[ Care and Use ManUal ]
XbrIdGe Columns
Thank you for choosing a Waters XBridge™ column. The XBridge™ packing
materials were designed to provid e excellent peak shape, high efficiency,
and excellent stability for acidic and basic mobile phases. The XBridge
packing materials are manufactured in a cGMP, ISO 9001:2000 certi-
™
fied plant using ultra pure reagents. Each batch of XBridge
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 Test Chromatogram is provided with each column along
with the Certificate of Acceptance.
material is
Contents
I. GettInG started
™
a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
II. Column use
a. Guard Columns
b. Sample Preparation
c. Operating pH Limits
d. Solvents
e. Pressure
f. Temperature
III. sCalInG up/down IsoCratIC methods
IV. troubleshootInG
V. Column CleanInG, reGeneratIon
and storaGe
a. Cleaning and Regeneration
b. Storage
XBridge™ Columns 1
VI. ConneCtInG the Column to the hplC
a. Column Connectors and System Tubing Considerations
b. Measuring System Bandspreading Volume
and System Variance
c. Measuring Gradient Delay Volume (or Dwell Volume)
VII. addItIonal InformatIon
a. Use of Narrow-Bore (3.0 mm i.d. Columns)
b. Impact of Bandspreading Volume on
2.1 mm i.d. Column Performance
c. Non-Optimized vs. Optimized LC/MS/MS System:
System Modification Recommendations
d. Waters Small Particle Size (2.5 µm) Columns –
Fast Chromatography
e. Getting Started with XBridge HILIC Columns
f. Getting Started with XBridge Amide Columns
[ Care and Use ManUal ]
I. GettInG started
Each XBridge™ column comes with a Certificate of Analysis and a Performance Test Chromatogram. The Certificate of Analysis, locat ed on the technical
information CD, is specific to each batch of packing material contained in
™
the XBridge
particles, analysis of bonded particles, and chromatographic results
and conditions. The Performance Test Chromatogram is specific to each
individual column and contains the information: batch number, column
serial number, USP plate count, USP tailing factor, retention factor, and
chromatographic conditions. This data data should be stored for future
reference.
column and includes the batch number, analysis of unbonded
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 5 µm pack-
ing in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based
upon the column i.d., length, particle size and backpressure of the XBridge
column being installed. See Scaling Up/Down Isocratic Separations section
for calculating flow rates when changing column i.d and/or length. See
Connecting the Column to the HPLC for a more detailed discussion on HPLC
connections
1. Purge the pumping system of any buffer-containing 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 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 1 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.
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.
b. Column Equilibration
XBridge™ 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 listing of empty column
volumes).
To avoid precipitating out 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 HPLC system
with 60% methanol in water prior to introducing 60% methanol/40%
buffer mobile phase).
For XBridge HILIC columns, flush with 50 column volumes of 50:50
acetonitrile:water with 10 mM final buffer concentration. For XBridge
™
HILIC Amide columns, flush with 50 column volumes of 60:40
acetonitrile:aqueous. 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 XBridge HILIC Columns”
or “Getting Started with XBridge HILIC Amide Columns” for additional
information.
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in the desired
application. Waters recommends using a suitable solute mixture, as found
in the “Performance Test Chromatogram,” to analyze the column upon
receipt.
2. Determine the n umber of theoret ical plates (N ) and use this value for p eriodic
comparisons.
3. Repeat the test at predetermined intervals to track column performance over time. Slight variations may be obtained on two different HPLC systems due to the quality of the connections, operating
environment, system electronics, reagent quality, column condition
and operator technique.
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.
XBridge™ Columns 2
[ Care and Use ManUal ]
Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column internal diameter (mm)
Column Length (mm) 1.0 2.1 3.0 4.6 7.8 10 19 30 50
20 – 0.07 0.14 0.33 – – – – –
30 – 0.10 0.21 0.50 – 2.4 8.5 – –
50 0.04 0.17 0.35 0.83 2.4 3.9 14 35 98
100 0.08 0.35 0.71 1.7 4.8 7.8 28 70 –
150 0.12 0.52 1.0 2.5 7.2 12 42 106 294
250 – 0.87 1.8 4.2 – 20 70 176 490
II. Column use
To ensure the continued high performance of XBridge™ columns, follow
these guidelines:
a. Guard Columns
Use a Waters g uard column of matc hing ch emistry and p article size bet ween
the injector and main column. It is important to use a high-performance
matching guard column to protect the main column while not compromising or changing the analytical resolution.
