[ CARE AND USE MANUAL ]
XBridge Columns
Thank you for choosing a Waters XBridge™ column. The XBridge™ packing materials were designed to provide 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 certified plant using ultra pure reagents. Each batch of XBridge™ material is 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.
Cont ent s
I. Get ting Start ed
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 Isoc ratic Methods IV. T roubleshooting
V.Column Cleaning, REGENERATION and Storage
a.Cleaning and Regeneration
b.Storage
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
2.1mm 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
X Bridge™ Columns |
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[ CARE AND USE MANUAL ]
I. Get ting Start ed
Each XBridge™ column comes with a Certificate of Analysis and a Performance Test Chromatogram. The Certificate of Analysis, located on the technical information CD, is specific to each batch of packing material contained in the XBridge™ column and includes the 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 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.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 5 µm packing 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.
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.
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 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 HPLC systems due to the quality of the connections, operating environment, system electronics, reagent quality, column condition and operator technique.
X Bridge™ Columns |
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[ CARE AND USE MANUAL ]
Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
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Column internal diameter (mm) |
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Column Length (mm) |
1.0 |
2.1 |
3.0 |
4.6 |
7.8 |
10 |
19 |
30 |
50 |
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20 |
– |
0.07 |
0.14 |
0.33 |
– |
– |
– |
– |
– |
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30 |
– |
0.10 |
0.21 |
0.50 |
– |
2.4 |
8.5 |
– |
– |
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50 |
0.04 |
0.17 |
0.35 |
0.83 |
2.4 |
3.9 |
14 |
35 |
98 |
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100 |
0.08 |
0.35 |
0.71 |
1.7 |
4.8 |
7.8 |
28 |
70 |
– |
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150 |
0.12 |
0.52 |
1.0 |
2.5 |
7.2 |
12 |
42 |
106 |
294 |
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250 |
– |
0.87 |
1.8 |
4.2 |
– |
20 |
70 |
176 |
490 |
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II. Column Use
To ensure the continued high performance of XBridge™ columns, follow these guidelines:
a. Guard Columns
Use a Waters guard column of matching chemistry and particle size between 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® solid-phase extraction cartridges/ columns or Sep-Pak® cartridges of the appropriate chemistry to clean up 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.
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 the solvent. Contact the filter manufacturer with solvent compatibility questions. 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 Table 3. Additionally, the column lifetime will vary depending upon the operating temperature, the type and concentration of buffer used.
X Bridge™ Columns |
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[ 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 |
Temperature Limits |
Surface |
Carbon Load |
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Low pH |
High pH |
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XBridge C |
18 |
2.5, 3.5, 5 µm |
130Å |
185 m2/g |
1-12 |
80 °C |
60 °C |
3.1 µmol/m2 |
18% |
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XBridge C |
8 |
2.5, 3.5, 5 µm |
130Å |
185 m2/g |
1-12 |
60 °C |
60 °C |
3.1 µmol/m2 |
13% |
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XBridge Phenyl |
2.5, 3.5, 5 µm |
130Å |
185 m2/g |
1-12 |
80 °C |
60 °C |
3.0 µmol/m2 |
15% |
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XBridge Shield RP18 |
2.5, 3.5, 5 µm |
130Å |
185 m2/g |
2-11 |
50 °C |
45 °C |
3.2 µmol/m2 |
17% |
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XBridge HILIC |
2.5, 3.5, 5 µm |
130Å |
185 m2/g |
1-9 |
45 °C |
45 °C |
- |
- |
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XBridge Amide |
3.5 µm |
130Å |
185 m2/g |
2-11 |
90 °C |
90 °C |
7.5 µmol/m2 |
12% |
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Table 3: Buffer Recommendations for Using XBridge™ Columns from pH 1 to 12
Additive/Buffer |
pKa |
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Buffer Range |
Volatility |
Used for Mass Spec |
Comments |
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(±1 pH unit) |
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TFA |
0.3 |
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Volatile |
Yes |
Ion pair additive, can suppress MS signal, used in the 0.02-0.1% range. |
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Acetic Acid |
4.76 |
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Volatile |
Yes |
Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range. |
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Formic Acid |
3.75 |
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Volatile |
Yes |
Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range. |
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Acetate (NH4CH2COOH) |
4.76 |
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3.76 |
– 5.76 |
Volatile |
Yes |
Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile. |
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Formate (NH4COOH) |
3.75 |
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2.75 |
– 4.75 |
Volatile |
Yes |
Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile. |
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Phosphate 1 |
2.15 |
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1.15 |
– 3.15 |
Non-volatile |
No |
Traditional low pH buffer, good UV transparency. |
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Phosphate 2 |
7.2 |
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6.20 |
– 8.20 |
Non-volatile |
No |
Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime. |
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Phosphate 3 |
12.3 |
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11.3 - 13.3 |
Non-volatile |
No |
Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime. |
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4-Methylmorpholine |
~8.4 |
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7.4 |
– 9.4 |
Volatile |
Yes |
Generally used at 10 mM or less. |
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Ammonia (NH4OH) |
9.2 |
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8.2 – 10.2 |
Volatile |
Yes |
Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with |
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Ammonium Bicarbonate |
10.3 (HCO |
-) |
8.2 – 11.3 |
Volatile |
Yes |
ammonium hydroxide or acetic acid. Good buffering capacity at pH 10 |
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3 |
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Note: use ammonium bicarbonate (NH |
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9.2 (NH +) |
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HCO |
), not ammonium carbonate ((NH |
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CO ) |
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4 |
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4 |
3 |
4 |
2 |
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3 |
Ammonium (Acetate) |
9.2 |
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8.2 – 10.2 |
Volatile |
Yes |
Used in the 1-10 mM range. |
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Ammonium (Formate) |
9.2 |
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8.2 – 10.2 |
Volatile |
Yes |
Used in the 1-10 mM range. |
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Borate |
9.2 |
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8.2 – 10.2 |
Non-volatile |
No |
Reduce temperature/concentration and use a guard column to maximize lifetime. |
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CAPSO |
9.7 |
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8.7 – 10.7 |
Non-volatile |
No |
Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor. |
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Glycine |
2.4, 9.8 |
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8.8 – 10.8 |
Non-volatile |
No |
Zwitterionic buffer, can give longer lifetimes than borate buffer. |
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1-Methylpiperidine |
10.2 |
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9.3 – 11.3 |
Volatile |
Yes |
Used in the 1-10 mM range. |
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CAPS |
10.4 |
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9.5 – 11.5 |
Non-volatile |
No |
Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor. |
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Triethylamine |
10.7 |
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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). |
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(as acetate salt) |
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Used as ion-pair for DNA analysis at pH 7-9. |
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Pyrrolidine |
11.3 |
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10.3 |
– 12.3 |
Volatile |
Yes |
Mild buffer, gives long lifetime. |
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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 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.
Degas 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. This 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.
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.
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 selectivity, 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 without issues (see recommended conditions in Getting Started with XBridge Amide section). See Table 2 for recommended pH and temperature limits.
X Bridge™ Columns |
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