[ Care and Use ManUal ] XterrA And XterrA PreP coLuMns

[ Care and Use ManUal ] XterrA And XterrA PreP coLuMns
[ Care and Use Manual ]
X T erra and X t erra p rep Columns
I. Int roduc t ion
Contents
Thank you for ordering a Waters XTerra® column. XTerra packing
materials are synthesized using Waters Hybrid Particle Technol-
I. INTRODUCTION
ogy. XTerra particles contain both inorganic (silica) and organic
(organosiloxane) components sharing the advantages of both. Hybrid
II. COLLUMN INSTALLATION
technology allows for the high efficiency of separation and improved
pH stability when compared to Silica based reversed phase packing
III. COLUMN USAGE
materials. The manufacture of XTerra columns starts with ultrapure
IV. SCALING UP/DOWN
reagents to control the chemical composition and purity of the Hybrid
particle. Every manufacturing step is conducted within narrow toler-
V. COLUMN CLEANING, REGENERATING AND STORAGE
ances and each column is individually tested. Certificates of Batch
Analysis and Column Efficiency are provided with each column.
VI. TROUBLESHOOTING
We recommend the use of Waters Sentry guard columns to extend
™
the life of your column and protect it from contaminants.
XTerra Columns
1
[ Care and Use Manual ]
II. COLUMN INSTALLATION
a. Connecting the Column to the HPLC Instrument
Handle the column with care. Do not drop or hit the column on a
Waters Ferrule Setting Parker Ferrule Setting
Figure 1: Waters and Parker Ferrule Types
hard surface as it may disturb the bed and affect its performance.
0.090 inches
c. The proper tubing/column connection
1. Correct connection of 1/16” outer diameter stainless steel tubing leading to and from the column is essential for high-quality
Tubing touches the bottom of the column endfitting, with no void
chromatographic results.
between them.
2. When using standard stainless steel compression screw fittings,
it is important to ensure proper fit of the 1/16” outer diameter
stainless steel tubing. When tightening or loosening the
compression screw, place the 5/16” wrench on the compression
Figure 2: Proper Tubing/Column Connection
screw and the other 3/8” wrench on the hex head of the column
A void appears if a tube with Parker ferrule setting is connected to a
endfitting.
Waters style column
Note: If one of the wrenches is placed on the column flat during
The presence of a void in the flow stream down grades the column
this process, the endfitting will be loosened and leak.
performance. There is only one way to fix the problem: Cut the end
of the tubing with the ferrule, put a new ferrule on the tubing and
3. If a leak occurs between the stainless steel compression screw
fitting and the column endfitting, a new compression screw
make the connection. Before tightening the screw, make sure that the
fitting, tubing and ferrule must be assembled.
tubing bottoms out in the endfitting of the column.
4. An arrow on the column identification label indicates correct
direction of solvent flow.
NOTE: It is important to realize that extra column peak broaden-
Void
ing can destroy successful separation. The choice of appropriate
Gap
column connectors and system tubing is discussed in detail
Figure 3: Parker Ferrule in a Waters Style Endfitting (left) & Waters Ferrule in a Parker Style Endfitting (right)
below.
If tubing with a Waters style ferrule setting is connected to a column
with Parker style endfitting, the end of the tubing will bottom out
b. Column connectors and system tubing considerations
before the ferrule reaches its proper sealing position. This will leave
Due to the absence of an industry standard, various column manu-
a gap creating a leak. There are two ways to fix the problem:
facturers have employed different styles of chromatographic column
1. Just tighten the screw a little bit more. The ferule moves
connectors. The chromatographic performance of your separation
forward, and reaches the sealing surface. Do not overtighten
can be negatively affected if the style of your column endfittings do
because this may end in breaking the screw.
not match the existing instrumentation tubing ferrule setting. This
page explains the difference between Waters style and Parker style
2. Cut the tubing, put a new ferrule on it and make the connection.
endfittings, which vary in the required length of the tubing protruding
An alternative is to replace the conventional compression screw
from the ferrule. The XTerra column is equipped with Waters style
fitting with an all-in-one PEEK TM fitting (Waters P/N PSL613315)
endfittings which require a 0.130” ferrule depth (see next section
that allows you to reset the ferrule depth. Another approach is to
for setting ferrule depth). If you are presently using a non-Waters
use a Keystone, Inc. Slipfree® fitting to always ensure the correct fit.
style column, it is critical that you reset the ferrule depth for optimal
The finger-tight Slipfree connectors automatically adjust to fit all
performance.
