Waters Acquity UPLC User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPLC Peptide CSH C18, 130 Å, 1.7 µm and XP 2.5 µm Columns
and ACQUITY PREMIER Peptide CSH C18, 130 Å, 1.7 µm Columns

CONTENTS

I. INTRODUCTION

II. GETTING STARTED

a. Column Connectors
b. Column Installation
c. Column Equilibration
d. Procedure for Using New,
Out-of-Box Columns
e. eCord Installation
f. Column QR Code
g. Initial Column Efficiency Determination
h. VanGuard Pre-Columns
i. Installation Instructions

III. COLUMN USE

a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature

IV. COLUMN CLEANING, REGENERATION,
AND STORAGE

a. Cleaning and Regeneration
b. Storage

V. eCORD INTELLIGENT CHIP TECHNOLOGY

VI. REPRESENTATIVE TEST CHROMATOGRAPH
AND CONDITIONS FOR SEPARATION OF
PROTEIN DIGEST

I. INTRODUCTION

Thank you for choosing a Waters™ ACQUITY™ UPLC™ and/
or ACQUITY PREMIER Peptide CSH™ Column. Both feature
Waters Charged Surface Hybrid (CSH) Technology which
provides excellent peak shape, high efficiency and loading
capacity for basic compounds when using acidic, low ionic
strength mobile phases. This same particle technology is
used in the XSelect™ Peptide CSH HPLC columns, thus
enabling seamless transferability between HPLC and UPLC
system platforms. The ACQUITY UPLC and ACQUITY
PREMIER Peptide CSH C18, 130 Å packing materials are
manufactured in a cGMP, ISO 9001 certified manufacturing
facility using ultra-pure reagents. Each batch of Peptide
CSH C18, 130 Å material is tested chromatographically with
acidic, basic, and neutral analytes as part of qualification
for use in peptide mapping. The results are held to narrow
specification ranges to assure excellent, reproducible
performance. ACQUITY UPLC and ACQUITY PREMIER
Peptide CSH C18, 130 Å batches are also QC tested with a
gradient separation of a tryptic digest of cytochrome c using

0.1% formic acid containing eluents. Finally, every shipped
column is individually tested for packed bed efficiency and a
Performance Chromatogram and Certificate of Batch Analysis
are provided on the eCord™ Intelligent Chip. ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C18, 130 Å Columns will
exhibit maximum chromatographic performance and benefits
ONLY when used on holistically-designed ACQUITY UPLC
Systems since these systems and columns were created and
designed to operate together.

VII. ADDITIONAL INFORMATION

VIII. CAUTIONARY NOTE

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[ CARE AND USE MANUAL ]

II. GETTING STARTED

Each ACQUITY UPLC and ACQUITY PREMIER Peptide CSH
C18, 130 Å 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 ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C18, 130 Å Column and
includes the gel batch number, analysis of unbonded particles,
analysis of bonded particles, and chromatographic results and
conditions. The 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.

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.

Note: Waters recommends using finger-tight fittings when
connecting Waters Peptide CSH C18, 130 Å, XP 2.5 µm column
to an HPLC. To ensure a proper connection to your XP 2.5 µm
column, Waters recommends p/n: 700003139 to connect to
the inlet and p/n: 700004841 to connect to the outlet of your
column. This will ensure correct seating of the HPLC system’s
connection tubing ferrules to your XP 2.5 µm column and
minimize undesired band broadening.

Optimized column inlet tubing (p/n: 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 ACQUITY
UPLC and AQCUITY PREMIER Column and tighten the
compression fitting using two 5/16-inch wrenches.

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.

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.

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

ACQUITY UPLC and ACQUITY PREMIER Peptide CSH C18,
130 Å 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).

Table 1. Empty column volumes in mL
(Multiply by 10 for flush solvent volumes)

Column length (mm) Internal diameter 2.1 mm

50 0.2 mL
100 0.4 mL
150 0.5 mL

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 information on the correct column outlet tubing, please
refer to the relevant detector section in the ACQUITY UPLC
System Operator’s Guide (p/n: 71500082502).

b. Column Installation

1. Purge the pumping system, with HPLC-grade water, 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 five minutes.

d. Procedure for Using New, Out-of-Box Columns

Prior to using a new column, it is important to confirm that
it produces reproducible chromatography and the desired
level of chromatographic resolution. To this end, it is useful
to benchmark column performance with a sample that is
representative of the intended application. The number of
injections necessary to achieve reproducible performance
may be dependent on sample characteristics and system
type. Method variables like pH, mass load, ionic strength, and
ion pairing, could also have impact. The ACQUITY PREMIER
Columns have MaxPeak™ High Performance Surfaces that
reduce the number of injections necessary to achieve desired
performance due to the improved hardware inertness.

2ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

[ CARE AND USE MANUAL ]

e. 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.

f. Column QR Code

The quick reference (QR) code that is located on the column
label provides column-specific information (i.e., the part and
serial numbers that are unique identifiers for the column), and
its encoding follows a widely adopted industry-standard.

ACQUITY UPLC Column VanGuard Pre-Column

Place wrench here

Ferrule

Collet

Place wrench here

Flow

1. Scan QR code using any device that is capable of

scanning QR codes (i.e., for smart phones and tablets,
use the built-in camera app).

2. Be directed to the column’s information hub

on waters.com.

3. Access technical and scientific information for the column

(i.e., certificate of analysis, application notes).

g. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as
found in the “Performance Test Chromatogram”, to
analyze the column upon receipt.

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.

Figure 1. VanGuard Pre- Column installation diagram.

i. Installation Instructions

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

2. Orient 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 ACQUITY UPLC and ACQUITY PREMIER Peptide
CSH C18, 130 Å Column perpendicular to work surface so
that column inlet is on the bottom (column outlet on top).

4. From below, insert VanGuard Pre-column into ACQUITY
UPLC and ACQUITY PREMIER Peptide CSH C18, 130 Å
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.

h. VanGuard Pre-Columns

VanGuard™ Pre-columns are 2.1 mm I.D. x 5 mm length guard
column devices designed specifically for use in the ACQUITY
UPLC Systems. VanGuard Pre-columns are packed with the
same chemistries and frits as our 2.1 mm I.D. ACQUITY UPLC
Peptide CSH C18, 130 Å Columns. VanGuard Pre-columns
are designed to be attached directly to the inlet side of an
ACQUITY UPLC Peptide CSH C18, 130 Å 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.

3ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns