BONDED PHASE

CHROMATOGRAPHY

Presented by

Dr. A.SUNEETHA

Dept of Pharmaceutical Analysis

Hindu College of Pharmacy

Bonded phase chromatography is comes under partition

chromatography.

The early forms of Partition Chromatography used Liquid –

Liquid column. These have been replacing in modern LC

systems by liquid – bonded phase columns. In Liquid –

Liquid chromatography the liquid was held in place by

physical adsorption. In bonded phase chromatography on

the other hand it is attached by chemical bonding resulting

in highly stable packing’s insoluble in the mobile phase.

Definition

A stationary phase chemically bonded to a support that

is used for the separation. It is the most commonly used

LC mode. The most popular support used is micro

particulate silica gel. An organosilane, such as

octadecyl (for reversed-phase chromatography), is the

most accepted type of bonded phase. Approximately 70

percent of all HPLC is carried out on chemically bonded

phases

In liquid chromatography, liquid-liquid systems are

unstable as, however small the solubility of the

stationary phase may be in the mobile phase, the

stationary liquid phase will be eventually stripped

from the column. It was therefore found necessary

to chemically attach the stationary phase to the

support to ensure a stable system and these

materials were called bonded phases.

Bonded phases

Different Classes of Bonded Phase

There are basically three classes of bonded phase

1. Brush phase

2. Oligomeric phase

3. Bulk phase

Mono-substituted silanes single layers of organic moieties could be bonded to the silica surface and these materials were called brush phases

alternately treating silica with di-substituted silanes and water in a heated fluidized-bed system, oligomericbonded phases

Employing tri-substituted silanes in the presence of water the organic moieties could be cross linked with ether groups and form a type of a polymer. These polymeric phases were strongly held to the silica matrix and were very stable and were given the name bulk phases

Charactersitics of Bonded Phases:

To render the silica dispersive in character, the interactive

surface must be chemically modified. For example,

appropriate hydrocarbon moieties could be chemically

linked to the surface hydroxyl groups.

The silicon-oxygen-carbon linkage is, very weak and regenerating the

original hydroxyl groups of the silica gel. An alternative bonding

method that involved the use of chlorsilane reagents. When a

chlorsilane reacts with a hydroxyl group of the silica gel surface, the

hydrocarbon chain is attached by the much stronger and stable

silicon-carbon link.

Bonded Phase Synthesis by Reaction in a Solvent:The solvents normally used in bonded phase synthesis are aromatic hydrocarbons e.g.,toluene that boils at 110˚C or mixed xylenes that boil 138-140˚C. The procedure varies a little depending on the size of the batch and the type of silanizing reagent

The Synthesis of Bonded Phases Although bonded phases are now sometimes

prepared form polylmeric material the most poplar and commonly used bonded phases are based on silica. The subject of bonded phase synthesis will therefore deal with bonded phases based on silica

First select appropriate base silica.

Select the particle size

High efficiencies can be obtained by using small particles but, as the particle size is reduced, the flow impedance of the column increases which requires higher inlet pressures

The choice of the pore size and surface area of the silica are also important.

The greater the pore size the less the surface area

The Carbon Content of Bonded Phases

The carbon content of the bonded phase is usually determined by microanalysis and the result expressed as %w/w of the combined bonded organic material and the silica gel

The Use of Alkoxysilane Reagents in Bonded Phase

Synthesis

The most reactive alkoxy reagents are the methoxy and

ethoxysilanes and their reaction with a hydroxyl group is

accompanied by the release of methanol or ethanol.

The Large Scale Preparation of Bonded Phases

chlorsilane reagents are used, the reaction is normally

carried out in an appropriately large flask fitted with a

reflux condenser and a water scrubber.

Apparatus for the Preparation of Bonded Phases

The Fluidized Bed Method for Bonded Phase Synthesis

The fluidization can best be described as a contact process in which particulate matter is transformed into a fluid-like state by a fluid stream that may be a gas or liquid. The fluidization is accomplished by the upward passage of a fluid through the bed of particles until a velocity is attained where the upward drag force acting on the surface of the particles is equal to the gravitational force downwards. Under these conditions, the particles move apart and become suspended by the fluid flow and the bed is said to be fluidized.

