Lit42D.qxd 11/29/2005 2:23 PM Page A Guidelines for Use and Care of Aminex Resin-Based Columns ® Instruction Manual For technical service call your local Bio-Rad office or in the U.S.
Lit42D.qxd 11/29/2005 2:23 PM Page 1 Table of Contents Section 1 Introduction to Resin-Based HPLC Columns ....1 Section 2 Column Set-Up ................................................2 2.1 Unpacking ..................................................................2 2.2 Preparing the Eluant ....................................................2 Section 3 Guard Columns ................................................2 3.1 Installing Guard Columns ............................................3 3.
Lit42D.qxd 11/29/2005 2:23 PM Page 1 Section 1 Introduction to Resin-Based HPLC Columns Resin-based HPLC columns use the mechanisms of ion exclusion, ion exchange, ligand exchange, size exclusion, reversed phase, and normal phase partitioning. These multiple modes of interaction offer a unique ability to separate compounds. The charge on the resin provides the capability for ion exclusion, while the polystyrene backbone allows hydrophobic interaction to take place.
Lit42D.qxd 11/29/2005 2:23 PM Page 2 efficiency. This guide for the care of resin-based columns will help to provide higher resolution, longer column life, and better reproducibility. Section 2 Column Set-Up 2.1 Unpacking While unpacking the column, check it carefully for evidence of shipping damage, rough handling, or leaking solvent. Save the shipping container to store the column.
Lit42D.qxd 11/29/2005 2:23 PM Page 3 Interferences caused by anions, cations, organics, salts, insolubles, and particulates can be reduced or eliminated using Micro-Guard cartridges. Micro-Guard cartridges are strongly recommended for use with Bio-Rad’s HPLC columns. The Micro-Guard HPLC column protection system consists of a disposable guard cartridge in a standard guard cartridge holder, or an anion and a cation cartridge in a double deashing holder.
Lit42D.qxd 11/29/2005 2:23 PM Page 4 Section 4 Connecting the Column Reduce the flow rate to 0.2 ml/min. Remove the end screws from the analytical column and attach the outlet end of the guard column tubing to the inlet of the analytical column. Connect the analytical column with the pump running at a slow flow rate, to exclude any air from the column inlet. Pass approximately 20 ml of degassed solvent through the column.
Lit42D.qxd 11/29/2005 2:23 PM Page 5 .300” .08” Valco .210” .09” Parker Fig. 1. Bio-Rad’s resin-based columns are manufactured using Parker end-fittings. Reverse nuts and ferrules are supplied with the columns and must be used to attach the column to the HPLC system if the system is not already equipped with Parker 1/16” reverse nuts or the equivalent.
Lit42D.qxd 11/29/2005 2:23 PM Page 6 5.2 Sample Preparation Some of the sample components may not be soluble in some solvents. To prevent any problems of this sort, always dissolve the sample in the mobile phase. Filter the sample solution through a 0.45 µm filter to remove particulates. 5.3 Pressure Checks The HPLC pump pressure limit device should be adjusted so that a pressure increase (10–20% above the standard operating pressure) will cause the pumps to turn off.
Lit42D.qxd 11/29/2005 2:23 PM Page 7 compounds adsorbing to the column matrix over a period of many injections. Occasionally, back pressure increases are caused by clogged frits. In any case, following the cleaning procedures and/or reversing the column flow may help to reduce the back pressure. If the guard system is in place and if the cartridge is changed according to directions, fewer contaminants adsorb to the matrix and higher column back pressures are delayed over the life of the column.
Lit42D.qxd 11/29/2005 2:23 PM Page 8 Bio-Rad’s columns are tailored for specific applications by optimizing several parameters, including resin ionic form and column configuration. The following tables compare the various resin-based analysis columns and provide operating guidelines. Table 1 provides column specifications and typical operating parameters. Table 2 provides data which can be useful for resin modification, as well as cleaning, regeneration, and storage information. Table 1.
Lit42D.qxd 11/29/2005 2:23 PM Page 9 Table 1. Specifications and Operating Guidelines for Aminex Columns (continued) Fast Carbohydrate Analysis Aminex HPX-42A Column, 100 x 7.8 mm Column, 300 x 7.8 mm Aminex HPX-87H Column, 300 x 7.8 mm Fast Acid Analysis Column, 100 x 7.
