3. CHROMATOGRAPHY
• Chromatography is an analytical
technique commonly used for
separating a mixture of chemical substances into
its individual components, so that the individual
components can be thoroughly analyzed.
• It is also defined as the process of separation of
the individual components of a mixture based on
their relative affinities towards stationary and
mobile phases.
4. • The mixture is dissolved in a fluid called
the mobile phase, which carries it
through a structure holding another
material called the stationary phase.
5. • Its name is derived from two words: "chromo" meaning
colour, and "graphy" meaning writing. in other words, colour
bands are formed in the procedure, which are then measured or
analysed. these bands are due to separation of individual
compounds at different lengths on the column, as seen in
column chromatography and on paper in paper chromatography.
ORIGIN OF NAME:-
6. • The word “chromatography” was firstly coined by
the italian-born scientist Mikhail tsvet in 1903 in
russia .
• He continued to work with chromatography in the
first decade of the 20th century, primarily for the
separation of plant pigments
• Chromatography technique developed
substantially as a result of the work of archer john
porter martin and richard laurence millington
synge during the 1940s and 1950s.
HISTROY:-
7. • Chromatography is based on the principle of separation of
compounds into different bands (color graphs) and then
identification of those bands.
• Basically, the samples are subjected to flow by mobile
liquid onto or through the stable stationary phase. the
sample components are separated into fractions based on
their relative affinity towards the two phases during their
travel.
• Thefraction with a greater affinity to stationary layer
travels slower and at a shorter distance, while that with a
lesser affinity travels faster and longer
PRINCIPLE:-
8. TYPES OF CHROMATOGRAPHY
Types of chromatography to
be discussed:-
• Paper Chromatography
• Column Chromatography
• Gel Filtration
Chromatography
• Ion Exchange
Chrmoatography
• Affinity Chromatography
9. • Paper chromatography is an analytical chemistry technique for
separating and identifying color mixtures.
• Substances are distributed between stationary phase and a mobile
phase.
• Stationary phase is usually a piece of filter paper and mobile phase is
colors that travels up stationary phase.
• Components of the samples will separate readily according to how
strongly they absorb on the stationary phase vs. how readily they
dissolve in the mobile phase.
PAPER CHROMATOGRAPHY:
12. • The principle involved is partition chromatography
wherein the substances are distributed or partitioned
between liquid phases. one phase is the water, which is
held in the pores of the filter paper used; and other is the
mobile phase which moves over the paper. the compounds
in the mixture get separated due to differences in their
affinity towards water (in stationary phase) and mobile
phase solvents during the movement of mobile phase under
the capillary action of pores in the paper.
PRINCIPLE:-
14. • Ink is a solution containing a number of different molecules.
These different molecules have different characteristics such
as size and solubility. Solubility is a molecule's ability to
dissolve in a particular solvent such as alcohol, water or nail
polish remover. Because of their different characteristics,
each molecule travels at a different speed when pulled along
the piece of paper toweling by the solvent. The lightest
particles, which are not necessarily the lightest coloured
particles, move more quickly and a greater distance than the
heavier particles. Thus, all of the pigments that make up an
ink sample are separated out.
WHAT HAPPEN IN PAPER
CHROMATOGRAPHY:-
15. • Paper chromatography is specially used for the
separation of a mixture having polar and non-polar
compounds.
• separation of amino acids.
• Determine organic compounds, biochemical in urine
• In the pharma sector it is used for the determination of
hormones, drugs, etc.
• Sometimes it is used for evaluation of inorganic
compounds like salts and complexes
USES AND APPLICATIONS:-
17. Column chromatography is a preparative technique used to
purify compounds depending on their polarity or hydrophobicity.
In column chromatography, a mixture of molecules is separated
based on their differentials partitioning between a mobile phase
and a stationary phase.
Stationary phase:- is confined to a glass or plastic tube
and is mostly silica gel
Mobile phase:- (a solvent or buffer) is allowed to flow
through the solid adsorbent.
