3. CONT ENT S
Chromatography
-- definition
-- History
-- Types of Chromatography
-- Uses of Chromatography
Column Chromatography
-- Types of Column
Chromatography
3
4. CONTENTS
Flash Chromatography
--definition & history
--Column VS Flash
Chromatography
--Theory
--Principle
--Selection of StationaryPhase
--Adsorbent
--Properties of FlashSolvents
--Selection of column
4
5. CONT ENTS
Instrumentation of Flash
Chromatography
--Parts of Flash Chromatography
--Pump Systems
--Sample Injection System
--Columns
--Pre Columns
--Fraction Collector
--Detectors
5
7. WHAT IS
CHROMATOGRAPHY
Chromatography is a Greek word chroma “colour” and graphein
“to write”.
And chromatography is a family of analytical chemistry
techniques for the separation of mixtures.
It was the Russian botanist “Mikhail Tsvet” who invented
the first chromatography technique in 1901.
7
9. USES OF
CHROMATOGRAPHY
It is used in crime scene investigations
In hospitals it can be used to detect alcohol levels in a patient's
blood stream
It is used for environmental agencies to determine the level of
pollutants in water supplies
It is used to purify chemicals needed to make a product in a
manufacturing plant
It is used by pharmacists to determine the amount of each
chemical found in each product.
9
10. COLU MN
CHROMATOGRAPHY
In column chromatography, the stationary phase, a solid
adsorbent, is placed in a vertical glass (usually) column.
The mobile phase, a liquid, is added to the top and flows down
through the column by either gravity or external pressure.
Column chromatography is separated into two categories,
depending on how the solvent flows down the column.
10
11. WHAT THEY?
Depending on how the solvent flows down the column.
Gravity column chromatography
Flash chromatography
If the solvent is allowed to flow down the column by gravity, or
percolation, it is called gravity column chromatography.
If the solvent is forced down the column by positive air pressure,
it is called flash chromatography,
11
12. FLASH CHROMATOGRAPHY
Flash chromatography, also known as medium pressure
chromatography.
“An air pressure driven hybrid of medium and short column
chromatography optimized for rapid separation"
It was popularized several years ago by Clark Still of Columbia
University.
An alternative to slow and often inefficient gravity-fed
chromatography
12
13. INTRODUCTION
Differs from the conventional technique in 2 ways:
Slightly smaller silica gel particles (250-400 mesh) are used,
and
Due to restricted flow of solvent caused by the small gel
particles, pressurized gas (10-15 psi) used to drive the solvent
through the column of stationary phase
The net result is a rapid “over in a flash” and high resolution
chromatography.
13
14. COLUMN VS FLASH CHROMTOGRAPHY
Cloumn Chromatography
Glass columns with silica gel
Separation is very slow
(typically many hours)
End of the run, silica gel must be
removed
Both time consuming and
hazardous
Flash Chromatography
Pre-packed plastic cartridges
Solvent is pumped through the
cartridge and separation is rapid
Much quicker and more
reproducible
Remaining solvent flushed out of
the column using pressurized gas
14
15. THEORY
Chromatography exploits the differences in partitioning behaviour
between a mobile phase and a stationary phase to separate the
components in a mixture.
Compounds of the mixture interact with the stationary phase
based on charge, relative solubility or adsorption.
The retention is a measure of the speed at which a substance
moves in a chromatographic system.
15
16. PRINCIPLE
The principle is that the eluent is, under gas pressure (normally
nitrogen or compressed air) rapidly pushed through a short glass
column.
The glass column is packed with an adsorbent of defined particle size
with large inner diameter.
The most used stationary phase is silica gel 40 –63 μm, but
obviously packing with other particle sizes can be used as well.
16
17. PRINCIPLE
Particles smaller than 25 μm should only be used with very low
viscosity mobile phases, because otherwise the flow rate would be very
low.
Normally gel beds are about 15 cm high with working pressures of 1.5
– 2.0
bars.
In the meantime, however, and parallel to HPLC, reversed phase
materials are used more frequently in flash chromatography.
The computerized system control the Working of Flash
chromatography.
17
18. SELECTION OF SATIONARY PHASE
The most important stationary phase in column chromatography is
silica.
Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly
used by the organic chemist for column chromatography.
Adsorbent particle size affects how the solvent flows through the
column.
18
19. Smaller particles (higher mesh values {70-230} ) are used for flash
chromatography; larger particles (lower mesh values {230- 400}) are
used for gravity chromatography.
The amount of silica gel depends on the Rf difference of the
compounds to be separated, and on the amount of sample.
SELECTION OF SATIONARY PHASE
19
20. ADSORBENTS WHICH ARE
MAINLY USED IN FLASH CHROMATOGRAPHY
Silica: Slightly acidic medium. Best for ordinary compounds,
good separation is achieved.
