Waters CORTECS 2.7 um User Manual
CORTECS 2.7 µm Columns
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Column Connection
b. Column Installation
c. Minimizing Band Spread Volume
d. Measuring Band Spread Volume
e. Measuring System Dwell Volume
f. Column Equilibration
g. eCord™ Installation
h. Functional Tests for Benchmarking a New Column
i. VanGuard™ Pre-Columns
III. COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
I. INTRODUCTION
Thank you for choosing a Waters CORTECS Column. CORTECS 2.7 µm
Columns are based on the same solid-core particle technology
as CORTECS UPLC® 1.6 µm Columns, enabling seamless method
transfer between HPLC, UHPLC and UPLC platforms. All CORTECS
Columns are manufactured in cGMP, ISO 9001:2000 certified
facilities. Strict quality procedures for the manufacture of
the solid-core particle, the bonded phase synthesis, and the
column packing result in a high quality and reproducible final
product. Every column is individually tested and a Performance
Chromatogram and Certificate of Batch Analysis are provided on
the eCord Intelligent Chip. The eCord is available on all 2.1 mm
and 3.0 mm column dimensions.
CORTECS 2.7 µm Columns will exhibit maximum chromatographic
®
performance when used on a member of the ACQUITY UPLC
System family.
IV. COLUMN CLEANING, REGENERATION
AND STORAGE
a. Cleaning and Regeneration
b. Storage for Reversed-Phase and HILIC columns
V. ECORD INTELLIGENT CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Column Use Information
VI. ADDITIONAL INFORMATION
a. Tips for Maximizing Column Lifetime
b. Getting Started with CORTECS® HILIC Columns
c. Troubleshooting Questions
II. GET TING STARTED
Each CORTECS 2.7 µm Column comes with a Certificate of
Analysis, which includes the bonded phase batch number and
the analytical test results for the unbonded and bonded particle.
The included Performance Test Chromatogram summarizes
the performance of each individual column and provides batch
number, column serial number, USP plate count, USP tailing
factor, retention factor, and chromatographic test conditions.
These data should be recorded and stored for future reference.
When available, the information can be accessed via the ACQUITY
UPLC Console using the attached eCord.
a. Column Connection
CORTECS 2.7 µm Columns are designed to operate on any HPLC,
UHPLC, or UPLC system. Due to the absence of an industry
standard, be aware that the type of fittings and connection on
each system will vary by manufacturer. All tubing connections
must be reseated to match the new column when it is installed.
If the style of the column endfitting does not properly match the
system connections, the chromatographic performance of your
new column will be negatively impacted, or leaking can occur.
c. Minimizing Band Spread Volume
Band spreading is the measurement of the system dispersion,
which impacts the chromatographic performance. Internal tubing
diameter and fluidic connections can significantly impact system
band spreading and chromatographic performance. Larger
tubing diameters cause excessive peak broadening and reduced
sensitivity (Figure 1).
0.005 inches
0.020 inches
0.040 inches
Diluted/Distorted Sample Band
Figure 1. Impact of Tubing Diameter on Band Spread.
d. Measuring Band Spread Volume
This test should be performed on a liquid chromatographic system
equipped with a UV detector.
b. Column Installation
Note. The flow rates given in the procedure below are described for
a 2.1 mm i.d. column. Adjust the flow rate up or down based on the
column pressure and system limits.
1. Purge the pumping system of any buffer-containing mobile
phases using 100 % HPLC-grade water.
2. Purge the pumping system to 100% organic mobile phase
(methanol or acetonitrile).
3. Connect the inlet of the column to the chromatographic system.
4. Flush the column with 100% organic mobile phase (methanol
or acetonitrile) by setting the pump flow rate to 0.1 mL/min.
Increase the pump flow rate to 0.5 mL/min over 5 minutes.
5. Once the mobile phase is flowing from the column outlet, stop
the flow.
6. Attach the column outlet to the detector. This prevents air
entering the detector flow cell.
7. Increase the flow rate as described in step 4.
8. Monitor until a steady backpressure and baseline have
been achieved.