Sample:- to be analyzed is layered on top of the
column.
“2.COLUMN CHROMATOGRAPHY”
18. • Russian-italian botanist Mikhail Tsvet.
• Tsvet applied his observations with filter paper extraction to the
new methods of column fractionation for separating the
components of petroleum.
“HISTORY”
19. • The rate at which the components of a mixture are
separated depends on the
• activity of the adsorbent
• polarity of the solvent.
• If the activity of the adsorbent is very high and polarity of
the solvent is very low, then the separation is very slow but
gives a good separation. On the other hand, if the activity
of adsorbent is low and polarity of the solvent is high the
separation is rapid but gives only a poor separation,
“PRINCIPLE”
20.
21. “PROCEDURE”
• Packing the column
• Loading the column
• Eluting The column
• Collecting the Eluent
• Detection of Eluting Components
26. • Analytes larger than the
pores cannot enter the
interior of the gel beads,
so they are limited to the
space between the beads.
As a result, they are not
slowed in their progress
through the column and elute rapidly in a single zone.
• Small molecules capable of diffusing in and out of the beads have a
much larger volume available to them. Therefore, they are delayed in
their journey through the column bed. Molecules of intermediate size
migrate through the column at a rate somewhere between those for
large and small molecules.
“PRINCIPLE”
27. 1. Select columns not greater
than 100 cm in length.
2. Selection the proper gel,
which act as stationary phase
3.Layer sample on the column.
4.Add buffer to wash proteins
through the column.
5. Eluting the sample
6. At th end collect the fraction.
“HOW DOES IT WORKS?”
28. • Fractionation of molecules and complexes within a predetermined
size range
• Size analysis and determination
• Removal of large proteins and complexes
• Desalting
• Removal of small molecules
• Assessment of sample purity
• purification.
• molecular weight determination and quantitative analysis of
molecular interactions.
“APPLICATIONS”:-
31. • Allows the separation of ions and polar molecules based on the
charge properties of the molecules.
• A type of column chromatography
• Useful in the separation of charge compounds like proteins
differing by only one charged amino acid.
(ion exchanger components)
(buffered sollution)
( positive/negative)
“INTRODUCTION”:-
32. Polymers that are capable of exchanging particular ions within the polymer with ions in a
solution that is passed through them
A-polystyrene: polymerisation reaction
Of styrene and divinylbenzene, useful
For separating small molecular weight
Compounds
B-cellulose: readily obtained in a high
Pure state,greater permeability to
Macromolecules
“COMPONENT OF ION EXCHANGE”:-
33. The choice of ion exchangers depends upon the stability,
molecular weight, and ionic strength of the sample components
Volume of exchanger 2.5 fold greater than to exchange with the
ion in the sample
Anion exchangers
Retains positively charged cations
Because the stationary phase displays
A negatively charged group
Cation exchangers
Retains anions using positively charged group
“COMPONENT OF ION EXCHANGE”:-
34. Ion exchange chromatography relies on the attraction between oppositely charged stationary
phase, known as an ion exchanger, and analyze.
To these covalently bound functional groups the oppositely charged ions are bounded (mobile
counter ion), which will be exchanged with like charge ions in the sample having charge
magnitude more than the ions bounded to the matrix.
Thus if anion exchange chromatography is performed, negatively charged sample
components will interact more with the stationary phase and will be exchanged for like
charged ions already bounded to the matrix.
“PRINCIPLE”:-
35. Positively charged molecules are attracted to a negatively charged
solid support. Commonly used cation exchange resins are s-resin,
sulfate derivatives; and CM resins, carboxylate derived ions
“TYPES OF ION EXCHANGE
CHROMATOGRAPHY”:-
36. Negatively charged molecules is attracted to a
positively charged solid support. Commonly used anion
exchange resins are q-resin and DEAE resin
“TYPES OF ION EXCHANGE
CHROMATOGRAPHY”:-
37. Step1
A sample introduced into a sample
Loop of known volume
Step 2 “Set the column (equlibrium phase)”
The mobile phase carries the sample
From the loop onto a column that contains
Some form of stationary phase material
Stationary phase material is a resin or gel matrix consisting of
cellulose beads with covalently bonded charged functional groups.