Guard columns need to be replaced at regular intervals as determined by
sample contamination. When system backpressure steadily increases above
a set pressure limit, it is usually an indication that the guard column should
be replaced. A sudden appearance of split peaks is also indicative of a
need to replace the guard column.
b. Sample Preparation
1. Sample impurities often contribute to column contamination. One option
®
to avoid this is to use Waters Oasis
®
columns or Sep-Pak
the sample before analysis.
2. It is preferable to prepare the sample in the operating mobile phase or
a mobile phase that is weaker (less organic modifier) than the mobile
phase for the best peak shape and sensitivity.
cartridges of the appropriate chemistry to clean up
solid-phase extraction cartridges/
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 filters to remove particulates. If the sample
is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile,
methanol, etc.) ensure that the filter material does not dissolve in t he solvent. Contact the filter manufacturer with solvent compatibility qu estions.
Alternatively, centrifugation for 20 minutes at 8,000 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 100% organic solvents (e.g.,
acetonitrile). See “Getting Started with XBridge HILIC Columns”
or “Getting Started with XBridge Amide Columns” for additional
information.
c. Operating pH Limits
The recommended operating pH limits for XBridge™ columns are listed in
Table 2. A listing of commonly used buffers and additives is given in Ta ble 3.
Additionally, the column lifetime will vary depending upon the operating
temperature, the type and concentration of buffer used.
XBridge™ Columns 3
[ Care and Use ManUal ]
Table 2: Recommended pH and temperature Limits for XBridge™ Columns at Ambient Temperatures
Name of Column Particle Size Pore Diameter Surface Area pH Limits
XBridge C
XBridge C
18
8
2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 80 °C 60 °C 3.1 µmol/m
2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 60 °C 60 °C 3.1 µmol/m
XBridge Phenyl 2.5, 3.5, 5 µm 130Å 185 m2/g 1-12 80 °C 60 °C 3.0 µmol/m
XBridge Shield RP18 2.5, 3.5, 5 µm 130Å 185 m2/g 2-11 50 °C 45 °C 3.2 µmol/m
XBridge HILIC 2.5, 3.5, 5 µm 130Å 185 m
XBridge Amide 3.5 µm 130Å 185 m
2
/g 1-9 45 °C 45 °C - -
2
/g 2-11 90 °C 90 °C 7.5 µmol/m
Temperature Limits
Low pH High pH
Surface Carbon Load
2
2
2
2
2
Table 3: Buffer Recommendations for Using XBridge™ Columns from pH 1 to 12
Additive/Buffer pKa
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
Formate (NH
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.
Phosphate 3 12.3 11.3 - 13.3 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
Ammonium Bicarbonate
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.
Borate 9.2 8.2 – 10.2 Non-volatile No Reduce temperature/concentration and use a guard column to maximize lifetime.
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.
Triethylamine
(as acetate salt)
Pyrrolidine 11.3 10.3 – 12.3 Volatile Yes Mild buffer, gives long lifetime.
COOH) 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.
4CH2
COOH) 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.
4
OH)
4
9.2
-
10.3 (HCO
)
3
+
9.2 (NH
)
4
10.7 9.7 – 11.7 Volatile Yes Used in the 0.1-1.0% range. Volatile only when titrated with acetic acid (not hydrochloric or phosphoric).
Buffer Range
(±1 pH unit)
8.2 – 10.2
8.2 – 11.3
Volatility Used for Mass Spec Comments
Volatile
Volatile
Yes
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
Used as ion-pair for DNA analysis at pH 7-9.
HCO3), not ammonium carbonate ((NH4)2CO3)
4
18%
13%
15%
17%
12%
Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.
d. Solvents
To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use. Pall Gelman
®
Laboratory Acrodisc
filters are recommended. Solvents containing
e. Pressure
XBridge™ columns can tolerate pressures of up to 6,000 psi (400 bar or 40
Mpa) although pressures greater than 4,000 – 5,000 psi should be avoided in
order to maximize column and system lifetimes.
