Waters XBridge BEH Glycan, 2.5 μm XP and 3.5 μm Columns and Standards - Manual (Page 3)

c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in
the desired application. Waters recommends using the solute
mixture and conditions described in the “Performance Test
Chromatogram” to test the column upon receipt.
2. Measure the retention of the test compounds and the number
of theoretical plates (N).
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different LC systems due to the quality of the connections,
operating environment, system electronics, reagent quality,
condition of column, and operator technique.
d. Useful Functional Tests for Benchmarking a New Column
We suggest use of Waters Glycan Performance Standard ( P/N
186006349 ) to benchmark your new column and monitor its
performance during use. Note: This same Glycan Performance
Standard is used by Waters manufacturing to quality control test
each batch of BEH Glycan material designed for this application
(See example in Figure 1).
To prepare the standard, add 100 µL of 100 mM ammonium
formate buffer pH 4.5 and 100 µL of acetonitrile directly to the
vial for a total volume of 200 µL.
Gently mix the sample by inversion.
Performance Test
The separation shown in Figure 1 was generated on an ACQUITY
UPLC
® whose total system volume and post column dispersion
characteristics make it ideally suited for this application. Note that
similar results can be obtained on an appropriately configured
HPLC System. It is also important to take note of the the injection
solvent and injection volume for this application. Larger glycans
have limited solubility in solutions that contain more than 50%
acetonitrile. Gradual precipitation and loss of these larger glycans
will be observed under these condtions. However, also note that
large injections of water-containing samples will distort the
peak shape in HILIC chromatography. Consequently, the optimum
injection volume for these applications is <3 μL.
LC Conditions:
LC Systems: Waters ACQUITY UPLC H-Class Bio System/
Waters Alliance 2695 HPLC
Detection: Waters ACQUITY UPLC FLR Detector/
Waters 2475 FLR Detector
Excitation: 330 nm
Emission: 420 nm
Scan Rate: 10 Hz
Time Const.: 0.2 sec
Gain: 1.00
Column: XBridge BEH Glycan, 2.5 µm XP, 130Å,
2.1 x 150 mm (P/N 186007265)
Column Temp.: 60 °C
Sample Temp.: 15 °C
Mobile Phase A: 100 mM Ammonium Formate, pH 4.5
Mobile Phase B: Acetonitrile (ACN)
Vials:
LCGC Certified Clear Glass 12 x 32 mm
Screw Neck Qsert Vial (P/N 186001126C)
Gradient: Time %A %B Flow Rate
(min) (mL/min)
0.00 22.00 78.00 0.34
56.62 44.10 55.90 0.34
58.09 80.00 20.00 0.17
65.44 80.00 20.00 0.17
68.38 22.00 78.00 0.34
75.53 22.00 78.00 0.34
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. 186006349).
e. Scalable UPLC and HPLC BEH Glycan Column Offerings
Waters BEH-based Glycan chemistry offerings are available in
three highly scalable particle sizes that address UPLC (i.e, 1.7 µm)
and HPLC-based (2.5 µm XP and 3.5 µm) application needs. (See
Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns Care and
Use Manual, literature code: 720003042EN). Figure 2 clearly
demonstrates the comparatively shorter analyses time with
improved component resolution as the particle size of the BEH
Glycan column chemistry is decreased. For this single variable
study, the same low system dispersion/volume ACQUITY UPLC
System was used with all three columns since use of different
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[ CARE AND USE MANUAL ]
XBridge BEH Glycan, 2.5 µm XP and 3.5 µm Columns and Standards