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[ Care and Use ManUal ] Waters Spherisorb Columns 1 Waters spherisorb columns contents i. GettinG started a. Column Installation 1. Reversed-Phase Columns 2. Normal-Phase Columns b. Column Equilibration 1. Reversed-Phase Columns 2. Normal-Phase Columns c. Initial Column Efficiency Determination ii. column use a. Guard Columns b. Sample Preparation c. pH Range d. Solvents e. Pressure f. Temperature iii. scalinG up/doWn isocratic methods iv. troubleshootinG v. column cleaninG, reGeneratinG and storaGe a. Cleaning and Regenerating 1. Reversed-Phase Columns 2. Normal-Phase Columns b. Storage 1. Reversed-Phase Columns 2. Normal-Phase Columns vi. connectinG the column to the hplc a. Column Connectors and System Tubing Considerations b. Band Spreading Minimization c. Measuring System Bandspreading Volume & System Variance d. Measuring System 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. Guard Cartridges and Columns Assembly Thank you for choosing a Waters Spherisorb® column. Spherisorb analytical columns are durable, high-efficiency chromatographic columns, featured in thousands of references in chromatographic literature. The wide range of column lengths and diameters offers you exceptional flexibility in optimizing methods and reducing solvent consumption. Follow the guidelines in this manual to obtain the best performance, reproducibility and durability from your analytical columns and cartridges. [ Care and Use ManUal ] Waters Spherisorb Columns 2 Chemistry Particle Shape Particle Size (µm) Pore Size (Å) Surface Area (m2/g) Pore Volume (cc/g) % Carbon Load Endcapped Silica Spherical 3, 5 and 10 80 220 0.50 n/a n/a ODS2 (C18) - Fully End Capped Spherical 3, 5 and 10 80 220 0.50 11.5 yes ODS1 (C18) - Partially End Capped Spherical 3, 5 and 10 80 220 0.50 6.2 no ODSB (C18) - Base De-activated Spherical 5 80 220 0.50 11.5 yes* C8 Spherical 3, 5 and 10 80 220 0.50 5.8 yes C6 Spherical 3, 5 and 10 80 220 0.50 4.7 yes C1 Spherical 3, 5 and 10 80 220 0.50 2.2 no Nitrile (CN) Spherical 3, 5 and 10 80 220 0.50 3.1 no Amino (NH2) Spherical 3, 5 and 10 80 220 0.50 1.9 no Phenyl Spherical 3, 5 and 10 80 220 0.50 2.5 no OD/CN (Mixed Mode) Spherical 5 80 220 0.50 5.0 yes SAX Spherical 3, 5 and 10 80 220 0.50 4.0 no SCX Spherical 3, 5 and 10 80 220 0.50 4.0 no i . Get t inG start ed Each Spherisorb column comes with a Performance Test Chromatogram. This Performance Test Chromatogram is specific to each individual column and contains the following information: gel batch number, column serial number, USP plate count, USP tailing factor, capacity factor, and chromatographic conditions. The performance test chromatogram should be stored for future reference. a. Column Installation Note: The flow rates given in the procedure below are for a typical 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 Spherisorb column being installed. See “Scaling Up/Down Isocratic Separations” 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. Reversed-Phase Columns 1. Purge the pumping system of any buffer-containing mobile phases and connect the inlet end of the column to the injector outlet. 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. 2. Normal-Phase Columns Note: It is assumed that your system has been used for reversed-phase chromatography. If this is not the case, you can start with step 3. 1. Purge the pumping system of any buffer containing mobile phases. 2. Flush the system thoroughly with acetonitrile. 3. Switch the system over to the mobile phase that you are planning to use in normal-phase chromatography. 4. Connect the column and equilibrate it with the mobile phase. Note: Equilibration with the mobile phase may require a larger amount of solvent than in reversed-phase chromatography. Table 1. Spherisorb Column Physical Characteristics * polar endcapping [ Care and Use ManUal ] Waters Spherisorb Columns 3 b. Column Equilibration Spherisorb columns are shipped in test mobile phase. 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 2 for a listing of empty column volumes). 1. Reversed-Phase Columns 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.) 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. 2. Normal-Phase (Spherisorb Silica, Amino, Cyano) Column Spherisorb normal-phase (NP) columns are delivered in 96% heptane / 4% isopropyl alcohol. Care should be taken not to pass any mobile phase through the column that might cause a precipitate. Spherisorb NP columns are compatible with water and all common organic solvents, provided that solvent miscibility is accounted for. Equilibrate normal-phase silica columns in the mobile phase. Very small quantities of water in the mobile phase can dramatically affect the activity of normal-phase packings. For good reproducibility, ensure that the mobile phase always has the same water content. It is difficult and usually unnecessary to completely eliminate the water from the mobile phase. Dry mobile phases can take a very long time to equilibrate the column. A water content of 50 percent of saturation is recommended for most applications. To equilibrate your column: 1. Starting at 0.0 mL/min, increase the flow rate in 0.1 mL/min increments to 1.0 minutes. 