[ CARE AND USE MANUAL ]
1
CONTENTS
I.
INTRODUCTION
II.
GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. Procedure for Using New,
Out-of-Box Columns
e. eCord Installation
f. Column QR Code
g. Initial Column Efficiency Determination
h. VanGuard Pre-Columns
i. Installation Instructions
III.
COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
IV.
COLUMN CLEANING, REGENERATION,
AND STORAGE
a. Cleaning and Regeneration
b. Storage
V.
eCORD INTELLIGENT CHIP TECHNOLOGY
VI.
REPRESENTATIVE TEST CHROMATOGRAPH
AND CONDITIONS FOR SEPARATION OF
PROTEIN DIGEST
VII.
ADDITIONAL INFORMATION
VIII.
CAUTIONARY NOTE
I. INTRODUCTION
Thank you for choosing a Waters™ ACQUITY™ UPLC™ and/
or ACQUITY PREMIER Peptide CSH™ Column. Both feature
Waters Charged Surface Hybrid (CSH) Technology which
provides excellent peak shape, high efficiency and loading
capacity for basic compounds when using acidic, low ionic
strength mobile phases. This same particle technology is
used in the XSelect™ Peptide CSH HPLC columns, thus
enabling seamless transferability between HPLC and UPLC
system platforms. The ACQUITY UPLC and ACQUITY
PREMIER Peptide CSH C
18, 130 Å packing materials are
manufactured in a cGMP, ISO 9001 certified manufacturing
facility using ultra-pure reagents. Each batch of Peptide
CSH C
18, 130 Å material is tested chromatographically with
acidic, basic, and neutral analytes as part of qualification
for use in peptide mapping. The results are held to narrow
specification ranges to assure excellent, reproducible
performance. ACQUITY UPLC and ACQUITY PREMIER
Peptide CSH C
18, 130 Å batches are also QC tested with a
gradient separation of a tryptic digest of cytochrome c using
0.1% formic acid containing eluents. Finally, every shipped
column is individually tested for packed bed efficiency and a
Performance Chromatogram and Certificate of Batch Analysis
are provided on the eCord™ Intelligent Chip. ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C
18, 130 Å Columns will
exhibit maximum chromatographic performance and benefits
ONLY when used on holistically-designed ACQUITY UPLC
Systems since these systems and columns were created and
designed to operate together.
ACQUITY UPLC Peptide CSH C
18, 130 Å, 1.7 µm and XP 2.5 µm Columns
and ACQUITY PREMIER Peptide CSH C
18, 130 Å, 1.7 µm Columns
2
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
II. GETTING STARTED
Each ACQUITY UPLC and ACQUITY PREMIER Peptide CSH
C
18, 130 Å 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 ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C
18, 130 Å Column and
includes the gel batch number, analysis of unbonded particles,
analysis of bonded particles, and chromatographic results and
conditions. The 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.
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.
Note: Waters recommends using finger-tight fittings when
connecting Waters Peptide CSH C
18, 130 Å, XP 2.5 µm column
to an HPLC. To ensure a proper connection to your XP 2.5 µm
column, Waters recommends p/n: 700003139 to connect to
the inlet and p/n: 700004841 to connect to the outlet of your
column. This will ensure correct seating of the HPLC system’s
connection tubing ferrules to your XP 2.5 µm column and
minimize undesired band broadening.
Optimized column inlet tubing (p/n: 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 ACQUITY
UPLC and AQCUITY PREMIER 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 (p/n: 71500082502).
b. Column Installation
1. Purge the pumping system, with HPLC-grade water, 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 five 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.
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.
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
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH C
18,
130 Å 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).
Table 1. Empty column volumes in mL
(Multiply by 10 for flush solvent volumes)
Column length (mm)
Internal diameter 2.1 mm
50
0.2 mL
100
0.4 mL
150
0.5 mL
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.)
d. Procedure for Using New, Out-of-Box Columns
Prior to using a new column, it is important to confirm that
it produces reproducible chromatography and the desired
level of chromatographic resolution. To this end, it is useful
to benchmark column performance with a sample that is
representative of the intended application. The number of
injections necessary to achieve reproducible performance
may be dependent on sample characteristics and system
type. Method variables like pH, mass load, ionic strength, and
ion pairing, could also have impact. The ACQUITY PREMIER
Columns have MaxPeak™ High Performance Surfaces that
reduce the number of injections necessary to achieve desired
performance due to the improved hardware inertness.
