[ Care and Use Manual ]

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1 Amino Acid Analysis Liquid Chromatography Column I. Introduction This manual provides the user with information necessary to obtain the maximum efficiency and life from the Waters Amino Acid Analysis Column. This column has been specifically designed for the separation and analysis of free amino acids. The column is a precision device. Waters Corporation uses very exacting manufacturing procedures to achieve a small average particle size, narrow range of particle size distribution and stable particle structure in the column. These procedures create the high efficiency column-to-column reproducibility and long separation life built into each amino acid column from Waters. However, care must be taken to maintain packing density and protect it from contamination by foreign material. The following information will aid you in obtaining the highest performance and longest life from your column. II. Background Information Although there are some hydrophobic interactions between the column and certain amino acids, the primary mode of separation is that of ion-exchange. The cation-exchange resin utilized has a large number of negatively charged (sulfonate) functional groups which are covalently bound to it. These negatively charged groups, in turn, readily associate with positively charged cations when they are present in solution. Before the amino acid sample is injected onto the column, the resin is equilibrated at a low ph with buffer containing a high concentration of sodium (Na+) ions. This places the column in the sodium form. As amino acids enter the column in this acidic buffer, they are strongly positively charged and readily exchange with the loosely bound sodium ions. As the buffer gradually changes to a higher ph during the gradient run, each amino acid becomes less positively charged reflecting its pk value and differentially elutes accordingly. Contents I. Introduction II. Background Information a. Elution System 1 b. Elution System 2 c. General Preparation Guide for One Liter of Eluent d. Use of Non-aqueous Solvents e. Backpressure f. Sample Considerations g. SEP-PAK C 18 Cartridge Clean Up for Amino Acid Samples h. Preparation Scheme III. Column Care & Use Considerations a. Column Care Considerations b. Column Storage c. Column Regeneration d. Troubleshooting e. Regeneration of Protein and Lipid Contaminated Column f. Regeneration of Heavy Metal Contaminated Column g. Available Service and Applications Information h. Chromatographic Troubleshooting i. Typical Separation IV. Column Performance Checks V. Column End Fittings a. End Fitting Removal Procedure b. Column Hookup c. Storage Appendix A. Amino Acid Analysis Column Specifications

2 Figure 1: Ion Exchange Mechanism of the halide ion requires immediate flushing of the system with distilled water containing 0.2% sodium azide or 0.1% trifluoroacetic acid before system shutdown. Allowing the halide buffers to sit static in the pumps overnight will lead to corrosion. a. Elution System 1: Waters Non-halide Buffer System Buffer Composition Buffer A-ES2 Sodium Citrate Dihydrate: 19.6 g Phenol (Preservative): 1.0 g HCI: to ph 3.00 Total Volume: 1 L Buffer B-ES1 Boric Acid: 1.5 g Sodium Nitrate: 21.0 g 6M NaOH: to ph 9.60 Total Volume: 1 L NOTE: Do not add phenol to Buffer B; it will decompose at this ph. CAUTION: Introduction of improperly prepared or contaminated buffers or samples onto the resin bed could irreversibly poison the column. Heavy metal ions can also poison the column. Column degradation will be indicated by band broadening loss in resolution and/or change in selectivity. All reagents to be used for high performance liquid chromatography (HPLC) analysis of amino acids must be of the highest purity. Water should be distilled and deionized with a purity equivalent to that obtained from a Milli-Q water purifica tion system. Impure buffers will lead to artifact peaks during analysis and shorten column lifetime. There are two elution systems available for analysis: 1) Elution System 1 is the Waters Non-halide Buffer System. It is recommended for use where possible since it employs no potentially corrosive halide ions. b. ELUTION SYSTEM 2: Halide Buffer System Buffer Composition Buffer Composition Buffer A-ES2 Sodium Citrate Dihydrate: 19.6 g Phenol (Preservative): 1.0 g HCI: to ph 3.00 Total Volume: 1 L Buffer B-ES2 Boric Acid: 1.5 g Sodium Chloride: g 6M NaOH: to ph 9.80 Total Volume: 1 L 2) Elution System 2 is a halide-containing buffer system and is recommended when maximum sensitivity is needed. Use of chloride enhances orthopthalaldehyde (OPA) fluorescence by approximately twofold as compared to the non halide elution system. The presence 2