2. Purge the column with the mobile phase until you obtain a stable baseline. 3. Verify that retention times and peak areas for a standard are stable by comparing 2-3 replicate consecutive injections Before you perform the first analysis on your new column, perform an efficiency test to confirm the performance of the column. Table 2. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes) Column Length Column Internal Diameter (mm) 1.0 2.1 3.0 4.6 10 20 20 mm - 0.07 0.14 0.33 - - 30 mm - 0.1 0.2 0.5 - - 50 mm 0.1 0.2 0.3 0.8 - - 100 mm 0.1 0.4 0.7 1.7 - - 150 mm 0.1 0.5 1.0 2.5 12 24 250 mm - 0.9 1.8 4 20 40 c. Initial Column Efficiency Determination 1. Perform an efficiency test on the column before using it. Waters recommends using a suitable solute mixture, as found in the “Performance Test Chromatogram”, to analyze the column upon receipt. However, if the column is used only for a single routine assay, it may be more convenient to test the column under these assay conditions. Keep a record of the initial column performance. 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. [ Care and Use ManUal ] Waters Spherisorb Columns 4 i i . column use To ensure the continued high performance of Spherisorb columns, follow these guidelines: a. Guard Columns Use a Waters Spherisorb guard column of matching chemistry and particle size between the injector and main column. It is important to use a 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 or other changes in chromatographic performance 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 Oasis® solid-phase extraction cartridges/ columns or Sep-Pak® cartridges of the appropriate chemistry to clean up the sample before analysis. Link to www.waters.com/sampleprep 2. It is preferable to prepare the sample in the operating mobile phase or a mobile phase that is weaker (less organic modifier in the case of reversed-phase chromatography, less polar modifier in the case of normal-phase chromatography or hydrophilic-interaction chro- matography, less salt in the case of ion exchange) 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. Filter sample with 0.2 μm membranes to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile, methanol, etc.) ensure that the membrane material does not dissolve in the solvent. Contact the membrane 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. c. pH Range The recommended operating pH range for Spherisorb columns is 2 to 8. A listing of commonly used buffers and additives is given in Table 3. Additionally, the column lifetime will vary depending upon the operating temperature, and the type and concentration of buffer used. For example, the use of phosphate buffer at pH 8 in combina- tion with elevated temperatures will lead to shorter column lifetimes. [ Care and Use ManUal ] Waters Spherisorb Columns 5 Table 3: Buffer Recommendations for Using Spherisorb Columns from pH 2 to 8 Additive or Buffer pKa Buffer Range (± pH unit) Volatility Used for Mass Spec? Comments Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range. Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range. Ammonmium Formate (NH4COOH) 3.75 2.75-4.75 Volatile Yes Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM) are recommended for UV applications. Note: sodium or potassium salts are not volatile. Trifluoroacetic Acid (TFA) 0.3 Volatile Low Conc. When used in LC/MS, due at signal suppression, it is generally recommended to use TFA at coencentrations <0.1%. Ammonium Acetate (CH3COONH4) 4.76 3.76-5.76 Volatile Yes Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM) are recommended for UV applications. Note: sodium or potassium salts are not volatile. 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 guard column to maxmize lifetime. 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. (Please refer to the filtration section of the Waters Chromatography Columns and Supplies Catalog or the Waters web site (www.waters.com) for additional product information.) 