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

[ CARE AND USE MANUAL ]

1

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

VII. 

ADDITIONAL INFORMATION

VIII. 

CAUTIONARY NOTE

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 C

18, 130 Å packing materials are 

manufactured in a cGMP, ISO 9001 certified manufacturing 
facility using ultra-pure reagents. Each batch of Peptide 
CSH C

18, 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 C

18, 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 C

18, 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. 

ACQUITY UPLC Peptide CSH C

18, 130 Å, 1.7 µm and XP 2.5 µm Columns 

and ACQUITY PREMIER Peptide CSH C

18, 130 Å, 1.7 µm Columns

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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

[ CARE AND USE MANUAL ]

II. GETTING STARTED
Each ACQUITY UPLC and ACQUITY PREMIER Peptide CSH 
C

18, 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 C

18, 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 C

18, 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.

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.

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 C

18, 

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.)

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.

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ACQUITY 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. 

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.

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 C

18, 130 Å Columns. VanGuard Pre-columns 

are designed to be attached directly to the inlet side of an 
ACQUITY UPLC Peptide CSH C

18, 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.

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 C

18, 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 C

18, 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.

Figure 1. VanGuard Pre-Column installation diagram.

ACQUITY UPLC Column

VanGuard Pre-Column

Place wrench here

Ferrule

Collet

Place wrench here

Flow

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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

[ CARE AND USE MANUAL ]

III. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC 
and ACQUITY PREMIER Peptide CSH C

18, 130 Å Columns, 

follow these guidelines:

a. Sample Preparation 
1.  Sample must be dissolved in a diluent compatiable 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 contained an organic modifier (e.g., acetronitrile, 
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 8000 rpm, 
followed by the transfer of the supernatant liquid to an 
appropriate vial, could be considered.

b. pH Range 
The recommended operating pH range for ACQUITY UPLC 
and ACQUITY PREMIER Peptide CSH C

18, 130 Å Columns is 

1 to 11. A listing of commonly used buffers and additives is given 
in Table 2. Additionally, the column lifetime will vary depending 
upon the operating temperature, the type and concentration of 
buffer used. For example, the use of phosphate buffer at pH 8 
or above in combination with elevated temperatures will lead 
to shorter column lifetimes.

Important Note: 

Waters ACQUITY UPLC Peptide CSH C

18, 130 Å material is 

produced from our ACQUITY UPLC Peptide CSH C

18, 130 Å 

particles that undergo a surface modification by the addition of a 
low concentration of weakly basic ionizable silanes, followed by 
C

18 bonding and end capping. The optimal surface concentration 

of the ionizable silane groups is more than an order of magnitude 
lower than that of the primary C

18 bonded phase. The weakly 

basic silane groups are protonated at a low pH and are neutral at 
pH greater than 7. Consequently, and while the ACQUITY UPLC 
Peptide CSH C

18, 130 Å particles can tolerate a pH range from 

1 to 11, it will not provide frequently desired positive surface 
charge benefits when used at a pH greater than six.

c. Solvents 
To maintain maximum column performance, use high quality 
chromat-ography grade solvents. Filter all aqueous buffers 
prior to use. Pall Gelman Laboratory Acrodisc® filters are 
recommended. 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 and ACQUITY PREMIER Peptide CSH 
C

18, 130 Å 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 
Temperatures up to 80 °C are recommended for operating 
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH C

18, 

130 Å Columns in order to enhance selectivity, lower solvent 
viscosity, and increase mass transfer rates. When operating 
at high pH, lower operating temperatures are recommended 
for longer column lifetime. Working at high temperatures 
(e.g, >70 °C) may also result in shorter column lifetimes. 

Note: Working at the extremes of temperature, pressure 
and/or pH will result in shorter column lifetimes.

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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

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Table 2. Buffer recommendations for using ACQUITY UPLC and ACQUITY PREMIER 

Peptide CSH C

18, 130 Å Columns from pH 1 to 11 

Additive/Buffer

pK

a

Buffer 

range  

Volatility 

(±1 pH unit)

Used for 

Mass 

Spec

Comments

TFA

0.3

–

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 
(NH

4CH2COOH)

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 (NH

4COOH)

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.

Phosphate 2

7.2

6.20–8.20 Non-volatile

No

Above pH 7, reduce temperature/concentration 
and use a guard column to maximize lifetime.

4–Methylmorpholine

~8.4

7.4–9.4

Volatile

Yes

Generally used at 10 mM or less.

Ammonia (NH

4OH)

9.2

8.2–10.2

Volatile

Yes

Keep concentration below 10 mM and 
temperatures  below 30 °C.

Ammonium 
Bicarbonate

10.3 (HCO

3

–

)

9.2 (NH

4

+

)

6.3 (H

2CO3)

6.8–11.3

Volatile

Yes

Used in the 5–10 mM range (for MS work keep 
source >150 °C ). Adjust pH with ammonium 
hydroxide or acetic acid. Good buffering 
capacity at pH 10. 

Note: Use ammonium bicarbonate (NH

4HCO3 ), 

not ammonium carbonate ((NH

4 )2CO3 ).

Ammonium (Acetate)

9.2

8.2–10.2

Volatile

Yes

Used in the 1–10 mM range.

Ammonium (Formate)

9.2

8.2–10.2

Volatile

Yes

Used in the 1–10 mM range.

CAPSO

9.7

8.7–10.7

Non-volatile

No

Zwitterionic buffer, compatible with acetonitrile, 
used in the 1–10 mM range. Low odor.

Glycine

2.4, 9.8

8.8–10.8 Non-volatile

No

Zwitterionic buffer, can give longer lifetimes 
than borate buffer.

