Waters CORTECS 1.6 um User Manual

CORTECS Columns

CONTENTS

I. GETTING STARTED

a. Column Connectors

b. Column Installation

c. Column Equilibration

d. eCord Installation

e. Initial Column Efficiency Determination

f. VanGuard Pre-Columns

II. COLUMN USE

a. Sample Preparation

b. pH Range

c. Solvents

d. Pressure

e. Temperature

III. COLUMN CLEANING, REGENERATING

AND STORAGE

a. Cleaning and Regeneration

b. Storage

Thank you for choosing a Waters CORTECS™ Column. The
CORTECS packing materials were designed specifically for use
with the Waters ACQUITY UPLC® System and are manufactured
in a cGMP, ISO 9001 certified plant using ultra pure reagents.
Each batch of CORTECS material is tested chromatographically with
acidic, basic and neutral analytes 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 are provided on

™

the eCord

CORTECS Columns were designed and tested specifically for use on
the ACQUITY UPLC System. CORTECS Columns will exhibit maximum
chromatographic performance and benefits when used on the
holistically-designed ACQUITY UPLC System since they were created
and designed to operate with it.

Intelligent Chip.

IV. INTRODUCING eCORD INTELLIGENT

CHIP TECHNOLOGY

a. Introduction

b. Installation

c. Manufacturing Information

d. Column Use Information

V. ADDITIONAL INFORMATION

a. Tips for Maximizing CORTECS Column Lifetimes

b. Getting Started with CORTECS HILIC Columns

I. GETTING STARTED

Each CORTECS Column comes with a Certificate of Analysis and
a Performance Test Chromatogram embedded within the eCord
intelligent chip. The Certificate of Analysis is specific to each batch
of packing material contained in the CORTECS Column and includes
the gel batch number, analysis of unbonded particles, analysis of
bonded particles, and chromatographic results and conditions. T he
Performance Test Chromatogram is specific to each individual
column and contains such information as: gel batch number,
column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data
should be stored for future reference.

Note: If mobile-phase additives are present in low concentrations
(e.g., ion-pairing reagents), 100 to 200 column volumes may be
required for complete equilibration. In addition, mobile phases that
contain formate (e.g., ammonium formate, formic acid, etc.) may
also require longer initial column equilibration times.

c. Column Equilibration

CORTECS Columns are shipped in 100% acetonitrile. 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 mobile phase to be used (refer to Table 1 for
a list of column volumes). The column may be considered thermally
equilibrated once a constant backpressure is achieved.

a. Column Connectors

The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.

Optimized column inlet tubing (Part Number 430001084) is supplied
with the ACQUITY UPLC System. The inject valve end of the tubing is
clearly marked with a blue shrink tube marker. Insert the opposite end
of the tubing into the CORTECS Column and tighten the compression
fitting using two 5/16-inch wrenches.

For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).

b. Column Installation

Note: The flow rates given in the procedure below are for a typical

1.6 µm, 2.1 mm ID by 50 mm length column. Scale the flow rate
up or down accordingly based upon the column pressure and
system limits.

1. Purge the pumping system of any buffer-containing mobile phases
and connect the inlet end of the column to the injector outlet.

2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.

3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.

Table 1. Column Volumes (mL)

Column Length (mm)

30 0.1 0.2

50 0.2 0.4

75 0.3 0.5

100 0.4 0.8

150 0.5 1.0

Internal Diameter

2.1 mm 3.0 mm

To avoid precipitating mobile-phase buffers on your column or
in your system, flush the column with five column volumes of a
water/organic solvent mixture, using the same or lower solvent
content as in the desired buffered mobile phase. (For example,
flush the column and system with 60% methanol in water prior to
introducing 60% methanol/40% buffer mobile phase).

For CORTECS HILIC Columns, flush with 50 column volumes of
50:50 acetonitrile/water with 10 mM final buffer concentration.
Prior to the first injection, equilibrate with 20 column volumes of
initial mobile-phase conditions (refer to Table 1 for a list of column
volumes). See “Getting Started with CORTECS HILIC Columns” for
additional information.

d. 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.

4. Gradually increase the flow rate as described in step 2.

5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.

2

CORT ECS Columns

e. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it. This
test may consist of:
a. An analyte test mixture that is commonly used in your
laboratory, and/or
b. An analyte mixture as found on the “Performance Test
Chromatogram” which accompanied your column.

Note: If b. is performed, the isocratic efficiencies measured in
your laboratory may be less than those given on the Waters
“Performance Test Chromatogram”. This is normal. CORTECS
Columns are QC tested on ACQUITY UPLC I-Class Systems
which have extremely low system volumes. This presents a
more challenging test of how well the column was packed and
guarantees the highest quality packed column.

2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.

3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different UPLC® Systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.

f. VanGuard Pre-Columns

VanGuard™ Pre-columns are 2.1 mm ID x 5 mm length guard
column devices designed specifically for use in the ACQUITY
UPLC System. VanGuard Pre-columns are packed with the same
CORTECS chemistries and frits as our 2.1 mm ID CORTECS
Columns. VanGuard Pre-columns are designed to be attached
directly to the inlet side of a CORTECS Column.

Note: In order to ensure void-free and leak-free connections, the
VanGuard Pre-column is shipped with the collet and ferrule NOT
permanently attached. Care must be taken when removing the
O-ring that holds these two pieces on the pre-column tubing.

CORT ECS Column

Place wrench here

Ferrule

Flow

VanGuard Pre-Column

Collet

Place wrench here

Installation Instructions

1. Remove the VanGuard Pre-column from its box and shipping
tube and remove plastic plug.

2. Orient the pre-column so that male end is facing up and
carefully remove rubber O-ring that holds collet and ferrule
in place during shipping (collet and ferrule are not yet
permanently attached).

3. Orient the CORTECS Column perpendicular to the work surface
so that column inlet is on the bottom (column outlet on top).

4. From below, insert the VanGuard Pre-column into the CORTECS
Column inlet and hand-tighten (collet and ferrule are not yet
permanently attached).

5. While pushing the VanGuard Pre-Column into the column
inlet, turn assembled column and pre-column 180˚ so that
pre-column is now on top.

6. Tighten with two 5/16” wrenches placed onto the CORTECS
Column flats and the VanGuard Pre-column hex nut (male end)
as shown above.

7. Tighten 1/4 turn to set collet and ferrule.

8. Check that the ferrule is set by loosening the connection and
inspecting the ferrule depth. A properly set ferrule depth will
resemble other connections in the ACQUITY UPLC System.

3

9. Reattach pre-column, apply mobile-phase flow and inspect
for leaks.

CORT ECS Columns