Waters ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

1

CONTENTS

I.  

INTRODUCTION

II. 

GETTING STARTED

a.  eCord Installation

b.  Column Installation

c.  Column Equilibration

d.  Initial  Column  Efficiency  Determination                               

e.  Column QR Code 

f.  VanGuard Pre-columns for 

  ACQUITY UPLC Columns

g.  Installation Instructions

h.  Replacing the VanGuard FIT Cartridge on  

  ACQUITY PREMIER VanGuard FIT 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

IV. 

eCORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction

b. Installation

c.  Manufacturing Information

d.  Column Use Information

V. 

ADDITIONAL INFORMATION

a.  Tips for Maximizing ACQUITY 

  HSS Column Lifetimes 

b.  Recommended Flow Rates and 

  Backpressures for Reversed-Phase 

  ACQUITY HSS Columns

VI. 

CAUTIONARY NOTE

I. INTRODUCTION
Thank you for choosing a Waters™ ACQUITY™ UPLC™ and/or 
ACQUITY PREMIER HSS Column. The ACQUITY HSS packing 
materials were designed specifically for use with the Waters 
ACQUITY UPLC System and are manufactured in a cGMP, ISO 
9001:2000 certified plant using ultra pure reagents. Each batch 
of ACQUITY HSS 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™ intelligent chip.

The ACQUITY PREMIER HSS Column is offered with or without 
a VanGuard™ Fully-Integrated Technology [FIT] Cartridge. 
To address the desire to extend the operating lifetimes of 
analytical columns, the VanGuard FIT Cartridge is designed 
to prevent the non-desired introduction of sample matrix or 
particulates onto the column without degrading the separation. 
It can be easily replaced to restore separation performance and 
extend the analytical column’s lifetime. 

In addition, the ACQUITY PREMIER HSS Columns utilize 
MaxPeak™ High Performance Surfaces, an innovative 
technology designed to increase analyte recovery, sensitivity, 
and reproducibility by minimizing analyte/surface interactions 
that can lead to sample losses.

ACQUITY UPLC and ACQUITY PREMIER HSS Columns

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ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

II. GETTING STARTED
A Certificate of Analysis and Performance Test Chromatogram 
are provided with each ACQUITY HSS Column, either in the 
column box or on the column’s eCord Intelligent Chip. The 
Certificate of Analysis is specific to each batch of packing 
material and includes the 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 the batch 
number, column serial number, USP tangent efficiency, USP 
tailing factor, retention factor, and chromatographic conditions. 
These data should be stored for future reference. For those not 
able to access the information on the eCord Intelligent Chip, the 
Certificate of Analysis and Performance Test Chromatogram 
are available on request at www.waters.com/coa. 

a. eCord Installation
(May not be available for all column configurations) 

The eCord Intelligent Chip button is designed for use on 
ACQUITY UPLC and ACQUITY Arc™ Systems, and should be 
attached to the side of the instruments’ column heater module. 
The eCord button is magnetized and does not require specific 
orientation. For more information on eCord Intelligent Chip 
functionality, see Section V.

b. Column Installation
(with or without a VanGuard FIT Cartridge) 

Note: Prior to handling ACQUITY HSS columns and any 
chemical, consult with your safety department and/or local 
regulations on the use of proper protective equipment. 

ACQUITY HSS columns are shipped in 100% acetonitrile. 
The flow rates given in the procedure below 
are for 2.1 mm I.D. columns.

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 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. 

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
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 Length (mm)

Internal Diameter

1.0 mm

2.1 mm

3.0 mm

30

–

0.1

0.2

50

0.04

0.2

0.4

100

0.08

0.4

0.8

150

0.12

0.5

1.0

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. 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.  The analyte mixture as found on the “Performance Test 

Chromatogram” that 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. The 
Waters isocratic column testing systems have been modified 
to achieve extremely low system volumes. This presents a 
more challenging test of how well the column was packed, 
and guarantees the highest quality packed column. These 
special testing systems have been modified to such an 
extent that they are not commercially viable and have limited 
method flexibility other than isocratic column testing.

