Care and use manual – Waters ACQUITY UPLC SEC Columns and Standards User Manual
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[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH SEC Columns
0.00
0.03
0.06
0.09
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0 min
1
3
2
4
Figure 3. Protein mixture separation on an ACQUITY UPLC BEH125 SEC, 1.7 µm column.
Table 2. BEH125 SEC Test Mix
Analyte
pI
MW
1. Thyroglobulin, 0.1 mg/mL
4.6
669,000
2. Ovalbumin, 0.3 mg/mL
4.5
44,200
3. Ribonuclease A, 0.3 mg/mL
9.6
13,700
4. Uracil, 0.05 mg/mL
N/A
112
Instrument:
ACQUITY UPLC System with Tunable UV (TUV) detector
Column:
ACQUITY UPLC BEH125 SEC, 4.6 x150 mm, 1.7 µm
Sample:
BEH125 SEC Protein Standard Mix
(P/N
186006519
)
BEH125 SEC Protein Standard Preparation:
Dissolve the BEH125 SEC Protein Standard in
500 μL of 100 mM sodium phosphate buffer pH
6.8. Once solubilized, do not freeze sample,
store at 2–8 °C for no more than a week.
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:
0.3 mL/min
Column Temp.:
30 °C
Detection:
UV @ 220 nm
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10 min
1
2
3
4
5
AU
Figure 4. Protein mixture separation on an ACQUITY UPLC BEH200 SEC, 1.7 µm column.
Table 3. BEH200 SEC Test Mix
Analyte
pI
MW
1. Thyroglobulin, 3 mg/mL
4.6
669,000
2. IgG, 2 mg/mL
6.7
150,000
3. BSA, 5 mg/m
L
4.6
66,400
4. Myoglobin, 2 mg/mL
6.8, 7.2
17,000
5. Uracil, 0.1 mg/mL
N/A
112
Instrument:
ACQUITY UPLC with Tunable UV (TUV) detector
Column:
ACQUITY UPLC BEH200 SEC, 4.6 x 150 mm, 1.7 µm
Sample:
BEH200 SEC Protein Standard (P/N
186006518
)
BEH200 SEC Protein Standard Preparation:
Dissolve the BEH200 SEC Protein Standard in
500 µL of 100 mM sodium phosphate buffer
pH 6.8. Once solubilized, do not freeze sample,
store at 2–8 °C for no more than a week.
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:
0.3 mL/min
Column Temp.:
30 ˚C
Detection:
UV @ 280 nm