Waters ACQUITY UPLC Peptide HSS T3 100A Guide

[ CARE AND USE MANUAL ]
1
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination
f. VanGuard Pre-columns
g. Installation Instructions
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
IV. eCORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
V. ADDITIONAL INFORMATION
a. Tips for Maximizing ACQUITY UPLC HSS T3
Column Lifetimes
VI. REPRESENTATIVE TEST CHROMATOGRAM
VII. CAUTIONARY NOTE
I. INTRODUCTION
Thank you for choosing a Waters® ACQUITY UPLC® Peptide
HSS T3, 100Å Column that contains our High Strength Silica
particle technology to deliver increased peptide retentivity
and different selectivity compared to results generated on
the Peptide CSH™ C18, 130Å or Peptide BEH C18, 130Å and
300Å Column offerings. The manufacture of our Peptide
HSS T3 particles begins with ultrapure reagents to control
the chemical composition and purity of the final product.
Peptide HSS T3 columns are manufactured in a cGMP, ISO
9001:2000 certified plant with each step being conducted
within narrow tolerances. Each batch of ACQUITY UPLC
HSS T3, 100Å material is chromatographically tested with
acidic, basic, and neutral analytes, as well part of Waters’
standard batch qualification procedure. In addition, each
batch of HSS T3, 100Å material is QC tested with a gradient
separation of a tryptic digest of cytochrome cand must pass
stringent performance criteria before acceptance for use in
an ACQUITY UPLC Peptide HSS T3, 100Å Column. A final
QC test for packed bed efficiency is also performed on each
column with this as well as the batch test results provided on
the column’s attached eCord™ Intelligent Chip.
ACQUITY UPLC Peptide HSS T3, 100Å Columns were designed
and tested specifically for use on ACQUITY UPLC Systems and
will deliver maximum chromatographic performance when
used on the holistically-designed ACQUITY UPLC System.
ACQUITY UPLC Peptide HSS T3, 100Å Columns

2ACQUITY UPLC Peptide HSS T3 Columns
[ CARE AND USE MANUAL ]
II. GETTING STARTED
Each ACQUITY UPLC Peptide HSS T3 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 Peptide HSS T3 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.
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 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 (p/n: 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for a typical 2.1 mm I.D.
by 50 mm length 1.8 µm column. Scale the flow rate up or down accordingly
based upon the flow rate and pressure guide provided in Section V
(Additional Information).
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.
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 Peptide HSS T3 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.
Table 1: Empty column volumes in mL
(multiply by 10 for flush solvent volumes)
Column internal diameter (mm)
Column length (mm) 1.0 2.1
50 0.04 0.2
100 0.08 0.4
150 0.12 0.5
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).
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.
e. 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.

