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[ Care and Use Manual ] Pico•Tag Column for Free Amino Acids I. Introduction Contents Waters Pico•Tag® Column for Free Amino Acids uses the exclusive bonding I.introduction and pack-ing processes developed by Waters for the original Pico•Tag column. This has resulted in a durable, high efficiency, 3.9 mm x 30 cm column ideally II.installation suited for the reversed-phase separation of phenylthiocarbamyl amino acids. a. Solvent Requirements Our stringent quality control procedures assure that this column, when used b. Sample and Solvent Preparation in conjunction with Waters Pico•Tag reagents, eluents, and HPLC system, c. Column and Cartridge Installation will provide the speed, resolution, and sensitivity needed for amino acid d. Equilibration analysis. III.Operating tips Use these procedures to ensure that you obtain quality results from your Pico•Tag column and take full advantage of the features it offers. IV. column efficiency WARNING: The ampules of phenylisothiocyanate which are contained in the a. Column Testing b. Troubleshooting Pico•Tag chemical package react on contact with strong acids, emitting highly toxic cyanide fumes and/or oxides of sulphur. Care must be taken to V. column storage assure that these ampules and contents are disposed of properly and that they do not come into contact with acids. vi. warranty/service information vii.ordering information The Pico•Tag Column for Free Amino Acids 1 [ Care and Use Manual ] II.installation Figure 1: Separations Method Using Pico•Tag Column For Free Amino Acids a. Solvent Requirements Abbreviation Component Name Retention Time Your Pico•Tag column is shipped containing an aqueous/organic mixture com- PSER Phosphoserine 3.05 patible with the Pico•Tag eluent. For best results use Pico•Tag Eluent 1 (P/N ASP Aspartic Acid 3.50 10960) and Waters Pico•Tag Eluent 2 (P/N 10965) with this column. GLU* Glutamic Acid 4.01 AAD α-Aminoadipic Acid 5.62 HYPRO Hydroxyproline 7.12 PEA Phosphoethanolamine 7.53 0.45 µm. Pico•Tag prefiltered eluents eliminate the need for this step SER* Serine 9.20 by providing a prepared, ready-to-use solvent to reduce the problem of ASN* Asparagine 9.50 plugged filters and preserve column life. GLY* Glycine 10.30 _-ALA β Aline 12.62 TAU Taurine 14.82 HIS* Histidine 17.00 GABA γ-Aminobutyric Acid 18.00 CIT Citrulline 18.62 NH2 Ammonia 19.62 THR Threonine 20.35 ALA Alanine 21.52 b. Sample and Solvent Preparation and Filtration • • Use LC grade solvents, filtered to remove microparticulate matter above Vacuum filtration or sonification may be used to remove dissolved gasses which could affect your solvent delivery system. • Waters Sample Clarification Kit, (P/N 26865) is recommended to filter prepared samples and prevent excessive pressure buildup. • Use of a Waters In-line Precolumn Filter (P/N 84560),is recommended. • Pico•Tag Diluent, (P/N 88119), is recommended for reconstitution of derivatized samples. BAIB B-Aminoisobutyric Acid 23.65 c. Column and Cartridge Installation CARN Carnosine 24.72 Remove the end plugs from your column with a 5/16” wrench. (Be sure ARG* Arginine 26.02 to replace the end plugs when the column is removed from the system for METSO2 Methionine Sulfone 27.00 storage.) The column outlet is indicated by an arrow on the label show- PRO Proline 30.15 1MH 1-Methyl Histidine 31.32 ing the direction solvent should flow. Tighten the fittings to turn. DO NOT 3MH 3-Methyl Histidine 32.25 AAB A-Aminobutyric Acid 37.99 TYR* Tyrosine 44.89 Follow the next three steps of this procedure if you must cut tubing to con- VAL* Valine 47.62 nect a new steel column or to improve the end connections on your existing MAT* Methionine 49.22 fittings. CYST Cystathionine 51.35 RG1 Reagent 1 52.90 CYS2* Cystine 52.99 RG2 Reagent 2 54.40 2. Grasp the tubing on both sides of the scribe mark with cloth-covered ILE Isoleucine 54.95 pliers (to prevent marring the tube surface) and gently work the tube LEU* Leucine 55.65 back and forth until it separates. HYLYS 1 Hydroxylsine 1 58.15 HYLYS 2 Hydroxylsine 2 58.90 PHE* Phenylanlanine 60.02 TRP* Tyrptophan 60.32 ORN Orninthine 60.77 LYS* Lysine 65.52 The Pico•Tag Column for Free Amino Acids OVERTIGHTEN - THIS WILL DAMAGE THE FITTING SEAT. A properly prepared and assembled compression fitting in good condition is all that is required. 