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 poor performance.
f. Temperature
Temperatures between 20 ˚C – 80 ˚C (up to 90 ˚C for XBridge Amide columns)
are recommended for operating XBridge columns in order to enhance selectivDegas all solvents thoroughly before use to prevent bubble formation in
the pump and detector. The use of an on-line degassing unit is also recommended. T his is especially important when running low pressure gradients
since bubble formation can occur as a result of aqueous and organic
solvent mixing during the gradient.
ity, lower solvent viscosity and increase mass transfer rates. However, any
temperature above ambient will have a negative effect on lifetime which will
vary depending on the pH and buffer conditions used. Under HILIC conditions
XBridge Amide columns can be used at high pH and at high temperatures with-
out issues (see recommended conditions in Getting Started wit h XBridge Amide
section). See Table 2 for recommended pH and temperature limits.
XBridge™ Columns 4
[ Care and Use ManUal ]
III. sCalInG up/down IsoCratIC methods
The following formulas will allow scale up or scale down, while maintaining
the same linear velocity, and provide new sample loading values:
If column i.d. and length are altered:
F
Load
Injection volume
= F1 (r2/r1)2
2
= Load1 (r2/r1)2 (L2/L1)
2
= Injection volume1(r2/r1)2 (L2/L1)
2
Where: r = Radius of the column
F = Flow rate
L = Length of column
1 = Original, or reference column
2 = New column
IV. Troubleshooting
Changes in retention time, resolution, or backpressure are often due to column
contamination. S ee the Column Cl eaning, Regeneration and Storage section of
this Care and Use Manual. 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 the Waters Corporation website for information on
seminars (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 or increa sing 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 each of HPLC-grade solvents (e.g., 80 mL total for
4.6 x 250 mm column) listed in Table 4. Incr easing mobile p hase temp erature to
35-55 ˚C incre ases cleaning eff iciency. If the col umn performance i s poor after
cleaning and regeneration, call your local Waters office for additional support.
Flush XBridge 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.
To clean polar contaminants from XBridge Amide columns, run a 25 minute
gradient from 0-100% water. Please note that as aqueous concentration
increases, backpressure will rapidly increase as well. Reduce flow rate when
operating at greater than 60% aqueous. Repeat if necessary.
Table 4: Cleaning and Regeneration Sequence or Options
Polar Sam ples Non-polar Samples Proteinaceous Samples
1. wa ter
2. meth anol 2. tetra hydrofura n (THF ) Option 2 : gradient of 10% to 90 %
3. tetra hydrofur an (TH F) 3. dichlor omethane
4. meth anol 4. h exane
5. water
6. mobil e phase 6. mo bile phase
1. isopropanol (or anappropriate
isopropanol/water mixture*)
5. isopropanol
(follow ed by an appro priate
isopropanol/water mixture*)
Optio n 1: Inject re peated
aliquot s of dimethyl su lfoxide
(DMSO)
B where:
A = 0.1% trifluor oacetic ac id
(T FA) in water
B = 0.1% trifluor oacetic ac id
(T FA) in acetonitril e (CH
Optio n 3: Flush col umn with 7M
guanid ine hydroc hloride, or 7 M urea
CN)
3
* Use low organic solvent content to avoid precipitating buffers.
b. Storage
For periods longer than four days at room temperature, store the reversed-
phase XBridge columns and XBridge Amide columns in 100% acetonitrile. Im-
mediately 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 (<20% organic) mobile phases, as this may promote bacterial growth.
If the mobile phase contained a buffer salt, flush the column with 10 column
vol umes of HPLC grade water (see Table 1 for common column volumes) and
replace with 100% acetonitrile for storage. Failure to perform this intermedi-
ate step could result in precipitation of the buffer salt in the column or system
when 100% acetonitrile is introduced. Run a gradient to 100% ACN in order
to flush all aqueous solvent from an XBridge Amide column prior to storage
in 100% ACN. Completely seal column to avoid evaporation and drying out
of the bed. For periods longer than four days, store XBridge 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).
