Care and use manual – Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns, Glycan Performance Test Standard User Manual
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[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Glycan, 1.7 �m Columns
4
Injection Volume:
1.5 µL
Injection Mode:
Partial loop (20 µL loop)
Column:
ACQUITY UPLC BEH Glycan,
2.1 x 150 mm , 1.7 µm
Eluent A:
100 mM ammonium formate, pH 4.5
Eluent B:
Acetonitrile
Weak Needle Wash:
Acetonitrile/HPLC grade water,
(90/10
v/v)
Strong Needle Wash:
Acetonitrile/HPLC grade water,
(10/90
v/v)
Seal Wash:
Acetonitrile/water (50/50 v/v)
Temperature:
60 °C
Detection:
Fluorescence: λex = 330 nm,
λem = 420 nm
Gradient:
Time
Flow Rate
(min) (mL/min) %A %B
Init. 0.50 22.0 78.0
38.5 0.50 44.1 55.9
39.5 0.25*
100.0 0
44.5 0.25*
100.0 0
46.5 0.50 22.0 78.0
Note: The peptide needle and additional mixers are not required to
perform the separations in this care and use manual. However, if
these components are already installed on the system from other
applications, it is not necessary to remove them. No changes in the
separation quality of N-linked glycans should be observed if these
components are installed.
Peak Identification
1. G0 (NGA2)
8. G1F+GN
2. G0F (NGA2F)
9. Man-6
3. Man-5
10. G2 (NA2)
4. G0F+GN
11. G2F (NA2F)
5. G1
12. G1F+SA
6. G1Fa (NA2G1F)
13. G2F+SA (A1F)
7. G1Fb (NA2G1F)
10
12
14
16
18
20
22
24
26
28
30 min
1
2
3
4
6
7
8 9
10
12
13
11
5
Figure 1: Typical chromatogram of 2-AB labeled human IgG N-linked
glycans using the Glycan Performance Test Standard (Part No.
186003649).
This chromatogram is typical of the results obtained in Waters
laboratories with the method described above, using an ACQUITY
UPLC BEH Glycan, 2.1 x 150 mm , 1.7 µm column. The retention
times will be decreased by 33% on a 100 mm length column and
67% on a 50 mm length column, and the resolution of component
peaks will also be reduced proportionately, as shown in Figure 2.
Since these columns are all designed to be used with an ACQUITY
UPLC instrument which has low dead volume, high pressure and high
sensitivity, chromatographic retention times will be quite similar.
Variances will be due to system volume (e.g. the mixer volume) and
dispersion, column oven, and detector cell. This test is exceptionally
valuable for monitoring the life of the column and for trouble-
shooting separation difficulties that may arise.
0
5
10
15
20
25
30
35
40 min
1
2
3
4
5
6
7
8 9
10
11
12
13
2.1 x 150 mm
2.1 x 100 mm
2.1 x 50 mm
3
3
3
4
4
4
Figure 2: Typical chromatogram of 2-AB labeled human IgG on
2.1 x 150 mm, 2.1 x 100 mm, and 2.1 x 50 mm columns.
1. 2AB
2. 2AB
3. 2AB
4. 2AB
5. 2AB
N-Acetylglucosamine
Manose
Galactose
Fucose
Sialic Acid
12. 2AB
13. 2AB
6,7. 2AB
10. 2AB
11. 2AB
9.
2AB
8.
2AB