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The column was initially hydrated with 0.5% acetonitrile and 99.5% of the phosphate buffer (NaH 2 PO 4 ) at a linear rate (Table 2) from 0.4 mL/min to 0.6 mL/min over 4 min, increased to 2% acetonitrile for 0.5 min at 1.25 mL/min and then to at 1.5 mL/min over 1.5 min, then changed linearly to 65% acetonitrile over 0.9 min and finally returned to 0.5% acetonitrile and 99.5% of the phosphate buffer at 1.5 mL/min for 2 min equilibration
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