Although the analysis of glucosamine hydrochloride according to USP method should be used a column packed with L8 (aminopropylsilane chemically bonded to totally porous silica gel), silica-based amino columns are often highly durable. In this application, Asahipak NH2P-50 4D, a polymer based amino column, and a silica-based amino column from other manufacturer was compared for durability. In the case of the silica-based amino column, it began to deteriorate around 200 injections, and the column deteriorated sharply after 300 injections. On the other hand, NH2P-50 4D has no significant change in the theoretical plate numbers and tailing factor after 3,000 injections, proving its excellence in durability.

[Note] This analysis requires column equilibration.
For column equilibration: First introduce 60 mM Phosphoric acid aq. at 0.5 mL/min for 15 minutes, and then switch to the eluent at 0.9 mL/min over 3 hours.



Sample : 3.8 mg/mL Glucosamine hydrochloride (in H₂O/CH₃CN=1/1), 10 μL
1. Glucosamine

Columns      : Shodex Asahipak NH2P-50 4D (4.6 mm I.D. x 150 mm)
               Silica-based amino column from other manufacturer (4.6 mm I.D. x 150 mm)
Eluent       : *Buffer (pH7.5)/CH3CN=30/70 (NH2P-50 4D)
               *Buffer (pH7.5)/CH3CN=25/75 (Silica-based amino column)              
Flow rate    : 0.9 mL/min (NH2P-50 4D)
               1.5 mL/min (Silica-based amino column)
Detector     : UV (195 nm)
Column temp. : 35 °C

*Buffer ; In a 1-L volumetric flask, dissolve 3.5 g K2HPO4 in water. 
          Add 0.25 mL Ammonium hydroxide (25 %), dilute with water to volume, and mix. 
          Adjusted with H3PO4 to a pH7.5