Waters XBridge Columns User Manual

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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
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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
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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
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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
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