Manual

ManualsBrandsWaters ManualsWater equipmentACQUITY UPLC SEC Columns and Standards

[ CARE AND USE MANUAL ]

ACQUITY UPLC BEH SEC Columns

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

VII. INTRODUCING eCord INTELLIGENT CHIP TECHNOLOGY

a. Introduction

b. Installation

c. Manufacturing Information

d. Column Use Information

VIII. ORDERING INFORMATION

I. INTRODUCTION

Thank you for choosing a Waters ACQUITY UPLC

BEH SEC guard,

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.

Summary of content (9 pages)

PAGE 1
[ CARE AND USE MANUAL ] ACQUITY UPLC BEH SEC COLUMNS AND STANDARDS CONTENTS I. INTRODUCTION I. INTRODUCTION Thank you for choosing a Waters ACQUITY UPLC® BEH SEC guard, column, and/or standard mix that are integral parts of Waters II. CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE IN SEC PROTEIN SEPARATIONS ACQUITY UPLC SEC System Solution. The BEH125 SEC offering is best suited for the analysis of peptides and proteins in the molecular a.
PAGE 2
[ CARE AND USE MANUAL ] II. CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE IN SEC PROTEIN SEPARATIONS 1 AU 0.15 4 2 ACQUITY UPLC BEH125 SEC 7 5 6 3 0.10 0.05 a. Calibrators 0.00 In order to obtain the best performance from your SEC column, it is ACQUITY UPLC BEH200 SEC important that your ACQUITY UPLC system is properly configured. It is recommended that only pre-cut tubing is used, and that the ID of 2 AU 0.12 all connecting tubing is 0.
PAGE 3
[ CARE AND USE MANUAL ] b. Column Connectors e. Useful Functional Tests for Benchmarking a New Column It is is recommended to use the 150 mm column stabilizer Waters recommends performing a benchmarking test upon receipt of (P/N 205000365) to connect the ACQUITY UPLC BEH SEC column your column and throughout the lifetime usage. By using a separation to the ACQUITY UPLC system.
PAGE 4
[ CARE AND USE MANUAL ] 0.22 3 0.09 0.20 2 4 0.18 1 0.06 2 0.16 4 0.14 3 5 0.12 AU 0.03 0.10 0.08 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.06 10.0 min 0.04 Figure 3. Protein mixture separation on an ACQUITY UPLC BEH125 SEC, 1.7 µm column. 0.02 0.00 Table 2. BEH125 SEC Test Mix Analyte 0.0 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.
PAGE 5
[ CARE AND USE MANUAL ] IV. COLUMN SPECIFICATIONS AND USE 3 0.12 To ensure the continued high performance of ACQUITY UPLC BEH SEC, 5 1.7 µm columns, follow these guidelines: 4 0.08 a. SEC Eluent and Needle Wash Preparation 6 2 0.04 1 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0  Use HPLC-grade buffers, water, and organic solvents when possible.  Filter solutions through a compatible 0.2 µm or smaller pore size 10.0 min filter.
PAGE 6
[ CARE AND USE MANUAL ]  Volume Load: < 20 μL for 4.6 x 150 mm  Recommended pH Range: 2 to 8. The column lifetime will vary with a single injection or whether it occurred over a series of injections. If the pressure gradually built up, it is likely that the column can be cleaned as described in Section VI. depending upon the operating temperature as well as the type If a single sample caused the pressure increase, it likely reflects and concentration of buffer used.
PAGE 7
[ CARE AND USE MANUAL ] b. Storage VI. COLUMN CLEANING, REGENERATION AND STORAGE For overnight storage, continuously flush the column with the mobile a. Cleaning and Regeneration phase at 10–20% of the maximum recommended flow rate. Store Changes in peak shape, such as increased tailing, shoulders on the column in the HPLC grade water when it will be used within 24 hrs the peak, shifts in retention, change in resolution, ghost peaks, or or in 20% methanol for long term storage.
PAGE 8
[ CARE AND USE MANUAL ] d. Column Use Information b. eCord inserted into The eCord chip provides the customer with column use data. The top side of column heater of the screen identifies the column including chemistry type, column dimensions and serial number. The overall column usage information includes the total number of samples, total number of injections, total sample sets, date of first injection, date of last injection, maximum Figure 7.
PAGE 9
[ CARE AND USE MANUAL ] VIII. ORDERING INFORMATION Description Particle Size Dimensions Part No. ACQUITY UPLC BEH125 SEC column 1.7 μm 4.6 x 150 mm 186006505 ACQUITY UPLC BEH125 SEC column 1.7 μm 4.6 x 300 mm 186006506 ACQUITY UPLC BEH125 SEC guard column kit 1.7 μm 4.6 x 30 mm 186006504 ACQUITY UPLC BEH200 SEC column 1.7 μm 4.6 x 150 mm 186005225 ACQUITY UPLC BEH200 SEC column 1.7 μm 4.6 x 300 mm 186005226 ACQUITY UPLC BEH200 SEC guard column kit 1.7 μm 4.