3 c. General Preparation Guide for One Liter of Eluent Dissolve salts in approximately 900 ml of purified water. Adjust ph with appropriate acid or base. Adjust to final one liter volume with water. Filter through the Solvent Clarification Kit using an 0.45 µm aqueouscompatible filter. Transfer buffer to an amber glass bottle and purge for 15 minutes with pure nitrogen. Transfer buffer to analyzer. Buffers prepared in this manner will be stable for at least one week at room temperature. d. Use of Non-aqueous Solvents For additional resolution of the early-eluting amino acids, small amounts of methanol, ethanol, or propanol can be added to Buffer A. This alcohol concentration should not exceed 10% High concentrations of a non-aqueous modifier will cause the resin to swell resulting in high backpressure. e. Backpressure With a new column flowing at 0.4 ml/min in Buffer A at 62 C. typical system pressures will range from 800 psi psi. As the column ages, pressure will slowly increase, although this increase is not usually associated with decreased efficiency. The most common causes of high backpressure are: particulates in the sample or buffer, proteinaceous material coating the resin packing, mold formation in the buffers or in the column. Reduced temperature, higher flow rate and high solvent viscosity will all yield higher pressure. To prevent excessive pressure, do not flow through the column with the temperature control module off. f. Sample Considerations Acidic protein hydrolyzates should be lyophilized and reconstituted in Buffer A. Convenient sample concentration range: nmol amino acid/5 µl - 50 µl injection volume. Sample volume and sensitivity requirements should be optimized for individual needs. Samples that contain protein, lipid or heavy metals (e.g., tissue extracts, food samples, feed samples, etc.) should not be introduced directly on the column. Pre-purification on a strong exchange resin or a Sep-Pak C 18 cartridge is recommended. g. Sep-Pak C 18 Cartridge Cleanup for Amino Acid Samples Prepare the following solutions: Solution 1: 0.1 % trifluoroacetic acid (TFA) in water Solution 2: 0.1 % TFA in H20:MeOH, 80:20 Solution 3: 0.1 % TFA in H2O:MeOH, 70:30 h. Preparation Scheme 1. Activate a new Sep-Pak C 18 cartridge with two 10 ml volumes of methanol. 2. Wash with two 10 ml volumes of Solution Wash with 10 ml of Solution Mix 1 ml sample with 2 ml Solution 3. Sample should be water or acid solubilized and free of particulate matter. 5. Pass the sample through the Sep-Pak C 18 cartridge. 6. Discard the first 1 ml of eluent and collect the next 2 ml. This fraction will contain all amino acids. Lipids and high molecular weight pro teins will be retained on the Sep-Pak C 18 cartridge. 3

4 III. Column Care & Use Considerations a. Column Care Considerations ALWAYS filter samples to prevent excessive pressure buildup due to particulate matter. Use a Sample Clarification Kit (Part No ) and, if necessary, centrifugation. For more details on the Waters Sample Clarification Kit call or write for Bulletin D97. ALWAYS filter buffers to remove microparticulate matter. This eliminates high pressures from plugged frits and also helps ensure long column life. Use a Solvent Clarification Kit (Part No ) to filter solvents. Vacuum filtration or sonification may be used to remove dissolved gasses which could affect your solvent delivery system. For more details on the Waters Solvent Clarification Kit call or write for Bulletin D97. ALWAYS use high purity solvents such as Waters solvents. For analytical work choose high quality Waters solvents. For more details on Waters solvents call or write for Bulletin M10. ALWAYS replace plugs in column fittings to prevent the column from drying out when not in use. b. Column Storage The column may be stored 1-2 days in Buffer A without flow. The presence of phenol in the buffer will prevent bacterial growth. For longer storage periods, 0.2 N NaOH is strongly recommended. c. Column Regeneration For protein hydrolyzate samples, regenerate the column at least once a week as follows: d. Troubleshooting This section discusses some possible problems and their solutions (refer to Table 1.) e. Regeneration of Protein and Lipid Contaminated Column Large proteins and some lipids can be removed following alkaline hydrolysis. Reverse direction of buffer flow through column. Elevate column heater temperature to C and pump 0.2 N NaOH solution through column for at least 2 hours. Particularly difficult contamination may require overnight treatment. Restore normal flow direction and temperature; reequilibrate column with Buffer A and test column with amino acid standard solution. If problem still exists, repeat the above treatment. Table 1: Typical Column Problems and Solutions Problem Cause Solution Excess pressure buildup Loss of resolution, broad peaks, low plate counts Column frits plugged with particulates or mold is growing in column or in buffers Sample and/or buffer precipitates on column Contaminated column Reverse flow on column. If this does not work, follow procedure for protein removal. If pressure is still high, carefully remove column inlet fitting being sure not to disturb resin. Clean in ultrasonic bath. Always filter solvents and samples. Follow procedure above. Follow procedures for metal and protein removal. Eluent: 0.2 N NaOH Flow: 0.5 ml/min Temperature: 62 C Flush Time: 30 min Crude samples may require more frequent regeneration. 4

