flash and chiral chromatography

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BY: Gauthami. K.B Roll no: 256213886009 M.pharmacy 1 st yr (pharmaceutics) Pharmaceutical Analytical Techniques Under the guidance of UTTAM sir FLASH CHROMATOGRAPHY AND CHIRAL CHROMATOGRAPHY

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BY: Gauthami. K.B

Roll no: 256213886009

M.pharmacy 1st yr (pharmaceutics)

Pharmaceutical Analytical Techniques

Under the guidance of

UTTAM sir

FLASH CHROMATOGRAPHY

AND

CHIRAL CHROMATOGRAPHY

FLASH CHROMATOGRAPHY

INTRODUCTION

• Flash chromatography, also known as medium pressure chromatography

was introduced by CLARK

• It is alternative to slow and often inefficient gravity-fed chromatography.

• Flash chromatography differs from the conventional technique in two ways:

1. first, slightly smaller silica gel particles (250- 400 mesh)are used

2. second, due to restricted flow of solvent caused by the small gel particles,

pressurized gas (ca.10-15 psi) is 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.

STEPS INVOLVED IN FLASH CHROMATOGRAPHY

• Selecting a Solvent System

• Determining the Quantity of Silica Gel Required

• Packing the Column

• Applying the Sample

• Eluting the Sample

• Selecting a Solvent System:

• The compound of interest should have a TLC Rf of ≈0.15 to 0.20

in the solvent system, 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 the eluent.

• The ratio of solvents determines the polarity of the solvent

system, and hence the rates of elution of the compounds to be

separated.

• Higher polarity of solvent increases rate of elution for ALL

compounds.

• Common binary solvent systems in order of increasing polarity

are dichloromethane/hexane, ether/hexane, hexane/ethyl

acetate, and dichloromethane/methanol.

CONTD…

• Some solvents list according to their increasing eluting

power 1) cyclohexane 2)pet. ether 3)Pentane 4)

Dichloromethane 5)Ethyl ether 6)Ethyl acetate

7)Ethanol 7)Water 8)Acetone 9)Acetic acid

10)methanol.

• If Rf is ≈0.2, you will need a volume of solvent ≈5X the

volume of the dry silica gel in order to run your column.

• Determining the Quantity of Silica Gel

Required

• The amount of silica gel depends on the Rf

difference of the compounds to be separated,

and on the amount of sample.

• For ngrams of sample, you should use 30 to

100 ngrams of silica gel. For easier

separations, ratios closer to 30 : 1 are

effective, for difficult separations, more silica

gel is often required.

• However, by using more silica gel, the length

of time required for the chromatography is

extended.

• Selecting the column and column diameter Plastic column :

• Recommended length 46cm, Diameter - depends on sample size and the difference in Rf value.

• Packing the Column:

• Obtain a glass column and make sure that it has either a glass frit or a plug of cotton wool directly above the stopcock to prevent the silica gel from escaping from the column through the stopcock.

• Next, put a ~1/2 in. layer of clean sand above the plug of glass

wool. Make sure the surface is flat. Then pour in the silica gel

using a funnel.

• Two methods for packing the column are:

1)wet packing

2)dry packing

• Applying the Sample:

• Loading the sample is important .

• Application of sample can be made by the small Pasteur pipette.

• Allow the solvent which remains above the silica to drain down

until it is flush with the surface of the silica.

• If the top surface of the silica gel is not flat, gently tap the side

of the column until it is.

• Dissolve sample into the minimum volume of the elution

solvent. Apply this to the top of the column, being careful not to

disturb the top of the silica.

• Add a small amount of sand to protect the top surface of the

silica when you add more solvent.

• Eluting the Sample:

• Add elution solvent to the column.

• Apply pressure to force the solvent through the column.

• The pressure should be the minimum necessary to keep a

steady stream coming out of the column.

• Begin collecting the eluted solvent into separate test tubes

(fractions).

• To maximize the efficiency of chromatography, the fractions

collected should not be more than about one tenth of the

column volume.

• Prepacked column Online uv-vis detection Automatic

Collection Sample Solvent reservoirs can be used.

• Locating the Sample:

• Use TLC to determine

which fractions contain

your compound.

• Combine the fractions that

contain your sample

together in a flask, then

concentrate the sample.

• Detection:

• Detection is usually done by UV-Vis detectors.

• Fully automated method with user-friendly software Online UV –

Vis detectors are used.

• Modern online FC systems have improved the method by

incorporating reusable plastic cartridges prepacked with the

sorbent, ultraviolet (UV) detection, computer software control,

mobile phase gradients, and automated fraction collection.

• Normal phase (NP) FC of polar compounds on silica gel

columns is still the most widely used mode, but reversed phase

(RP), ion exchange, and other types of sorbents are becoming

used more frequently.