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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
e. 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.
f. Column QR Code
The quick reference (QR) code that is located on the column
label provides column-specific information (i.e., the part and
serial numbers that are unique identifiers for the column), and
its encoding follows a widely adopted industry-standard.
1. Scan QR code using any device that is capable of
scanning QR codes (i.e., for smart phones and tablets,
use the built-in camera app).
2. Be directed to the column’s information hub
on waters.com.
3. Access technical and scientific information for the column
(i.e., certificate of analysis, application notes).
g. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as
found in the “Performance Test Chromatogram”, to
analyze the column upon receipt.
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.
h. VanGuard Pre-Columns
VanGuard™ Pre-columns are 2.1 mm I.D. x 5 mm length guard
column devices designed specifically for use in the ACQUITY
UPLC Systems. VanGuard Pre-columns are packed with the
same chemistries and frits as our 2.1 mm I.D. ACQUITY UPLC
Peptide CSH C
18, 130 Å Columns. VanGuard Pre-columns
are designed to be attached directly to the inlet side of an
ACQUITY UPLC Peptide CSH C
18, 130 Å 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.
i. Installation Instructions
1. Remove VanGuard Pre-column from box and shipping
tube and remove plastic plug.
2. Orient 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 ACQUITY UPLC and ACQUITY PREMIER Peptide
CSH C
18, 130 Å Column perpendicular to work surface so
that column inlet is on the bottom (column outlet on top).
4. From below, insert VanGuard Pre-column into ACQUITY
UPLC and ACQUITY PREMIER Peptide CSH C
18, 130 Å
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.
Figure 1. VanGuard Pre-Column installation diagram.
ACQUITY UPLC Column
VanGuard Pre-Column
Place wrench here
Ferrule
Collet
Place wrench here
Flow
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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
III. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C
18, 130 Å Columns,
follow these guidelines:
a. Sample Preparation
1. Sample must be dissolved in a diluent compatiable with
initial strength of mobile phase.
2. Sample must be completely in solution and free
of particulates.
3. To remove particulates the sample may be filtered with a
0.2 µm membrane. If the sample is dissolved in a solvent
that contained an organic modifier (e.g., acetronitrile,
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.
b. pH Range
The recommended operating pH range for ACQUITY UPLC
and ACQUITY PREMIER Peptide CSH C
18, 130 Å Columns is
1 to 11. A listing of commonly used buffers and additives is given
in Table 2. Additionally, the column lifetime will vary depending
upon the operating temperature, the type and concentration of
buffer used. For example, the use of phosphate buffer at pH 8
or above in combination with elevated temperatures will lead
to shorter column lifetimes.
Important Note:
Waters ACQUITY UPLC Peptide CSH C
18, 130 Å material is
produced from our ACQUITY UPLC Peptide CSH C
18, 130 Å
particles that undergo a surface modification by the addition of a
low concentration of weakly basic ionizable silanes, followed by
C
18 bonding and end capping. The optimal surface concentration
of the ionizable silane groups is more than an order of magnitude
lower than that of the primary C
18 bonded phase. The weakly
basic silane groups are protonated at a low pH and are neutral at
pH greater than 7. Consequently, and while the ACQUITY UPLC
Peptide CSH C
18, 130 Å particles can tolerate a pH range from
1 to 11, it will not provide frequently desired positive surface
charge benefits when used at a pH greater than six.
c. Solvents
To maintain maximum column performance, use high quality
chromat-ography grade solvents. Filter all aqueous buffers
prior to use. Pall Gelman Laboratory Acrodisc® filters are
recommended. 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.
d. Pressure
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH
C
18, 130 Å Columns can tolerate operating pressures up
to 18,000 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 up to 80 °C are recommended for operating
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH C
18,
130 Å 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, >70 °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.
