CONT ENTS
I. INT RODUCTION
II. GET TING START ED
a. eCord™ Installation
b. Column Connectors
c. Column Installation
d. Column Equilibration
e. Initial Column Efficiency Determination
f. Conditioning of Previously Unused Columns
and LC System Considerations
g. Useful Functional Tests for Benchmarking
a New Column: Glycoprotein Performance
Test Standard
III. COLUMN USE
a. Sample Preparation
b. Operating pH Limit
c. Solvents
d. Pressure
e. Temperature
IV. T ROUBLESHOOTING
V.
COLUMN CLEANING, REGENERATION,
AND STORAGE
a. Cleaning and Regeneration
b. Storage
VI. INT RODUCING
eCORD INT ELLIGENT
CHIP T ECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
VII. CAUTIONARY NOT E
VIII. ORDERING INFORMATION
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns
and Glycoprotein Performance Test Standard
I. INT RODUCTION
Thank you for choosing a Waters ACQUITY UPLC® Glycoprotein
BEH Amide, 300Å, 1.7 µm Column that has been purposefully
designed for intact glycoprotein profiling, subunit fragment
analysis, and glycopeptide mapping using hydrophilic interaction
chromatography (HILIC). These orthogonal analytical techniques
provide additional means of characterizing protein glycosylation
by assisting in glycan identification or in elucidating sites of
glycan occupancy as documented in Waters Application Notes
Entitled: “Developing High Resolution HILIC Separations of Intact
Glycosylated Proteins Using a Wide-Pore Amide-Bonded Stationary
Phase” (720005380EN), “Mapping IgG Subunit Glycoforms Using
HILIC and a Wide-Pore Amide Stationary Phase” (720005385EN),
and “HILIC Glycopeptide Mapping with a Wide-Pore Amide
Stationary Phase” (720005409EN).
The high resolving power of this amide-based, UPLC® Column is
due in part to the small particle size (1.7 μm) of the fully-porous
packing material when used on an appropriately configured UPLC
System. Outstanding chemical and mechanical stability of the
column, that translates into long column life, are the result of
using Waters ethylene bridged hybrid (BEH) particle coupled to
innovative Amide bonding technology.
See Pages
10–13 for
Tips & Tricks
[ CARE AND USE MANUAL ]
To help ensure column-to-column consistency, each batch of
Waters ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm
material is quality control tested with Waters Glycoprotein
Performance Test Standard (p/n 186008010). This same
glycoprotein standard (i.e, ribonuclease glycoforms) is included
with each shipped column to assist users in obtaining optimal
performance with a new column, and monitoring column and
system performance over time.
The ACQUITY UPLC Glycoprotein BEH Amide, 300Å Column
is based on the 1.7 µm particles that are characteristic of
UltraPerformance LC®. The small particle size packing reduces
dispersion and band broadening so that improved resolution,
sensitivity, and speed are obtained in glycan separations. It is
commonly expected that very high system backpressures will be
observed with such small particles. The high pressure capability
of the ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY
UPLC H-Class Bio Systems is required for UPLC glycan analysis.
With HILIC, back pressure increases with the increasing water
content during the gradient. If an aqueous wash is chosen before
re-equilibration, it might be necessary to use a lowered flow rate
to prevent excessive back pressure.
Note: ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns are
optimally designed for use with the ACQUITY UPLC, ACQUITY UPLC H-Class,
and ACQUITY UPLC H-Class Bio Systems. The expected separations will not be
obtained on a conventional HPLC system because of excessive dispersion and
pressure limitations.
II. GET TING START ED
Each ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm
Column has a Certificate of Analysis and a Performance Test
Chromatogram. The Certificate of Analysis is specific to each
batch of packing material and includes the batch number and
analyses of the physical and chemical properties of the particle.
Particle size and pore structure are analyzed prior to bonding.
The carbon and nitrogen content of the bonding are measured
to insure consistent coverage. The selectivity of each batch is
also assessed with the chromatographic separation of Waters
Glycoprotein Performance Test Standard that consists of a
mixture of Ribonuclease A and Ribonuclease B glycoforms
(p/n 186008010). The retention times and resolution of
selected components are used as the quality control test for each
batch of packing material. The Performance Test Chromatogram
is specific to each individual column and contains the following
information: batch number, column serial number, backpressure,
USP plate count, reduced plate height (RPH), USP tailing factor,
retention factor (k’), peak width, and chromatographic conditions.