XBridge™ Columns 5
[ Care and Use ManUal ]
Void
Note: If a column has been run with a mobile phase that contains for mate
(e.g., ammonium formate, formic acid, etc.) and is then flushed with 100%
acetonitrile, slightly longer equilibration times may be nec essary when the
column is re-installed and run again with a formate-containing mobile phase.
slightly longer equilibration times may be necessary when the column is
re-installed and run again with a formate-containing mobile phase.
VI. ConneCtInG the Column to the hplC
a. Column Connectors and System Tubing Considerations
Tools needed:
•3/8inchwrench
•5/16inchwrench
Handle the column with care. Do not drop or hit the column on a hard
surface as it may disturb the bed and affect its performance.
1. Correct connection of 1/16 inch outer diameter stainless steel tubing lead-
ing to and from the column is essential for high-quality chromatographic
results.
2. When using standard stainless steel compression screw fittings, it is
important to ensure prop er fit of the 1/16 inch outer diameter stainless steel
tubing. When tightening or loosening the compression screw, place a 5/16
inch wrench on the compression screw and a 3/8 inch wrench on the hex
head of the column endfitting.
Note: If one of the wrenches is placed on the column tube flat during this
process, the endfitting will be loosened and leak.
3. If a leak occurs between the stainless steel compression screw fitting
and the column endfitting, a new compression screw fitting, tubing and ferrule must be assembled.
4. An arrow on the column identification label indicates correct direction
of solvent flow.
Correct connection of 1/16 inch outer diameter stainless steel tubing leading
to and from the column is essential for high-quality chromatographic results. To
obtain a void-free connection, the tubing must touch the bottom of the column
endfitting. It is important to realize that extra column peak broadening due to
voids can destroy an otherwise successful separation. T he choice of appropriate
column connectors and system tubing is discussed in detail below.
Figure 1: Waters and Parker Ferrule Types
Waters Ferrule Setting Parker Ferrule Setting
.130” .090”
Due to the absence of an industry standard, various column manufactur-
ers have employed different types of chromatographic column connectors.
The chromatographic separation can be negatively affected if the style
of the column endfittings does not match the existing tubing ferrule
settings. This section explains the differences between Waters style
and Parker style ferrules and endfittings (Figure 1). Each endfitting style
varies in the required length of the tubing protruding from the ferrule. The
™
XBridge
column is equipped with Waters style endfittings that require a
0.130 inch ferrule depth. If a non-Waters style column is presently being
used, it is critical that ferrule depth be reset for optimal performance
™
prior to installing an XBridge
column.
In a proper tubing/column connection (Figure 2), the tubing touches the bottom
of the column endfitting, with no void between them.
Figure 2: Proper Tubing/Column Connection
Th e presence of a void in the f low stream
reduces column performance. This can
occur if a Parker ferrule is connected to
a Waters style endfitting (Figure 3).
Note: A void appears if tubing with a Parker ferrule is connected to a Waters
style column.
Figure 3: Parker Ferrule in a Waters Style Endfitting
There is only one way to fix this problem: Cut the end of the tubing with
the ferrule, place a new ferrule on the
tubing and make a new connection.
Before tightening the screw, make
sure that the tubing bottoms out in the
endfitting of the column.
Conversely, if tubing with a Waters ferrule is connected to a column with
Parker style endfitting, the end of the tubing will bottom out before the
ferrule reaches its proper sealing position. This will leave a gap and create
a leak (Figure 4).
t
XBridge™ Columns 6
Note: The connection leaks if a Waters ferrule is connected to a column with
a Parker style endfitting.
[ Care and Use ManUal ]
Figure 4: Waters Ferrule in a Parker Style Endfitting
Gap
There are two ways to fix the problem:
1. Tighten the screw a bit more. The ferrule moves forward, and reaches
the sealing surface. Do not overtighten since this may end in breaking the screw.
2. Cut the tubing, replace the ferrule and make a new connection.
Alternatively, replace the conventional compression screw fitting with
an all-in-one PEEK fitting (Waters part number PSL613315) that allows
resetting of the ferrule depth. Another approach is to use a Keystone,
®
Inc. SLIPFREE
SLIPFREE
connector to always ensure the correct fit. The fingertight
®
connectors automatically adjust to fit all compression screw
type fittings without the use of tools (Figure 5).