“PROCEDUE”:-
38. Step3 “sample application into the column”
The target analytes (anions or cations) are retained on the stationary phase
Step4 “elution”
When we increased the concentration of a similarly charged species it will displace
the analyte ions from the stationary phase.
The analytes of interest must then be detected by some means, typically by
conductivity or uv/visible light absorbance.
Step5 “regeneration”
Cation exchanger = by acid then washed with water
Anion exchanger =by alkali then washed with water
39.
40. 1. Separating the proteins
Separates proteins according to their net charge, which is
dependent on the composition of the mobile phase(by
adjusting the ph or the ionic concentration)
Proteins are charged molecules. At specific ph, it can exist in
anionic (-), cationic (+) or zwitterion (no net charge) stage.
cationic pH =pI anionic
pH increase
“APPLICATIONS”:-
41. Hardness due to the presence of ca2+.Mg2+
Removed by passing hard water to cation exchanger changed
with na+
nar+nca2+ car + nna+
Resin hard water resin solution
Canr2 + 2nna+ nca2+ +2nanr
Resin solution resin regenerated
“APPLICATIONS”:-
42. Separation of similar ions from one another
antibody purification monoclonal antibodies
Hemoglobins seperation
Separation of vitamins
Separation of inorganic cations and anions
“APPLICATIONS”:-
43.
44. • Also known as bioselective adsorption
• It is a protein purification technique developed thirty
years ago.
• It is the isolation of a particular protein on a bioligand
that is attached to an inert matrix by a spacer arm.
• It relies on the specificity of a protein binding site for a
particular ligand
“INTRODUCTION”:-
46. • Specificity is based on three aspects:
Matix: for ligand attachment
Spacer arm: bind ligand to matrix
Ligand: bind with protein to purify it
“SPECIFICITY”:-
47. • Principle of affinity chromatography
is that the matrix combines with the
specific ligand due to specific
functional groups present on matrix .
• as the mixture of proteins is passed
through the chromatography
column, those proteins that have a
binding site for the immobilised
ligand will bind to the matrix,
while all other proteins will be
eluted from the column.
“PRINCIPLE”:-
48. •Step 1
Attachment of ligand to matrix
•Step 2
Attachment of proteins to complex
•Step 3
After washing proteins that are
unable to bind separated
•Step 4
Finally elution occurs and protein is
purified
“PROCEDURE”:-
50. Advantages
• Purify and concentrate protein
• Used in genetic engineering
• Production of vaccines
• The binding sites of biological molecules can be investigated
“ADVANTAGES”:-
Separating proteins Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. 15
Affect of pH in the separation of proteins By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not. 16
.Softening of hard water
We are aware of the fact that the hardenes of water is due to the presence of Ca2+.Mg2+ and other devalent ions these ions may be removed by passing hard water to cation exchanger changed with Na+ when the following exchange reaction takes place
NaR+nCa+2 CaR+ nNa+
resin hard water resin solution
the ca+ and Mg+ ions retained in the column wherease the sodium ions passes through the solution these na ions are harmless for washing purpose .after using ion exchange for a long time it becomes inactive its activity can be revived by perculating sodium chloride solution through the reaction
CanR2+ 2nNa+ nCa2+ +2NanR
Resin solution resin regenerated
Monoclonal antibodies produced in mouse ascites can be separated from other components of the ascites fluid.
Immunoglobulins can be separated from albumin, transferrins and proteases.
Hemoglobins can be separated by high-performance cation-exchange chromatography using the Vydac strong cation-exchange column.