These data can be found on the eCord supplied with each column
and should be stored for future reference.
a. 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.
b. Column Connectors
The ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC
H-Class Bio Systems utilize tubing and gold plated compression
screws which have been designed to meet stringent tolerance
levels and to minimize system dispersion.
For the ACQUITY UPLC System, columns should be attached to
the injector with a column stabilizer, of which there are 4 types:
205000291 50 or 100 mm column
205000365 150 mm column
205000489 HTCH 50 or 100 mm
205000494 HTCH 150 mm
The first two parts are for the original heater and differ in the
tubing arrangement to allow 150 mm columns to be used with
a VanGuard™ pre-column or in-line filter while stabilizing the
solvent temperature. The second two parts are for the newer
high temperature column heater (HTCH). Optimized column inlet
tubing 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 Column and tighten the compression fitting using two
5/16-inch wrenches (or finger tighten the knurled nut).
If this column will be used on an ACQUITY UPLC H-Class
or ACQUITY UPLC H-Class Bio System, simply connect the
column to the active preheater supplied on the system using the
gold fingertight fittings. There is only one configuration of
column stabilizer on the ACQUITY UPLC H-Class and
ACQUITY UPLC H-Class Bio Systems.
For more information on fittings and connective tubing, please
refer to the relevant sections of the operator’s guides for the
ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC
H-Class Bio Systems.
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[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
c. Column Installation
Note: The flow rates given in the procedure below are for a typical 1.7 μm
packing in a 2.1 mm I.D. column.
1. Purge the solvent delivery system of any buffer-containing
or water-immiscible mobile phases and connect the inlet
end of the column to the injector outlet. An arrow on the
column identification label indicates the correct direction
of solvent flow.
2. Flush the column with 100% organic mobile phase
(acetonitrile) by setting the pump flow to 0.1 mL/min and
increase the flow to 0.25 mL/min over 3 minutes. Increase
the aqueous phase to 90% over 10 minutes. Note the
backpressure. Decrease aqueous phase to starting
conditions (15% aqueous in the test chromatogram).
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 from 0.25 to 0.5 mL/min
over 3 minutes.
5. Once a stable backpressure and baseline have been
achieved, proceed to the next section.
d. Column Equilibration
Glycoprotein 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 column volumes).
Table 1. Empty column volumes in mL
(multiply by 10 for flush solvent volumes)
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 higher
acetonitrile content as in the desired buffered mobile phase.
For example, flush the column and UPLC system with 50%
acetonitrile in water prior to introducing 50% acetonitrile/50%
buffered mobile phase.
Column equilibration may be judged initially by stable pressure
and by a stable detector baseline. For a specific application, it is,
however, necessary to test the required duration of equilibration.
The criteria for adequate equilibration include reproducibility of
retention time for major and minor peaks, resolution for critical
pairs, and consistent baseline characteristics.
Note: Low concentration mobile-phase additives, particularly those with minimal
buffering capacity, may require extended equilibration and re-equilibration
between gradient analyses.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it
in the desired application. Waters recommends using the
solute mixture and conditions described in the “Performance
Test Chromatogram” to test the column upon receipt. These
conditions can be found on the eCord attached to the column.
2. Measure the retention of the test compounds and the number
of theoretical plates (N).
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, condition of column, and operator technique.
Column length
Volume in mL (2.1 mm I.D.)
50
0.17
100
0.35
150
0.52
3
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
f. Conditioning of Previously Unused Columns
and LC System Considerations
New (previously unused) ACQUITY UPLC Glycoprotein BEH
Amide, 300Å, 1.7 µm Columns should be conditioned, before
actual test sample analyses. This can be accomplished via
sequential injections of a representative sample until a stable
chromatographic profile is achieved. Alternatively two 40 µg
injections of Glycoprotein Performance Test Standard (10 µL
at 4 mg/mL in 0.1% TFA, 80% ACN) could be used.
