Fractionation of PEG-labeled
amino acids was performed manually employing the LC method described
above but injecting 10 μL. Up to 15 50 μL fractions were
collected, evaporated until dryness using a Concentrator 5301 at 30
°C (Eppendorf; Hamburg, Germany), and reconstituted in 100 μL
of 20% acetonitrile (HPLC-S gradient grade; Biosolve; Valkenswaard,
The Netherlands). Due to the noncomplete LC separation some of the
fractions contained more than one PEG-labeled amino acid. Flow injection
analysis was implemented on the LC–MS system described above
without the use of a column. In each analysis, five injections of
1 μL of sample were preceded and followed by a 1 μL injection
of a blank (ultrapure water containing 20% acetonitrile and 0.1% formic
acid). The mobile phase consisted of 3%, 10%, 20%, 40%, 60%, or 80%
acetonitrile and 0.1% (v/v) formic acid in ultrapure water at a flow
rate of 50 μL/min.
amino acids was performed manually employing the LC method described
above but injecting 10 μL. Up to 15 50 μL fractions were
collected, evaporated until dryness using a Concentrator 5301 at 30
°C (Eppendorf; Hamburg, Germany), and reconstituted in 100 μL
of 20% acetonitrile (HPLC-S gradient grade; Biosolve; Valkenswaard,
The Netherlands). Due to the noncomplete LC separation some of the
fractions contained more than one PEG-labeled amino acid. Flow injection
analysis was implemented on the LC–MS system described above
without the use of a column. In each analysis, five injections of
1 μL of sample were preceded and followed by a 1 μL injection
of a blank (ultrapure water containing 20% acetonitrile and 0.1% formic
acid). The mobile phase consisted of 3%, 10%, 20%, 40%, 60%, or 80%
acetonitrile and 0.1% (v/v) formic acid in ultrapure water at a flow
rate of 50 μL/min.