Waters ACQUITY APC XT 45 Guide

[ CARE AND USE MANUAL ]

CONTENTS

I. INTRODUCTION

II. GETTING STARTED

a. Preparing the System

b. Column Connections

c. Column Installations

d. eCord Installation

e. Column Equilibration

III. COLUMN USE

a. Guidelines

b. Calibration

c. Useable Flow-Rate Ranges

d. Minimizing Band Spread

IV. TROUBLESHOOTING

V. COLUMN CLEANING, REGENERATION

AND STORAGE

a. Cleaning and Regeneration

b. Storage

VI. eCORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction

b. Installation

c. Column Use Information

I. INTRODUCTION

Thank you for choosing an ACQUITY APC™ Column. The

ACQUITY APC Columns are designed to achieve maximum

separation performance when used with the ACQUITY™

Advanced Polymer Chromatography™ (APC™) System. Please

take a few moments to read this manual carefully. Following

the recommendations in this manual will prolong column life

and enhance chromatographic reproducibility.

ACQUITY APC Columns are packed with sub-3-µm ethylene

bridged hybrid (BEH) particles that provide mechanical

strength, packed-bed stability and high separation efficiency.

They deliver superior chromatographic performance for all

polymer classes, including low molecular weight aqueous

and organic soluble polymers, up to a molecular weight of

2,000,000 g/mole (Table 1).

To maximize separation selectivity and performance

ACQUITY APC Columns are available in two bonding

technologies optimized for each solvent class:

ACQUITY APC XT: Packed with a high-coverage trimethyl

silane bonded to a BEH substrate. These columns are

recommended for extended temperature (< 90 °C)

separations in organic solvents.

ACQUITY APC AQ: Feature unbonded BEH substrate.

Recommended for room temperature (< 45 °C) separations

in aqueous solvents.

ACQUITY APC Columns are manufactured in an ISO 9001

facility and are held to narrow specification ranges to ensure

reproducible performance. Every column is individually tested

and a Certificate of Batch Analysis is provided on the attached

eCord™ Intelligent Chip.

ACQUITY APC Columns

2ACQUITY APC Columns

[ CARE AND USE MANUAL ]

II. GETTING STARTED

a. Preparing the System

Before attaching the column, the system must be prepared

as follows:

1. Remove the old columns and connect the tubing ends with

a zero-dead-volume connector.

2. Convert the system to the solvent required for the

separation by flushing the system and injector

pathway to remove old solvents.

For additional information, refer to the ACQUITY APC

System’s operator guide.

b. Column Connections

The ACQUITY APC System uses tubing and connectors

that have been designed to meet stringent tolerances to

minimize extra-column volume within the system. It is highly

recommended that you use the column connection hardware

that is supplied with the system, and, when needed, replace

with original manufacturer’s hardware. For applications that

require a bank of columns connected in series, a u-shaped

column-joining tube (which has been optimized to fit within

the column heater compartment) is available separately.

Table 1. Physical Characteristics

Recommended

Separation

Solvent

Temperature

Limit

(°C)

pH Range Pore Size

(Å)

(µm)

Linear Range

(g/mole)*

ACQUITY APC XT 45 Organic 90 1-11 45 1.7 200-5,000

ACQUITY APC XT 125 Organic 90 1-11 125 2.5 1,000-30,000

ACQUITY APC XT 200 Organic 90 1-11 200 2.5 3,000-70,000

ACQUITY APC XT 450 Organic 90 1-11 450 2.5 20,000-400,000

ACQUITY APC XT 900 Organic 90 1-11 900 2.5 300,000-2,000,000

ACQUITY APC AQ 45 Aqueous 45 1-9 45 1.7 200-5,000

ACQUITY APC AQ 125 Aqueous 45 1-9 125 2.5 1,000-30,000

ACQUITY APC AQ 200 Aqueous 45 1-9 200 2.5 3,000-70,000

ACQUITY APC AQ 450 Aqueous 45 1-9 450 2.5 20,000-400,000

*Linear range based on polystyrene standards

c. Column Installations

Generally, analytical results are independent of the sequence

in which a column bank is arranged. However, to improve

resolution and column life, arrange the columns in order of

decreasing pore size, with the largest pore size closest to the

injector. This is recommended because the species with the

highest molecular weight in the sample contributes the most to

the viscosity of the sample. If the largest species are separated

first, the viscosity of the sample plug decreases more quickly,

placing less strain on the column bank. In the case of higher

molecular weight polymers, there is less chance of shear

degradation of the polymer sample.