Alumina: Basic or neutral medium. Can be effective for
easy separations, and purification of amines.
Reverse phase silica: The most polar compounds elute
fastest,
the most non-polars lowest.
20
21. THE PROPERTIES OF COMMONLY USED
FLASH SOLVENTS :
The compound of interest should have a TLC Rf of ≈0.15 to 0.20 in
the solvent system you choose.
Binary (two component) solvent systems with one solvent having a
higher polarity than the other are usually best since they allow for easy
adjustment of the average polarity of theeluent.
Higher polarity of solvent increases rate of elution for allcompounds
If your Rf is a≈0.2, you will need a volume of solvent ≈5X the
volume of the dry silica gel in order to run your column
21
22. SOLVENT SYSTEMS
Flash column chromatography is usually carried out with a
mixture of two solvents, with a polar and a nonpolar component.
1.Hydrocarbons: pentane, petroleum ether , hexanes.
2. Ether and dichloromethane (very similarpolarity)
3. Ethyl acetate
22
23. THE MOST COMMON TWO -COMPONENT
SOLVENT SYSTEM
Ether/Petroleum Ether
Ether/Hexane
Ether/Pentane
Ethyl Acetate/Hexane
Methanol/Dichloromethane
23
25. PARTS OF FLASH
CH ROMATOGRAPHY
1.Pump Systems.
-- Pump Controller.
2. Sample Injection Systems.
3.Glass Columns, Filling Sets & Column Valves.
4. Pre columns.
5.Fraction Collector.
6. Detectors and Chart Recorders.
7.Computerize LCD Display.
25
26. PUMP SYSTEMS
Pump Controller :-
Apressure range up to either 10 bar or 50 bar gives
optimum
separation results for a broad range of applications.
The pump modules can be controlled by three different
units.
Pump Controller C610
Pump Manager C615
Control Unit C620
26
27. PUMP CONTROLLER C- 610
The Pump Controller C-610 for one Pump Module C-601 is
designed for isocratic separations.
The flow rate can be easily adjusted by turning a knob and is
indicated by a large illuminated LCD-display.
Delivered with a overpressure sensor for maximum safety.
27
28. PUMP MANAGER C- 615
The Pump Manager C-615 is designed for both isocratic
and gradient separations.
Fast operation, easy programming and a large graphical
display allows a quick and easy set up.
The unit has Input/Outputs for 2 solvent valves and level
sensors
and includes a pressure sensor and mixing chamber.
28
29. CONTROL UNIT C- 620
The Control Unit C-620 in combination with Sepacore Control provides
precise control of the chromatography system.
The following components can be connected to the Control Unit C- 620
2 to 4 Pump Modules C-601 or C- 605
Up to 2 Fraction Collectors
Up to 8 Detectors e. g. UV,RIS equential Modules C-623 or C-625
for automatic sequential chromatography on up to 5 columns
29
30. COLUMNS
Glass Columns :-
A wide range of columns offer maximum flexibility for every
situation.
Depending on the nature and the quantity of the sample offers a
series of column types which vary in form, size and performance.
30
31. COLUMN
Plastic +Glass Column :-
Plastic+Glass-coated Glass Columns are available for larger
amounts of samples and higher pressure applications on a high
safety level.
31
32. PRE COLUMNS
Pre column are minimizing dead volumes and enhance the life
time of the main column by trapping contaminants.
The small Pre column, fits to Glass Columns of inner diameter of
ID 15, 26, 36 and 49 mm.
The large Pre column, fits to Glass Columns of ID 70 and 100
mm inner diameter.
32
33. FILLING SETS FOR GLASS COLUMNS
Dry Filling Set :-
The Dry Filling Set is employed for filling glass columns with
silica gel using compressed gas.
Silica gel in the size range of 25 –200micro meters can be packed
with this method.
33
34. Slurry Filling Set :-
The Slurry Filling Set is used for wet filling and conditioning
of glass columns with silica gelparticles smaller than 25 micro
meter.
34
35. DETECTORS AND
RECORDERS / SOFTWARE
For most applications one of the robust UV/Vis detectors would
be sufficient for the systems detection needs.
Both detectors are delivered in combination with a preparative
flow cell.
UV Monitor C-630
UV Photometer C-635
35
36. ADVAN TAGES
Fast and economic methods for the synthesis laboratory.
Ideal for the separation of compounds up to gram quantities.
No expensive equipment required.
ideal waytransfers results from TLC to CLC.
Automated changes between normal phase and reversed phase
chromatography.
36
37. APPLICATIONS OF FLASH
CHROMATOGRAPHY:-
Natural Products/Nutraceuticals Application:
Separation and Isolation of α-Santalol and β-Santalol from
Sandalwood
Extraction.