Figure 5: Single and Double SLIPFREE
®
Connectors
Table 5: Waters Part Numbers for SLIPFREE® Connectors
SLIPFREE® Type Tubing Internal Diameter
Tubing Length 0.005” 0.010” 0.020”
Single 6 cm PSL 618000 PSL 618006 PSL 618012
Single 10 cm PSL 618002 PSL 618008 PSL 618014
Single 20 cm PSL 618004 PSL 618010 PSL 618016
Double 6 cm PSL 618001 PSL 618007 PSL 618013
Double 10 cm PSL 618003 PSL 618009 PSL 618015
Double 20 cm PSL 618005 PSL 618001 PSL 618017
Band Spreading Minimization
Figure 6 shows the influence of tubing internal diameter on system band
spreading and peak shape. As can be seen, the larger tubing diameter
causes excessive peak broadening and lower sensitivity.
Figure 6: Effect of Connecting Tubing on System
0.005 inches
0.020 inches
0.040 inches
SLIPFREE® Connector Features:
• Tubingpushedintoendfitting,therebyguaranteeinga
void-free connection
• Connector(s)come(s)installedontubing
• VarioustubingIDsandlengthsavailable
• Fingertightto10,000psi–neverneedswrenches
• Readjuststoallcolumnendfittings
• Compatiblewithallcommerciallyavailableendfittings
• Uniquedesignseparatestube-holdingfunctionfrom
sealing function
XBridge™ Columns 7
Diluted/Distorted Sample Band
b. Measuring System Bandspreading Volume
and System Variance
This test should be performed on an HPLC system with a single wavelength
UV detector (not a Photodiode Array (PDA)).
1. Disconnect column from system and replace with a zero dead volume
union.
2. Set flow rate to 1 mL/min.
3. Dilute a test mix in mobile phase to give a detector sensitivity
of 0.5 - 1.0 AUFS (system start up test mix can be used which
contains uracil, ethyl and propyl parabens; Waters part number
WAT034544).
4. Inject 2 to 5 µL of this solution.
[ Care and Use ManUal ]
5. Measure the peak width at 4.4% of peak height (5-sigma method):
5-sigma Bandspreading (µL) = Peak Width (min) x
Flow Rate (mL/min) x (1000 µL/1 mL)
System Variance (µL
2
) = (5-sigma bandspreading)2 / 25
Figure 7: Determination of System Bandspreading Volume Using 5-Sigma
Method
System Volume
5
4.4 %h
In a typical HPLC system, the Bandspreading Volume should be no
2
greater than 100 µL ± 30 µL (or Variance of 400µL
± 36µL2).
In a microbore (2.1mm i.d.) system, the Bandspreading Volume should be no
2
greater than 20 to 40 µL (or Variance no greater than 16µL
to 64µL2).
c. Measuring Gradient Delay Volume (or Dwell Volume)
For successful gradient-method transfers the gradient delay volumes should be
measured using t he same method on bot h HPLC systems. T he proce dure below
describes a method for determining the gradient delay volumes.
1. Replace the column with a zero dead volume union.
2. Prepare mobile phase A (pure solvent, such as methanol) and mobile phase
B (mobile phase A with a UV absorbing sample, such as (v/v) 0.1% acetone
in methanol).
3. Equilibrate the system with mobile phase A until a stable baseline is
achieved.
4. Set the detector wavelength to the absorbance maximum of the probe (265
nm for acetone).
5. Program a 0-100% B linear gradient in 10 min at 2 mL/min (the exact
conditions are not critic al; just make sure the gradient volume is at least 20
mL) with a hold at 100% B.
Figure 8: Determination of Gradient Delay Volume
1.0
0.8
0.6
Au
0.4
0.2
0.0
1/2 Vertical
Distance
Time
t
1/2
6. Determine the dwell time by first locating the time at the midpoint of the
formed gradient (t
) (half the vertical distance between the initial and final
1/2
isocratic segments as shown in Figure 8).