The main advantage of the fluidized bed system was found to be, the reproducibility of the product, its ease of operation

and automation and, its greater flexibility

The Fluidized Bed Apparatus for the Synthesis of Bonded Phases

Normal & Reversed phase Packings: Bonded phase packings are classified as reversed

phase when the bonded coating is non-polar in character and as normal phase when the coating contains polar functional groups.

The R group of the Siloxane in these coatings is a C8

chain (n – octyl) or a C18 chain (n – octyldecyl).

Aqueous solution containing various concentrations of such solvents as CH3OH, ACN, or THF. In this mode care must be taken to avoid PH values greater than about 7.5 because the silica can form soluble silicate species, causing dissolution of the stationary phase.

In normal phase bonded packings R in the siloxane structure is a polar functional group such as cyano –C2H4CN; diol – C3H6OCH2CHOHCH2OH; amino –C3H6NH2 & dimethyl amino C3H6N(CH3)2. Cyano type for least polar & amino type compounds. Diol packings are intermediate in polarity

Column selection in partition chromatographic separations:

The polarities of the various analyte functional groups increase in the following order

Hydrocarbons < Ethers < Esters < Ketones < Aldehydes < Amides < Amines < Alcohols

Often in choosing a column for a partition chromatographic separation the polarity of the stationary phase is matched roughly with that of the analytes: a mobile phase of considerably different polarity is then is used for elution.

Mobile phase selection in partition chromatography:

In LC the retention factor K is experimentally the most

easily manipulated of the 3 because of the strong

dependence of this constant on the composition of the

mobile phase. For optimal performance K should be in

the ideal range 2 and 10; for complex mixtures this range

must often be expanded to perhaps 0.5 to 20.

Effect of solvent strength on retention factors: Solvents

that interact strongly with solutes are often termed as

strong solvents. Solvent strength depends on the nature

of the analyte & stationary phase.

Applications of partition chromatography:

Derivative Formation:

It is useful to covert the components of a sample to a derivative before or after chromatographic separation. Such treatment may be desirable

To reduce the polarity of the species so that partition rather than adsorption or Ion-exchange columns can be used

To increase the detector response, and thus sensitivity for all of the sample components

To selectively enhance the detector response to certain components of the sample.

Ion pair chromatography

Ion pair chromatography some times called paired ion chromatography is a subset of reversed phase chromatography in which easily ionizable species are separated on reversed phase columns.

In this an organic salt containing a large organic counter ion, such as quaternary ammonium ion or alkyl sulfonate is added to the mobile phase as an ion pairing reagent.

Two mechanisms for separation.

1. First the counter ion forms an uncharged ion pair with a solute ion of opposite charge in the mobile phase. This ion pair then partitions into the non-polar stationary phase giving differential retention of solutes based on the affinity of the ion pair for the two phases.

2. Alternatively the counter ion is retained strongly by the normally neutral stationary phase & imparts a charge to this phase .

Separation of organic solute ions of the opposite charge

then occurs by formation of reversible ion pair

complexes wit h more strongly retained solutes forming

the strongest complexes with the stationary phase.

Separation of ionic & Non-ionic compounds using alkyl

sulfonates of various chain lengths as ion pairing

agents. Note that a mixture of C5 & C7 alkyl sulfonates

gives the best separation results.

Chiral Chromatography

• Compounds that are non superimposable mirror

images of each other are called chiral compounds. Such

mirror images are called Enantiomers.

• Either Chiral mobile phase additives or Chiral stationary

phase are required for these separations. Preferential

complexation between the chiral resolving agent & one

of the isomers results in a separation of the enantiomer.

The chiral resolving agent must have chiral character

itself to recognize chiral nature of the solute.

• A chiral agent is immobilized on the surface a solid

support. Several different modes of interaction can occur

between the chiral resolving agent & the solute.

• In one type the interactions are due to attractive forces

such as those between π bonds, hydrogen bonds or

dipoles. In another the solute can fit in to chiral cavities

in the stationary phase to form inclusion complexes.

References

Instrumental Analysis by Skoog

Bonded phases by RPW Scott

www.wikepedia.com.

Thank You