Isolation and Purification of Flavonoids from Ginkgo Biloba
Leaves Extract.
Isolation and Purification of Catechins from Green Tea Extract .
In Purification of GallaChinensis.
Isolation and Purification of Ginsenosides from Red Panax
Ginseng Extract.
37
38. Carbohydrate Application
Purification of Conjugated Quercetin and Rutinose.
Impurity Isolation of Valproic Acid from Cyclodextrine During
Encapsulation.
Isolation ofAminosugar and Acarbose .
Isolation of AminoglycosideAntibiotics.
38
39. Lipids Application:
1.Purification of Fatty Acid Methyl Esters (FAMEs).
2. Purification of a Mixture of Glycerides, Mono- , Di-, and
Tristearin.
3. Purification of Sterols.
39
40. Pharmaceutical/Small Molecules Application
In Anti-malarial Drug Purification in Drug Discovery.
Mestranol Purification During Chemical Synthesis.
In Impurity Isolation During Drug Purification.
Bile Acid Purification During Lead Generation in Drug
Discovery.
40
41. CONCLUSION
Purification of drug is an important step in any branch of
research.
Preparative chromatography is used to separate the components
of a mixture for more advanced use and is thus a form of
purification.
Flash Chromatography can be alternative to preparative HPLC as
it saves time and solvent.
41
42. REFERENCES
Yvesrubin.files.wordpress.com. (2019). [online] Available
at:https://yvesrubin.files.wordpress.com/2011/03/flash_chromatography.pdf [Accessed 28
Mar. 2019].
Pharmatutor.org. (2019). [online] Available at:
https://www.pharmatutor.org/pdf_download/pdf/Vol.%202,%20Issue%205,%20May%2020
14,%20PharmaTutor,%20Paper-8.pdf [Accessed 28 Mar. 2019].
Web.mit.edu. (2019). [online] Available at:
http://web.mit.edu/5.32/www/Appendix_3_Flash_Cromatography_03.pdf [Accessed 28
Mar. 2019].
Ocw.mit.edu. (2019). [online] Available at: https://ocw.mit.edu/courses/chemistry/5-301-
chemistry-laboratory-techniques-january-iap-
2012/labs/MIT5_301IAP12_FlashHandout.pdf [Accessed 28 Mar. 2019].
42
45. • Ion exclusion chromatography (IEC) is a relatively old separation
technique.
• IEC provides a useful technique for the separation of ionic from non
ionic compounds and to separate mixture of acids using an ion
exchange stationary phase in which ionic substances are rejected by
the resin while non ionic or partially ionized substances are
retained and separated by partition between the liquid inside the
resin particles and the liquid outside the particles.
• The ionic substances therefore pass quickly through the column,
but non ionic (molecular) or partially ionized substances are held
up and are eluted more slowly.
• IEC is also referred to by several other names, including ion
exclusion partition chromatography, ion chromatography-
exclusion mode, and Donnan exclusion chromatography.
ION EXCLUSION
CHROMATOGRAPHY
45
46. The characteristic feature of ion exclusion
chromatography is that the sign of the electric charge of
the dissociated functional groups on the ion exchange
resin is the same as that on the ionic compound analysed.
It follows that negatively charged ions, e.g. dissociated
acidic compounds, are separated on cation-exchange
resins with anionic(usually sulphonic) functional groups.
By analogy, positively charged species are separated on
anion- exchange resins containing cationic ( tetra alkyl
ammonium functional groups)
46
47. • The same column can be used for both ion exchange chromatography
and ion exclusion chromatography
• The eluents used are usually water, water/ organic solvent
mixtures, dilute (high conductivity) aqueous solutions of a strong
acid, or dilute (low conductivity) aqueous solutions of a weak
acid.
• A conductivity detector is commonly used to monitor the column
effluent and, when the eluent conductivity is extremely high, a
suitable suppressor system is generally used.
• Using IEC, it is possible to separate weakly ionized anions such as
Sulfide, phosphate, nitrite, aliphatic carboxylic acids, aromatic
carboxylic acids, bicarbonate, borate, aliphatic alcohols, sugars,
amino acids, water, and others, as well as ammonium, amines, and
others, based on a combination of the separation mechanisms of ion-
exclusion, adsorption, and/or size exclusion.
47
48. • For specific requirement of ion exclusion chromatography,
large ion exchange capacity is preferential.
• Columns capacity is increased by increasing its
dimensions, maximizing the concentration of functional
groups on the support, and using a strong ion-exchanger.
• The usual supports are based on macro-porous copolymers
of styrene and divinyl benzene in which the degree of
cross-linking is characterized by the concentration of
divinyl benzene in the reaction mixture.