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ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

Figure 1. VanGuard Pre-Column installation diagram.

ACQUITY UPLC Column

VanGuard Pre-Column

Place wrench here

Ferrule

Collet

Place wrench here

Flow

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 observed 
due to differences in the quality of the connections, 
operating environment, system electronics, reagent 
quality, column condition, and operator technique.

e. 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).

f. VanGuard Pre-columns for ACQUITY UPLC Columns
VanGuard Pre-columns are designed to work with ACQUITY 
UPLC Columns. They 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. 

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 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 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 ACQUITY 

UPLC 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.

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

inspect for leaks.

h. Replacing the VanGuard FIT Cartridge 
The VanGuard FIT Cartridge is designed specifically for 
ACQUITY PREMIER Columns. 

For the ACQUITY PREMIER Columns that have a VanGuard 
FIT Cartridge, the VanGuard FIT Cartridge may be replaced 
using two 3/8" wrenches. Simply apply the wrenches to the 
flats on the guard and column end nut and turn in a counter 
clockwise direction (Figure 1). This will allow the VanGuard FIT 
Cartridge to be removed and appropriately discarded when 
following good laboratory practices.

A new VanGuard FIT Cartridge can now be used to replace the 
discarded one. Extra cartridges can be obtained separately, 
as needed. Hand-tighten the new cartridge in a clockwise 
direction, then tighten using two 3/8" wrenches. Proper 
sealing should not require more than a 1/4 turn past the 
hand-tightened position.

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ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

Table 2. Buffer Recommendations for Using ACQUITY HSS Columns

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

4CH3CO2)

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

4HCO2)

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.

II. COLUMN USE
To ensure the continued high performance of ACQUITY HSS 
columns, follow these guidelines:

a. Sample Preparation
1.  Sample impurities often contribute to column contamination. 

One option to avoid this is to use Waters Oasis™ Solid-
Phase Extraction Cartridges/Columns or Sep-Pak™ 
Cartridges of the appropriate chemistry to clean-up 
the sample before analysis. For more information, visit 
www.waters.com/sampleprep.

2.  It is preferable to prepare the sample in the operating 

mobile phase or a mobile phase that is weaker than the 
mobile phase for the best peak shape and sensitivity.

3.  If the sample is not dissolved in the mobile phase, ensure 

that the sample, solvent and mobile phases are miscible in 
order to avoid sample and/or buffer precipitation.

Filter sample with 0.2 µm membranes to remove particulates. 
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 
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 HSS 
columns is 2–8 for the HSS T3, HSS C

18 SB, HSS Cyano and 

HSS PFP chemistries, and 1–8 for the HSS C

18 chemistry. 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.

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. See Section V for 
more information.

d. Pressure
ACQUITY HSS 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.

Flats used to tighten 

guard to column

Figure 2. Recommended 3/8” wrench placement to remove the VanGuard 

FIT Cartridge from the ACQUITY PREMIER VanGuard FIT Column.

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ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

e. Temperature
Temperatures between 20–45 °C are recommended for 
operating ACQUITY HSS 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., >40 °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.

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 
(Table 4). 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.

Table 3. Recommended pH and Temperature Limits for ACQUITY HSS Columns

Column

Particle 

Size

Pore 

Diameter

Surface 

Area

pH Limits

Temperature Limits

Surface

Carbon %

Low pH

High pH

HSS C

18

1.8 µm

100 Å

230 m2/g

1–8

45 °C

45 °C

230 µmol/m2

15

HSS T3

1.8 µm

100 Å

230 m2/g

2–8

45 °C

45 °C

230 µmol/m2

11

HSS C

18 SB

1.8 µm

100 Å

230 m2/g

2–8

45 °C

45 °C

230 µmol/m2

8

HSS PFP

1.8 µm

100 Å

230 m2/g

2–8

45 °C

45 °C

230 ��mol/m2

7

HSS CN

1.8 µm

100 Å

230 m2/g

2–8

45 °C

45 °C

230 µmol/m2

5

 
Table 4. Reversed-Phase Column Cleaning Sequence

Polar Samples

Non-polar Samples*

Proteinaceous Samples

1. Water

1.  Isoproanol (or an appropriate 

isopropanol/ water mixture**)

