Waters XBridge Protein BEH SEC Columns and Standards - Manual (Page 3)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH SEC Columns and Standards
d. Useful Functional Tests for Benchmarking LC System and
XBridge Protein BEH SEC Column
Waters recommends performing a benchmarking test upon
receipt of your column and throughout the lifetime usage. By
using a separation of common proteins with an appropriate
method, you can:
■ ■
Verify the performance of the column upon receipt.
■ ■
Monitor the condition of the columns for extended use.
■ ■
Troubleshoot separation difficulties that may arise.
Waters BEH200 SEC Protein Standard Mix ( P/N 186006518 )
and BEH450 SEC Protein Standard Mix ( P/N 186006842 ) were
specifically designed for this purpose with carefully chosen
proteins and/or peptides to provide a good representation of the
intended application.
The information below details how to successfully prepare and
use the included BEH SEC Protein Standard for benchmarking or
troubleshooting purposes. Figures 2 and 3 provide separation
conditions and results you should expect to obtain.
Buffer Preparation
Chemicals:
■ ■
Sodium phosphate monobasic, monohydrate
■ ■
Sodium phosphate dibasic, anhydrous
■ ■
HPLC-grade water
Preparation of 100 mM sodium phosphate buffer (500 mL):
1. Weigh out 500 ± 0.02 g of water into a 500 mL beaker.
2. Weigh out 3.55 ± 0.02 g of sodium phosphate dibasic and add
to the 500 mL beaker.
3. Weigh out 3.45 ± 0.02 g of sodium phosphate monobasic and
add to the 500 mL beaker.
4. Stir for a minimum of 30 minutes then filter the solution
through a 0.2 µm filter.
5. Take the pH and record the value for reference purposes
(approximate pH: 6.8).
AU
0.000
0.012
0.024
0.036
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
1.) Thyroglobulin, 2.) IgG FU, 3.) BSA, 4.) Myoglobin, 5.) Uracil
1
2 3
4
5
Figure 2: Protein Mixture separation on XBridge Protein BEH SEC 200,
3.5 µm 7.8 x 150
Analyte pl MW
1. Thyroglobulin, 3 mg/mL 4.6 669,000
2. IgG, 2 mg/mL 6.7 150,000
3. BSA, 5 mg/mL 4.6 66,400
4. Myoglobin, 2 mg/mL 6.8, 7.2 17,000
5. Uracil, 0.1 mg/mL N/A 112
Table 2: BEH200 SEC Test Mix
Conditions:
Instrument:
ACQUITY TUV with Tunable UV detector
Column: XBridge Protein BEH SEC, 200Å, 3.5 μm,
7.8 x 150 mm
Sample: BEH200 SEC Protein Standard Mix
( P/N 186006518 )
Mobile phase: 100 mM sodium phosphate, pH 6.8
Weak needle wash: 100% Milli-Q ® water
Strong needle wash: 100% Milli-Q water
Seal wash: 90/10 water/methanol
Injection type: Full loop
Injection volume: 2 µL
Flow rate: 0.86 mL/min
Column temp.: Ambient
Detection: UV @ 280 nM