XTerra Columns
compression screw type fittings without the use of tools.
2
[ Care and Use Manual ]
3. Dilute a test mix in mobile phase to give a detector sensitiv-
d. SLIPFREE Connectors
ity 0.5-1.0 AUFS (can use the system start up test mix
A SLIPFREE Connector guarantees a void-free connection because
which contains uracil, ethyl and propyl parabens; Waters
it pushes the tubing into the endfitting. This design comes
P/N WAT034544.)
installed on the tubing. Fingertight to 10,000 psi – never needs
wrenches. Readjusts to all column endfittings. Compatible with all
4. Inject 2 to 5 µL of this solution.
commercially available endfittings. Unique design separates tube-
5. Using 5 sigma method measure the peak width at 4.4% of peak
holding function from sealing function.
height:
Bandspread (µL) = PW (seconds)/60 (see Figure 6)
Typical LC system should be 100 µL ± 30 µL
Microbore (2.1mm i.d. and smaller) system should be no
greater than 20-40 µL
Figure 4: Single and Double SLIPFREE Connectors
System Volume
Waters Part Numbers for SLIPFREE Pre-assembled Tubing
Tubing Length
0.005” i.d.
0.010” i.d.
0.020” i.d.
Single SLIPFREE® 6 cm
PSL 618000
PSL 618006
PSL 618012
Single SLIPFREE® 10 cm
PSL 618002
PSL 618008
PSL 618014
Single SLIPFREE® 20 cm
PSL 618004
PSL 618010
PSL 618016
Double SLIPFREE® 6 cm
PSL 618001
PSL 618007
PSL 618013
Double SLIPFREE® 10 cm
PSL 618003
PSL 618009
PSL 618015
Double SLIPFREE® 20 cm
PSL 618005
PSL 618011
PSL 618017
5
4.4 %h
Minimization of band spreading
Figure 6: Determination of System Bandspread Volume using the
5-Sigma Method
The following figure shows the influence of tubing internal
g. Measuring Gradient Delay Volume
diameter on system band spreading and peak shape. As can be
seen, the larger tubing diameter causes excessive peak broadening
1. Replace the column with a zero dead volume union.
and lower sensitivity.
2. Determine the gradient-delay or dwell volume for your system
by performing the following test. Prepare eluent A (pure solvent,
0.005 inches
such as methanol) and eluent B (solvent plus sample, such as
0.020 inches
5.6 mg/mL propylparaben in methanol).
0.040 inches
3. Equilibrate the system with eluent A until a stable baseline is
achieved. Switch to 100% eluent B and record the half height of
the step. Refer to Figure 7 for an illustration. The dwell volume
Diluted/Distorted Sample Band
should be less than 1 mL. If this is not the case, see section on
Figure 5: Effect of Connecting Tubing on System
System Modifications to reduce your system volume.
f. Measuring System Bandspread Volume
1. Disconnect column from system and replace with a zero dead
volume union
2. Flow rate 1 mL/min. This should be performed on a single
wavelength detector (not a PDA/DAD)
XTerra Columns
3
[ Care and Use Manual ]
j. Modification Guidelines
Figure 7: Determination of Dwell Volume
1. Use a microbore detector flow cell with the 2.1 mm columns.
1.0
Recall that due to the shorter pathlength, detector sensitivity is
0.8
reduced to achieve lower band spread volume.
0.6
2. Injector sample loop should be reduced to minimum.
1/2 Vertical
Au
Distance
0.4
3. Use 0.009” (0.25 mm) tubing between pump and injector.
t1/2
0.2
4. Use 0.009” (0.25 mm) tubing for rest of connections in
standard systems and 0.005” (0.12 mm) tubing for narrowbore
0.0
Time
(≤2.1 mm i.d.) systems.
h. Use of Narrow-Bore Columns – ( ≤3.0 mm i.d.)