7. Subtract half the gradient time (1/2 tg) (10 min/2 = 5 min in this
example) from the gradient midpoint (t
8. Convert the dwell time (t
) to the dwell volume (VD) by multiplying
D
) to obtain the dwell time (tD).
1/2
by the flow rate (F ).
Dwell Volume VD = (t
—1/2 tg) x F
1/2
For fast gradient methods, the gradient delay volume (or dwell volume)
should be less than 1 mL. If the gradient delay volume is greater than 1
mL, see System Modification Recommendations section on how to reduce
system volume.
VII. addItIonal InformatIon
a. Use of Narrow-Bore (3.0 mm i.d.) Columns
This section describes how to minimize extra column effects and provides
guidelines on maximizing the performance of a narrow-bore column in an
HPLC system. A 3.0 mm i.d. narrow-bore column usually requires no system modifications. A 2.1 mm i.d. column, however, requires modifications
to the HPLC system in order to eliminate excessive system bandspreading
volume. Without proper system modifications, excessive system bandspreading volume causes peak broadening and has a large impact on peak
width as peak volume decreases.
XBridge™ Columns 8
[ Care and Use ManUal ]
b. Impact of Bandspreading Volume on 2.1 mm i.d. Column
Performance
System with 70 µL bandspreading: 10,000 plates
System with 130 µL bandspreading: 8,000 plates (same column)
Note: Flow splitters after the column will introduce additional
bandspreading.
System optimization, especially in a system that contains a flow
splitter, can have dramatic effects on sensitivity and resolution.
Optimization includes using correct ferrule depths and minimizing
tubing inner diameters and lengths. An example is given in Figure 9
where system optimization resulted in a doubling of sensitivity and
resolution of the metabolite in an LC/MS/MS system.
Figure 9: Non-Optimized vs. Optimized LC/MS/MS System
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00
7.00 7.50 8.00
c. Non-Optimized vs. Optimized LC/MS/MS System: System
Modification Recommendations
1. Use a microbore detector flow cell with 2.1 mm i.d. columns.
Note: Detector sensitivity is reduced with the shorter flow cell path length in
order to achieve lower bandspreading volume.
d. Waters Small Particle Size (2.5 µm) Columns –
Fast Chromatography
Waters columns that contain 2.5 µm particles provide faster and more efficient separations without sacrificing column lifetime. This section describes
five parameters to consider when performing separations with columns
containing 2.5 µm particles.
Note: Columns that contain 2.5 µm particles have smaller outlet frits to retain
packing material. These columns should not be backflushed.
1. Flow Rate—Compared with the 5 µm columns, columns with 2.5 µm particles have higher optimum flow rates. These columns are used when high
efficiency and short analysis times are required. These higher flow rates,
however, lead to increased backpressure.
Note: Use a flow rate that is practical for your system.
2. Backpressure—Backpressures for columns with 2.5 µm particles are higher
than for 5 µm columns with the same dimensions. Waters suggests using a
shorter column to compensate for increased backpressure and to obtain a
shorter analysis time.
3. Temperature —Use a higher tem perature to reduc e backp ress ure c au sed b y
smal ler pa rticl e sizes . Th e rec ommend ed temperature range for XBridge
columns is 20 °C to 60 °C. See Column Use section for a discussion of
™
elevated temperature use with XBridge
4. Sampling Rate—Use a sampling rate of about 10 points per second
or higher. A minimum of 20 points across the earliest eluting peak of
interest is needed for optimum reproducibility.
5. Detector Time Constant—Use a time constant of 0.1 seconds or lower
for fast analyses.
columns.
e. Getting Started With XBridge HILIC Columns
™
2. Minimize injector sample loop volume.
3. Use 0.009 inch (0.25 mm) tubing for rest of connections in standard
systems and 0.005 inch (0.12 mm) tubing for narrowbore (2.1 mm
i.d.) systems.
4. Use perfect (pre-cut) connections (with a variable depth inlet if using columns from different suppliers).
5. Detector time constants should be shortened to less than
0.2 seconds.