5 f. Regeneration of Heavy Metal Contaminated Column Often metals can be removed by chelation under basic and acidic conditions. Reverse direction of buffer flow through column. Elevate column heater temperature to C and pump 0.2 N NaOH solution containing 0.25 g EDTA/liter through the column for minimum of 4 hours. Switch buffer tube to second container and pump 2 N HNO, solution containing 0.25 g EDTA/liter through the column for another 2-4 hours. The more advanced Waters HPLC System allows for the generation of a continuous gradient using only two buffers. This is equivalent to an infinite step gradient. Superior resolution is achieved without baseline disturbances. The constant ionic strength, ph gradient (0.2N Na +, ph 3.00 to ph 9.60) allows for rapid reequilibration without NaOH regeneration. i. Typical Separation Figure 2: Amino Acid Analysis Continuous Gradient Restore normal flow direction and temperature. Reequilibrate column with Buffer A and test column with amino acid standard solution; If problem still exists, repeat the above basic/acidic EDTA treatment. CAUTION: Never attempt to clean the column using hydrochloric acid (HCI) or sulphuric acid (H2SO,) solutions. Such acids are harmful to stainless steel. g. Available Service and Applications Information The Waters Corporation staff of trained and e perienced service specialists provides maintenance for Waters instruments on preventive and/or corrective levels. Contact Waters Corporation at or your local Waters representative for further details. For answers to specific chromatography questions in areas such as methods development, applications and quality control, contact your local Waters representative or Waters home office applications laboratory. As well as the standard hydrolyzate amino acids, unusual amino acids can be separated by the two-buffer, gradient system. Note cysteic acid, hydroxyproline, methionine sulfoxide, hydroxylysine, and tryptophan. Slight modifications of the ph and/or constituency of the A or B buffers can quickly be used to optimize new separations. h. Chromatographic Troubleshooting When a new column is installed, it is recommended that a separation of standard hydrolyzate amino acids be performed using Elution System 1. A typical separation is shown on the following page (Figures 3 & 4). 5

6 Figure 3: Amino Acid Analysis Special Amino Acids IV. Column Performance Checks 1. Cysteic Acid 2. Methionine Sulfoxide 3. Hydroxy Proline 4. Aspartic Acid 5. Threonine 6. Serine 7. Glutamic Acid 8. Proline 9. Glycine 10. Alanine 11. Cystine 12. Valine 13. Methionine 14. Isoleucine 15. Leucine 16. Tyrosine 17. Phenylalanine 18. Histidine 19. Hydroxy Lysine 20. Tryptophan 21. Lysine 22. Ammonia 23. Arginine Column: CAT EX Resin Column (0.4 cm x 25 cm) Sample: 1 NMOL AA STDS Injection Volume: 10 µl Flow Rate: 0.4 ml/min Mobile Phase: A. 0.2 N Na citrate, ph 3.0 B. 0.2 N Na borate, ph % B, curve 6, 48 min Detector: Model 420 ex: 338 nm em: 425 nm gain: 4 Table 2 provides performance check information. Use the table in conjunction with the following procedure to determine the condition of your column. If you feel it has become contaminated, refer to Troubleshooting section for appropriate column cleaning procedures. Table 2: Performance Check for Columns Column: Buffer: Flow rate: Waters Amino Acid Analysis Column (Part No ) A-ES1 Temperature: 62 C Chart Speed: Detection: 0.4 ml/min 0.25 cm/min Fluorescence (o-phthalaldehyde) Sample: Sample Amount: Results: Amino acid mixture contain ing equalmolar concentra tion aspartic acid, threonine, serine, and glutamic acid 1-2 n mole of each amino acid in 5-20 µl volume Chromatogram should show a minimum resolution of 85% for all amino acids as determined by the following equation: Figure 4: Resolution Variations in results as compared to the expected chromatogram can usually be explained by improper buffer preparation (weighing/volume error) or improper ph. The following guidelines describe the effect of ph: 1. If Buffer A is 0.20 ph units too high, there will be a loss of THR-SER resolution and OH-PRO will coelute with ASP. 2. If Buffer A is 0.30 ph units too low, there will be broadening of the neutral amino acid peaks and ASP will elute later than expected. 3. If Buffer B is 0.20 ph units too high, ARG will coelute with NH. If Buffer B is 0.20 units too low, ARG will elute minutes later than expected. Increased column temperature will lead to a decrease in the elution volume for all amino acids and a decrease in the THR-SER resolution. 6