5. Use perfect (pre-cut) connections (with a variable depth inlet if
This section describes how to minimize extra column effects and
using columns from different suppliers).
gives some guidelines on how to maximize the advantages of your
narrow-bore column. The 3.0 mm i.d. narrow-bore column usually
6. Time constants should be shortened <0.2.
requires no system modifications. With the 2.1 mm i.d. column,
to eliminate excessive system bandspread volume. Without proper
k. Waters Small Particle Size (2.5 µm and 3.5 µm) Columns
– Fast Chromatography
system modifications, excessive system bandspread volume causes
The Waters columns with 2.5 µm and 3.5 µm packings provide faster
peak broadening and has a large impact on peak width as peak
and more efficient separations without sacrificing column lifetime.
volume decreases.
This section describes five parameters to consider when performing
however, modifications to your HPLC system may be required in order
separations on the 2.5 µm and 3.5 µm columns.
i. Impact of bandspreading on column performance
Note: All 3.5 µm and 2.5 mm materials have smaller outlet frits to
(2.1 mm i.d. column)
retain packing material. These columns should not be backflushed.
System with 70 µL bandspread >> 10,000 plates
1. Flow Rate — Compared with the 5 µm columns, the 2.5 µm and
System with 130 µL bandspread >> ~8,000 plates (same column)
3.5 µm columns have a higher optimum flow rate. These columns
Figure 8: Determination of Gradient Delay Volume
are used for high efficiency and short analysis times. The higher
flow rates, however, lead to increased backpressure. Use a flow
rate that is practical for your system.
2. Backpressure — The backpressures on the 2.5 µm and 3.5
µm columns are higher than for the 5 µm columns of the same
dimension. Use a shorter column to compensate for increased
backpressure and obtain a shorter analysis time.
7.00
7.50
8.00
Non-optimized LC/MS/MS System
7.00
7.50
8.00
3. Temperature — Use a higher temperature to reduce backpres-
Optimized System
sure caused by smaller particle sizes (see Column Care and Use
Note flow splitters after the column will introduce additional
temperatures recommended in Section III. e.).
bandspreading. Optimizing a system, especially one using flow split-
4. Sampling Rate — Use a sampling rate of about 10 points per
ters can have a dramatic effect on sensitivity and resolution. Use of
second.
correct ferrule depth connectors and minimizing tubing diameter and
5. Detector Time Constant – Use a time constant of 0.1 seconds for
lengths showed a doubling of sensitivity and enabled resolution of the
fast analysis.
metabolite on this LC/MS/MS system.
XTerra Columns
4
[ Care and Use Manual ]
l. Column Performance Validation
3. When the mobile phase is changed, gradually increase the flow
Each pre-packed column has an individual quality control report that
rate of the new mobile phase from zero mL/min to 1.0 mL/min
provides significant information about the column. This report is available
in 0.1 mL/min increments.
as a ready reference and should be kept in your files. It indicates the column
4. Once a steady backpressure and baseline have been achieved,
specifics: gel lot, column dimensions, bonding chemistry type, particle
the column is ready to be used.
shape, particle size, porosity, and chromatographic test conditions.
Note: If mobile phase additives are present in low concentrations (such as
1. Perform an efficiency test on your column before you use it.
ion-pairing reagents, at 5 to 10 mmol/L) 100 to 200 column volumes may
Waters recommends using a suitable solute mixture, such as
be required for complete equilibration.
found in the ”Column Test Report”, to immediately analyze the
column once you receive it. Determine the number of theoretical
Column Length
plates (N) and use for periodic comparison. Repeat the test
Column internal diameter (mm)
1.0 2.1 3.0 3.9 4.6 7.8 10 19
periodically to track column performance over time. Slight
30 mm
-
0.1
0.2
variations may be obtained on two different HPLC systems due
50 mm
0.1
0.2
to the quality of the connections, operating environment, system
100 mm
0.1
0.4
electronics, reagent quality, column condition and operator
150 mm
0.1
technique. Please report any column problems observed upon
250 mm
receipt of the column.