XBridge™ Columns 9
1. Because XBridge HILIC columns do not posses a bonded phase,
the pH operating range is 1 to 9, 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 column is first received, flush in 50% acetonitrile: 50% water
with 10 mM final buffer concentration for 50 column volumes.
[ Care and Use ManUal ]
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 5% polar solvent in the mobile phase or gradient (e.g., 5% aqueous/5% methanol or 2% aqueous/3% methanol,
etc.). This ensures that the XBridge particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in your
mobile phase or gradient.
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 at least
10 mM in your mobile phase/gradient at all times.
Injection Solvents
1. If possible, injection solvents should be 100% organic solvent of
the initial mobile phase conditions. Water must be eliminated or minimized. Choose weak HILIC solvents such as acetonitrile, isopropanol,
methanol, etc.
Miscellaneous Tips
1. As compared to Atlantis® HILIC Silica HPLC columns, the XBridge
HILIC columns are approximately 20% less retentive for gradient
analysis and 35 to 65% less retentive for isocratic analysis. This
is due to the lower residual surface silanol concentration of the BEH
particle.
2. In HILIC, it is important to remember that water is the strongest solvent. Therefore, it must be eliminated or minimized in the injection
solvent.
3. For initial scouting conditions, run a gradient from 95% acetonitrile
to 50% acetonitrile. If no retention occurs, run isocratically with
95:3:2 acetonitrile:isopropanol:aqueous buffer.
4. Alternate polar solvents such as methanol, ethanol or isopropanol
can also be used in place of water to increase retention.
f. Getting Started with XBridge Amide Columns
Operating Ranges
1. XBridge Amide Columns can be used routinely under HILIC conditions
between pH 2 to 11, and they can be operated at temperatures up to
90 °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 column is first received, flush in 60% acetonitrile: 40% aqueous
(or initial starting conditons) for 50 column volumes.
2. A generic injection solvent is 75:25 acetonitrile:methanol. This is
a good compromise between analyte solubility and peak shape. If
solubility is still poor, 0.2% formic acid can be added.
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.
XBridge™ Columns 10
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.
[ Care and Use ManUal ]
Mobile Phase Considerations
1. Always maintain at least 5% polar solvent in the mobile phase or
gradient (e.g., 5% aqueous, 5% methanol or 2% aqueous/3% met hanol, etc.).
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in your
mobile phase or gradient.
3. At aqueous concentrations greater than 60%, lower flow rates
should be used due to high backpressure. This includes all aqueous
wash procedures.
4. Avoid phosphate salt buffers to avoid precipitation in HILIC mobile
phases. Phosphoric acid is OK.
Injection Solvents
1. If possible, injection solvents should be as close to the mobile
phase composition as possible (if isocratic) or the starting gradient
conditions.
2. A generic injection solvent is 75:25 acetonitrile:methanol. This is a
good compromise between analyte solubility and peak shape. When
separating saccharides with limited solubility in in organic solvents,
higher concentrations of aqueous solvent in the sample are acceptable. 50:50 acetonitrile:water can provide satisfactory results.
3. The injection solvent’s influence on peak shape should be determined
experimentally. In some cases, injections of water (or highly aqueous
solutions) may not adversely affect peak shape.
Miscellaneous Tips
1. 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.
2. Alternate polar solvents such as methanol, acetone or isopropanol
can also be used in place of water to increase retention.
2. If separating reducing sugars, please review the following information.
3. Reducing sugars can undergo mutarotation which produces the undesired separation of the α and β ring forms (anomers).
4. Collapsing anomers into one peak is accomplished through the use of
a combination of elevated temperature and high pH:
a. Use of 35 °C with high pH (0.2% triethylamine (TEA) or 0.1%
ammonium hydroxide (NH
b. Use of >80 °C with 0.05% TEA high temperature (>80 °C)
5. When separating reducing sugars (e.g., fructose, glucose, maltose,
lactose, arabinose, glyceraldehyde, etc.) please pay attention to the
following suggestions. Failure to do so will result in the appearance
of split peaks (anomer separation) for these analytes:
a. Operate at a slow flow rate to facilitate anomer collapse.
b. With longer columns, increased flow rates can be used. As with
all LC separations, optimal flow rates should be determined
experimentally.