• Application :

These systems are applied In

1. sample cleanup,

2. Natural products purification,

3. Organic synthesis,

4. Combinatorial chemistry,

5. Drug discovery,

6. Pharmaceutical intermediate purification, and many

other areas.

CHIRAL CHROMATOGRAPHY

INTRODUCTION

• Chiral Chromatography is a branch of chromatography that is

oriented towards the exclusive separation of chiral

substances.

• Certain stereoisomers that differ only in the spatial

arrangement of their atoms and in their capacity for rotating

the plane of polarized light are termed optically active or

chiral and the individual isomers are called enantiomers.

• Enantiomeric separations are achieved in chiral

chromatography by the judicious use of chiral phases.

• The mobile phase can be a gas or liquid giving rise to chiral

gas chromatography and chiral liquid chromatography.

PRINCIPLE OF ENANTIOMER SEPARATION

• On transfer of a pair of enantiomers into asymmetric

environment, two diastereomeric species are formed

with distinct physicochemical property profile. On the

basis of which physical separation into individual

enantiomers may be achieved.

• Chiral selector (SO):-

• Capable of undergoing covalent or non-covalent

interaction with the individual enantiomer (Selectand

SAs)

Depending on the nature of the interaction stabilizing

the respective diastereomer. SA-SO species.

• Enantiomer separation strategies:-

• Indirect enantiomer separation

• Direct enantiomer separation

• Indirect enantiomer separation:- Chiral Derivatization

agent (CDAs):- Transformation of the SAs of interest into

covalent diastereomer by conversion with suitably

reactive SOs. Followed by separation of diastereomeric

product with achiral chromatographic techniques.

• Applicable only to enantiomer presenting a single or more

but selectively addressable functional group suitable for

derivatization.

• Direct enantiomer separation:- 1) Chiral mobile phase

additive 2) chiral stationary phase mode

• Chiral mobile phase additive (CMPA): A combination of

an chiral stationary and a chiral mobile phase is

employed.

• On introduction of a mixture of enantiomers into this

system, the individual of enantiomers form diastereomeric

complex with the chiral mobile phase additive.

• This diastereomeric complex may exhibit distinct

association / dissociation rate, thermodynamics stability,

and physiochemical property therefore may be separated

on an appropriate a chiral stationary phase..

• chiral stationary phase mode: Consists of an inert

chromatographic support matrix incorporating

chemically or physically immobilized SO species.

• CSPs may be created by a variety of SO immobilization

technique.

1. Covalent attachment on to the surface of suitably

prefunctionalized carrier materials.

2. Physical fixation employing coating technique.

3. Incorporation into polymeric network by

copolymerization or combination of this procedures

• CSPs provide several operational advantages over

CMAs based enantiomers separation:

1. stability of CSPs more and flexibility with respect to

method optimization parameter.

2. Used for wide range of mobile phase solvent and

modifiers.

3. also used for gradient elution and variable temp.

protocol

• Classification of Chiral Stationary Phase:

• Type-1 Organic Polymer -Pure -Polymer coating on inorganic support -Grafted polymer.

• Type-2 Carrier material modified with Chiral moieties -inorganic material mainly silica gel modified on the surface. -organic polymer network grafted with chiral molecules.

• Type-3 Imprinted material -imprinted polymer -inorganic material imprinted on the polymer.

DETECTORS

• Commonly detectors used in chiral

chromatography:

• Polarimeter

• Optical rotatory dispersion detector

• Circular dichroism detector.

• Applications:-

• Quinine and Quinidine

• Atropine and Hyoscyamine

• Cetrizine and Levo-cetrizine

• Omeprazole and Esomeprazole(more effective in

GERD)

• Dopamine and levodopamine

• D-Amphetamine and Amphetamine

• Dextromethorphan and Levo-methorphan

CONCLUSION

• Modern flash chromatography is applicable to a wide

range of compound types.

• Saves time and solvents.

• HPLC linear gradients can be transformed into step

gradients in flash chromatography.

• Flash chromatography can be a reliable and cost-

effective alternative to preparative HPLC.

CONTD..

• Chiral chromatography has become a preferred method

for rapidly separation enantiopure compounds in the

pharmaceutical industry, largely owing to the speed

with which a chromatographic method can be

developed and executed as well as the comparatively

small labour requirements of the chromatographic

approach.

• The use of chiral chromatography within the field of

organic synthesis can be expected to increase as the

technique becomes more familiar to synthetic chemists.

REFERENCES

• Peter Atkins & Julio de Paula, Aitkin's Physical Chemistry 7 the Ed., Oxford, New York

(2002) Chapter 22 AR Genaro

• Remington: The Science and Practice of Pharmacy 20 the Ed.

• Lippincott Williams & Wilkins (2000) Part 4 DG Peters, JM Hayes, GM Hefted, Chemical

Separations and Measurements, Saunders, Philadelphia(1974) Chapter 17

• http://www.chromatography.amershambiosciences.com.

• Chiral chromatography by T.E.Beesely

THANK YOU