necessary.
o The sample is applied on one corner of a square piece of paper and after
development with the first solvent, the paper is dried , rotated 90o and
developed in the second direction.
o Usually, different types of solvents systems are used in each direction. It
is essential that the first solvent be completely volatile.

19.  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.

20. 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

21. 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.

22. o Water softening: Removal of Ca2+, Mg2+ & other multivalent ions
causing hardness of water by filtration through a layer of strong cation
resin.
o Water demineralization: Removal of cations & anions dissolved in
water. Usually carried by the two step technique in which two columns of
strongly acid cation exchanger in [H+] form & strongly basic anion
exchanger in [OH-] form are used in sequence.
o Neutralization: Cationic exchanger in [H+] can be used to neutralize
alkali hydroxide & anionic exchanger in [OH+] form to neutralize the
acidity.
o Separation of electrolytes from non-electrolytes.
o Separation of carbohydrates & their derivatives:

23. 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.

24.  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.

25. Affinity chromatography
Insert matrix
Antibody
Enantiomer with
Low affinity to the
antibody
Enantiomer with
high affinity to the
antibody
Antibody-antigen
For biomolecule
separation

26.  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.

27.  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.


28.  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

29. 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.

30. • 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)

31. Yellow is more polar compound , Red less polar compound
,Blue is nonpolar compound

32. 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!

35. • 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)

36. 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

37. 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

38. 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

41. 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

42.  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 .

44.  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

45. 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.

46. Spectroscopic
Techniques

47. 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.

48. 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.

49.  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

50.  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.

55.  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

57.  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.

58.  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.

59. Mass Spectrometry used in conjunction with chromatographic methods can
provide a powerful tool for identifying the components of complex mixture, e.g
pharmaceuticals

61.  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.

62.  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.

66. Microscopy

68. 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.

69. 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.

70. 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.

71. 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)

73. Depth of field
Depth of field: The range of heights (distances) on the specimen
surface for which the image is in focus
Optical
microscope
SEM

74. 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.

75. 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.

78. THANK YOU