September 2002 Meeting

Immobilized a1-Acid Glycoprotein and Cellobiohydrolase (CBH) as Chiral Selectors in Chiral Chromatography

The September 2002 Connecticut Separation Science Council was held at the University of Connecticut in Storrs, CT.

Adsorption studies together with chromatographic competition studies have demonstrated that AGP has two binding sites, one high and one low affinity site. All types of compounds, independently of the nature, are bound to and compete for the same sites.

The AGP column has an extremely broad applicability.  It resolves drugs and related compounds from a very wide variety of compound classes such as amines (primary, secondary, tertiary, quaternary ammonium compounds), weak and strong acids and non-protolytes of different character such as alcohols, amides, esters, sulphoxides etc. One reason for the broad applicability is that the chiral bonding properties of the column can be affected in a dynamic way by changing the mobile phase composition. The AGP column is used in the reversed-phase mode, which means that the retention and the enantioselectivity can be regulated in many different ways, for example by the pH of the mobile phase, the concentration and the nature of an uncharged organic modifier (2-propanol, acetonitrile), the nature and the concentration of the buffer and also by using charged organic modifiers like N,N-dimethyloctylamine (DMOA) and octanoic acid.  However, pH is the most important tool for the regulation of the retention and the enantioselectivity. By changing the pH, the net charge of the protein is affected. Thus, the degree of ionic bonding of the solute can be affected, giving strong effects on both retention and enantioselectivity. For example, chromatography of hydrophobic amines at a pH between 4-5 normally gives high enantioselectivity and a low retention compared to the results obtained at pH 7. This is due to the lower degree of ionic bonding of the positively charged amine obtained at pH 4 compared to pH 7, which means that low organic modifier concentrations can be used, resulting in high enantioselectivity and low retention. More hydrophilic basic drugs are resolved at a pH of about 7 where they can be retained.

Also the uncharged modifiers can strongly affect the enantioselectivity and the retention. It has been demonstrated that enantioselectivity can be induced by adding certain organic solvents, like 1- and 2-propanol and acetonitrile, to the mobile phase. Chromatography of hexobarbital or some of the anti-inflammatory drugs of the profen type in a pure buffer as the mobile phase, gives no enantioselectivity. However, addition of low concentrations of a certain organic solvent, can induce the chiral selectivity, resulting in high resolution of the enantiomers. These effects are most likely caused by reversible changes of the secondary structure of the selector, which is indicated by CD studies. The most dramatic changes of the enantioselectivity have been obtained by the charged modifiers, DMOA (for acidic analytes) and aliphatic carboxylic acids (for basic analytes). For example, the separation factor of the anti-inflammatory drug compound naproxen could be increased from about 1.7 to about 16 by adding 1 mM DMOA to the mobile phase. However, the charged organic modifiers are only used to a limited extent, due to the fact that the majority of the compounds can be resolved using a simple mobile phase composition, i.e. a buffer and an uncharged modifier like 2-propanol or acetonitrile.

The extremely broad applicability of the CHIRAL-AGP column will be demonstrated by the resolution of  drugs from many different classes of compounds.

Cellobiohydrolase (CBH) is, as AGP, used as chiral selector in liquid chromatography (CHIRAL-CBH) [4]. CBH is regarded as the key enzyme in fungal cellulose degradation. The molecular weight of CBH is 64000 and it has an isoelectric point of  3.9 and contains 497 amino acid residues. It is structurally organized in three main regions, a core with the catalytically active site, a hinge region and the cellulose binding domain. The active site is located in a 50 Å long tunnel in the core region. The enzymatically active site is also responsible for the major chiral recognition ability of CBH.

 The CBH column is also a reversed-phase column and the retention and the enantioselectivity can be regulated by the pH, the nature and the concentration of an uncharged modifier and the nature and the concentration of the buffer in the mobile phase. The CBH column has a more narrow applicability compared to the AGP column. However, the CBH column is particularly good for the resolution of amino alcohols of different types. The column also resolves other amines containing, in addition to the basic nitrogen, other hydrogen bonding groups than an alcohol, for example a carbonyl group etc. The CBH column normally gives very high a-values.

 References:

1. J. Hermansson, J. Chromatogr., Trends Anal. Chem., 8 (1989) 251
2. J. Hermansson and I. Hermansson, J. Chromatogr. A, 666 (1994) 181
3. J. Hermansson and A. Grahn, J. Chromatogr. A, 694 (1995) 57
4. J. Hermansson and A. Grahn, Trends Anal. Chem., 687 (1994) 45

Prof. Hermansson is the author of numerous scientific papers and books in various fields associated with liquid chromatography. He is also active in editorial advisory boards of scientific journals. 

Prof. Hermansson is one of the pioneers in chiral chromatography, with more than twenty-five years in the field. He is the inventor of chiral columns of which the most well known is the column based on a1-acid glycoprotein (AGP) as the chiral selector. Today the AGP range of columns are among the most widely used chiral separation tools worldwide. In 1988 Prof. Hermansson received the Jubilee Medal from the British Chromatographic Society for his contribution to the field of chiral chromatography.

Besides chiral chromatography Prof. Hermansson is also very active in HPLC bioanalysis, where his research interest is focused on the development of restricted access media columns, enabling determination of drugs and metabolites by direct injection of biological fluids, like serum/plasma, on the HPLC system.

From I-84 East:  Take Exit 68.  At the bottom of the exit ramp, take a right onto Route 195.  Follow Route 195 straight to the University of Connecticut Storrs Campus.  Take a right onto Mansfield Road (Look for a large pond on your right.  Mansfield Road will be at the next set of traffic lights.).  At the first stop sign, take a left onto Gilbert Road.  Then continue left onto Gilbert Extension (Across from Alumni Residence Hall).  The Nathan Hale Inn is straight ahead.  Park in the gated lot behind the hotel.

From I-84 West:  Take Exit 68.  At the bottom of the exit ramp, take a left onto Route 195.  Follow Route 195 straight to the University of Connecticut Storrs Campus.  Take a right onto Mansfield Road (Look for a large pond on your right.  Mansfield Road will be at the next set of traffic lights.).  At the first stop sign, take a left onto Gilbert Road.  Then continue left onto Gilbert Extension (Across from Alumni Residence Hall).  The Nathan Hale Inn is straight ahead.  Park in the gated lot behind the hotel.

From I-384 West:  Follow I-384 until it becomes Route 44 (East).  Follow Route 44 until you reach Route 195 (Four Corners).  Take a right onto Route 195 South and follow Route 195 straight to the University of Connecticut Storrs Campus.  Take a right onto Mansfield Road (Look for a large pond on your right.  Mansfield Road will be at the next set of traffic lights.).  At the first stop sign, take a left onto Gilbert Road.  Then continue left onto Gilbert Extension (Across from Alumni Residence Hall).  The Nathan Hale Inn is straight ahead.  Park in the gated lot behind the hotel.

From Route 32 North:  Follow Route 32 North until it intersects with Route 275 (South Eagleville Road).  Take a right onto Route 275, and continue straight until it reaches Route 195.  Take a left onto Route 195 North.  Continue straight on Route 195 until you reach Mansfield road on the left (If you pass a large pond on the left, you have gone too far.).  At the first stop sign, take a left onto Gilbert Road Then continue left onto Gilbert Extension (Across from Alumni Residence Hall).  The Nathan Hale Inn is straight ahead.  Park in the gated lot behind the hotel.

University of Connecticut
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