To install the columns:

1. Remove the end plugs from each column and save them.

2. Connect the first column to the injector outlet tubing. Note

the direction of flow. A flow direction arrow is etched on

the inlet side end nut of the column.

3. Finger-tighten the fitting, then tighten with a wrench by

another turn using the flats machined into the column end

nut. Do not use a wrench on the column tubing. Figure 1

shows a proper tubing-to-column connection.

4. Connect the next column to the previous column using

u-shaped tube connectors. Ensure that the solvent flow

continues in the direction shown on the column end

fittings. Thread the inlet and outlet fittings of the u-shaped

tube until finger tight, then tighten with a wrench.

5. Repeat Step 4 until all columns in the bank are connected.

6. Connect the last column to the detector inlet tubing.

3ACQUITY APC Columns

[ CARE AND USE MANUAL ]

Figure 1. Proper Tubing Column Connection.

Figure 2. Parker Ferrule in a Waters Style Endfitting.

In a proper tubing/column connection (Figure 1), the tubing

touches the bottom of the column endfitting, with no void

between them.

The presence of a void in the flow stream reduces column

performance. This can occur if a Parker ferrule is connected

to a Waters style endfitting (Figure 2).

d. eCord Installation

Attach the eCord button for each column to the side of the

column heater module noting their sequence. Up to four

columns can be installed at one time. The eCord button is

magnetized and does not require specific orientation. Once

connected, column identification and usage will be available

though the instrument’s control software.

e. Column Equilibration

ACQUITY APC Columns are shipped dry giving the user full

option of the solvent needed for the separation. Equilibrate

the column with a minimum of 20-column volumes, or until

a stable detector baseline is achieved. For a column bank,

use the sum of the column volumes in series to determine

the total equilibration volume. Refer to Table 2 for a listing

of column volumes.

Table 2. Empty Column Volumes in mL

Column Length

(mm)

Column Internal

Diameter (mm)

Volume

(mL)

30 4.6 0.50

50 4.6 0.83

75 4.6 1.25

150 4.6 2.50

The rigid hybrid particle bed used for the ACQUITY APC

Columns allows the user to rapidly switch solvents without

damaging the column packing material. Changing solvents

works best between compatible solvents. For example, when

changing between two immiscible solvents, an intermediate

solvent/co-solvent that is miscible in both initial and final

conditions should be used. For highly viscous solvents,

reduce the flow rate to avoid over pressuring the system.

Once the exchange is complete, equilibrate the column using

the final solvent conditions with a minimum of 20-column

empty volumes, or until a stable detector baseline is achieved.

III. COLUMN USE

a. Guidelines

ACQUITY APC Columns have a finite lifetime directly related to

their care and use. Column lifetime is reduced by contamination

from samples and eluents; improper handling and storage; and

exceeding operational conditions. To maximize ACQUITY APC

Column lifetime, pay attention to these guidelines:

 For best resolution and maximum column life, do not exceed

the flow rate recommendations found in Section III c.

 Protect the column from vibration and mechanical shock.

 Avoid precipitation by dissolving samples in the mobile

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

ensure that the sample, solvent and mobile phase are

miscible to avoid precipitation.

 Always use high-quality, particle-free, HPLC-grade solvents.

 Dedicate, whenever possible, the column to specific

applications. Frequent switching of samples and solvents

accelerates column deterioration and loss of resolution.

 Exceeding the upper temperature limit of the column.

 For aqueous mobile phases, take steps to avoid bacterial

contamination. For additional information refer to

“Controlling Contamination in UPLC™-MS and HPLC-MS

Systems”, Waters part number 715001307.

