CORTECS Columns
CONTENTS
I. GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination
f. VanGuard Pre-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
Thank you for choosing a Waters CORTECS™ Column. The
CORTECS packing materials were designed specifically for use
with the Waters ACQUITY UPLC® System and are manufactured
in a cGMP, ISO 9001 certified plant using ultra pure reagents.
Each batch of CORTECS 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
CORTECS Columns were designed and tested specifically for use on
the ACQUITY UPLC System. CORTECS Columns will exhibit maximum
chromatographic performance and benefits when used on the
holistically-designed ACQUITY UPLC System since they were created
and designed to operate with it.
Intelligent Chip.
IV. INTRODUCING eCORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
V. ADDITIONAL INFORMATION
a. Tips for Maximizing CORTECS Column Lifetimes
b. Getting Started with CORTECS HILIC Columns
I. GETTING STARTED
Each CORTECS Column comes with a Certificate of Analysis and
a Performance Test Chromatogram embedded within the eCord
intelligent chip. The Certificate of Analysis is specific to each batch
of packing material contained in the CORTECS Column and includes
the gel batch number, analysis of unbonded particles, analysis of
bonded particles, and chromatographic results and conditions. T he
Performance Test Chromatogram is specific to each individual
column and contains such information as: gel batch number,
column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data
should be stored for future reference.
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
CORTECS 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
a list of column volumes). The column may be considered thermally
equilibrated once a constant backpressure is achieved.
a. Column Connectors
The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.
Optimized column inlet tubing (Part Number 430001084) 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 CORTECS Column and tighten the compression
fitting using two 5/16-inch wrenches.
For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for a typical
1.6 µm, 2.1 mm ID by 50 mm length column. Scale the flow rate
up or down accordingly based upon the column pressure and
system limits.
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 5 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
and gives more rapid baseline equilibration.
Table 1. Column Volumes (mL)
Column Length (mm)
30 0.1 0.2
50 0.2 0.4
75 0.3 0.5
100 0.4 0.8
150 0.5 1.0
Internal Diameter
2.1 mm 3.0 mm
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).
For CORTECS HILIC Columns, flush with 50 column volumes of
50:50 acetonitrile/water with 10 mM final buffer concentration.
Prior to the first injection, equilibrate with 20 column volumes of
initial mobile-phase conditions (refer to Table 1 for a list of column
volumes). See “Getting Started with CORTECS HILIC Columns” for
additional information.
d. 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.
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.
2
CORT ECS Columns
e. 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. An analyte mixture as found on the “Performance Test
Chromatogram” which 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. CORTECS
Columns are QC tested on ACQUITY UPLC I-Class Systems
which have extremely low system volumes. This presents a
more challenging test of how well the column was packed and
guarantees the highest quality packed column.
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 obtained
on two different UPLC® Systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.
f. VanGuard Pre-Columns
VanGuard™ Pre-columns are 2.1 mm ID x 5 mm length guard
column devices designed specifically for use in the ACQUITY
UPLC System. VanGuard Pre-columns are packed with the same
CORTECS chemistries and frits as our 2.1 mm ID CORTECS
Columns. VanGuard Pre-columns are designed to be attached
directly to the inlet side of a CORTECS 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.
CORT ECS Column
Place wrench here
Ferrule
Flow
VanGuard Pre-Column
Collet
Place wrench here
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 CORTECS 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 CORTECS
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 CORTECS
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.
3
9. Reattach pre-column, apply mobile-phase flow and inspect
for leaks.
CORT ECS Columns
Table 2: Buffer Recommendations for Using CORTECS Columns
Additive/Buffer pKa
TFA 0.30 Volatile Yes
Acetic Acid 4.76 Volatile Yes
Formic Acid 3.75 Volatile Yes
Acetate
(NH
COOH)
4CH2
Formate
(NH
COOH)
4
Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.
Phosphate 2 7.20 6.20– 8.20 Non-volatile No
4.76 3.76 – 5.76 Volatile Yes
3.75 2.75 – 4.75 Volatile Yes
Buffer
Range
Volatility
(±1 pH unit)
Used for
Mass Spec
Comments
Ion pair additive, can suppress MS signal, used in the
0.02-0.10% range.
