Waters ACQUITY APC Columns User Manual
ACQUITY APC Columns
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, packedbed 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.
Table 1. Physical Characteristics
Recommended
Separation Solvent
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
Temperature Limit
(°C)
pH Range
Pore Size
(Å)
dp
(µm)
Linear Range
(g/mol e)*
II. GET TING 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 extracolumn 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.
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.
2
ACQUITY APC Columns
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.
In a proper tubing/column connection (Figure 1), the tubing touches
the bottom of the column endfitting, with no void between them.
Figure 1. Proper Tubing Column Connection.
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).
Table 2. Empty Column Volumes in mL
Column Length
(mm)
30 4.6 0.50
50 4.6 0.83
75 4.6 1.25
150 4.6 2.50
Column Internal Diameter
(mm)
Volume
(mL)
The rigid hybrid particle bed used for the ACQUIT Y 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
Figure 2. Parker Ferrule in a Waters Style Endfitting.
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.
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.
3
ACQUITY APC Columns
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.
Loading...