3ACQUITY UPLC Peptide HSS T3 Columns
[ CARE AND USE MANUAL ]
f. 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® System. VanGuard Pre-columns are packed with the
same UPLC Chemistries and frits as our 2.1 mm I.D. UPLC
Columns. VanGuard Pre-columns are designed to be attached
directly to the inlet side of an ACQUITY UPLC 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.
5. While pushing the VanGuard Pre-column into the column
inlet, turn assembled column and pre-column 180˚ so that
the pre-column is now on top.
6. Tighten with two 5/16"wrenches placed onto the
ACQUITY UPLC Column flats and the VanGuard
Pre-column hex nut (male end) as shown.
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.
9. Reattach pre-column, apply mobile-phase flow, and
inspect for leaks.
II. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC
Peptide HSS T3 Columns, follow these guidelines:
a. Sample Preparation
1. Sample must be dissolved in a diluent compatible
with initial strength of mobile phase.
2. Sample must be completely in solution and free
of particulates.
3. To remove particulates, the sample may be filtered
with a 0.2 µm membrane.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
membrane material does not dissolve in the solvent. Contact
the membrane manufacturer with solvent compatibility
questions. Alternatively, centrifugation for 20 minutes at
15,000 rpm, followed by the transfer of the supernatant
liquid to an appropriate vial, could be considered.
ACQUITY UPLC Column VanGuard Pre-Column
Place wrench here
Ferrule
Collet
Place wrench here
Flow
g. 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 ACQUITY UPLC Peptide HSS T3 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
ACQUITY UPLC Peptide HSS T3 Column inlet and hand-
tighten (collet and ferrule are not yet permanently attached).
Table 2. Buffer Recommendations for Using ACQUITY UPLC Peptide HSS T3, 100Å Columns from pH 2 to 8
Additive/Buffer pKa Buffer range Volatility
(±1 pH unit)
Used for
mass spec Comments
TFA 0.30 —Volatile Yes Ion pair additive, can suppress MS signal, used in the 0.02–0.1% range.
Acetic acid 4.76 —Volatile Yes Maximum buffering obtained when used with ammonium
acetate salt. Used in 0.1–1.0% range.
Formic acid 3.75 —Volatile Yes Maximum buffering obtained when used with ammonium
formate salt. Used in 0.1–1.0% range.
Acetate (NH4CH2COOH) 4.76 3.76–5.76 Volatile Yes Used in the 1–10 mM range. Note that sodium or potassium salts are not volatile.
Formate (NH4COOH) 3.75 2.75–4.75 Volatile Yes Used in the 1–10 mM range. Note that sodium or potassium salts are not volatile.
Phosphate 1 2.15 1.15–3.15 Non-volatile No Traditional low pH buffer, good UV transparency.

4ACQUITY UPLC Peptide HSS T3 Columns
[ CARE AND USE MANUAL ]
b. pH Range
The recommended operating pH range for ACQUITY UPLC
Peptide HSS T3 Columns is pH 2 to 8. A listing of commonly
used buffers and additives is given in Table 2. Additionally,
the column lifetime will vary depending upon the operating
temperature, and the type and concentration of buffer used.
Note: Working at the extremes of pH, temperature and/or pressure will
result in shorter column lifetimes.
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
to use through a 0.2 µm filter. Solvents containing suspended
particulate materials will generally clog the outside surface of
the inlet distribution frit of the column. This will result in higher
operating pressure and poorer performance.
d. Pressure
ACQUITY UPLC Peptide HSS T3 Columns can tolerate
operating pressures up to 18,000 psi (1241 bar or 124 MPa).
Note: Working at the extremes of pressure, pH and/or temperature will
result in shorter column lifetimes.
e. Temperature
The maximum recommended temperature for ACQUITY UPLC
Peptide HSS T3 Columns is 60 °C. Higher temperatures can
be used, but may result in shorter column lifetimes. When
operating with mobile phases that are close to the lower pH
limit, using temperatures above 60 °C may result in shorter
column lifetimes due to hydrolysis of the bonded phase. High
temperatures should also be avoided when operating close
to the upper pH limit, where the dissolution of silica particles
starts to occur. To maximize column lifetime near the upper
pH limit, temperatures lower than 45 °C are recommended.
The rate of silica dissolution also varies with the type and
concentration of the buffer, with carbonate and phosphate
buffers giving some of the highest rates. As a result, when
using these buffers near the upper pH limit, lower temperatures
and lower concentrations should be considered.
Note: Working at the extremes of temperature, pressure and/or pH will
result in shorter column lifetimes.
III. COLUMN CLEANING, REGENERATING,
AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the
peak, shifts in retention, change in resolution or increasing
backpressure may indicate contamination of the column.
Flushing with a neat organic solvent, taking care not to
precipitate buffers, is usually sufficient to remove the
contaminant. If the flushing procedure does not solve the
problem, purge the column using the following cleaning
and regeneration procedures.
Use the cleaning routine that matches the properties of the
samples and/or what you believe is contaminating the column
(see Table 3). Flush columns with 20 column volumes of solvent.
Increasing column temperature increases cleaning efficiency.
If the column performance is poor after regenerating and
cleaning, call your local Waters office for additional support.
b. Storage
For periods longer than four days at room temperature, store
reversed-phase ACQUITY UPLC HSS Columns in 100%
acetonitrile. For elevated temperature applications, store
immediately after use in 100% acetonitrile for the best column
lifetime. Do not store columns in buffered eluents. If the mobile
phase contained a buffer salt, flush the column with 10 column
volumes of HPLC grade water (see Table 1 for common column
volumes) and replace with 100% acetonitrile for storage. Failure
to perform this intermediate step could result in precipitation
of the buffer salt in the column when 100% acetonitrile is
introduced. Completely seal column to avoid evaporation
and drying out of the bed.
Note: If a column has been run with a mobile phase that contains formate
(e.g., ammonium formate, formic acid, etc.) and is then flushed with
100% acetonitrile, slightly longer equilibration times may be necessary
when the column is re-installed and run again with a formate-containing
mobile-phase.
Table 3. Reversed-Phase Column Cleaning Sequence
Proteinaceous Samples
1. Water Option 1: Inject repeated 100 µL aliquots of dimethylsulfoxide (DMSO) using a reduced flow rate delivering 50% Eluent A and 50% Eluent B
2. Methanol Option 2: Gradient of 10% to 90% B where: A = 0.1% trifluoroacetic acid (TFA) in water, B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH3CN)
3. Isopropanol Option 3: Flush column with 7M guanidine hydrochloride, or 7 M urea
Note: To avoid potentially damaging precipitation within your column (e.g., if your separation eluent contains phosphate buffer), be certain to flush column with
5 to 10 column volumes of water BEFORE using suggested organic eluent column wash procedures.

