User guide

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[ Care and Use ManUal ]

XBridge

™

Columns 1

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 certi-

fied 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.

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

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

XbrIdGe

Columns

Summary of content (12 pages)

PAGE 1
[ Care and Use Manual ] XBridge Columns Thank you for choosing a Waters XBridge ™ column. The XBridge ™ packing Cont ent s materials were designed to provide excellent peak shape, high efficiency, I. and excellent stability for acidic and basic mobile phases. The XBridge ™ Get t ing Sta rt ed a. Column Installation packing materials are manufactured in a cGMP, ISO 9001:2000 certi- b. Column Equilibration fied plant using ultra pure reagents. Each batch of XBridge ™ material is c.
PAGE 2
[ Care and Use Manual ] I. Get t ing Sta rt ed b. Column Equilibration Each XBridge column comes with a Certificate of Analysis and a Perfor- XBridge™ columns are shipped in 100% acetonitrile. It is important to ensure mance Test Chromatogram. The Certificate of Analysis, located on the technical mobile phase compatibility before changing to a different mobile phase sys- information CD, is specific to each batch of packing material contained in tem.
PAGE 3
[ 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 3.
PAGE 4
[ 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 C18 2.5, 3.5, 5 µm 130Å 185 m2/g XBridge C8 2.5, 3.5, 5 µm 130Å XBridge Phenyl 2.5, 3.5, 5 µm XBridge Shield RP18 Temperature Limits Surface Carbon Load 60 °C 3.1 µmol/m2 18% 60 °C 60 °C 3.1 µmol/m2 13% 1-12 80 °C 60 °C 3.0 µmol/m2 15% 185 m2/g 2-11 50 °C 45 °C 3.
PAGE 5
[ Care and Use Manual ] III. Scaling U p/ Dow n Isoc rat ic Met hods contaminants. If this flushing procedure does not solve the problem, purge the The following formulas will allow scale up or scale down, while maintaining column with 5:95 acetonitrile:water. the same linear velocity, and provide new sample loading values: To clean polar contaminants from XBridge Amide columns, run a 25 minute gradient from 0-100% water. Please note that as aqueous concentration If column i.d.
PAGE 6
[ Care and Use Manual ] Figure 1: Waters and Parker Ferrule Types Note: If a column has been run with a mobile phase that contains formate Waters Ferrule Setting (e.g., ammonium formate, formic acid, etc.) and is then flushed with 100% Parker Ferrule Setting acetonitrile, slightly longer equilibration times may be necessary 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 .
PAGE 7
[ Care and Use Manual ] Figure 4: Waters Ferrule in a Parker Style Endfitting 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 Tighten the screw a bit more.
PAGE 8
[ Care and Use Manual ] 5. Figure 8: Determination of Gradient Delay Volume Measure the peak width at 4.4% of peak height (5-sigma method): 5-sigma Bandspreading (µL) = Peak Width (min) x 1.0 Flow Rate (mL/min) x (1000 µL/1 mL) 0.8 System Variance (µL2) = (5-sigma bandspreading)2 / 25 0.6 Figure 7: Determination of System Bandspreading Volume Using 5-Sigma Method 1/2 Vertical Au Distance 0.4 System Volume t1/2 0.2 0.0 Time 6.
PAGE 9
[ Care and Use Manual ] b. Impact of Bandspreading Volume on 2.1 mm i.d. Column Performance d. Waters Small Particle Size (2.5 µm) Columns – Fast Chromatography System with 70 µL bandspreading: 10,000 plates Waters columns that contain 2.5 µm particles provide faster and more effi- System with 130 µL bandspreading: 8,000 plates (same column) cient separations without sacrificing column lifetime.
PAGE 10
[ Care and Use Manual ] 2. Miscellaneous Tips Equilibrate with 20 column volumes of initial mobile-phase conditions before making first injection. 3. 4. 1. If gradient conditions are used, equilibrate with 8-10 column volumes HILIC columns are approximately 20% less retentive for gradient between injections. analysis and 35 to 65% less retentive for isocratic analysis.
PAGE 11
[ Care and Use Manual ] Mobile Phase Considerations 2. If separating reducing sugars, please review the following information. 1. 3. Reducing sugars can undergo mutarotation which produces the unde- Always maintain at least 5% polar solvent in the mobile phase or sired separation of the α and β ring forms (anomers). gradient (e.g., 5% aqueous, 5% methanol or 2% aqueous/3% methanol, etc.). 2. 3. 4. Maintain at least 40% organic solvent (e.g.
PAGE 12
[ Care and Use Manual ] g. b. For HPLC/MS separations of more complex sugar mixtures (e.g., polysaccharides), using XBridge Amide columns typical gradi- i. Weigh out sample (~3 g) into 50 mL centrifuge tube organic mobile phase reservoirs): ii. Add 25 mL of 50:50 ACN/H2O and homogenize (mechanically) i. Gradient going from 75% to 45% ACN with 0.1% NH4OH, 35 °C iii. Centrifuge at 3200 rpm for 30 minutes iv. Collect supernatant and filter using 0.45 µm or 0.