Option 1: Inject repeated aliquots 

of dimethylsulfoxide (DMSO)

2. Methanol

2. Tetrahydrofuran (THF)

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)

3. Tetrahydrofuran (THF)

3. Dichloromethane

4. Methanol

4. Hexane

5. Water

5. Isopropanol (followed by an appropriate 

isopropanol/water mixture**)

Option 3: Flush column with 7 M guanidine 

hydrochloride or 7 M urea

6. Mobile phase

6. Mobile phase

*  Prior to using THF or hexane, ensure your system is compatible with these solvents. THF or hexane should only be considered when the column cannot be 

cleaning by running neat, reversed-phase organic solvents such as acetonitrile. Reduce flow rate, lower operating temperatures, and limit system exposure 

to THF and/or hexane.

**  Use low organic solvent content to avoid precipitating buffers.

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ACQUITY UPLC and ACQUITY PREMIER HSS Columns

[ CARE AND USE MANUAL ]

b. Normal-phase Conditions
The HSS Cyano column can be used for both reversed-phase 
separations as well as normal-phase separations. The column 
is originally shipped in acetonitrile and is ready to use for 
reversed-phase conditions.

If you intend to use the column for normal-phase 
applications, you will need to condition the column 
with the following procedure:

1.  Flush the column with a minimum of 20 column 

volumes of 100% methanol using a low flow rate to 
avoid overpressuring the LC system. Refer to Table 1 
for minimum solvent volume.

2.  Flush the column with a minimum of 20 column 

volumes of 100% isopropanol using a low flow rate 
to avoid overpressuring the LC system. Refer to 
Table 1 for the minimum solvent volume.

3.  Flush the column with a minimum of 20 column volumes 

of 100% dichloromethane using a low flow rate to avoid 
overpressuring the LC system. Refer to Table 1 for the 
minimum solvent volume.

4.  Flush the column with the intended mobile-phase 

conditions until a stable baseline is achieved.

c. Storage
For periods longer than four days at room temperature, 
store reversed-phase ACQUITY 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.

d. Post Normal-phase Use
For rapid equilibration upon startup, it is recommended that 
you store the HSS Cyano column in the mobile phase that is 
commonly used for your normal-phase separation. Completely 
seal the column to avoid evaporation and drying out of the bed.

IV. eCORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction
The eCord Intelligent Chip 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.

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.

Figure 3. eCord Intelligent Chip.

eCord Intelligent Chip

Figure 4. eCord inserted into side of column heater. 

eCord inserted into 

side of column heater

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ACQUITY UPLC and ACQUITY PREMIER HSS 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 allowing the user to access column 
information on their desktop.

c. Manufacturing Information

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 met basic 
system suitability requirements. Up to 50 sample sets can be 
stored on the eCord chip.

Figure 6. 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 5. The eCord chip provides the user with 

an overview of the bulk material QC test results.

Figure 7. An example of column use information provided by the eCord chip.

V. ADDITIONAL INFORMATION

a. Tips for Maximizing ACQUITY HSS 
Column Lifetimes
1.  To maximize ACQUITY 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.

[ 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, Arc, Oasis, Sep-Pak, eCord, and VanGuard are 
trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2020 Waters Corporation. Produced in the U.S.A.  October 2020  715001429  Rev F  IH-PDF

■

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 ACQUITY 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?

VI. CAUTIONARY NOTE 
Depending on the 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 rests entirely with 
the purchaser and user. The Safety Data Sheet (SDS) for this 
product is available at www.waters.com/sds.