Capital HPLC

Type V HPLC Chiral Phases

TYPE V HPLC Chiral Phases

These phases consist of proteins, which are naturally occurring chiral high molecular weight
polymers, immobilized on silica. At present, the two most useful chiral protein columns are based
on human a-acid glycoprotein (AGP) and bovine serum albumin (BSA).
The work of Hermansson has lead to the development of two commercially available
phases based on AGP immobilized on silica. The first known as Enantiopac, has now been largely
superseded by the much more stable and versatile CHIRAL AGP phase. Whereas the work
of Allenmark, the first to covalently bind BSA to silica, was used as the basis for the development
of the commercially available phase RESOLVOSIL-BSA.
Due to the complex nature of the proteins utilised, the chiral recognition mechanism is largely
unknown, but involves mainly hydrophobic and electrostatic interactions, although hydrogen
bonding and charge transfer interactions can play a significant role. Type V HPLC-chiral stationary
phases have a wide applicability in the field of drug bioanalysis, as they undergo stereoselective
interactions with a large number of pharmacologically active compounds. This is aided by the fact
that the phases are used in reverse phase, and can be used in conjunction with column switching.
RESOLVOSIL-BSA main applications are for hydrophobic molecules and anionic compounds.
The solute must contain aromatic and polar moieties, but steric effects play an important role.
The standard mobile phase used with RESOVOSIL-BSA, is a phosphate buffer (0.01M to 0.20M)
at a pH of 4.5 to 8.0, modified with 1-propanol (0 to 6%). Methanol and acetonitrile should not
be used as they denature the protein.

CHIRAL AGP is primarily used to resolve a wide range of enantiomeric cationic molecules,
but a number of anionic chiral molecules have been resolved.

The magnitude of the stereochemical resolution is highly dependant on the structure of the solute,
but often enantioselectivity can be induced by changing the mobile phase composition.
Mobile phases used with this phase are typically 0.01 M phosphate buffer, at a pH of 3.0 to 7.5,
modified with an organic solvent such as methanol, acetonitrile, ethanol, 2-propanol and 1- propanol.
Mobile phase additives such as dimethyloctylamine and octanoic acid have also been employed, but
these can be difficult to completely remove.
While type V chiral stationary phases offer high selectivities for a wide variety of enantiomeric solutes,
they have a low load capacity which severely limits their usefulness in preparative separations.
The use of guard and pre- columns is recommended, especially for biological applications.

Resolvosil-BSA

The protein bovine serum albumin (BSA), is bonded covalently to a 7μm wide pore
NUCLEOSIL material. This chiral stationary phase has many applications, covering a wide
range of compound classes, some of which are shown below.
Others include lactams and reduced folates. 

Chiral AGP

This is the second generation chiral stationary phase based on the immobilization of the a-acid
glycoprotein (AGP) on porous spherical silica. It offers much higher stability, efficiency and capacity
than its predecessor. It is capable of separating racemic acids, amines and nonprotolytic compounds,
without derivatization.

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                                        ExamplesofenantiomericcationicdrugsresolvedonCHIRAL AGP

CHIRAL MOBILE PHASE ADDITIVES

Many racemic compounds have been separated, on conventional-achiral chromatography systems
(TLC, HPLC), by the use of chiral additives in the mobile phase. This approach has the following
advantages:

1 less expensive conventional phases (eg.C18, NH2) can be used.

2 Chiral additives and their concentration can easily be changed.

3 there is a wide range of possible additives.

4 the selectivity of the additive (with its free orientation) and the immobilised species
(with its more rigid structure) are sometimes different.

5 the additive does not have to be optically pure, as in the case of chiral derivatization,
where it would lead to false results for enantiomeric purity. The optical purity of the
chiral additive will only affect (a) selectivity.

The two main types of mobile phase additives, are chiral counter ions, and those that form
inclusion-complexes (eg. cyclodextrins, crown ethers). When the above are used in combination
they can lead to significant improvements in selectivity, and also can be used to investigate
simultaneously ionic and non-ionic solutes.

The high cost of many of these additives maybe overcome in part, by the use of microbore columns,
which also have the added advantage of increased sensitivity. Capital HPLC Limited supply
most phases in high efficiency narrow bore columns and cartridges, with internal diameters
of 2.1 and 1.0mm.

Chiral Mobile Phase Additives Ordering Information
For current prices, please consult our price list page 17-18.

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