1. Using a three-cornered file with a cutting edge scribe the circumference of the tubing at the desired break. 2 [ Care and Use Manual ] IV. column efficiency 3. Slide the compression fitting, followed by the ferrule (large end of the taper first) over the tube. Be cer-tain to bottom the tube in the fitting Liquid chromatography columns have a finite life which is directly related seat to assure a leak-free connection. to the care and use they receive. Column life is influenced by the number of Note: Attach a union in place of the column and flush the lines free of previous injections, sample and solvent cleanliness, frequency of solvent changeover, solvents before attaching the column. and handling and storage procedures. Figure 2: Ferrule and Compression Assembly If a change is observed in the: • Retention of a particular compound • Resolution between two compounds • Peak shape Take immediate steps to determine the reason for the changes. Until the cause of the change is determined, the results of any separation using the column d. Equilibration must not be relied upon. A necessary step to successful use of your column is the initial solvation (or wetting) of the packing. Purge the column in a column heater and bring Follow generally accepted procedures for quality control and methods devel- the temperature to 46 °C. Purge the column with 30 mls of Eluent 2, then opment when using these columns. equilibrate with Eluent 1. Equilibration between the mobile phase and pack- Note: Before running the first analysis on your new column perform the test ing is established when a stable baseline can be produced. If your result is sample separation given in the test conditions section. unsatisfactory repeat the equilibration process. a. Column Testing The w5 sigma method shown in Figure 3 is used to measure column efficiency. III.Operating tips • Normal recommended pressure should not exceed 3500 psi. • For all silica based packing materials, stay within a pH range of 2-8 Unlike the tangent method used to determine system efficiency, this stringent method considers naturally occurring peak asymmetry. Figure 3. 5 Sigma Test Method (avoid using concentrated acids or bases). Maximum column life (pH) 3.5 - 6.5. • Filter all aqueous buffers (Pico•Tag eluents are prefiltered). Avoid using turbid or cloudy buffers. Be sure that any solutions containing buffers, salts, etc. are compatible with the wetted surfaces of the column and equipment. • Columns are thoroughly tested in our quality control laboratories for adher- Protect column from vibration, mechanical shock and rapid changes in ence to our specifications. Since slight variations in your results will occur pressure. Column packings are based on a highly porous and delicate depending on the equipment used, test sample makeup and equipment set- silica gel alignment. Any thermal, physical or chemical shock (such as tings and conditions, perform the test sample run given here for your new changing solvents rapidly or at high flow rates) can cause the particles column and record the results (retention time and the settings used) before to shift and may result in a loss of efficiency. • attempting the first analysis. Use these results for comparison throughout the When using water, distill or treat with a Milli-Q or equivalent system. life of your column. De-ionized water is not acceptable because it contains organic com- Note: Be sure to record results and instrument settings (and configurations) pounds which alter column selectivity. • to allow exact reproduction and comparison in the future. Protect the column from rapid changes in solvent composition. DO NOT change the flow rate faster than 0.5 ml/min increments. The Pico•Tag Column for Free Amino Acids 3 [ Care and Use Manual ] Test Sample: Acenaphthene (0.05% in Acetonitrile) vi. warranty/service information Solvent: Pico•Tag Eluent B (P/N 88112)* a. Service and Applications Assistance Flow Rate: 1.0 ml/min Chart Speed: 10 cm/min Injection Volume: 8 µl Waters’ staff of experienced service specialists provide