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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
Table 2. Buffer recommendations for using ACQUITY UPLC and ACQUITY PREMIER
Peptide CSH C
18, 130 Å Columns from pH 1 to 11
Additive/Buffer
pK
a
Buffer
range
Volatility
(±1 pH unit)
Used for
Mass
Spec
Comments
TFA
0.3
–
Volatile
Yes
Ion pair additive, can suppress MS signal,
used in the 0.02–0.1% range.
Acetic acid
4.76
–
Volatile
Yes
Maximum buffering obtained when used with
ammonium acetate salt. Used in 0.1–1.0% range.
Formic acid
3.75
–
Volatile
Yes
Maximum buffering obtained when used with
ammonium formate salt. Used in 0.1–1.0% range.
Acetate
(NH
4CH2COOH)
4.76
3.76–5.76
Volatile
Yes
Used in the 1–10 mM range.
Note that sodium or potassium salts are not volatile.
Formate (NH
4COOH)
3.75
2.75–4.75
Volatile
Yes
Used in the 1–10 mM range.
Note that sodium or potassium salts are not volatile.
Phosphate 1
2.15
1.15–3.15 Non-volatile
No
Traditional low pH buffer, good UV transparency.
Phosphate 2
7.2
6.20–8.20 Non-volatile
No
Above pH 7, reduce temperature/concentration
and use a guard column to maximize lifetime.
4–Methylmorpholine
~8.4
7.4–9.4
Volatile
Yes
Generally used at 10 mM or less.
Ammonia (NH
4OH)
9.2
8.2–10.2
Volatile
Yes
Keep concentration below 10 mM and
temperatures below 30 °C.
Ammonium
Bicarbonate
10.3 (HCO
3
–
)
9.2 (NH
4
+
)
6.3 (H
2CO3)
6.8–11.3
Volatile
Yes
Used in the 5–10 mM range (for MS work keep
source >150 °C ). Adjust pH with ammonium
hydroxide or acetic acid. Good buffering
capacity at pH 10.
Note: Use ammonium bicarbonate (NH
4HCO3 ),
not ammonium carbonate ((NH
4 )2CO3 ).
Ammonium (Acetate)
9.2
8.2–10.2
Volatile
Yes
Used in the 1–10 mM range.
Ammonium (Formate)
9.2
8.2–10.2
Volatile
Yes
Used in the 1–10 mM range.
CAPSO
9.7
8.7–10.7
Non-volatile
No
Zwitterionic buffer, compatible with acetonitrile,
used in the 1–10 mM range. Low odor.
Glycine
2.4, 9.8
8.8–10.8 Non-volatile
No
Zwitterionic buffer, can give longer lifetimes
than borate buffer.
1–Methylpiperidine
10.2
9.3–11.3
Volatile
Yes
Used in the 1–10 mM range.
CAPS
10.4
9.5–11.5
Non-volatile
No
Zwitterionic buffer, compatible with acetonitrile,
used in the 1–10 mM range. Low odor.
Note: Working at the extremes of pH, temperature, and/or pressure will result in shorter column lifetimes.
6
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
IV. 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 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 3). Flush columns with 20-column volumes of
HPLC-grade solvents. Increasing mobile-phase temperature
to 35–55 °C increases cleaning efficiency. If the column
performance is poor after regenerating and cleaning, call
your local Waters office for additional support.
Table 3. Column Cleaning Sequence
Polar Samples Proteinaceous Samples
Water
Option 1: Inject repeated 100 µL aliquots
of dimethylsulfoxide (DMSO) using a
reduced flow rate delivering 50% Eluent
A and 50% Eluent B
Methanol
Option 2: Gradient of 10% to 90% B where:
A = 0.1% trifluoroacetic acid (TFA) in water,
B = 0.1% trifluoroacetic acid (TFA) in
acetonitrile (CH
3CN)
Isopropanol
Option 3: Flush column with 7 M
guanidine hydrochloride, or 7 M urea
Note: To avoid potentially damaging precipitation within your
column (e.g., if your separation eluent contains phosphate
buffer), be certain to flush column with 5 to 10 column volumes
of water BEFORE using suggested organic eluent column
wash procedures.
b. Storage
For periods longer than four days at room temperature, store
the column 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.