1. Disconnect the column from the system and replace with a zero
dead volume union.
2. Set the flow rate on the pumping system to 1 mL/min.
3. Use a test mixture (dissolved in the mobile phase conditions)
that delivers a maximum peak height of less than 0.5 AU.
4. Inject 2–5 µL of this solution.
5. Using the 5-Sigma method, measure the peak width in minutes
at 4.4% of peak height:
System Volume
4.4% h
Figure 2. Determination of System Band Spread Volume using 5-Sigma method.
Band Spreading (µL) = Peak Width (min) x Flow Rate (µL/min)
(For example, If peak width = 0.1 min and flow rate = 1000 µL/min,
band spread = 100 µL)
CORT ECS 2.7 µm Columns
2
Table 1. Expected System Band Spread Volumes for Waters
LC Systems
Recommended
CORTECS Column i.d.
System
Alliance
Band
Spread
®
2695 HPLC 29 µL 4.6 mm 3.0 mm
1st
Choice
2nd
Choice
ACQUITY UPLC 12 µL 2.1 mm 3.0 mm
ACQUITY UPLC H-Class 9 µL 2.1 mm 3.0 mm
ACQUITY UPLC I-Class (FTN) 7.5 µL 2.1 mm 3.0 mm
ACQUITY UPLC I-Class (FL) 5.5 µL 2.1 m m 1.0 mm
Non-Waters UHPLC System 15–25 µ L 3.0 mm 2.1 mm
e. Measuring System Dwell Volume
Dwell volume is different than system band spreading. System
dwell volume is a measurement of the volume it takes for the
initial gradient conditions to reach the head of the column. This
value is necessary when transferring a method between different
chromatographic systems.
Programmed time = 5.00 minutes
0.70
0.65
0.60
0.55
0.50
0.45
0.40
AU
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Figure 3. Measuring System Dwell Volume.
50%
0 2 4 6 8 10 12 14 16 18 20 min
Flow Rate = 1.5 mL/min
5.69 minutes
- 5.00 minutes
0.69 minutes
Time = 5.69 minutes
System Volume:
0.69 min x 1.5 mL/min = 1.04 mL
Table 2. Expected System Dwell Volumes for Waters Systems
System Dwell Volume
Alliance 2695 HPLC 900 µL
ACQUITY UPLC 120 µ L
ACQUITY UPLC H-Class 350 µL
ACQUITY UPLC I-Class (FTN) 100 µ L
ACQUITY UPLC I-Class (FL) 95 µL
1. Disconnect the column from the system and replace with a zero
dead volume union.
2. Use acetonitrile as mobile phase A, and acetonitrile containing
0.05 mg/mL uracil as mobile phase B.
3. Use the flow rate in the original method and the intended flow
rate on the target instrument.
4. Collect detector baseline using 100% mobile phase A for
5 minutes.
5. At 5.00 minutes, program a step to 100% mobile phase B,
and collect data for an additional 5 minutes.
6. Measure absorbance difference between 100% A and 100% B.
7. Measure time at 50% of that absorbance difference.
8. Calculate time difference between start of step and 50% point.
9. Multiply time difference by the flow rate to calculate
system volume.
f. Column Equilibration
CORTECS 2.7 µm Columns are shipped in 100% acetonitrile. It is
important to ensure mobile phase compatibility before changing
to a different mobile phase system. To avoid precipitating mobile
phase buffers within the column or system, flush the column with
five column volumes of a waters/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).
Note. If mobile phase additives (i.e., ion-pairing reagents) are
present in low concentrations (<0.2% v/v), 100 to 200 column
volumes may be required for complete equilibration. In addition,
mobile phases that contain formate (i.e., ammonium formate,
formic acid) may require extended equilibration times.
For reversed-phase separations, equilibrate the column with a
minimum of 10 column volumes of the mobile phase to be used
(refer to Table 3 for a list of column volumes). The column may be
considered fully equilibrated once a constant backpressure and a
stable detector baseline is achieved.
CORT ECS 2.7 µm Columns
3
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