EU
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
6
EU
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
8
10
12
14
16
18
20
22 min.
4
6
8
10
12
14
16
18
20
22 min.
4
Chromatography after a single, 40 µg
protein column conditioning injection
Chromatography after two, 40 µg
protein column conditioning injections
Figure 1. Conditioning of a previously unused ACQUITY UPLC Glycoprotein BEH
Amide, 300Å, 1.7 μm Column. Top: Separation of Glycoprotein Performance Test
Standard after a single column conditioning with 40 μgs of same Glycoprotein
Performance Test Standard. Bottom: Overlay of two sequential chromatograms
of the Glycoprotein Performance Test Standard after two, 40 μg injections
(i.e., 80 μgs total protein) of same Glycoprotein Performance Test Standard.
It should also be noted that even if the Glycoprotein BEH
Amide column is to be used with an alternative mobile phase
system, such as ammonium formate, it can still prove useful to
first condition it with a gradient and mobile phases containing
0.1% TFA. TFA can be effective in cleaning a column’s station-
ary phase by both neutralizing and ion pairing contaminants
that in their ionic form might otherwise strongly adsorb to a
HILIC stationary phase.
In addition, LC systems might require passivation in order to
achieve optimal HILIC separations. It is advised to use metallic
flow paths where ever possible, including post column tubing
that extends to optical detectors and/or the divert valve of a
mass spectrometer. Stainless steel and MP35N alloy materials
have been found to show better compatibility with large
molecule HILIC than PEEK.
g. Useful Functional Tests for Benchmarking a New Column:
Glycoprotein Performance Test Standard
We suggest use of Waters Glycoprotein Performance Test
Stardard (p/n 186008010) to benchmark your new column and
monitor its performance during use.
Stability: Please store the standard in the original packaging at
-20 °C upon arrival for long-term storage before solubilization.
Once solubilized, the standard can be aliquoted and stored frozen
for up to 3 months or stored at 4-10 °C for no more than a week,
but avoid any freeze/thaw that could lead to degradation.
Recommended Reconstitution: Reconstitute the contents of
each vial with 25 μL of 0.1% TFA in 80% ACN. For conditioning
a previously unused 2.1 mm ID ACQUITY UPLC Glycoprotein
BEH Amide, 300Å, 1.7 μm Column, perform two sequential
gradient separations of the standard injected at a volume of
10 μL. For benchmarking and monitoring the performance of
a 2.1 mm ID ACQUITY UPLC Glycoprotein BEH Amide, 300Å,
1.7 μm Column, perform gradient separations of the standard
injected at a volume of 0.5 μL.
Generic Chromatographic Method
The conditions shown below are for use on an ACQUITY UPLC
H-Class or ACQUITY UPLC H-Class Bio System, wherein 70/30
acetonitrile/water is used for the purge and wash solvents.
Note that the ACQUITY UPLC H-Class and ACQUITY UPLC
H-Class Bio Systems do not have a strong and weak needle
wash. Instead, they have one purge and one wash solvent, both
of which should be in 70/30 acetonitrile/water.
It might be necessary to avoid high organic diluents for some
samples due to the propensity for proteins to precipitate
under ambient conditions. A 2.1 mm ID column can typically
accommodate up to a 1.2 µL aqueous injection before
chromatographic performance is negatively affected.
High ACN diluents can sometimes be used in intact protein
HILIC, but care must be taken to enhance the solubility of the
protein sample through either the use of TFA ion pairing at
concentrations between 0.2 to 1%, the combined application of
TFA and hexafluoroisopropanol (HFIP), or by use of co-solvents,
such as dimethylsulfoxide (DMSO).