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.
V. eCORD
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.
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. The eCord provides a
solution to easily track the history of column usage.
Figure 1. eCord Intelligent Chip.
eCord Intelligent Chip
Figure 2. eCord inserted into side of column heater.
eCord inserted into
side of column heater
7
ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
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 Empower™ and MassLynx™
Software allowing the user to access column information on
their desktop.
c. Manufacturing Information
Figure 4. 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.
Figure 3. The eCord chip provides the user with
an overview of the bulk material QC test results.
Figure 5. An example of column use information provided by the eCord chip.
d. Customer Use Information
The eCord will automatically capture 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 if the
column met basic system suitability requirements.
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ACQUITY UPLC and ACQUITY PREMIER Peptide CSH Columns
[ CARE AND USE MANUAL ]
VI. REPRESENTATIVE TEST CHROMATOGRAPH AND CONDITIONS
FOR SEPARATION OF A CYTOCHROME c TRYPTIC DIGEST
1.20
1.10
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
2
3
4
5
6
7
8
9
10
11
12
min.
Chromatographic Conditions:
Column:
ACQUITY UPLC Peptide CSH C
18,
130 Å, 1.7 µm, 2.1 x 50 mm
(p/n: 186006936)
Mobile phase A:
0.1% formic acid in 100% water
Mobile phase B:
0.085% formic acid in 75% acetonitrile
reconstituted w/ 200 µL of MPA,
5.0 µL injection
Sample:
Tryptic digest of bovine cytochrome c
(p/n: 186006371)
Flow rate:
0.2 mL/min
Gradient:
1–21% B in 5 min; 21–31.4% B in 2.5 min;
31.4–95% B in 2.5 min; 95% B for 1.5 min;
1% B for 2.5 min
Temp.:
40 °C
UV detection:
214 nm
Peak identification
T1 N-AcGDVEK
T13-T14 KYIPGTK
T14 YIPGTK
T4 IFVQK
T9-T10 KTGQAPGFSYTDANK
T10 TGQAPGFSYTDANK
T8 TGPNLHGLFGR
T15 MIFAGIK
T19C EDLIAY
T19 EDLIAYLK
T12-T13 GITWGEETLMEYLENPKK
T12
GITWGEETLMEYLENPK
T5
CAQCHTVEK (heme attached)
Retention time (min)
Result
T19C 6.71
Retention time difference (min)
Result
T4 and T14
0.30
T10 and T9-T10
0.77
T19 and T19C
0.47
T12 and T12-T13
0.28
VII. ADDITIONAL INFORMATION
Tips for maximizing ACQUITY UPLC and ACQUITY PREMIER
Peptide CSH C
18, 130 Å Column lifetimes.
1. To maximize ACQUITY UPLC and ACQUITY PREMIER
Peptide CSH C
18, 130Å 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. When 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.
[ CARE AND USE MANUAL ]
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 ]
Waters, The Science of What’s Possible, ACQUITY, UPLC, XSelect, Empower, MassLynx, CSH, eCord,
and VanGuard are trademarks of Waters Corporation. Acrodisc is a trademark of Pall Life Sciences.
All other trademarks are the property of their respective owners.
©2020 Waters Corporation October 2020 720004544EN Rev D IH-PDF
■
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” or adding “new” mobile phase to “old”
mobile phase.
■
Old aqueous mobile phases. Remember to rinse bottles
thoroughly and prepare fresh every 24–48 hours.
■
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
back pressure 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
6. Remember: UPLC flow rates are often much lower and,
therefore, mobile phases last much longer (only prepare
what you need or store excess refrigerated).
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 hours?
■
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 a 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?
VIII. CAUTIONARY NOTE
Depending on user’s 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 rest
entirely with the purchaser and user. The Safety Data Sheet
(SDS) for this product is available at www.waters.com/sds.