300 mm
30
50
-
0.5
-
2.4
8
-
-
0.3
-
0.8
2.4
4
14
35
98
0.7
1.2
1.7
5
8
28
70
-
0.5
1.0
1.8
2.5
7
12
42
106
294
-
0.9
1.8
-
4
-
20
70
176
490
-
-
-
-
-
14
24
85
212
589
Table 2. Volume of standard columns (mL), multiply by 10 for flush
solvent volume
m. Sample Preparation
1. It is preferable to prepare sample in the mobile phase or a
III. Column Usage
weaker solvent than the mobile phase.
2. If the sample is not dissolved in the mobile phase, ensure
sample, solvent and mobile phases are miscible to avoid sample
To ensure the continued high performance of your columns and cartridges,
or buffer precipitation.
follow these guidelines:
3. Filter sample with 0.2 µm membrane to remove particulates.
a. Guard columns
Samples: Sample impurities very often contribute to column contami-
n. Column Equilibration
nation. Two ways to avoid this are:
Waters delivers the column in 100% acetonitrile. It is important to ensure
solvent compatibility before changing to a new solvent. Equilibrate your
1. Use of Waters Oasis® solid-phase extraction sample clean-up
column with a minimum of 10 times its internal volume with the mobile
cartridges or columns or Sep-Pak cartridges of the appropriate
phase to be used (refer to Table 2 for some standard column volumes).
chemistry to clean up your sample before analysis.
1. Purge your pumping system and then connect the inlet end
2. Use of a Waters guard cartridge of matching chemistry and par-
of the column to the injector outlet. Turn on the pump flow at
ticle size between the injector and main column. It is important
0.1 mL/min. and increase to 1 mL/min over 5 minutes.
to use a high-performance matching guard column to protect the
main column while not compromising analytical resolution.
2. When the solvent is flowing freely from the column outlet,
attach the column to the detector.This procedure prevents
entry of air into the detection system and gives more rapid
equilibration.
XTerra Columns
5
[ Care and Use Manual ]
b. pH Range
XTerra columns have a widened useable pH range over silica based columns. The pH range is pH 1-12 for the MS columns and 2-12 for the RP
columns. Lifetime although greater is still finite and will vary depending upon what buffers are used, the concentration of those buffers and the
temperature at which they are used. Here is a table of recommended and non-recommended buffers to be used as a guideline when developing
methods. Note that high pH use of Phosphate is not recommended even though it will generally give longer lifetimes than silica based columns.
For a table on appropriate buffers to use, please see Tables 3, 4 and 5.
Table 3: XTerra buffers for use from pH 1-7
Additive or Buffer
pka
Buffer Range
(± 1 pH unit)
Volatile or
Non-Volatile
Recommended Use with XTerra® Packagings
TFA
<1.0
Volatile
Yes (0.02-0.1%)
Acetic Acid
4.76
Volatile
Yes (0.1-1.0%)
Formic Acid
3.75
Acetate (Ammonium)
4.76
3.76-5.76
Volatile
Yes (0.1-1.0%)
Volatile
Yes (1-10mM) note Na+, K+ salts are not volatile
Formate (Ammonium)
3.75
Phosphate 1
2.15
2.75-4.75
Volatile
Yes (1-10mM) note Na+, K+ salts are not volatile
1.15-3.15
Non-Volatile
Yes
Phosphate 2
7.2
6.20-8.20
Non-Volatile
pH’s >7.0 lifetime decreases significantly with this buffer. See
also note on Phosphate 3 below. The lower the temperature and
buffer molarity, the longer the column lifetime achievable.
