High Performance Strong Anion Exchange Liquid Chromatography

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Ion exchange chromatography separates molecules on the basis of their charge. In anion exchange chromatography, the stationary phase or matrix possesses positive charge and interacts with analytes of negative charge. The positive charge on the matrix is due to a strong base derivatized on to a support medium, which remains positively charged across the pH range of 1-14. The sample mixture is introduced into the mobile phase and will bind ionically to the positively charged matrix. The strength of this interaction is dependent on the number of sulphate groups present in the oligosaccharide and the environmental pH of the mobile phase. The pH of the mobile phase must be above the pKa of the sulphate and carboxyl groups to de-protonate the acidic groups. If the pH is less than the pKa of the acidic groups, they will not interact with the column matrix and will pass through the column (although this is unlikely for heparin and heparan sulphate oligosaccharides).

In order to elute the oligosaccharide molecules from the column in a manner, which separates them based on their charge, a counter ion must be introduced that can compete for the basic groups present on the support matrix. The counter ion used for elution is dependent not only on the anion exchange column, but also the charge state of the analytes.

A reliable strong anion exchange column for separation of heparin and heparan sulphate oligosaccharides is the Propac PA1 column (Dionex). This column is polymer based and does not suffer from the steady deterioration problems encountered with its silica-based counterparts (short column life, peak broadening and inconsistent retention times). The Propac PA1 column provides the consistent elution times, which are necessary for the disaccharide analysis described earlier coupled with column longevity (consistent over 250 runs in a 2-year period) (12). Sample loads of between 0.5 and 1 mg can be carried out on a 4 mm x 250 mm analytical column, or up to as high as 5 mg on a semi-prep scale (9 mm x 250 mm) column.

A general separation, which is common in heparin or heparan sulphate structure determination, is disaccharide analysis. This involves the de-polymerization of the glycosaminoglycan chains into their constituent disaccharide components. This is achieved by a cocktail of bacterial lyase enzymes (heparitinase I, heparitinase II and heparinase (see Table 1)) (13). Note that structural information with regard to the hexuronic acid epimer present at the nonreducing terminus (formerly iduronic or glucuronic acid) is lost by the creation of a C=C bond between C4 and C5. A reference chromatogram of structurally defined disaccharide standards can be used to identify the disaccharides present in the sample mixture.

With regards to column choice for disaccharide analysis, the Propac PA1 column separates all eight common disaccharide standards with baseline resolution and consistent and predictable elution times. In order to perform this analysis a linear chloride counter ion gradient, 0-100%, 1 M NaCl, pH 3.5, is used for example over a 45-min period at a flow rate of 1 ml/min. The sample is loaded in double-distilled water adjusted to pH 3.5. An example of the resolution of eight disaccharide standards on Propac PA1 is shown in Fig. 2. Sensitivity is approximately 50 pmol per peak, so a minimum starting sample of about 1 p,g is required for a clear compositional analysis.

An alternative to the Propac column is provided by the silica based C18 Hypersil column (Agilent) for disaccharide analysis. This column is derivatized prior to use with cetyltrimethylammonium hydroxide in order to provide the

Table 1 Enzymes for Depolymerising and Sequencing Heparan Sulphate

Enzyme

Substrate specificity:

Polysaccharide lyases

Heparitinase I (Heparinase III) Heparitinase II (Heparinase II) Heparitinase III (Heparinase I)

GlcNR(± 6S)al-4GlcA GlcNR(± 6S)al-4GlcA/IdoA GlcNS(± 6S)al-4IdoA(2S)

Exosulphatases

Iduronate-2-sulphatase

Glucosamine-6-sulphatase

Sulphamidase (glucosamine-A'-sulphatase)

Glucuronate-2-sulphatase

Glucosamine-3-sulphatase

GlcNS03(3S)

GlcNSOa GlcA(2S)

IdoA(2S)

GlcNAc(6S), GlcNS03(6S)

Exoglycosidases Iduronidase Glucuronidase a-/V-acetylglucosaminidase

IdoA GlcA GlcNAc

Bacterial exoenzymes

A4,5-Glycuronate-2-sulphatase A4,5-Glycuronidase

AUA(2S) AUA

*The specificities are shown as the linkage specificity (for the bacterial polysaccharide lyases), and as the nonreducing terminal group recognized by the enzymes (for the exoenzymes). Sulphatases remove only the sulphate group whereas the glycosidases cleave the whole nonsulphated monosaccharide.

basic group used for anion exchange (14). The column does not appear to have the longevity of the Propac PA1 column in our hands, requiring re-derivitization after 34 months. This column does however provide better resolution than the Propac PA1 for disaccharides. This can be seen by the resolution of a- and (3-anomers with some of the disaccharide standards. The CI 8 CTA derivatized column has a mobile phase of double-distilled water brought to pH 3 with methane sulphonic acid. The counter ion is ammonium methane sulphonate and a gradient of 0-100%, 0-2 M ammonium methane sulphonate, pH 2.5 is used over 74 min at a flow rate of 0.22 ml/min. Another advantage of this column is the UV transparency of the mobile phase. This column has also been coupled to gel permeation columns to give a two-dimensional separation (14). An example of the disaccharide separation profile is shown in Fig. 3.

The separation of disaccharides derived from chemical degradation using nitrous acid cleavage poses more of a challenge. This cleavage occurs between the A'-sulphatcd glucosamine residue and the hexuronic acid leaving a reducing end anhydromannose residue, which is usually reduced to anhydromannitol (15). One advantage is that the hexuronic acid residue remains intact and can thus be identified. Detection can be achieved by reducing end labeling with radioactivity (3H-borohydride) as there is no C=C double bond at the nonreducing terminus as after lyase digestion. For separation, a silica based column has been used conventionally (e.g., SAX Partisil), but this column appears to give inadequately resolved, broad peaks. This hampers quantitation, identification, and the sensitivity of detection.

Standard 1

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