4ACQUITY APC Columns

[ CARE AND USE MANUAL ]

Figure 4. Effective Molecular Weight Range for ACQUITY APC Columns.

10 10

Effective Molecular Weight Range

ACQUITY APC (AQ/XT) 45

ACQUITY APC (AQ/XT) 125

ACQUITY APC (AQ/XT) 200

ACQUITY APC (AQ/XT) 450

ACQUITY APC (XT) 900

Figure 3. Calibration Curves for ACQUITY APC XT and ACQUITY APC AQ Columns (4.6 x 150 mm)

Using Polystyrene Standards.

100

1,000

10,000

100,000

1,000,000

10,000,000

0.50 1.00 1.50 2.00

Polystyrene Molecular Weight (Da)

Elution volume (mL)

ACQUITY APC

AQ/XT 450

ACQUITY APC

AQ/XT 200

ACQUITY APC

AQ/XT 900

ACQUITY APC

AQ/XT 125

ACQUITY APC

AQ/XT 45

b. Calibration

Whenever replacing a single column of a complete column bank, generate a new calibration

curve to ensure the reproducibility of the application. Figure 3 shows typical calibration curves

for each column. The calibration curves were obtained with polystyrene standards. Figure 4

shows the expected linear range based on the calibration data.

c. Useable Flow-Rate Ranges

Excessive mobile-flow rates can create backpressure that can damage the column packing

material. For any given particle size, as the pore volume increases, the base particle loses

mechanical strength due to the reduction in particle wall thickness surrounding the open pore.

Table 3 shows the maximum flow rate recommendation for a single column for each pore size,

independent of column length. When connecting multiple columns in series, the column that

specifies the lowest flow rate will dictate the maximum flow rate for the column bank. Higher

viscosity solvents will limit the flow rate due to flow restriction. Table 4 provides viscosity data

for common solvents.

5ACQUITY APC Columns

[ CARE AND USE MANUAL ]

Figure 5. Effect of Connecting Tubing on System.

Diluted/Distor ted Sample Band

0.005 inches

0.020 inches

0.040 inches

For example, if you were to select a three-column bank using

45Å, 200Å and 450Å columns and require that DMF to be

used as the mobile-phase solvent, you would be limited to

a maximum flow rate of 1.0 mL/min. In this case, DMF has a

solvent viscosity of 0.92 cP at 25 °C. Under these conditions

the 450Å column limits the flow to 1.0 mL/min, even though

the other columns can support a higher flow.

Note: When connecting multiple columns in series, the maximum flow rate

may not be achievable due to pressure limitations of the instrumentation.

Please refer to the system’s owner manual for more information.

Table 3. Recommended Mobile Phase

Flow Rate for a Single ACQUITY APC Column

Pore Size

(Å)

Maximum Flow Rate

at Solvent Viscosity

< 0.6 cP

Maximum Flow Rate

at Solvent Viscosity

> 0.6 cP

45 1.8 mL/min 1.1 mL/min

125 2.0 mL/min 1.6 mL/min

200 2.0 mL/min 1.4 mL/min

450 1.7 mL/min 1.0 mL/min

900 0.90 mL/min 0.60 mL/min

Table 4. Viscosity of Common Solvents

at Different Temperatures **

Solvent Viscosity (cP)

20 °C 40 °C

Acetone 0.32 0.26

Chloroform 0.57 0.45

Dimethyl formamide (DMF) 0.92 0.68

Dimethyl sulfoxide (DMSO) 2.00 1.50

Ethyl Acetate 0.44 0.36

Hexane 0.33 0.26

Methanol 0.60 0.44

Methylene chloride 0.44 N/A

N-Methylpyrrolidone (NMP) 1.70 1.30

Tetrahydrofuran (THF) 0.55 0.39

Toluene 0.59 0.46

Water 1.00 0.67

**References:

1. Reid, R.C., Prausnitz, J.M., Poling, B.E. The Properties of Gases

and Liquids, 4th Edition, McGraw Hill, 1987, Table 9-8.