4
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
m/z
1000
1500
2000
2500
3000
3500
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
aglycosylated
+Man5
+Man6
+Man7
+Man8
+Man9
6+
7+
8+
9+
5+
KETAA AKFER QHMDSSTSAA
CNQMMKSRNL
TKDRC KPVNT
AVCSQ KNVAC KNGQT
NCYQSYSTMS ITD CR ETGSS KYPNC AYKTT QANKH
IIVA C EGNPYVPVHF DASV
Disulfides
Injection volume:
1.0 µL
Column:
ACQUITY UPLC Glycoprotein
BEH Amide, 300Å, 1.7 μm
2.1 x 150 mm (p/n 186007963)
Eluent A:
0.1% (v/v) TFA, H2O
Eluent B:
0.1% (v/v) TFA, ACN
Wash and purge solvents: Acetonitrile/HPLC grade water,
(70/30
v/v)
Temperature:
45 °C
Native Fluorescence
Detection:
Fluorescence: λex = 280 nm,
λem = 320 nm
UV Detection:
214 / 280 nm, 2 Hz
Flow:
0.2
mL/min
Gradient:
Time
%A
%B
Curve
0.0 15.0 85.0 6
0.5 15.0 85.0 6
1.0 33.0 67.0 6
21.0 40.0 60.0 6
22.0 100.0 0.0 6
24.0 100.0 0.0 6
25.0 15.0 85.0 6
35.0 15.0 85.0 6
EU
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
5
10
15
20 min
1
2
3
4
5
6
Glycoprotein
Performance
Test Standard
Peak
Species
Rs
1
RNase A
-
2
RNase B (+Man 5) 21.2
3
RNase B (+Man 6)
3.5
4
RNase B (+Man 7)
2.7
5
RNase B (+Man 8)
2.6
6
RNase B (+Man 9)
3.1
Figure 2. Separation of the Glycoprotein Performance Test Standard
(RNase A + RNase B glycoforms) using an ACQUITY UPLC Glycoprotein BEH Amide,
300Å, 1.7 μm, 2.1 x 150 mm Column. Fluorescence detection at Ex 280 nm and Em
320 nm and a column temperature of 45 ˚C were employed in this example.
Additive/Buffer
pKa
Buffer
range (±1
pH unit)
Volatility
Used for
Mass
Spec
Comments
Trifluoroacetic acid
0.23
–
Volatile
Yes
TFA ion pairing is often essential in large molecule HILIC separations as it improves the
solubility of proteins in high ACN mobile phase and shields the proteinaceous residues from
interacting with the amide stationary phase. TFA ion pairing therefore improves peak shape,
selectivity and resolution of the hydrophilic modifications on proteins and peptides
(Waters Application Note 720005380EN)
Formic acid
3.75
–
Volatile
Yes
Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range.
Table 2. Buffer Recommendations for Using ACQUITY UPLC Glycan BEH Amide, 300Å, 1.7 μm Columns
Species
MAverage (Da)
Aglycosylated (4 disulfides)
13682.3
RNase B (4 disulfides), Man5
14899.4
RNase B (4 disulfides), Man6
15061.6
RNase B (4 disulfides), Man7
15223.7
RNase B (4 disulfides), Man8
15385.8
RNase B (4 disulfides), Man9
15548.0
Figure 3. Sequence Information and MS Data for the Glycoprotein Performance
Test Standard.
5
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
III. COLUMN USE
To ensure the continued high performance of the ACQUITY UPLC
Glycoprotein BEH Amide, 300Å, 1.7 µm Columns, observe the
following guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
Samples should be free of particles before injection into
the system.
2. If the sample is not dissolved in the mobile phase or
solvent combinations specified in this manual, ensure
that the sample, solvent, and mobile phases are miscible
in order to avoid sample and/or buffer precipitation. It is
good practice to remove any precipitate or particulates by
centrifugation at >10,000 rpm for more than 2 minutes.
b. Operating pH Limits
The recommended operating pH range for the ACQUITY UPLC
Glycoprotein BEH Amide, 300Å, 1.7 µm Column is 2 to 10. A
listing of commonly used buffers and additives is given in Table
2. Additionally, the column lifetime will vary depending on the
operating temperature as well as the type and concentration of
buffer used.
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. If filtering, Acrodisc® filters
are recommended. Solvents containing suspended particulate
materials can damage the fluidic components of the UPLC
System and will generally clog the inlet distribution frit of
the column. This will result in higher operating pressure and
poor performance.
d. Pressure
The ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm
Columns will have greatly increased backpressure when
operated in 90-100% aqueous mobile phases. When employing
long column lengths or investigating high flow rates, it might
be necessary to be mindful of this increase in pressure and to
potentially reduce flow rates during the aqueous regeneration
step of the HILIC separation. ACQUITY UPLC Glycoprotein BEH
Amide, 300Å, 1.7 µm Columns can tolerate pressures of up to
15,000 psi (1034 bar or 103 Mpa).
