Biology Reference
In-Depth Information
5.1
Separation by ion-pairing chromatography
Prepare mobile phase A by mixing 0.01 mol (1.85 g or 2.38 mL) tributylamine
with 0.015 mol (0.90 g or 0.855 mL) acetic acid. Subsequently, add 50 mL
methanol (LC-MS grade) and mix thoroughly. Then, fill to a total volume of 1 L
with nanopure water of an electrical resistance greater than 16 M
Ω
. Unless
working with an inline degasser, degas mobile phase A and mobile phase B
(isopropanol).
Connect the column (Waters Acquity T3, 150 mm
m, Waters
Corporation, Milford, MA, USA) with pre-filter (KrudKatcher Ultra,
Phenomenex, Torrance, CA, USA) and place in column oven set to 40
C.
Prior to first use, wash the column for 30 min with 100% mobile phase A,
followed by a gradient to 100% mobile phase B over 30 min, then 30 min
at 100% mobile phase B and finally, a gradient to 100% mobile phase A.
Then, pre-condition the column with 10 injections of 10
2.1 mm
1.8
m
Lof10mM
potassium phosphate buffer (pH 7) to improve the peak shape of phosphorylated
analytes.
Equilibrate the column with mobile phase A, starting at a flow rate of
0.15 mL min
1
. Slowly increase the flow rate to 0.4 mL min
1
. Take care to keep
the back pressure below 1000 bar.
Connect the ultrahigh performance liquid chromatography system to the ESI
source.
Check the backpressure to determine if the liquid path (in particular, the column
and the pre-filter) is clean; typical values for a clean system are between 600 and
750 bar.
Check the mass spectrometry signal at 60
m
/
z
(the M
m
1 isotopologue of acetate
in negative mode) to ensure proper operation of the ESI source. The signal should
be stable and within 10% of the value obtained directly after optimizing the
settings of the ESI source.
Separation is obtained by a combined mobile phase and flow rate gradient.
þ
Flow (mL min
1
)
Time (min)
B (%)
0
0
0.4
5
0
0.4
10
2
0.4
11
9
0.35
16
9
0.25
18
25
0.25
19
50
0.15
25
50
0.15
26
0
0.15
32
0
0.4
36
0
0.4