Lit42D.qxd 11/29/2005 2:23 PM Page 10 Table 2. Cleaning, Regeneration, and Storage Guidelines Aminex HPX-87C Column, 300 x 7.8 mm Aminex HPX-42C Column, 300 x 7.8 mm Aminex HPX-87P Column, 300 x 7.
Lit42D.qxd 11/29/2005 2:23 PM Page 11 Table 2. Cleaning, Regeneration, and Storage Guidelines (continued) Fast Carbohydrate Analysis Column, 100 x 7.8 mm Aminex HPX-42A Column, 300 x 7.8 mm Aminex HPX-87H Column, 300 x 7.8 mm Fast Acid Analysis Column, 100 x 7.
Lit42D.qxd 11/29/2005 2:23 PM Page 12 Table 2. Cleaning, Regeneration, and Storage Guidelines (continued) Fermentation Monitoring Column, 300 x 7.8 mm Aminex HPX-72-O Column, 300 x 7.8 mm Aminex HPX-72S Column, 300 x 7.
Lit42D.qxd 11/29/2005 2:23 PM Page 13 Table 2. Cleaning, Regeneration, and Storage Guidelines (continued) Aminex HPX-87N Column, 300 x 7.8 mm Aminex HPX-87K Column, 300 x 7.8 mm Aminex HPX-87C Column, 250 x 4 mm Glyphosate Analysis Columns, 100-300 x 7.
Lit42D.qxd 11/29/2005 2:23 PM Page 14 Section 6 Regeneration Procedures 6.1 Fluffing the Resin Bed Ion exchange resins are resilient. Gentle backwashing can fluff a collapsed bed back to its original configuration or allow entrained air spaces to redissolve. If bed compression is severe, the column may not return to original performance. 1. Turn off the pump and let the column bed relax for about 15 min. 2. Reverse the flow direction and backwash the column at 0.
Lit42D.qxd 11/29/2005 2:23 PM Page 15 To avoid contaminating the detector flow cell when cleaning or regenerating the column, disconnect the tubing between the column and the detector. Allow the appropriate solvent to pass directly to waste. 6.3 Reconverting a Column to its Original Ionic Form Ionic exchange resins shrink and swell with changes in ionic form.
Lit42D.qxd 11/29/2005 2:23 PM Page 16 6.5 Rectifying Instrument/Connecting Tubing Problems Many instances of poor resolution are not the result of column problems, but rather the result of hardware problems elsewhere in the system. The following list can help to point out troublesome areas and provide ideal specifications. 1. Injector Sample volume Loop volume 100 µl maximum 500 µl maximum 2. Connecting Tubing Fittings not deteriorated Tube end cut square Tube bottoms in fitting Tube ID 0.
Lit42D.qxd 11/29/2005 2:23 PM Page 17 Section 8 Column Storage Always be certain to exclude air when closing column end fittings. For prolonged storage, the columns may be refrigerated to prevent drying out. However, they should never be frozen. If the column will not be used for several days, it should be stored according to one of these procedures. 8.1 Long Term Storage 1. Replace the mobile phase with the appropriate storage/shipping solvent as shown in Table 2.
Lit42D.qxd 11/29/2005 2:23 PM Page 18 Table 3. Troubleshooting Guide Problem Cause Characteristics High back pressure 1. Bed collapse from excessive flow rate Sudden catastrophic pressure increase at flow above recommended maximum 2. Chemical contamination Gradual increase in pressure during use 3. Trapped air Column returned to use after storage 4. Microbial contamination Column returned to use after storage 5. Change in ionic form Rapid pressure increase after buffer change 6.
Lit42D.qxd 11/29/2005 2:23 PM Page 19 Table 3. Troubleshooting Guide (continued) Regeneration Procedure Solution 1. Turn off pump. Allow 15 min relaxation, then operate at suitable flow rate 6.1 2. Reverse flow direction and backwash 6.2 3. 6.1 — 4. Reverse flow direction and backwash 6.2 5. — 6.3 6. — 6.1 1. — 6.2 2. — 6.3 3. Shut off pump. Allow 15 min relaxation before operating at suitable flow. 6.1, 6.4 4. — 6.1, 6.4 5. — 6.2 6. Check all instrument fittings 6.
Lit42D.qxd 11/29/2005 2:23 PM Page 20 Bio-Rad Laboratories, Inc. Life Science Group Web site www.bio-rad.
