for IonPac AG11-HC IonPac AS11-HC

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1 for IonPac AG-HC IonPac AS-HC

2 IonPac AS-HC Manual Document No Page of 45 PRODUCT MANUAL for the IONPAC AG-HC GUARD COLUMN (4 x 5 mm, P/N 59) ( x 5 mm, P/N 593) IONPAC AS-HC ANALYTICAL COLUMN (4 x 5 mm, P/N 59) ( x 5 mm, P/N 59) Dionex Corporation, Revision 7 8 August 8

3 IonPac AS-HC Manual Document No Page of 45 TABLE OF CONTENTS SECTION - INTRODUCTION... 5 SECTION - COMPARISON OF ION CHROMATOGRAPHY SYSTEMS... 7 SECTION 3 - INSTALLATION System Void Volume System Requirements System Requirements for -mm Operation System Requirements for 4-mm Operation Installing the CR-ATC Trap Column for Use with EGC II KOH Cartridge The Sample Concentrator The Injection Loop The -mm System Injection Loop, - 5 μl The 4-mm System Injection Loop, - 5 μl The IonPac AG-HC Guard Column Eluent Storage Anion Self-Regenerating Suppressor Requirements Anion MicroMembrane Suppressor Requirements Using Displacement Chemical Regeneration (DCR) with the Chemical Suppression Mode Using AutoRegen with the ASRS ULTRA II or the AMMS III in the Chemical Suppression Mode Detector Requirements Using the EG4 or EG5 with AS-HC... SECTION 4 - OPERATION General Operating Conditions IonPac AS-HC Operation Precautions Chemical Purity Requirements Inorganic Chemicals Deionized Water Making Eluents that Contain Solvents Solvents... 4

4 IonPac AS-HC Manual Document No Page 3 of Regenerant Preparation for the AMMS III Sample Concentration... 5 SECTION 5 - EXAMPLE APPLICATIONS Recommendations for Optimum System Performance Eluent Preparation Production Test Chromatograms Test Chromatograms at Room Temperature Gradient Elution of a Large Number of Inorganic Anions and Organic Acid Anions Using NaOH Gradient Gradient Elution of a Large Number of Inorganic Anions and Organic Acid Anions Using a NaOH Gradient without Solvent or with Solvent Method Optimization for Food and Beverage Applications Using NaOH gradient with a Solvent Step Change or a Solvent Gradient Analysis of a Beer Sample Analysis of Apple Juice Samples Analysis of Orange Juice Samples Analysis of a Wine Sample Gradient Elution of Organic Acid Anions, Common Inorganic Anions, and Hydrophobic Inorganic Anions Gradient Analysis using Large Loop Injection Clean-up After Humic Acid Samples SECTION - TROUBLESHOOTING GUIDE High Back Pressure Finding the Source of High System Pressure Replacing Column Bed Support Assemblies High Background Preparation of Eluents A Contaminated Trap Column, CR-ATC, ATC-HC, or ATC A Contaminated Guard or Analytical Column Contaminated Hardware A Contaminated ASRS ULTRA II or AMMS III Suppressor Poor Peak Resolution Loss of Column Efficiency Poor Resolution Due to Shortened Retention Times Loss of Front End Resolution Spurious Peaks... 4

5 IonPac AS-HC Manual Document No Page 4 of 45 APPENDIX A - QUALITY ASSURANCE REPORT... 4 A. IonPac AS-HC 4-mm QAR... 4 A. IonPac AS-HC -mm QAR APPENDIX B - COLUMN CARE B. Recommended Operation Pressures B. Column Start-Up B.3 Column Storage B.4 Column Cleanup B.4. Choosing the Appropriate Cleanup Solution B.4. Column Cleanup Procedure... 45

