Waters XSelect XP Guida

[ CARE AND USE MANUAL ]

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. XSelect XP Columns

— VanGuard Protection

g. Replacing the

VanGuard FIT Cartridge

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™ XSelect XP and/or XSelect

Premier HSS Column. The XSelect HSS packing materials

are manufactured in a cGMP, ISO 9001:2000 certified plant

using ultra pure reagents. Each batch of XSelect 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 XSelect 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 XSelect 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.

XSelect XP and XSelect Premier 2.5 µm HSS Columns

2XSelect XP and XSelect Premier 2.5 µm HSS Columns

[ CARE AND USE MANUAL ]

II. GETTING STARTED

A Certificate of Analysis and Performance Test Chromatogram

are provided with each XSelect 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 XSelect HSS columns and any

chemical, consult with your safety department and/or local

regulations on the use of proper protective equipment.

XSelect 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)

Internal Diameter

Column

Length (mm) 1.0 mm 2.1 mm 3.0 mm 4.6 mm

30 –0.1 0.2 0.3

50 0.04 0.2 0.4 1.0

100 0.08 0.4 0.8 2.0

150 0.12 0.5 1.0 2.5

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. 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.

3XSelect XP and XSelect Premier 2.5 µm HSS Columns

[ CARE AND USE MANUAL ]

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. XSelect XP Columns — VanGuard Protection

For XSelect HSS XP Columns, a separate guard holder

and guard cartridge can be easily attached to the inlet

of the column.

For more information on VanGuard products,

visit www.waters.com/vanguard.

g. Replacing the VanGuard FIT Cartridge

The VanGuard FIT Cartridge is designed specifically

for XSelect Premier Columns.

For the XSelect 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.

Flats used to tighten

guard to column

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

FIT Cartridge from the XSelect Premier VanGuard FIT Column.

Figure 1. Connection of the VanGuard Cartridge Column,

VanGuard Cartridge Holder, and analytical column.

Place 3/8-inch wrench here

Place 7/16-inch wrench here

Place 5/16-inch wrench here

VanGuard

Cartridge

VanGuard

Cartridge Holder

HPLC Column

4XSelect XP and XSelect Premier 2.5 µm HSS Columns

[ CARE AND USE MANUAL ]

Table 2. Buffer Recommendations for Using XSelect HSS Columns

Additive/Buffer pKa

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

(NH4CH3CO2)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

(NH4HCO2)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 XSelect 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 XSelect HSS

Columns is 2–8 for the HSS T3, HSS C18 SB, HSS Cyano and

HSS PFP chemistries, and 1–8 for the HSS C18 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

1. The 2.1 mm I.D. XSelect XP and XSelect Premier 2.5 µm

Columns can tolerate operating pressures up to 18,000 psi

(1241 bar or 124 MPa).

2. The 3.0 mm I.D. XSelect XP Columns can tolerate operating

pressures up to 18,000 psi (1241 bar or 124 MPa).

3. The 4.6 mm I.D. XSelect XP and XSelect Premier 2.5 µm

Columns can tolerate pressures up to 10,000 psi

(700 bar or 70 MPa).

Note: Working at the extremes of pressure, pH and/or

temperature will result in shorter column lifetimes.

5XSelect XP and XSelect Premier 2.5 µm HSS Columns

[ CARE AND USE MANUAL ]

e. Temperature

Temperatures between 20–45 °C are recommended for

operating XSelect 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 XSelect HSS Columns

Column Particle

Size

Pore

Diameter

Surface

Area pH Limits Temperature Limits Surface Carbon %

Low pH High pH

HSS C18 2.5 µm 100 Å 230 m2/g 1–8 45 °C 45 °C 230 µmol/m215

HSS T3 2.5 µm 100 Å 230 m2/g 2–8 45 °C 45 °C 230 µmol/m211

HSS C18 SB 2.5 µm 100 Å 230 m2/g 2–8 45 °C 45 °C 230 µmol/m28

HSS PFP 2.5 µm 100 Å 230 m2/g 2–8 45 °C 45 °C 230 µmol/m27

HSS CN 2.5 µm 100 Å 230 m2/g 2–8 45 °C 45 °C 230 µmol/m25

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 (CH3CN)

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.

6XSelect XP and XSelect Premier 2.5 µm 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 XSelect 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.

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.

Figure 3. eCord Intelligent Chip.

eCord Intelligent Chip

Figure 4. eCord inserted into side of column heater.

eCord inserted into

side of column heater

7XSelect XP and XSelect Premier 2.5 µm HSS Columns

[ CARE AND USE MANUAL ]

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 XSelect HSS

Column Lifetimes

1. To maximize 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 Cartridge.

■ 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, XSelect, ACQUITY, UPLC, Arc, Oasis, Sep-Pak, eCord, and VanGuard

are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

■ 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.

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