maintenance assistance on both preventative and/or corrective levels. For complete information and assistance, please call Waters Service Department at 1-800-252-HPLC. For solutions to particular applications questions Waters team of technical support personnel are available to help you with specialized support. They may Detection:254 nm @ 0.2 AUFS be contacted at 1-800-252-HPLC in Milford. *or equivalent made by mixing 600 ml of HPLC grade acetonitrile with 400 b. Warranty ml Milli-Q° water. Warrants its high performance liquid chromatography columns in accordance b. Troubleshooting with the following terms and conditions: Table 1: Typical Column Problems and Solutions Problem Excess pressure buildup Cause • Filters plugged with Particulates • Sample precipitates on column (sample not soluble in mobile phase) Loss of resolution, • Filters partially broad peaks, low plugged plate counts • Contaminated colum, insufficent equilibration • Column collapse and void formation Waters will repack or replace (at our discretion) without cost any steel col- Solution • Clean in an ultrasonic bath or replace. • Always alter mobile phases and samples. • Slowly purge with a strong mobile phase that is both appropriate to dissolve the contaminate and compatible with the column. • Replace or clean inlet and outlet filters in an ultrasonic bath. umn that fails to perform satisfactorily if notified within 90 days from your receipt. Any column that is returned must have a Return Authorization Number granted by the Waters Customer Ser-vice Department. Approval is subject to the following exclusions: • Physical damage to the column because of misuse or abuse. • Chemical damage to the packing material because of use with incompatible solvents or buffers, or at an incorrect pH. • rect temperatures or pressures. • or sample filtration practices. Leaving the column unused for less than 72 hours does not generally require vii.ordering information storage procedures. Follow the procedures below to store column for periods of more than 72 hours. • Item Part Number DO NOT store the column in water alone; this practice will promote Phenylisothiocyanate (PITC) 88120 microbial contamination. Store the column in Pico•Tag Eluent 2. Triethylamine (TEA) 88121 Physiological Standard A/N 10948 Physiological Standard B 10949 Pico•Tag Eluent 1 10960 Pico•Tag Eluent 2 10965 Sample Clarification Kit 26865 In-Line Precolumn Filter Kit 84560 Pico•Tag Diluent 88119 DO NOT allow buffers or other potentially harmful materials to remain in the system when not being used. Flush and replace with Pico•Tag Eluent 2 or 10% organic in water mixture. • Particulate buildup or precipitation in the column or end fittings causing high internal pressure which has occurred because of improper solvent V. column storage • Physical damage to the packing material because of operation at incor- Return the column to its box with the end plugs firmly in place for storage. Allowing steel columns to dry out can result in poor chromatography performance. The Pico•Tag Column for Free Amino Acids 4 [ Care and Use Manual ] Sales Offices: Austria and European Export (Central South Eastern Europe, CIS and Middle East) 431 877 18 07 Australia 2 9933 1777 Belgium 32 2 726 1000 Brazil 55 11 5094 3788 Canada 800 252 4752 China 8621 6495 6999 CIS/Russia +7 495 3367000 Czech Republic 42 02 617 11384 Denmark 45 46 59 8080 Finland +358 9 5659 6288 France (33) 1 30 48 72 00 Germany 49 6196 400600 Hong Kong 852 29 64 1800 Hungary 36 1 350 5086 India and India Subcontinent 91 80 2 837 1900 Ireland 353 1 448 1500 Italy 39 02 274 211 Japan (81) 3 3471 7191 Korea (82) 2 820 2700 Mexico 5255 5200 1860 The Netherlands +31 (0)76-50 87 200 Norway 47 63 84 60 50 Poland (48) 22 833 4400 Puerto Rico 787 747 8445 Singapore 65 6273 1221 Spain 34 93 600 93 00 Sweden 46 8 555 11500 Switzerland 41 62 889 2030 Taiwan 886 2 2543 1898 United Kingdom 44 208 238 6100 ©2007 Waters Corporation. Waters, The Science of What’s Possible, and PicoTag are trademarks of Waters Corporation. MilliQ is a trademark of Millipore Corporation. November 2007 WAT010954 Rev 4 VW-PDF The Pico•Tag Column for Free Amino Acids 5 Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com