Buffer Range
(± 1 pH unit)
Volatile or
Non-Volatile
Recommended Use with XTerra® Packagings
Volatile
Yes (10mM)
Table 4: XTerra buffers for use from pH 7-12
Additive or Buffer
pka
4-Methyl-Morpholine
~8.4
Ammonia
9.2
Ammonium BIcarbonate
10.3 (HCO3)
9.3-11.3
9.2 (NH4)
8.2-10.2
Volatile
<10 mM and <30 °C
Volatile
Yes 5-10 mM (keep source >150 °C)
(do not use carbonate)
(total pH range 6.-11.3, natural pH=8.4) adjust pH with either
ammonium hydrocide or acetic acid
Yes (1-10mM)
7.8 (H2CO3)
6.8-8.8
Ammonium (Acetate) or
(Formate)
9.2
8.2-10.2
Volatile
Borate
9.2
8.2-10.2
Non- Volatile
1-Methyl- Piperidine
(Acetate or Formate)
10.3
9.3-11.3
Volatile
Yes
Triethylamine (Acetate or
Formate)
10.7
9.7-11.7
Volatile
Yes (0.1-1%)
Pyrrolidine
11.3
10.3-12.3
Volatile
Phosphate 3
12.3
11.3-13.3
Non- Volatile
Table 5: XTerra buffers for use from pH 9-12 with alternative buffers
Additive or Buffer
pka
Buffer Range
(± 1 pH unit)
Glycine
9.8
8.8-10.8
Yes
CAPSO
9.7
8.7-10.7
Yes (0.1-1.0mM)
CAPS
10.5
9.5-11.5
Yes (0.1-1.0mM)
XTerra Columns
Volatile or
Non-Volatile
6
Recommended Use with XTerra® Packagings
[ Care and Use Manual ]
v. Column Cl eaning, Regenerat ing and Storage
c. Solvents
To maintain maximum column performance, use high quality
a. Cleaning and Regeneration
chromatography grade solvents. Filter all buffers before use. Pall
Gelman Laboratory Acrodisc filters are recommended. Solvents
®
A shift in retention or resolution may indicate contamination of the
containing suspended particulate materials will generally clog the
column. Flushing with a neat organic solvent is usually sufficient to
outside surface of the inlet distribution frit of the column. This will
remove the contaminant. If the flushing procedure does not
result in higher operating pressure and poorer performance. Degas
solve the problem, purge the column with a sequence of
all solvents thoroughly before use to prevent bubble formation in
progressively more nonpolar or hydrophobic solvents. For example,
the pump and detector.
switch from water to tetrahydrofuran (THF) to methylene chloride.
d. Pressure
Return to the standard mobile phase conditions by reversing the
sequence.
All XTerra columns, regardless of dimension, can be operated at
pressures up to 6000 psi, 400 bar or 40 Mpa.
Guard columns need to be replaced at regular intervals as
determined by sample contamination. When system backpressure
e. Temperature
steadily increases above a set pressure limit, it is usually an
Temperatures between 20 – 60˚C are recommended for
indication that the guard column should be replaced.
operating Waters XTerra columns to enhance selectivity, lower
b. Storage
solvent viscosity and increase mass transfer rates. However, any
temperature rise above ambient will have a negative effect on
For periods longer than four days store the column in 100%
lifetime which will vary dependingon the pH and buffer conditions
acetonitrile. Do not store columns in buffered, acidic or basic
used.
eluents. If the mobile phase contained a buffer salt flush the column
with 10 column volumes of HPLC grade water (see Table 2) and
replace with 100% acetonitrile. Completely seal column to avoid
IV. Scaling Up/ Dow n
evaporation and drying out of the bed.
The following formulas will allow scale up or scale down, while
VI. Troubleshooting
maintaining the same linear velocity (retention time), and provide new
sample loading values:
If only column i.d. is changed:
X = (r2/r1)2
Changes in retention time, resolution, or backpressure are often due
to column contamination. See the Column Cleaning, Regeneration
If both column i.d. and length are altered: F2 = F1(r /r1)
2
2
and Storage section of this instruction sheet. Information on column
Load2 = Load1(r2/r1) (L2/L1)
2
Where: X = Factor by which original flow must be modified (also adjusts sample load)
L = Length of column, in mm
r = Radius of the column, in mm
F = Flow rate, in mL/min.
1 designates the original, or reference column
2 designates the new dimension column.
XTerra Columns
troubleshooting problems may be found in HPLC Columns Theory,
Technology and Practice, U.D. Neue, (Wiley-VCH, 1997) or the Waters
HPLC Troubleshooting Guide (Literature code # 720000181EN).
7
[ Care and Use Manual ]
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©2007 Waters Corporation. Waters, The Science of What’s
Possible, Atlantis, Sentry, Oasis, and SepPak are trademarks
of Waters Corporation. Slipfree is a registered trademark of
Keystone, Inc. Aerodisc is a registered trademark of Pall Gelman
laboratory.
October 2007 736000269 Rev 7 VW-PDF
XTerra Columns
8
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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