7 All peaks should be sharp and symmetrical. Backpressure should be less than 2,000 psi. If you suspect that the quality of your column has decreased, the above test is a rapid method of determining the efficiency of your separation. Peaks which are not symmetrical could in dicate partially plugged frits are at fault or foreign material has accumulated on the resin packing. Generally this phenomenon is also ac companied by increased backpressure (refer to Table 2 for corrective action). Alternatively, resin packing material may have shifted, voided or become uneven. Careful removal of top column packing nut and visual inspection of the resin bed is the best diagnostic procedure to determine whether this is the cause. Since removal of the nut could, itself, disturb the packing, this should be a last resort measure. If resin bed is not smooth and completely full, please contact your nearest Waters representative for instructions. If backpressure is normal but resolution is poor or peaks are very broad to nonexistent, the problem could be heavy metal contamination of the resin (refer to Table 1 for corrective action). It should always be kept in mind that column efficiency is affected by the liquid chromatography (LC) system as well as buffers and reagents. Care should be taken to make sure the LC system is working normally, that tubing and post-column reaction coil(s) are properly connected and leak free, and that buffers and reagents are fresh, filtered and tree from contamination. Whenever problems persist after making the above tests and checks on your system, con tact your local Waters representative for further assistance. V. Column End Fittings The column is symmetrical, having the same kind of fittings and frits at both ends. Flow of buffer can occur in either direction without harmful effects. However, the end bearing and column label should be considered the inlet since this was the direction of flow during packing and testing procedures. Use of the same flow direction for analysis is encouraged, and it will prove helpful during any necessary cleaning procedures. Both end fittings are made up of a smaller and a larger hex nut. The larger hex nuts contain a non-removable stainless steel frit to both retain the resin packing and to catch particulates before they reach the resin. In normal operation particulates from the sample or buffer should be filtered out using a 0.45 µm membrane filter (refer to Section III d.). If the backpressure of the column should rise above 2500 psi, reversing the direction of flow on the column for a few minutes will usually flush particulates away. If flushing and cleaning procedures (see Troubleshooting, Section III d.) do not work, the frits may be cleaned ultrasonically following their removal from the column. a. End Fitting Removal Procedure CAUTION: Removal of end fittings may disturb the resin packing material and cause skewed or broad peaks. Therefore, this procedure should be used only as a last resort measure. 1. Work only in a clean area where dust, dirt and debris will not contaminate the exposed packing material. 2. Secure column in a vice. Hold in an upright position by clamping lightly (do not over tighten) onto the smaller hex fitting. 3. Remove only one fitting at a time to avoid loss of packing material. Always begin by removing column inlet (end with column label) first. 4. Use 5/8 inch (16 mm) wrench, turning counterclockwise slowly until fitting is removed. 5. With a clean spatula or similar tool, remove any resin packing which has adhered to the frit and place smoothly and carefully back on to resin bed at end of column tube. 6. Fritted fitting may now be cleaned in ultrasonic cleaner using appropriate solvent. 6 N nitric acid (HNO,) often works well but be very cautious to avoid contact with skin. 7. Column fittings should be reassembled as soon as possible after inspection and cleaning to avoid drying and contamination of resin. 7

8 b. Column Hookup 1. Direction of flow is indicated on the column. 2. Connect one adaptor to the inlet and one adaptor to the outlet of the column. 3. Connect the tubing from the injector to the column inlet union. Be sure to use inch (0.23 mm) i.d. tubing for this purpose. 4. Connect the outlet adaptor to Mixing Tee #1 of the post column reaction system. c. Storage If you do not intend to use this column for a period of a week or longer, it is best to store it in 0.2 N sodium hydroxide (NaOH). Using your HPLC, pump the filtered NaOH solution through the column at 0.4 ml/min for at least 30 min. To reduce solution viscosity, column heater should be on at normal analysis temperature (60-70 C). At the end of this period turn off pump and disconnect inlet and outlet tubing. Replace the plug fittings on the column, screwing them in tightly to prevent evaporation and drying out of the resin. Appendix A: Amino Acid Analysis Column Specifications Column Size Length: 25 cm Diameter: 0.46 cm i.d. Packing Material Styrene - divinyl benzene copolymer with sulfonic acid functionality in Na+ form Particle Size 9 ± 0.5 Nm in diameter Separation Method Strong Cation Exchange ph Range 1-14 Pressure Limit 3000 psi Shipping Solvent 0.2 M NaOH (Na + form) The HPLC should now be flushed out with filtered water before it is stored or used for any other pur pose. A stainless steel jumper tube which takes the place of the column will prove useful for this purpose. 8

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