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 ™ tested chromatographically with acidic, basic and neutral analytes and fied plant using ultra pure reagents. Each batch of XBridge ™ material is packing materials are manufactured in a cGMP, ISO 9001:2000 certi- c. Initial Column Efficiency Determination the results are held to narrow specification ranges to assure excellent, with the Certificate of Acceptance. Performance Test Chromatogram is provided with each column along reproducible performance. Every column is individually tested and a III. Scaling U p/ Dow n Isoc rat ic Met hods IV. T roubl eshoot ing V. Column C l eaning, REGENERAT ION and Storage V I. Connec t ing t he Column to t he HPLC a. Column Connectors and System Tubing Considerations b. Measuring System Bandspreading Volume and System Variance c. Measuring Gradient Delay Volume (or Dwell Volume) V II. Addit ional Informat ion b. Impact of Bandspreading Volume on 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 Am

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. Equilibrate the column with a minimum of 10 column volumes of the the XBridge column and includes the batch number, analysis of unbonded mobile phase to be used (refer to Table 1 for a listing of empty column particles,...

Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes) Column internal diameter (mm) Column Length (mm) 3. If the sample is not dissolved in the mobile phase, ensure that the sample, To ensure the continued high performance of XBridge columns, follow solvent and mobile phases are miscible in order to avoid sample and/or these guidelines: 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, Use a Waters guard column of matching chemistry and particle...

Table 2: Recommended pH and temperature Limits for XBridge™ Columns at Ambient Temperatures Name of Column Particle Size Pore Diameter Surface Area XBridge Phenyl XBridge Shield RP18 XBridge HILIC Temperature Limits Carbon Load XBridge Amide 3.5 µm 130Å 185 m /g Table 3: Buffer Recommendations for Using XBridge™ Columns from pH 1 to 12 2 Buffer Range (±1 pH unit) Used for Mass Spec Ion pair additive, can suppress MS signal, used in the 0.02-0.1% range. Acetic Acid Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range. Formic Acid Maximum buffering obtained when...

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. and length are altered: increases, backpressure will rapidly increase as well. Reduce flow rate when operating at greater than 60% aqueous....

Figure 1: Waters and Parker Ferrule Types Note: If a column has been run with a mobile phase that contains for ate m 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 necssary when the e column is re-installed and run again with a formate-containing mobile phase. slightly longer equilibration times may be necessary when the column is .130” re-installed and run again with a formate-containing mobile phase. Due to the absence of an industry standard, various column manufactur-...

Figure 4: Waters Ferrule in a Parker Style Endfitting Table 5: Waters Part Numbers for SLIPFREE® Connectors SLIPFREE® Type Tubing Internal Diameter Tubing Length Tighten the screw a bit more. The ferrule moves forward, and reaches the sealing surface. Do not overtighten since this may end in break- There are two ways to fix the problem: 1. Band Spreading Minimization Cut the tubing, replace the ferrule and make a new connection. Figure 6 shows the influence of tubing internal diameter on system band Alternatively, replace the conventional compression screw fitting with spreading and peak shape....

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 Flow Rate (mL/min) x (1000 µL/1 mL) Figure 7: Determination of System Bandspreading Volume Using 5-Sigma Method System Volume 6. Determine the dwell time by first locating the time at the midpoint of the formed gradient (t1/2) (half the vertical distance between the initial and final isocratic segments as shown in Figure 8). 7. Subtract half the gradient time (1/2 tg) (10 min/2 = 5 min in this 4.4 %h example) from the gradient midpoint...

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: 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. This section describes five parameters to consider when performing separations with columns Note: Flow splitters after the column will introduce additional Note: Columns that contain 2.5 µm particles have smaller outlet frits to retain System...