1–Methylpiperidine

10.2

9.3–11.3

Volatile

Yes

Used in the 1–10 mM range.

CAPS

10.4

9.5–11.5

Non-volatile

No

Zwitterionic buffer, compatible with acetonitrile, 
used in the 1–10 mM range. Low odor. 

Note: Working at the extremes of pH, temperature, and/or pressure will result in shorter column lifetimes.

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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

[ CARE AND USE MANUAL ]

IV. COLUMN CLEANING, REGENERATION, 

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 
HPLC-grade solvents. Increasing mobile-phase temperature 
to 35–55 °C increases cleaning efficiency. If the column 
performance is poor after regenerating and cleaning, call 
your local Waters office for additional support.

Table 3. Column Cleaning Sequence

Polar Samples Proteinaceous Samples

Water

Option 1: Inject repeated 100 µL aliquots 
of dimethylsulfoxide (DMSO) using a 
reduced flow rate delivering 50% Eluent 
A and 50% Eluent B

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 (CH

3CN)

Isopropanol

Option 3: Flush column with 7 M 
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.

b. Storage 
For periods longer than four days at room temperature, store 
the column 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.

V. eCORD

a. Introduction
The eCord Intelligent Chip provides the history of a 
column’s performance throughout its lifetime. The eCord 
is 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.

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. The eCord provides a 
solution to easily track the history of column usage. 

Figure 1. eCord Intelligent Chip.

eCord Intelligent Chip

Figure 2. eCord inserted into side of column heater. 

eCord inserted into 

side of column heater

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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns

[ CARE AND USE MANUAL ]

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 in Empower™ and MassLynx™ 
Software allowing the user to access column information on 
their desktop. 

c. Manufacturing Information 

Figure 4. 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.

Figure 3. The eCord chip provides the user with 

an overview of the bulk material QC test results.

Figure 5. An example of column use information provided by the eCord chip.

d. Customer Use Information

The eCord will automatically capture 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 met basic system suitability requirements.

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[ CARE AND USE MANUAL ]

VI. REPRESENTATIVE TEST CHROMATOGRAPH AND CONDITIONS 

FOR SEPARATION OF A CYTOCHROME c TRYPTIC DIGEST

1.20
1.10

1.00
0.90
0.80

0.70

0.60
0.50

0.40

0.30
0.20

0.10

0.00

2

3

4

5

6

7

8

9

10

11

12 

min.

Chromatographic Conditions: 
Column: 

ACQUITY UPLC Peptide CSH C

18, 

130 Å, 1.7 µm, 2.1 x 50 mm 
(p/n: 186006936) 

Mobile phase A: 

0.1% formic acid in 100% water

Mobile phase B: 

0.085% formic acid in 75% acetonitrile  
reconstituted w/ 200 µL of MPA, 
5.0 µL injection 

Sample: 

Tryptic digest of bovine cytochrome c 
(p/n: 186006371)

Flow rate: 

0.2 mL/min

Gradient: 

1–21% B in 5 min; 21–31.4% B in 2.5 min;  
31.4–95% B in 2.5 min; 95% B for 1.5 min;  
1% B for 2.5 min 

Temp.: 

40 °C 

UV detection:  

214 nm

Peak identification
T1 N-AcGDVEK
T13-T14 KYIPGTK
T14 YIPGTK
T4 IFVQK
T9-T10 KTGQAPGFSYTDANK
T10 TGQAPGFSYTDANK
T8 TGPNLHGLFGR
T15 MIFAGIK
T19C EDLIAY
T19 EDLIAYLK
T12-T13 GITWGEETLMEYLENPKK
T12  

GITWGEETLMEYLENPK

T5 

CAQCHTVEK (heme attached)

Retention time (min) 

Result

T19C 6.71

Retention time difference (min) 

Result

T4 and T14 

0.30

T10 and T9-T10 

0.77

T19 and T19C 

0.47

T12 and T12-T13 

0.28

VII. ADDITIONAL INFORMATION
Tips for maximizing ACQUITY UPLC and ACQUITY PREMIER 
Peptide CSH C

18, 130 Å Column lifetimes.

1.  To maximize ACQUITY UPLC and ACQUITY PREMIER 

Peptide CSH C

18, 130Å 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.

[ 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, ACQUITY, UPLC, XSelect, Empower, MassLynx, CSH, eCord, 
and VanGuard are trademarks of Waters Corporation. Acrodisc is a trademark of Pall Life Sciences. 
All other trademarks are the property of their respective owners.

©2020 Waters Corporation  October 2020  720004544EN  Rev D  IH-PDF

■

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.

■

Consider sample preparation (e.g., solid-phase 
extraction, filtration, etc).

4.  Avoid (where possible):

■

 100% aqueous mobile phases (if possible).

■

 HPLC-grade bottled water.

■

“Topping off” or adding “new” mobile phase to “old” 
mobile phase.

■

Old aqueous mobile phases. Remember to rinse bottles 
thoroughly and prepare fresh every 24–48 hours.

■

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 

back pressure 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: UPLC flow rates are often much lower and, 

therefore, mobile phases last much longer (only prepare 
what you need or store excess refrigerated).

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 hours?

■

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 a 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?

VIII. CAUTIONARY NOTE
Depending on user’s application, these products may be 
classified as hazardous following their use and as such are 
intended to be used by professional laboratory personnel 
trained in the competent handling of such materials. 
Responsibility for the safe use and disposal of products rest 
entirely with the purchaser and user. The Safety Data Sheet 
(SDS) for this product is available at www.waters.com/sds.