2. http://www.wolframalpha.com

3. Yang, J. Chem Eng Data (2008), 53, 1639-1642.

d. Minimizing Band Spread

The ACQUITY APC System was designed to minimize band

spreading. Deviation from Waters specified tubing could result

in deterioration of chromatographic performance. Figure 5

shows the influence of tubing inner diameter. Using larger

tubing causes excessive peak broadening, lower sensitivity,

and loss in resolution.

IV. TROUBLESHOOTING

Changes in retention time, resolution or backpressure are

often due to column contamination. See the Column Cleaning,

Regeneration and Storage section of the care and use

manual. Information on column troubleshooting problems

may be found in HPLC Column Theory, Technology and

Practice, U.D. Neue, (Wiley-VCH, 1997); Waters HPLC

Troubleshooting Guide (Literature Code # 720000181EN);

or by visiting www.waters.com

V. COLUMN CLEANING, REGENERATION

AND STORAGE

Assuming that there is no damage to the column bed,

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 high concentrations of 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.

a. Cleaning and Regeneration

Use a cleaning routine that matches the properties of the

samples and/or what you believe is contaminating the

column. The columns are stable using a wide range of

6ACQUITY APC Columns

[ CARE AND USE MANUAL ]

Figure 7. Installing the eCord Intelligent Chip.

eCord Fob

Figure 6. eCord Intelligent Chip.

solvents to improve the dissolution of the contaminant.

Before beginning any cleaning procedure it is best to isolate

the column from downstream connections to protect further

contamination post column. Increasing column temperature

increases cleaning efficiency. If the column performance is

poor after cleaning and regenerating, call your local Waters

office for additional support.

If the inlet for the first column in the column bank series is

plugged with precipitated material or sample, it is possible

to disconnect the column and reverse the flow to dislodge

the blockage. This must be done extremely carefully at a low

flow rate (not to exceed 0.1 mL/min) to prevent disruption of

the packed sorbent bed. Once the blockage is removed, the

column must be returned to its proper flow direction. If inlet

plugging is a concern, a column in-line filter unit is available

(Waters part number 205000343).

Note: The addition of an in-line filter increases the likelihood of shear

degradation, especially for large molecular weight polymer species.

If the column performance is poor after cleaning

and regenerating, call your local Waters office for

additional support.

b. Storage

If you will be using the column again within 24 hours, special

storage procedures are not necessary. Maintain operating

temperature and reduce the flow rate to 0.1 mL/min when

columns are not in use for up to 24 hours. For maximum

column life, avoid temperature cycling. Do not leave a column

at elevated temperature without solvent flow.

For longer storage periods of APC XT columns, return the

column to its box with the end plugs firmly in place.

For longer storage periods of APC AQ columns, flush existing

mobile phase from the column with 10 to 20-column volumes

of HPLC grade and filtered water to remove any buffer and

salts. Next, flush the APC AQ column with 10 to 20-column

volumes of 100% acetonitrile. The column can then be safely

stored at room temperature with end plugs firmly in place.

Note: It is important that all salt and/or buffer is flushed from the APC AQ

column before storage in acetonitrile. Failure to remove salt and/or buffer

may cause precipitation in the column when exposed to organic solvent,

resulting in column clogging.

VI. eCORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction

The eCord Intelligent Chip will provide 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.

b. Installation

Install the column into the ACQUITY APC Column Manager.

Plug the eCord into the side of the column heater noting the

order of the attachment point. Once the eCord is inserted into

the column heater (see Figure 7) the identification and overall

column usage information will be available allowing the user

to access column information on their desktop. Up to four

columns can be connected at one 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, Empower, UPLC, Advanced Polymer Chromatography,

ACQUITY APC, ACQUITY, APC, and eCord are registered trademarks of Waters Corporation.

c. Column Use Information

The eCord Chip provides the customer with column use data, 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. In addition, the eCord provides two-way communications

between the eCord Chip and Empower™ Software.

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