Manual
2
[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH SEC Columns
II. CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE IN
SEC PROTEIN SEPARATIONS
a. Calibrators
In order to obtain the best performance from your SEC column, it is
important that your ACQUITY UPLC system is properly configured. It
is recommended that only pre-cut tubing is used, and that the ID of
all connecting tubing is 0.005” or less for optimal chromatographic
performance.
Size-exclusion chromatography may require modifications to an
existing ACQUITY UPLC system. Please refer to “Size-Exclusion and
Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC
System” (P/N 715002147A) for specific recommendations that can
be obtained at www.waters.com/chemcu
The sample loop used may affect the performance of your separation.
Optimally, select the smallest volume sample loop that is required
for the application. Sample loops larger than 20 µL with the ACQUITY
UPLC BEH SEC column are not recommended.
III. GETTING STARTED
A Performance Test Chromatogram and a Certification of Analysis
(COA) are available for each shipped ACQUITY UPLC BEH SEC column.
The COA is specific to each batch of packing material and includes the
batch number and chromatographic separation obtained using defined
protein standards. This document is available upon request at
www.waters.com/chemcu
ACQUITY UPLC BEH450 SEC,
2.5 µm: 100K–1500K Daltons
ACQUITY UPLC BEH200 SEC,
1.7 µm: 10K–450K Daltons
ACQUITY UPLC BEH125 SEC,
1.7 µm: 1K–80K Daltons
Protein standards; T: 30°C; Mobile phase: 100mM sodium phosphate, pH=6.8
MW
100
1000
1,000,000
100,000
10,000
Normalized Retention Volume (Vr/ VC)
0.4 1.00.80.6
Uracil (112 Da)
Allantoin (158 Da)
Angiotensin frag 1-7 (899 Da)
Aprotinin (6.5 kDa)
RNase A (13.7 kDa)
Myoglobin (17 kDa)
Ovalbumin (44 kDa)
Conalbumin (75 kDa)
Amylglucosidase (97 kDa)
IgG (150 kDa)
Thyroglobulin (669 kDa)
Blue Dextran
IgM (900 kDa)
Figure 1. Calibration Curves on ACQUITY UPLC BEH125, BEH200, and
BEH450 SEC Columns
Figure 2. Separation of Protein and Peptide Standards on ACQUITY UPLC
BEH125, BEH200, and BEH450 SEC Columns
a. eCord Installation
The eCord button should be attached to the side of the ACQUITY UPLC
column heater module. The eCord button is magnetized and does not
require specific orientation.
0.00
0.04
0.08
0.12
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0.10
0.15
2 3 4 5 6 7 8 min
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7
AU AU AU
0.00
0.02
0.04
0.06
0.08
0.10
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1
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4 5
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7
Thyroglobulin dimer
(Approx. 1.4 million Daltons)
1. T hyroglobulin (670K)
2. BSA (66K)
3. Ovalbumin (44K)
4. Carbonic Anhydrase (29K)
5. Myoglobin (17K)
6. Angiotensin Frag 1–7 (899)
7. Uracil (112)
ACQUITY UPLC
BEH450 SEC
Note: Stds 4 and 6 above
were not included in the
BEH450 Column test mix
ACQUITY UPLC
BEH200 SEC
ACQUITY UPLC
BEH125 SEC
Columns
ACQUITY UPLC BEH125, 1.7 µm
ACQUITY UPLC BEH200 1.7 µm
ACQUITY UPLC BEH450, 2.5 µm
Column
Configuration
4.6 x 150 mm
Mobile Phase 100 mM Sodium Phosphate Buffer, pH 6.8
Weak
Needle Wash
100% Milli-Q
®
Water
Strong
Needle Wash
100% Milli-Q Water
Seal wash 90/10 water/methanol
Samples:
Diluted in
mobile phase
Thyroglobulin 0.3 mg/mL
BSA 0.3 mg/mL
Ovalbumin 0.3 mg/mL
Carbonic Anhydrase 0.3 mg/mL
Myoglobin 0.3 mg/mL
Angiotensin Frag. 1–7 0.1 mg/mL
Uracil 0.1 mg/mL
Thyroglobulin 3 mg/mL
BSA 5 mg/mL
Ovalbumin 3 mg/mL
Myoglobin 2 mg/mL
Uracil 0.1 mg/mL
Injection Vol. 2 µL, Full Loop
Flow Rate 0.3 mL/min
Column Temp. Ambient
Detection
Wavelength
UV @ 220 nm UV @ 280 nm
Compounds: