Waters ACQUITY UPC2 Trefoil Columns User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPC2 Trefoil Columns

CONTENTS

I. INTRODUCTION

II. GETTING STARTED

a. Safety Considerations

b. Column Connectors and Installation

c. eCord Installation

d. Column Equilibration

e. Initial Column Efficiency Determination

III. COLUMN USE

a. Sample Preparation

b. Solvents

c. Additives

d. Pressure

e. Band Spreading Minimization

IV. TROUBLESHOOTING

V. COLUMN CLEANING, ADDITIVES, AND STORAGE

a. Cleaning

b. Effects of Additives

c. Storage

I. INTRODUCTION

Thank you for choosing an ACQUITY UPC

ACQUITY UPC2 Trefoil Columns are specifically designed to use

the complete range of capabilities of the ACQUIT Y UPC2 System

to achieve fast, robust chiral separations. ACQUITY UPC2 Trefoil

Columns are uniquely designed for the ACQUITY UPC2 System

to enable both selectivity and speed in chiral separations and to

reduce method development time. ACQUIT Y UPC2 Trefoil Columns

consist of modified polysaccharide coated stationary phases for

broad-spectrum chiral selectivity. T he higher order structure of

the polysaccharides have specific spatial orientations that impart

chiral selectivity through well-chosen modifications or selectors.

ACQUITY UPC2 Trefoil AMY1, CEL1, and CEL2 column

chemistries are complementary to each other and independently

offer different retention characteristics for separating chiral

compounds. Selectivity can be further enhanced by using

co-solvents and additive blends that most favorably modulate

chiral recognition. These columns are designed to separate

enantiomers, stereoisomers, metabolites, degradants, and

impurities with greater resolution and speed.

Trefoil™ Column.

VI. INT RODUCING ECORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction

b. Installation

c. Manufacturing Information

d. Column Use Information

VII. ACQUITY UPC2 COLUMN FAMILY

[ CARE AND USE MANUAL ]

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min

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min

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min

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ACQUITY UPC2 Trefoil Columns for Chiral Separations

ACQUITY UPC2 Trefoil AMY1

2.5 µm Columns

Amylose tris-(3,5-dimethylphenylcarbamate)

min

Flavanone

ACQUITY UPC2 Tref oil C EL1

2.5 µm Columns

Cellulose tris-(3,5-dimethylphenylcarbamate)

ACQUITY UPC2 Trefoil CEL2

2.5 µm Columns

Cellulose tris-(3-chloro-4-methylphenylcarbamate)

Chiral separations were all run using the 2-minute screening method.

Trefoil Column: 2.1 x 50 mm Co-solvent: 1:1 MeOH:IPA w/20 mM NH4OH Gradient: 3–60% in 1.5 min, 60% for 0.5 min Flow rate: 1.2 mL/min Temperature: 40 °C ABPR: 3,200 psi UV Detection: 220 nm

The ACQUITY UPC2 packing materials are designed for use

with the ACQUITY UPC2 Systems and are manufactured in an

ISO-certified plant using ultra-pure reagents. Each batch of

ACQUITY UPC2 material is tested and the results are held to

narrow specification ranges to assure excellent, reproducible

performance. Every column is individually tested and a

Performance Chromatogram and Certificate of Batch Analysis

is provided on the eCord™ Intelligent Chip.

ACQUITY UPC2 Columns are designed and tested specifically for

use on ACQUITY UPC2 Systems. ACQUITY UPC2 Columns will

exhibit maximum chromatographic performance and benefit

ONLY when used on holistically-designed ACQUITY UPC2 Systems

since these systems and columns were created and designed to

operate together. For these reasons, Waters cannot support the

use of ACQUITY UPC2 Columns on any system other than an

ACQUITY UPC2 System.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

II. GETTING STARTED

Each ACQUITY UPC2 Column comes with a Certificate of Analysis

and a Performance Test Chromatogram. The Certificate of Analysis

is specific to each batch of packing material and includes the gel

batch number, physical characterization, analysis of unbonded

particle, analysis of bonded particles, and a SFC chromatographic

batch test. The Performance Test Chromatogram is specific to each

individual column and contains the following information: gel

batch number, column serial number, USP plate count, USP tailing

factor, capacity factor, and chromatographic conditions under

normal-phase LC conditions. These data should be stored

for future reference.

a. Safety Considerations

An SFC column, while in use, is under pressure with CO2 and

possible modifiers as a supercritical fluid. A major safety concern

is frostbite caused by adiabatic cooling when CO2 decompresses

from a fluid to a gas at atmospheric pressure. Pay attention to any

frosting on the column or system connections. This indicates a

leak, usually with temperatures far below 0 °C.

min

Oxprenolol

Tetramisole

H N

ACQUITY UPC2 Trefoil Columns Care & Use

[ CARE AND USE MANUAL ]

Any small leak could produce a situation where the LEL (lower

exposure limit) is reached. Laboratories should be equipped with

CO2 and/or O2 sensors when carbon dioxide (CO2) is in use.

b. Column Connectors and Installation

ACQUITY UPC2 Systems utilize tubing and connectors which

have been designed to meet stringent tolerance levels and to

minimize extra-column volume. For information on system tubing

and connectors, please refer to the ACQUITY UPC2 System

Operator’s Guide (Part Number 720004226EN can be found

on www.waters.com/chemcu).

Note: Scale the flow rate up or down accordingly based upon

the column i.d., length, particle size, and backpressure of the

ACQUITY UPC2 Column being installed.

1. Make sure your co-solvent pump is primed and has an adequate

solvent/modifier supply before performing injections. It is

recommended to use CO2 with purity level of 99.97% (food

grade) and use high-quality chromatography grade solvents

(see section IIIb).

2. Connect both the inlet and outlet of the column to the

UPC2 System.

3. If the column is still filled with a solvent, use a low flow rate

and back pressure setting (100 bar) to start pumping CO2 and

modifier through the column.

4. If you see frosting on the column at the inlet or outlet, tighten

the finger-tight fitting or compression screw on that side. If you

continue to see frosting, turn off the CO2 and vent the system.

Allow the column to depressurize fully before disconnecting

the inlet or outlet to troubleshoot the leaking issue.

c. eCord Installation

The eCord button should be attached to the side of the column

heater module. The eCord button is magnetized and does not

require specific orientation.

e. Initial Column Efficiency Determination

1. Before use, perform an efficiency test on the column,

using suitable analytes dissolved in a weaker injection solvent

to avoid peak distortion from strong injection solvent effects

(see section IIIb). It is recommended to use Waters Quality

Control Reference Material (QCRM) (p/n 186007950)

which contains a mixture of standards specifically chosen

for the ACQUITY UPC2 Trefoil and Torus Columns. For more

information, please review the ACQUIT Y UPC2 Columns

Brochure (literature code 720004275EN).

ACQUITY UPC2 Quality Control Reference Materials (QCRM)

Intended Use Contents

Provides convergence chromatographic performance information for both chiral and achiral modes

2. Determine the number of theoretical plates (N) and use this

value as a benchmark for periodic comparisons.

3. Repeat the test at predetermined intervals to track

column performance over time. (Slight variations may

occur if performance tests are performed on two different

ACQUITY UPC2 Systems due to the quality of the connections,

operating environment, system electronics, reagent quality

and column condition.)

Empty Column

Volumes (m L)

Column Length 2.1 mm 3.0 mm

50 mm 0.2 0.4

150 mm 0.5 1.0

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

1. 0.50 mg/mL (+/-) trans-Stilbene oxide

2. 0.50 mg/mL Thymine

3. 0.50 mg/mL Sulfamethoxazole

4. 0.50 mg/mL Sulfamethizole

In a 1 mL solution of 75:25 ACN:MeOH

Column Internal Diameter (mm)

d. Column Equilibration

ACQUITY UPC2 Columns are shipped dry. Equilibrate the column

with a minimum of 10-column volumes of the mobile phase prior

to use. (Refer to Table 2 for a listing of empty column volumes.)

Thermal and chemical equilibration are equally important

chromatography. Ensure that the mobile phase is preheated before

entering the column to prevent a thermal miss-match. This can be

done by enabling active preheating on a UPC

instrument.

ACQUITY UPC2 Trefoil Columns Care & Use

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Waters ACQUITY UPC2 Trefoil Columns User Manual

Specifications and Main Features

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User Manual