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 poorer 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 Spherisorb columns can tolerate pressures of up to 6,000 psi (400 bar or 40 MPa) although long-term, routine operating pressures greater than 4,000 – 5,000 psi should be avoided in order to maximize column and system lifetimes. f. Temperature Temperatures between 20 – 45 ˚C are recommended for operating Spherisorb columns in order to enhance selectivity, lower solvent viscosity and increase mass transfer rates. However, any temperature above ambient may have a negative effect on lifetime which will vary depending on the pH and buffer conditions used. i i i . scalinG up/doWn isoc rat ic met hods 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: F2 = F1(r2/r1)2 or Injection volume1 = Injection volume2 (r2/r1)2(L2/L1) Where: r = radius of the column, in mm F = flow rate, in mL/min L = length of column, in mm 1 = original, or reference column 2 = new column [ Care and Use ManUal ] Waters Spherisorb Columns 6 iv. t roubleshoot inG Changes in retention time, resolution, or backpressure are often due to column contamination. See “Column Cleaning, Regenerating and Storage”. Information on column troubleshooting problems may be found in the current Waters Chromatography Columns and Supplies Catalog. You can also download a copy of the HPLC Troubleshooting Guide at www.waters.com, and in the Search Box, enter WA20769. v. column cleaninG, reGenerat inG and storaGe a. Cleaning and Regenerating Changes in peak shape, peak splitting, shoulders on the peak, shifts in retention, change in resolution or increasing backpressure may indicate contamination of the column. Changing the guard column being used will often restore column performance. If not (or if no guard column is being used), follow the procedures detailed below. To prevent potential contamination from affecting detector performance, it is recommended that any detector(s) be disconnected from the effluent flow of the column during cleaning. Reversing the direction of the flow through the column (backflushing) may sometimes improve the effectiveness of any cleaning procedure. 1. Reversed-Phase Columns Flushing with a neat organic solvent, taking care not to precipitate buffers, is usually sufficient to remove most 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 of HPLC-grade solvents (e.g., 80 mL total for 4.6 x 250 mm column). Increasing mobile-phase temperature to 35-55 ˚C increases cleaning efficiency. If the column performance is poor after regenerating and cleaning, call your local Waters office for additional support. Table 4: Column Sequence or Options Polar Samples Non-polar Samples Proteinaceous Samples 1. Water 1. Isopropanol (or an appropriate isopropanol/ water mixture*) Option 1: Inject repeated aliquots of dimethyl sulfoxide (DMSO) 2. Methanol 2. Tetrahydrofuran (THF) Option 2: gradient of 10-90% B where: A= 0.1% trifluoroacetic acid (TFA) in water B= 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH3CN) 3. Tetrahydrofuran 3. Dichloromethane 4. Methanol 4. Hexane 5. Water 5. Isopropanol (followed by an appropriate isopropanol/ water mixture*) Option 3: Flush column with 7M guanidine hydrochloride or 7M urea 6. Mobile Phase 6. Mobile Phase * Use low organic solvent content to avoid precipitating buffers. 2. Normal-Phase Columns To regenerate, pump 20-30 column volumes each of dichloromethane and isopropanol through the column. Other wash solvents such as tetrahydrofurane (THF) may also be selected based on the suspected contamination. 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. Storage Completely seal the column to avoid evaporation and drying out of the bed. 1. Reversed-Phase Columns For periods longer than four days at room temperature, store the column (with the exception of cyano chemistry columns) in 100% acetonitrile at room temperature. For elevated temperature applica- tions, store immediately after use in 100% acetonitrile for the best column lifetime. Do not store columns in buffered eluents. If the mobile phase contained a buffer salt, flush the column with 10 col- umn volumes of HPLC grade water (see Table 2 for common column volumes) and replace with 100% acetonitrile for storage. Failure to perform this intermediate step could result in precipitation of the [ Care and Use ManUal ] Waters Spherisorb Columns 7 buffer salt in the column when 100% acetonitrile is introduced. Note: The exception of the storage recommendations above are cyano (CN) columns used for reversed-phase methods (CN-RP). These columns should never be stored in 100% intermediate polarity solvents (acetonitrile, methanol, IPA). Store CN-RP columns in 0.1 M ammonium acetate/acetonitrile 50/50. 2. Normal-Phase Columns For rapid equilibration upon start-up, store your normal-phase column in the mobile phase that is commonly used. Completely seal column to avoid evaporation and drying out of the bed. v i. connect inG t he column to t he hplc a. Column Connectors and System Tubing Considerations All Spherisorb column and cartridges have Parker style endfittings. Tools needed for Spherisorb analytical column: 1/2 inch wrench 9/16 inch wrench Tools needed for Spherisorb analytical cartridge column: 1/4 inch wrench 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 leading to and from the column is essential for high quality chromato- graphic results. 2. When using standard stainless steel compression screw fittings, it is important to ensure proper 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 1/2 inch wrench on the hex head of the column endfitting. Note: If one of the wrenches is improperly placed on