5ACQUITY UPLC Peptide HSS T3 Columns
[ CARE AND USE MANUAL ]
IV. eCORD INTELLIGENT CHIP TECHNOLOGY
a. Introduction
The eCord Intelligent Chip Technology provides the history
of a column’s performance throughout its lifetime. The eCord
will be permanently attached to the column to assure that the
column’s performance history is maintained in the event that
the column is moved from one instrument to another.
Figure 1. Waters eCord Intelligent Chip.
Figure 2. eCord inserted into side of column heater.
At the time of manufacture, tracking and quality control
information will be downloaded to the eCord. Storing this
information on the chip will eliminate the need for a paper
Certificate of Analysis. Once the user installs the column,
the software will automatically download key parameters
into a column history file stored on the chip. In this manual,
we explain how the eCord will provide a solution for easily
tracking the history of the columns, reduce the frustration of
paperwork trails, and give customers the reassurance that a
well-performing column is installed onto their instruments.
b. Installation
Install the column into the column heater. Plug the eCord into
the side of the column heater. Once the eCord is inserted into
the column heater the identification and overall column usage
information will be available allowing the user to access column
information on their desktop.
c. Manufacturing Information
Waters eCord Intelligent Chip
eCord inserted into
side of column heater
The eCord chip provides the user with an overview of the bulk material
QC test results.
The eCord chip provides the
user with QC test conditions and
results on the column run by the
manufacturer. The information
includes mobile phases, running
conditions, and analytes used to
test the columns. In addition, the
QC results and acceptance is
placed onto the column.
d. Column Use Information
The eCord Chip provides the customer with column use
data. The top 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 pressure, and
temperature. The information also details the column history
by sample set including date started, sample set name, user
name, system name, number of injections in the sample set,
number of samples in the sample set, maximum pressure,
and temperature in the sample set and if the column.

