Waters ACQUITY UPLC SEC Columns and Standards User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPLC BEH SEC COLUMNS AND STANDARDS

CONTENTS

I. INTRODUCTION

II. CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE

IN SEC PROTEIN SEPARATIONS

a. Calibrators

III. GETTING STARTED

a. eCord™ Installation

b. Column Connectors

c. Column Installation

d. Column Equilibration

e. Useful Functional Tests for Benchmarking a New Column

IV. COLUMN SPECIFICATIONS AND USE

a. SEC Eluent and Needle Wash Preparation

b. Sample Preparation

c. Column Specification

V. TROUBLESHOOTING

VI. COLUMN CLEANING, REGENERATION AND STORAGE

a. Cleaning and Regeneration

b. Storage

I. INTRODUCTION

Thank you for choosing a Waters ACQUITY UPLC

column, and/or standard mix that are integral parts of Waters

ACQUITY UPLC SEC System Solution. The BEH125 SEC offering is

best suited for the analysis of peptides and proteins in the molecular

weight range from 1,000–80,000 Daltons that include insulin and

its aggregates. The BEH200 SEC column was designed to characterize

proteins ranging in molecular weight range from 10,000–450,000

Daltons that include monoclonal IgG monomers from aggregates,

while our BEH450 SEC is best suited for the analyses of proteins

and conjugates that range from 100,000–1.5 million Daltons

(Fig 1). All of these SEC chemistries are based on Waters Ethylene

Bridged Hybrid (BEH)-based particle technology and diol-bonded

surface that provide a stable chemistry with minimal non-desired

secondary interactions for proteins and peptides. The columns are

manufactured in a cGMP, ISO 9001 certified plant using ultra

pure reagents. Each batch of Protein Separation Technology SEC

packing material has been tested and qualified using a protein

test mixture (that is also available for purchase), and the results

are held to narrow specification ranges to ensure reproducible

performance. Every column is also individually tested for efficiency.

A Performance Test Chromatogram along with a Certification of

Analysis are available with each column.

BEH SEC guard,

VII. INTRODUCING eCord INTELLIGENT CHIP TECHNOLOGY

a. Introduction

b. Installation

c. Manufacturing Information

d. Column Use Information

VIII. ORDERING INFORMATION

ACQUITY UPLC BEH SEC Columns

[ CARE AND USE MANUAL ]

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

1,000,000

100,000

10,000

1000

100

IgM (900 kDa)

Ovalbumin (44 kDa)

Myoglobin (17 kDa)

RNase A (13.7 kDa)

Blue Dextran

Conalbumin (75 kDa)

Aprotinin (6.5 kDa)

Angiotensin frag 1-7 (899 Da)

Thyroglobulin (669 kDa)

IgG (150 kDa)

Amylglucosidase (97 kDa)

Allantoin (158 Da)

Uracil (112 Da)

ACQUITY UPLC BEH125 SEC,

1.7 µm: 1K–80K Daltons

ACQUITY UPLC BEH200 SEC,

1.7 µm: 10K–450K Daltons

ACQUITY UPLC BEH450 SEC,

2.5 µm: 100K–1500K Daltons

0.15

0.10

0.05

0.00

0.12

0.08

0.04

0.00

Thyroglobulin dimer (Approx. 1.4 million Da ltons)

0.10

0.08

0.06

AU AU AU

0.04

0.02

0.00

2 3 4 5 6 7 8 min

4 5

ACQUITY U PLC BEH125 SEC

ACQUITY U PLC BEH200 SEC

ACQUITY U PLC BEH450 SEC

Note: Stds 4 and 6 above were not included in the

BEH450 Column test mix

Compounds:

1. Thyroglobulin (670K)

2. BSA (66K)

3. Ovalbumin (44K)

4. Carbonic Anhydrase (29K)

Columns

Column Configuration

ACQUITY U PLC BEH125, 1.7 µm

ACQUITY U PLC BEH200 1.7 µm

5. Myoglobin (17K)

6. Angiotensin Frag 1–7 (899)

7. Uracil (112)

ACQUITY U PLC BEH450, 2.5 µm

4.6 x 150 mm

Mobile Phase 100 mM Sodium Phosphate Buffer, pH 6.8

Weak Needle Wash

Strong Needle Wash

100% Milli-Q® Water

100% Milli-Q Water

Seal wash 90/10 water/methanol

Thyroglobulin 0.3 mg/mL

Samples: Diluted in mobile phase

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

Figure 2. Separation of Protein and Peptide Standards on ACQUITY UPLC BEH125, BEH200, and BEH450 SEC Columns

0.4 1.00.80.6

Normalized Retention Volume (Vr/VC)

Protein standards; T: 30°C; Mobile phase: 100mM sodium phosphate, pH=6.8

Figure 1. Calibration Curves on ACQUITY UPLC BEH125, BEH200, and BEH450 SEC Columns

ACQUITY UPLC BEH 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.

[ CARE AND USE MANUAL ]

b. Column Connectors

It is is recommended to use the 150 mm column stabilizer

(P/N 205000365) to connect the ACQUITY UPLC BEH SEC column

to the ACQUITY UPLC system. A reusable fitting is supplied with the

ACQUITY UPLC column stabilizer.

c. Column Installation

1. Prior to placing the column on the system, purge the solvent

delivery system of any organic or water-immiscible mobile phases.

When connecting the column, orient it in the proper direction as

noted by the arrow on the column inlet side which indicates the

correct direction of solvent flow.

2. Flush column with 100% aqueous buffer, by pumping at a flow

rate of 0.2 mL/min.

3. Ensure that the mobile phase is flowing freely from the column

outlet. Attach the column outlet to the detector using .004” ID

tubing (P/N 430001562). Monitor the system pressure to

ensure the column is within its pressure limitations.

e. Useful Functional Tests for Benchmarking a New 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.

The BEH125 SEC Protein Standard Mix (P/N 186006519), BEH200

SEC Protein Standard Mix (P/N 186006518), and BEH 450 SEC

Protein Standard Mix (P/N 1860 06 842) were specifically designed

for this purpose with carefully chosen proteins and/or peptides

to provide a good representation of the intended application.

Figure 3 is an example of the Performance Test Chromatogram using

Part Number 18600 6519, Figure 4 is an example of the Performance

Test Chromatogram using Part Number 186006518. Figure 5 is an

example of the Performance Test Chromatogram using Part Number

186006842. These are typical results obtained in a waters laboratory

using 4.6 x 150 mm columns on an ACQUITY UPLC system.

4. Gradually increase the flow rate, by not more than 0.1mL/min at a

time, as described in Step 2.

5. Once the system pressure has stabilized, ensure that there are no

leaks at either the column inlet or outlet.

d. Column Equilibration

ACQUITY UPLC BEH SEC columns are shipped in 20% methanol

in water. It is important to ensure mobile-phase compatibility before

changing to a different mobile-phase system. Equilibrate the column

with a minimum of 10 column volumes of the buffer to be used

(refer to Table 1 for column volumes).

Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volume)

Column Dimension Approximate Volume

4.6 x 150 mm 2.5 mL

4.6 x 300 mm 5.0 mL

Buffer Preparation for Standard:

Chemicals:

- Sodium Phosphate Monobasic, Monohydrate

- Sodium Phosphate Dibasic, Anhydrous

- Milli-Q Water

Preparation of 100mM Sodium Phosphate Buffer (500 mL)

- Weigh out 500 ± 0.02 grams of H2O into a 500 mL beaker.

- Weigh out 3.55 ± 0.02 grams of Sodium Phosphate dibasic and

add to the 500 mL beaker.

- Weigh out 3.45 ± 0.02 grams of Sodium Phosphate monobasic

and add to the 500 mL beaker.

- Stir for a minimum of 30 minutes then filter the solution

through a 0.2 µm Millipore GV filter.

- Take the pH and record the value for reference purposes

(approximate pH: 6.8).

ACQUITY UPLC BEH SEC Columns

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