OraChrom, Inc.

APPLICATION NOTE

AN121799-10

Fast Separation of Protein Isoforms with Fully Porous Hard Gel Media: Hemoglobins.

Separation of nearly identical protein variants (isoforms) is a very difficult challenge, whether for characterization, quantitation, and/or purification.

Ion exchange chromatography with highly porous polymeric hard gels can provide a high degree of resolution far more efficiently than traditional soft gel or reversed phase chromatography.

The chromatogram below shows hemoglobin (Hb) isoforms separated on a narrow bore DEAE STYROSä column. The structure of Hb A (the principal isoform in the adult) is a heterotetramer : a₂b₂.
Hb A2, a second Hb species in the adult, varies at the three d-chain aminoacids (diagram). Hb S, the pathologic variant in sickle cell anemia, differs only in the substitution of Glutamic acid by Valine in the b-chain. It is nevertheless sharply separated by STYROSä.

The chromatographic conditions are summarized in the following table.

HPLC System.	HP 1100
Column	STYROS™ DEAE/NB 250x2.1mm
Mobile Phase	A: 20 mM Tris, pH = 8.2 B: A + 1 M NaCl
Flow rate	1 ml/min (1,800 cm/hr)
Gradient	1 to 15 % B in 18 Column Volume
Temperature	30°C
Detection	280 nm
Injection volume	5 ml
Samples	Hb A, Hb S (2.5 mg/ml each) Hb A2 (as impurity)

Note: the linear flow rate is 1,800 cm/hr, to be compared with the usual rate of 180 cm/hr available with soft gel media, a 10 x advantage.

Separation is complete: there is essentially no loss of resolution compared with that at lower flow rates.

The high dynamic capacity (80 mg/ml) of STYROSä media avoids the saturation artifacts commonly seen with lower capacity media.

Regeneration and re-equilibration of the STYROSä column requires less than 5 minutes. The useful life of the column is 3-5 times longer than stationary phases based on silica or soft gels.

Revalidation requires proportionally less time.

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