48
49. • In conventional IEC of ionic and non ionic substances, a
poly(styrene)divinylbenzene (PS-DVB) based strongly acidic
cation exchange resin in the hydrogen form is used exclusively
as the separation column. The resin bed can be considered to
consist of three distinct components:
1. a solid resin network with charged functional groups (the
membrane);
2. occluded liquid with in the resin beads(the stationary phase);
and
3. the mobile liquid between the resin beads (the mobile
phase or eluent).
• The ion exchange resin acts as a hypothetical semipermeable
membrane (a Donnan membrane) separating the two liquid
phases .This membrane is permeable only for non ionic
substances.
SEPERATION MECHANISM 49
50. • When the column is filled with water, which is pumped through
as mobile phase, the water soluble molecules build up
hydration spheres around the dissociated functional groups of
the support.
• water contained in the pores of the support and in the hydration
spheres is immobilized thus forming the stationary phase
• The basic mechanism is that the neutral and uncharged
molecules can penetrate the resin, whereas similarly charged
co-ions are repelled owing to the presence of dissociated
functional group immobilized in the stationary phase.
RETENTION MECHANISM
50
51. • Because the concentration of the ions in the stationary phase
exceeds that in the mobile phase, osmotic forces tend to
drive water into the resin causing it to swell.
• This swelling is less for more highly cross-linked stationary
phases and for mobile phases containing high concentration
of ions.
• The ratio of the concentration of ionized to neutral form of
an analyzed compound is determined by its dissociation
constant and is equivalent to solute effective charge.
• Solute retention therefore depends on this constant.
• Strong acids that are completely dissociated are
electronically repulsed . As a consequence they are eluted
un separated in the column dead volume ( VM) , which
corresponds to the volume of the mobile phase in the
column.
51
52. • On the other hand un dissociated molecules are able to enter the
resin network.
• They are eluted together with a retention volume equal to the sum
of the inner and dead column volumes.
• The inner column volume means just the column of the volume
of stationary phase.
• Only electrolytes of the intermediate strength can be separated by
this method.
• For these electrolytes higher retention volumes are expected for
species with higher pka value
52
54. To optimize an IEC separation, careful selection of the
following experimental parameters must
be made:
1. The type of matrix used as the stationary phase (PS-DVB,
polymethacrylate or silica);
2. The nature of the functional group (e.g. strong or weak
acid);
3. The ion exchange capacity (low or high);
4. The nature of the eluent (e.g. strong or weak acids);
5. The pH of the eluent
6. The amount of organic modifier present in the eluent; and
7. The type of detector used (universal or selective).
OPTIMIZATION OF ION EXCLUSION
CHROMATOGRAPHIC SEPARATION
54
58. SUPPRESSOR
Reduce the background conductivity of the eluent and enhance
the conductivity of the analytes.
Minimizes baseline noise
Increases the detection sensitivity of the measurement system
Optimizes the signal-to-noise ratio
58
61. Detectors with UV-Visible spectrophotometry
Used in cases where the component is absorbed in the UV-
Visible range.
Eg: iodide, nitrite, nitrate, iodate or chromate ions.
Detector is photodiode and the cell is a quartz cuvette.
Deuterium and tungsten lamps are used as a source of
light.
61
62. Fluorescence-based detectors
Components of the sample are excited by a given
wavelength light and the components emit light.
Detection of biological samples.
62
65. Determination of weakly ionized
inorganic anions.
Fluoride, nitrite, phosphate, sulfite, arsenite,
arsenate, bicarbonate, borate and cyanide
from seawater and waste waters.
Separation of bicarbonate in tap waters by
IEC with conductimetric detection by elution
with water.
(A) Raw tap water (10-fold dilution);
(B)tap water after softening treatment;
(C)tap water (10-fold dilution).
Peaks: 1, strong acid anions; 2, bicarbonate ion.
65
66. Strong InorganicAcids
Sulfate, nitrate and chloride ions, and strong base cations such
as sodium, ammonium potassium, magnesium and calcium ions
commonly found in acid rainwater.
66
68. Determination of water in some
organic solvents.
PS-DVB based cation exchange resin in the
hydrogen form.
Eluent - methanolcontaininga small amount of
strong acid.
Spectrophotometric detection.
Peak at at 310nm.
68
69. Amino acids and amino acid
derivatives.
were determined using
and orotic acids were
Hippuric and orotic acid
photodiode array detection.
The two peaks of hippuric
monitored at 210 and 280 nm.
69
70. 1. Głód, Bronisław. (1997). Principles and Applications
of Ion Exclusion Chromatography. Acta
Chromatographica. 7. 72-87.
2. Pdfs.semanticscholar.org. (2019). [online] Available
at:
https://pdfs.semanticscholar.org/6e3d/e7af8a62e18f84
d8b4b46dad72cc510fdb79.pdf [Accessed 2 Apr.
2019].
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