Maximum buffering obtained when used with ammonium acetate
salt. Used in 0.1-1.0% range.
Maximum buffering obtained when used with ammonium formate
salt. Used in 0.1-1.0% range.
Used in the 1-10 mM range. Note that sodium or potassium salts
are not volatile.
Used in the 1-10 mM range. Note that sodium or potassium salts
are not volatile.
Above pH 7, reduce temperature/concentration and use a guard
column to maximize lifetime.
II. COLUMN USE
To ensure the continued high performance of CORTECS 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 cleanup 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.
4. 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.
5. For Hydrophilic Interaction Chromatography (HILIC) separations, the samples must be prepared in a high percentage of organic
solvents (e.g., 95% acetonitrile). See “Getting Started with CORTECS HILIC Columns”.
b. pH Range
The recommended operating pH range for CORTECS Columns is 2 to 8 for the C
See Table 2 for more characteristic details of CORTECS chemistries.
+
and C18 chemistries, and 1 to 5 for the HILIC chemistry.
18
4
CORT ECS Columns
A listing of commonly used buffers and additives is given in Table 2. Additionally, the column lifetime will vary
OSi
O
O
OSi
O
O
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.
Table 3: CORTECS Chemistry Characteristics
C
18
C18+ HILIC
Chemistry
General purpose, high-efficiency,
Intended Use
reversed-phase column. Balanced
retention of acids, bases and
neutrals at low and mid-range pH.
Ligand Type Trifunctional C
General purpose, high-efficiency,
reversed-phase column. A positively
charged surface delivers excellent
peak shape for basic compounds
at low pH.
18
Trifunctional C
18
High-efficiency column designed
for retention of extremely polar
analytes. Offers ort hogonal
selectivity vs. C
None
Surface Charge Modification None + None
Endcap Style Proprietary Proprietary None
Carbon Load 6.6% 5.7% Unbonded
Ligand Density 2.7 µmol/m
2
2.4 µmol/m
2
N/A
pH Range 2-8 2-8 1-5
Temperature Limits
1
Low pH = 45 °C
High pH = 45 °C
Low pH = 45 °C
High pH = 45 °C
Low pH = 45 °C
High pH = 45 °C
columns.
18
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
CORTECS Columns can tolerate operating pressures up to 18000 psi (1241 bar or 124 MPa).
Note: Working at the extremes of pressure, pH and/or temperature will result in shorter column lifetimes.
e. Temperature
Temperatures between 20–45 °C are recommended for operating CORTECS 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.
5
CORT ECS Columns
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
(see 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.
Flush CORTECS HILIC Columns with 50:50 acetonitrile/water to
remove polar contaminants. If this flushing procedure does not
solve the problem, purge the column with 5:95 acetonitrile/water.
Table 4. Reversed-Phase Column Cleaning Sequence
Polar Samples
1. water
2. methanol
3. tetrahydrofuran
(THF)
4. methanol 4. hexane
5. water
6. mobile phase 6. mobile phase
Non-polar
Samples**
1. isoproanol (or
an appropriate
isopropanol/water
mixture*)
2. tetrahydrofuran
(THF)
3. dichloromethane
5. isopropanol (followed by an appropriate isopropanol/
water mixture*)
Proteinaceous Samples
Option 1: Inject
repeated aliquots of
dimethylsulfoxide
(DMSO)
Option 2: gradient of
10% to 90% B where:
A = 0.1% trifluoroacetic
acid (T FA) in water
B = 0.1% trifluoroacetic
acid (T FA) in acetonitrile
CN)
(CH
3
Option 3: Flush column
with 7M guanidine
hydrochloride, or 7M
urea
b. Storage
For periods longer than four days at room temperature,
store reversed-phase and CORTECS HILIC 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.
For periods longer than four days, store CORTECS HILIC Columns
in 95:5 acetonitrile/water. Do not store in buffered solvent. If
the mobile phase contained a buffered salt, flush the column with
10 column volumes of 95:5 acetonitrile/water (see Table 1 for
common column volumes).
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.
* Use low organic solvent content to avoid precipitating buffers.
** Unless a Hexane Tetrahydrofuran Compatibility Kit (P/N 205000464) has been
installed, running solvents such as 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.