Note: Working at the extremes of pressure, pH and/or temperature will result in
shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C – 90 °C are recommended for
operating ACQUITY UPLC Glycoprotein BEH Amide, 300Å,
1.7 µm Columns in order to enhance selectivity, lower solvent
viscosity, and increase mass transfer rates. However, higher
temperature will have a negative effect on lifetime that will vary
depending on the pH and buffer conditions used.
IV. T ROUBLESHOOTING
The first step in systematic troubleshooting is comparing
the column, in its current state, to the column when it was
functioning properly. The method suggested in Section II for
measuring plate count is an essential first step. This technique
detects physical changes to the packed bed and chemical
changes in the bonded-phase surface. The functional test
with the ribonuclease A and B glycoforms in the Glycoprotein
Performance Test Standard may reveal more subtle changes in
surface chemistry that affect the application.
There are several common symptoms of change in the column.
1. An increase in pressure is often associated with lost
performance in the application. The first step in diagnosis
is to ensure that the elevated pressure resides in the
column rather than somewhere else in the system. This
is determined by measuring pressure with and without
the column attached to the instrument. If the system is
occluded, the blockage should be identified and removed.
If the pressure increase originates from the column, it
is helpful to know whether the problem was associated
with a single injection or whether it occurred over a
series of injections. If the pressure gradually built up,
it is likely that the column can be cleaned as described
below (Section V). For future stability, it may be useful to
incorporate a stronger regeneration step in the method.
If a single sample caused the pressure increase, it likely
reflects particulates or insoluble components. Cleaning
is still an option, but using the more aggressive methods.
The sudden pressure increase suggests that the user
should consider some sample preparation, such as high
speed centrifugation.
6
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
2. Loss of retention can reflect a change in the column
surface chemistry. Before proceeding with diagnostic or
corrective measures, check that the mobile phases have
been correctly prepared and the correct method has been
selected. Then repeat the plate count test and a separation
of the Glycoprotein Performance Test Standard. If both the
plate count and glycoprotein test show loss of retention, it
is likely that a significant fraction of the bonded phase has
been lost, and the column will require replacement. If the
changes are small and reflected only for some glycans,
one of the cleaning procedures may be effective.
3. Change in peak shape, resolution, or relative retention
of peaks. Follow the same steps as for loss of retention
(Section II).
4. Carryover and memory effects are defined as the appearance
of the constituents of one sample in the next gradient
analysis. First determine whether the column or the system
is the source of carryover. Define a gradient method that
includes an “internal gradient”. That is, the analytical
gradient is repeated within a single method. If the peaks
appear in both gradients, at the same time after start, the
carryover came from the column in what is often described
as a “memory effect”. If the peaks only appear when an
injection is made, they likely originated from adsorption
to some system component. In that case follow the
instrument manufacturer’s recommendations. Memory
effects as a source of carryover may be reduced or
eliminated in several ways. First, raising the temperature
of the separation reduces the possibility of non-specific
adsorption. Second, memory effects may be more pronounced
with steep gradients. Keep the gradient slope at 1% per
column volume or less. Finally, apparent memory effects
may actually reflect the solubility of the sample in the
mobile phase. Reducing the amount injected may eliminate
the effect.
Note: Useful general information on column troubleshooting problems may be
found in HPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH,
1997), the Waters HPLC Troubleshooting Guide (Literature code # 720000181EN),
or visit www.waters.com.
V. 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, carryover, ghost peaks,
or increasing backpressure may indicate contamination of the
column. Choose a cleaning option that may be expected to
dissolve the suspected contaminant.
1. All cleaning procedures will be more effective at higher
temperatures. It is reasonable to conduct cleaning at 70 °C.
2. It may be useful to conduct cleaning procedures at one-
half the flow rate typically used with that column. In this
way, the possibility of high pressure events is reduced.
3. The first and simplest cleaning procedure is to run a series
of gradients from 0–100% water. Be sure to reduce the
flow rate for gradients with higher than 75% aqueous
content. Columns of 150 mm length should be operated
at 250 µL per minute or less during washes. The gradients
can be as short as 5 column volumes and 3–5 repetitions
may be effective.
4. Regeneration steps and flushing procedures using
100% aqueous mobile phase can help to maintain the
optimal peak shape and selectivity of a HILIC separa-
tion. Additionally, an analyst can perform gradients
with mobile phases containing 0.1% TFA as a means to
maintain or recover the performance of a Glycoprotein
BEH Amide column. TFA can be effective in cleaning
a column’s stationary phase by both neutralizing and
ion pairing contaminants that in their ionic form might
otherwise strongly adsorb to a HILIC stationary phase.
5. Several different cleaning solutions may be injected to
strip strongly adsorbed material or particulates from the
column. Make the largest injection possible with the system
configuration. With such strong cleaning solutions, it is best to
disconnect the detector from the column and to direct the flow
to waste.
7
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
At the time of manufacture, tracking and quality control
information will be downloaded to the eCord. This includes the
conditions and results for the Performance Test Chromatogram.
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 column, 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 in the ACQUITY UPLC
Console, allowing the user to access column information on
their desktop.
eCord Fob
Figure 5. Installing the eCord Intelligent Chip.
c. Manufacturing Information
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.
6. Flow reversal or backflushing is often suggested as part
of a cleaning procedure. This should be reserved as a last
resort. It may further damage the column or provide a
short-lived improvement in performance.
b. Storage
For periods longer than four days at room temperature, store
the column in 100% acetonitrile. Immediately after use with
elevated temperatures and/or at pH extremes, store in 100%
acetonitrile for the best column lifetime. Do not store columns
in highly aqueous (<50 % organic) mobile phases. 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 or system when 100% acetonitrile
is introduced. Completely seal the column to avoid evaporation
and drying out the bed.
VI. INT RODUCING eCORD INT ELLIGENT
CHIP T ECHNOLOGY
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.
Figure 4. eCord Intelligent Chip.
8
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
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: 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.
VII. CAUTIONARY NOT E
Depending on users 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.
VIII. ORDERING INFORMATION (Partial listing. For more information visit www.waters.com).
Description
Pore size
Particle size
Dimensions
Part no.
ACQUITY UPLC Glycoprotein BEH Amide
300Å
1.7 µm
2.1 x 5 mm Guard 3/pk with Std
176003699
ACQUITY UPLC Glycoprotein BEH Amide
300Å
1.7 µm
2.1 x 50 mm with Std
176003700
ACQUITY UPLC Glycoprotein BEH Amide
300Å
1.7 µm
2.1 x 100 mm with Std
176003701
ACQUITY UPLC Glycoprotein BEH Amide
300Å
1.7 µm
2.1 x 150 mm with Std
176003702
ACQUITY UPLC Glycoprotein BEH Amide
300Å
1.7 µm
2.1 x 100 MVK, 3/pk with Std
176003703
Glycoprotein Performance Test Standard
186008010
9
[ CARE AND USE MANUAL ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
Condition Your New Column
■
■
Column conditioning is a common practice used to optimize
performance via passivation of secondary interactions.
■
■
HILIC of large molecules is a new frontier! Conditioning of new
columns is as important as ever.
■
■
The amide bonding should be primed for its interaction with the
analyte, yet once it is conditioned it need not be re-conditioned
even after long-term storage.
TIP
■
■
Before using a new column, run 2 injections of your intended
sample type at a mass load 10x greater than would be used in
a normal analytical run.
■
■
Per conditioning run, load 20-100 μg protein or
50-250 pmoles of released glycan.
■
■
If the column is for mAbs, use a mAb; tetrasialylated glycans,
use a labeled/unlabeled fetuin N-glycan pool; etc.).
■
■
Run 1 blank and continue with your analyses.
Figure 6. Conditioning of a previously unused ACQUITY UPLC Glycoprotein BEH
Amide, 300Å, 1.7 μm Column. Shown in the lower panel is an overlay of two
analytical runs.