6ACQUITY UPLC Peptide HSS T3 Columns
[ CARE AND USE MANUAL ]
V. ADDITIONAL INFORMATION
a. Tips for Maximizing ACQUITY UPLC Peptide HSS T3
Column Lifetimes
1. To maximize ACQUITY UPLC Peptide HSS T3 Column
lifetime, pay close attention to:
 Water quality (including water purification system)
 Solvent quality
 Mobile-phase preparation, storage, and age
 Sample, buffer, and mobile-phase solubilities
 Sample quality and preparation.
2. When problems arise, often only one improper practice
must be changed.
3. Always remember to:
 Use in-line filter unit or, preferably, a VanGuard
Pre-column.
 Discourage bacterial growth by minimizing the use
of 100% aqueous mobile phases where possible.
 Change aqueous mobile phase every 24–48 hours
(if 100% aqueous mobile phase use is required).
 Discard old 100% aqueous mobile phases every
24–48 hours to discourage bacterial growth.
 Add 5%–10% organic modifier to mobile
phase A and adjust gradient profile.
 Filter aqueous portions of mobile phase through
0.2 µm filter.
 Maintain your water purification system so that
it is in good working order.
 Only use ultra pure water (18 megohm-cm) water
and highest quality solvents possible. HPLC grade
water is not UPLC grade water.
4. Avoid (where possible):
 100% aqueous mobile phases (if possible)
 HPLC-grade bottled water
 “Topping off” your mobile phases
 Old aqueous mobile phases. Remember to
rinse bottles thoroughly and prepare fresh
every 24 to 48 hrs
 Using phosphate salt buffer in combination with high
ACN concentrations (e.g., >70%) due to precipitation.
5. Don’t: assume a “bad” column is the culprit when
high backpressure or split peaks are observed:
 Investigate cause of column failure
 Backpressure
 Mobile phase(s), bacteria, precipitation,
and/or samples
 Peak splitting
 Sample quality
 Injection solvent strength.
6. Remember: the diameter of UPLC Columns (1.0, 2.1, and
3.0 mm I.D.) are often lower than that of a conventional
HPLC column and therefore, mobile phases last
much longer. To reduce the chances of mobile-phase
contamination or degradation, only prepare what you
need for analysis or store excess bulk quantities in a
refrigerated environment.
7. Mobile-phase-related questions to ask:
 Am I using 100% aqueous mobile phases?
Am I able to add a small amount of organic
modifier to my mobile phase A?
 Do I filter my aqueous mobile phases
through 0.2 µm filters?
 How old is my mobile phase? Do I label the
bottle with preparation date?
 Do I “top off” or do I prepare fresh mobile phases
every 24–48 hrs?
 What is the quality of my water? Has the quality
recently changed? How is my water purification
system working? When was it last serviced?
 Am I working with pH 7 phosphate buffer (which is
VERY susceptible to bacterial growth)?
8. Sample-related questions to ask:
 If I inject neat standards prepared in mobile phase
do I observe these problems?
 If I prepare my standards in water and prepare
them like samples (e.g. SPE, filtration, etc.) do I
still observe these problems?
 Has the quality of my samples changed over time?

[ CARE AND USE MANUAL ]
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
[ CARE AND USE MANUAL ]
Waters, The Science of What’s Possible, and ACQUITY UPLC are registered trademarks of Waters Corporation. CSH, eCord,
and VanGuard are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2017 Waters Corporation April 2017 720005940EN Rev A IH-PDF
Figure 3. Separation of tryptic digest of cytochrome c on an ACQUITY UPLC HSS T3, 1.8 µm Column.
Column: ACQUITY UPLC Peptide HSS T3, 100Å,
1.8 µm, 2.1 × 150 mm (p/n: 186008756)
Sample: Cytochrome cDigestion Standard
(p/n: 186006371)
Inject: 7.5 µL
Mobile phase A: 0.045% TFA in water
Mobile phase B: 0.045% TFA in acetonitrile
Temp.: 35 °C
Wavelength: 214 nm
T1
T13T14
T14
T4
T9T10
T10
T19C
T8
T15
T19
T5
T12T13
T12
AU
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 min
Gradient: Time Flow %A %B1 Curve
0 0.2 100 0 –
9.0 0.2 85 15 6
39.0 0.2 64 36 6
43.0 0.2 40 60 11
58.0 0.2 100 0 11
67.0 0 100 0 11
VI. REPRESENTATIVE TEST CHROMATOGRAM
IX. CAUTIONARY NOTE
Some products may be hazardous during and after use and
are to be used by professional laboratory personnel trained in
the competent handling of such materials. The responsibility
for the safe use of products rests entirely with the purchaser
and user. The safety data sheets (SDS) for these products are
available at www.waters.com/sds.