c. Add triethylamine (TEA) or ammonium hydroxide (NH4OH)
modifiers to aqueous and organic mobile phase reservoirs at
equal concentrations.
d. For HPLC separations of mono- and/or disaccharides using
XBridge Amide columns typical isocratic conditions include:
i. 75% acetonitrile (ACN) with 0.2% TEA, 35 °C
ii. 77% acetone with 0.05% TEA, 85 °C
e. For HPLC separations of more complex sugar mixtures (e.g.,
polysaccharides) using XBridge Amide columns typical gradient
conditions include (add TEA modifier to both mobile phases A
and B):
i. Gradient going from 80% to 50% ACN with 0.2% TEA,
35 °C,
OH)) and/or
4
Tips for Separating Sugars/Saccharides/Carbohydrates
1. If separating sugars or sugar-containing compounds that do not
include reducing sugars (see below) follow generic ‘Getting Started
with XBridge Amide Columns’ recommendations described above.
XBridge™ Columns 11
ii. 80%-55% acetone with 0.05% TEA, 85 °C
f. For HPLC/MS separations of mono- and disaccharides using
XBridge Amide columns typical isocratic conditions include:
i. 75% ACN with 0.1% NH4OH, 35 °C
[ Care and Use ManUal ]
g. For HPLC/MS separations of more complex sugar mixtures (e.g.,
polysaccharides), using XBridge Amide columns typical gradient conditions include (add NH
OH modifier to aqueous and
4
organic mobile phase reservoirs):
i. Gradient going from 75% to 45% ACN with 0.1% NH4OH, 35 °C
6. More complex sample mixtures may require the use of gradient
conditions and/or longer column lengths.
8. Typical sample preparation suggestions for samples that contain
sugars/saccharides/carbohydrates:
a. Liquid Samples
i. Dilute with 50:50 ACN/H
O
2
ii. Filter using 0.45 µm or 0.22 µm syringe filter (if necessary)
b. Solid Samples
i. Weigh out sample (~3 g) into 50 mL centrifuge tube
ii. Add 25 mL of 50:50 ACN/H
O and homogenize (mechanically)
2
iii. Centrifuge at 3200 rpm for 30 minutes
iv. Collect supernatant and filter using 0.45 µm or 0.22 µm
syringe filter (if necessary)
c. Depending on sample and /or analyte concentrations, additional
sample dilutions may be necessary.
d. More complex samples and/or lower analyte concentrations
may require additional sample preparation steps and/or procedures such as solid phase extraction (SPE).
e. Consider guard columns for column protection.
Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07, Australia 61 2 9933 1777, Belgium 32 2 726 1000, Brazil 55 11 4134 3788,
Canada 1 800 252 4752, China 86 21 6156 2666, Czech Republic 420 2 617 11384, Denmark 45 46 59 8080, Finland 358 9 5659 6288, France 33 1 30 48 72 00,
Germany 49 6196 400 600, Hong Kong 852 2964 1800, Hungary 36 1 350 5086, India and India Subcontinent 91 80 2837 1900, Ireland 353 1 448 1500, Italy 39 02 265 0983,
Japan 81 3 3471 7191, Korea 82 2 6300 4800, Mexico 52 55 52 00 1860, The Netherlands 31 76 508 7200, Norway 47 6 384 6050, Poland 48 22 101 5900, Puerto Rico 1 787 747 8445,
Russia/CIS 7 495 727 4490/ 290 9737, Singapore 65 6593 7100, Spain 34 93 600 9300, Sweden 46 8 555 115 00, Switzerland 41 56 676 7000, Taiwan 886 2 2501 9928,
XBridge™ Columns 12
United Kingdom 44 208 238 6100, All other countries: Waters Corporation U.S.A. 1 508 478 2000/1 800 252 4752
©2012 Waters Corporation. Waters, The Science of W hat’s Possible,
Oasis, SepPak and XBridge are trademarks of Waters Corporation.
Acrodisc is a trademark of Pall Life Sciences. SLIPF REE is a trademark
of Keystone, Inc.
Produced in the U.S.A.
June 2012 720001366EN Rev 7 VW-P DF
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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