EU
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
6
EU
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
8
10
12
14
16
18
20
22 min.
4
6
8
10
12
14
16
18
20
22 min.
4
After 1x
40 g Load
Conditioning
After 2x
40 g Load
Conditioning
HILIC FOR LARGE MOLECULES: P RACTICAL CONSIDERATIONS
Understand the Role of Ion Pairing
■
■
TFA ion pairing is often essential in large molecule HILIC
separations not only because it improves the solubility of
proteins in high ACN mobile phase but because it shields
proteinaceous residues from interacting with the amide
stationary phase.
■
■
TFA ion pairing therefore improves peak shape, selectivity
and resolution of the hydrophilic residues and modifications
of proteins and peptides.
■
■
The larger the proteinaceous analyte the more critical TFA
ion pairing.
■
■
For smaller analytes, like peptides/glycopeptides, some TFA
can be replaced with formic acid. However, peaks are likely to be
more strongly retained and to exhibit additional broadening due
to more complicated, heterogenous analyte to stationary phase
interactions. Selectivity between aglycosylated and glycosylated
peptides will also be affected.
TIP
■
■
Use mobiles phases modified with 0.1% TFA whenever possible.
0.025
0.015
0.005
-0.005
8
9
10
11
12
13
14
15
16
17
18 min.
50 mM Ammonium Formate
pH 4.4
0.5% FA
0.1% TFA
TFA
Ion Pairing
Figure 7. Effect of mobile phase additive on the separation of ribonuclease B.
10
[ TIPS & TRICKS ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
Consider Your Diluents and Sample Solubility
■
■
It is common practice to match the composition of the
sample diluent to your initial chromatographic conditions.
HILIC of large molecules requires that this be given careful
consideration, because some analytes may not be soluble
in high organic concentrations. 70-90% ACN could be a
condition that initiates the precipitation of your sample.
■
■
Understand that HILIC can in fact be performed on aqueous
samples. The only requirement is that very minimal volumes of
sample be injected (see next section).
■
■
High ACN diluents can be used, but care must be taken to
enhance the solubility of the sample.
TIP
■
■
Add TFA to the sample at concentrations between 0.2 to 1%.
■
■
Use additional additives, like heptafluorobutyric acid (HFBA) or
hexafluoroisopropanol (HFIP).
■
■
Use a co-solvent, such as dimethylsulfoxide (DMSO), in
concentrations ranging between 1 and 25%.
F
F
F
F
OH
O
F
F
F
O
S
H3C
H3C
H
H
H
C
C
N
F
O
F
F
OH
OH
F
F
F F F F
H
H
O
TFA
Ion Pairing
HFIP
DMSO
HFBA
How do you deal
with so much
organic solvent?
Can you load from
aqueous diluent?
If high ACN…
Figure 8. Additives to consider if using ACN/organic solvent diluents.
Minimize Injection Volumes
■
■
In most situations, HILIC injection volumes must be much
lower than what can otherwise be used in reversed-phase
chromatography.
■
■
100% AQUEOUS Samples
■
■
For a 2.1 mm ID column, maximum injection volumes are
usually between 0.4 and 1.2 μL (depending on the dispersion
of your LC).
■
■
ACETONITRILE Containing Samples
■
■
Adding ACN to a sample increases the allowable injection
volume but can create solubility issues as noted earlier.
■
■
High ACN initial conditions can facilitate sample loading and the
mixing of sample to higher ACN content before it sees the head
of the column. Better band formation can thereby be achieved.
TIP
■
■
Empirically determine your maximum injection volume.
■
■
Inject varying volumes, i.e. 1, 3, and 10 μL.
■
■
Select an injection volume that does not show signs of
breakthrough or solvent effects.
Figure 9. Determining the optimal volume load for an aqueous sample of ribonuclease
B. An initial gradient condition to improve band formation (shown in red).
0.2
0.15
0.1
0.05
0
5
10
15
20 min.