6
CORT ECS Columns
IV. INTRODUCING 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
Figure 1. Waters eCord Intelligent Chip
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
eCord inserted into
side of column heater
history of the columns, reduce the frustration of paperwork trails, and give
customers the reassurance that a well-performing column is installed onto
Figure 2. eCord Inserted into Side of Column Heater
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.
Waters eCord -
intelligent chip
c. Manufacturing Information
The eCord chip provides the user with an overview of the bulk material QC test results.
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 the column.
The eCord chip provides 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.
7
CORT ECS Columns
V. ADDITIONAL INFORMATION
a. Tips for Maximizing CORTECS Column Lifetimes
1. To maximize CORTECS 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. W hen 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.
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.
Consider sample preparation (e.g., solid-phase extraction,
filtration, etc).
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.
8
CORT ECS Columns
6. Remember: the diameter of CORTECS Column (2.1 and 3.0 mm
ID) 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?
b. Getting Started with CORTECS HILIC Columns
1. Due the fact that CORTECS HILIC Columns do not possess a
bonded phase, the pH operating range is 1 to 5, and they can be
operated at temperatures up to 45 °C.
2. As with any LC column, operating at the extremes of pH,
pressures and temperatures will result in decreased column
lifetime.
Column Equilibration
1. When the column is first received, flush in 50% acetonitrile/
50% water with 10 mM final buffer concentration for
50 column volumes.
2. Equilibrate with 20 column volumes of initial mobile-phase
conditions before making first injection.
3. If gradient conditions are used, equilibrate with 8-10 column
volumes between injections.
4. Failure to appropriately equilibrate the column could result in
drifting retention times.
Mobile-Phase Considerations
1. Always maintain at least 3% polar solvent in the mobile
phase or gradient (e.g., 3% aqueous/3% methanol or 2%
aqueous/1% methanol, etc.). This ensures that the CORTECS
particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in
your mobile phase or gradient.
Miscellaneous Tips
1. CORTECS HILIC Columns are designed to retain very polar
bases. Acidic, neutral and/or non-polar compounds will have
limited retention.
2. Optimal flow rates for small (<200 daltons) very polar bases
are in the 0.4 to 0.8 mL/min range with the CORTECS HILIC
Columns.
3. Avoid phosphate salt buffers to avoid precipitation in HILIC
mobile phases. Phosphoric acid is okay.
4. Buffers such as ammonium formate or ammonium acetate
will produce more reproducible results than additives such
as formic acid or acetic acid. If an additive (e.g., formic acid,
etc.) must be used instead of a buffer, use 0.2% (v:v) instead
of 0.1%.
5. For best peak shape, maintain a buffer concentration of 10 mM
in your mobile phase/gradient at all times.
Injection Solvents
1. If possible, injection solvents should be 95% acetonitrile.
The polar solvent (i.e., water, methanol, isopropanol) should
be minimized to 25% of the total volume.
2. A generic injection solvent is 75:25 acetonitrile/methanol.
This is a good compromise between analyte solubility and
peak shape.
3. Avoid water and dimethylsulfoxide (DMSO) as injection solvents. These solvents will produce very poor peak shapes.
4. Exchange water or DMSO with acetonitrile by using
reversed-phase solid-phase extraction (SPE). If this is not
possible, dilute the water or DMSO with organic solvent.
3. In HILIC, it is important to remember that water is the strongest
solvent. Therefore, it must be eliminated or minimized in the
injection solvent.
4. For initial scouting conditions, run a gradient from
95% acetonitrile to 50% acetonitrile. If no retention occurs, run
isocratically with 95:3:2 acetonitrile/methanol/aqueous buffer.
5. Alternate polar solvents such as methanol, acetone or isopropanol
can also be used in place of water to increase retention.
6. If using an ACQUITY UPLC System, the weak needle wash
should closely match the % organic present in the initial
mobile-phase conditions, otherwise, analyte peak shape or
retention may suffer.
©2013 Waters Corporation. Waters, ACQUITY UPLC, UPLC, Oasis and
SepPak are registered trademarks of Waters Corporation. CORT ECS,
VanGuard, eCord and The Science of What’s Possible are trademarks
of Waters Corporation. All ot her trademarks are the property of their
respective owners.
June 2013 720004750EN Rev. A VW-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
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