Mass Load
1 µg
10 µL
2 µL
1 µL
0.5 µL
Peak distortion due
to solvent effects
Time (min)
%A
%B
0
33.0
67.0
20.0
40.0
60.0
21.0
100.0
0.0
23.0
100.0
0.0
24.0
33.0
67.0
34.0
33.0
67.0
Time (min)
%A
%B
0
15.0
85.0
0.5
15.0
85.0
1.0
33.0
67.0
21.0
40.0
60.0
22.0
100.0
0.0
24.0
100.0
0.0
25.0
15.0
85.0
35.0
15.0
85.0
11
[ TIPS & TRICKS ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
Figure 10. Using flow restriction post-column, pre-detector to eliminate the
on-column aggregation of an intact mAb (trastuzumab).
Take Advantage of Temperature, Additives, and High
Pressures to Avoid On-Column Aggregation
■
■
With HILIC of large molecules, on-column aggregation can
sometimes be encountered in the form of distorted, spurious
peaks comprised of multiple analyte forms.
■
■
Choose method conditions that enhance the solubility of
analytes in ACN.
■
■
Proteins are most soluble in ACN mixtures when in the
presence of hydrophobic, strong acid mobile phase additives.
■
■
Elevated column temperatures improve solubility.
■
■
Ultrahigh back pressures can also be used to
minimize aggregation.
TIP
■
■
Use hydrophobic mobile phase additives.
■
■
TFA and/or HFIP.
■
■
Avoid HFBA if performing LC-MS (adduct formation).
■
■
Explore elevated column temperatures if needed (i.e., 50 to 80 ˚C).
■
■
Use long column lengths, coupled columns and/or flow
restrictors if possible.
0.2
0.1
0
20 min.
*Pressure at retention time of the mAb
15
10
7300 psi
Increasing
pressure
4500 psi
3200 psi
Aggregate peak
Flow restriction
25 µm I.D. PEEK tubing
post-column
Increasing column pressure
minimizes on-column aggregation
Disrupt Non-Covalent Interactions in Your Sample
■
■
The denaturing conditions of HILIC chromatography cannot
always be relied upon to disrupt non-covalent interactions.
■
■
It is best practice to disrupt these interactions in the sample,
prior to making an injection.
■
■
Non-covalent complexes can sometimes otherwise be separated
to the detriment of accurately profiling a sample.
TIP
■
■
Add GuHCl to your sample (up to 6 M GuHCl into a fully
aqueous sample).
Low Concentration
Surfactant
6M GuHCl
No interference from
non-covalent interactions
0.45
0.40
0.35
0.30
0.25
0.20
A
214
0.15
0.10
0.05
0.00
15 min.
10
5
Heat denaturation
Self associating heavy chain subunits
Figure 11. HILIC profiling of reduced mAb samples prepared from various conditions.
12
[ TIPS & TRICKS ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
Use a Flow Through Needle When Possible
■
■
With the possible solubility issues of proteins under HILIC
conditions, it is best to use a flow through needle autosampler so
that sample immediately goes to the LC flow path and is quickly
pressurized via the pump and the column backpressure.
■
■
If restricted to a fixed loop system, use a full loop injection
mechanism to eliminate issues with precipitation, which could
occur as analyte is diluted at low pressure with needle wash
during a partial loop injection.
■
■
Weak Needle Wash: 0.1% TFA, 85% ACN.
■
■
Strong Needle Wash : 0.1% TFA, water.
TIP
■
■
Use flow through needle injection mechanisms whenever possible.
Figure 12. Flow through needle injections.
Bottle tray
Detector
Column heater
Sample manager -
flow through needle
Solvent manager
Wash
Solvent
Purge
Solvent
Waste
Union
To
column
Inject valve
inject position
From solvent
manager
Flow Through
Needle
Wash pump
Solenoid
valve
Sample
To waste
Injection port
Sample compartment
IN
OUT
Pressure
transducer
Sample
syringe
13
[ TIPS & TRICKS ]
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard
Waters, ACQUITY UPLC, UPLC, UltraPerformance LC, and The Science of What’s Possible are registered trademarks of Waters
Corporation. eCord, VanGuard, GlycoWorks, and RapiFluor are trademarks of Waters Corporation. Acrodisc is a registered
trademark of Pall Corporation. All other trademarks are the property of their respective owners.
©2016 Waters Corporation. February 2016 Rev D 720005408EN IH-PDF
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 ]