6 IonPac AS-HC Manual Document No Page 5 of 45 SECTION - INTRODUCTION The lonpac AS-HC -mm (P/N 59) and 4-mm (P/N 59) Analytical Columns are specifically designed to resolve a large number of inorganic anions and organic acid anions from a single sample injection in one gradient run using hydroxide eluent systems. The AS-HC is a high capacity column with selectivity similar to the AS. The high capacity allows injection of more concentrated samples without overloading and peak broadening. Hydroxide is normally used for gradient elution to minimize background shift. Because of high background conductance, sodium carbonate/bicarbonate eluents are not appropriate for gradient analysis but can be used for isocratic applications. By using a hydroxide gradient, strongly retained trivalent ions, such as phosphate and citrate, are efficiently eluted in the same run that also gives baseline resolution of the weakly retained monovalent anions; fluoride, lactate, acetate, formate, and butyrate. Another benefit of using the AS-HC column is the ability to easily change the order of elution of ions with different valencies simply by changing the gradient profile. For example, if citrate is present in high enough concentration to interfere with chromate, the chromate peak can be moved ahead of the citrate peak by using a slightly different gradient. AS-HC columns are stable between ph and 4 and are compatible with eluents containing -% organic solvents. The AG-HC guard column is packed with a microporous resin with a lower capacity. The microporous resin ensures optimum long term performance of the guard column. Table IonPac AS-HC/AG-HC Packing Specifications Column AS-HC (4 x 5 mm) AG-HC (4 x 5 mm) Particle Diameter µm Substrate X- Linking Latex Diameter nm Latex X- Linking % Column Capacity µeq/column Functional Group 9 55% 7 9 Alkanol quaternary ammonium 3 55% 7 7 Alkanol quaternary ammonium Hydrophobicity Medium-Low Medium-Low AS-HC ( x 5 mm) AG-HC ( x 5 mm) 9 55% Alkanol quaternary ammonium 3 55% 7.75 Alkanol quaternary ammonium Medium-Low Medium-Low a macroporous ( Å) divinylbenzene/ethylvinylbenzene polymer b microporous divinylbenzene/ethylvinylbenzene polymer Table AS-HC/AG-HC Operating Parameters Column IonPac AS-HC Analytical Column (4 x 5 mm) IonPac AG-HC Guard Column (4 x 5 mm) Typical Back Pressure psi (Mpa) Standard Flow Rate ml/min Maximum Flow Rate < 9 (3.).5 3. < 5 (.3).5 3. AS-HC + AG-HC 4-mm < 5 (4.3).5 3. IonPac AS-HC Analytical Column < 8 (.4) ( x 5 mm) IonPac AG-HC Guard Column < 5 (.3) ( x 5 mm) AS-HC + AG-HC -mm < 95 (3.44).38.75

7 IonPac AS-HC Manual Document No Page of 45 Always remember that assistance is available for any problem that may be encountered during the shipment or operation of Dionex instrumentation and columns through the Dionex North America Technical Call Center at -8-DIONEX- ( ) or through any of the Dionex Offices listed in, Dionex Worldwide Offices.

8 IonPac AS-HC Manual Document No Page 7 of 45 SECTION - COMPARISON OF ION CHROMATOGRAPHY SYSTEMS The proper configuration of -mm system injection volume, mass loading, system void volume and flow rate is based on the ratio of the -mm to 4-mm column cross-sectional area which is a factor of /4. CONFIGURATION -mm 4-mm Eluent Flow Rate.38 ml/min.5 ml/min SRS ASRS ULTRA II (-mm) (P/N 5) ASRS ULTRA II (4-mm) (P/N 5) MMS AMMS III (-mm) (P/N 575) AMMS III (4-mm) (P/N 575) NOTE Do not run suppressors over 4 C. If application requires a higher temperature, place suppressor outside of chromatographic oven. Injection Loop - 5 µl - 5 µl Use the Rheodyne Microinjection Valve, Model No. 9 DIONEX P/N 4497) for full loop injections <5 µl. System Void Volume Eliminate switching valves, couplers and the GM-3 Gradient Mixer. Use only the -mm GM-3 Mixer (P/N 4349). Minimize dead volumes. Switching valves, couplers can be used. Use the GM-, GM-3 or recommended gradient mixers. Pumps Use the GS5/GP5/GP4/IP/IP5 in Microbore Configuration with a Microbore GM-4 (- mm) Gradient Mixer. Use the GP4/GP5/GS5/IP/IP5 in Standard-Bore Configuration. Chromatographic Module No External Gradient Mixer is required for GS5/GP5/GP4 Pump when performing gradient analysis The GPM- can be used for - mm isocratic chromatography at flow rates of.5 ml/min or greater but cannot be used for -mm gradient chromatography NOTE: Use of an EG4 (P/N 539) or EG5 (P/N 585) with an EGC II KOH cartridge (P/N 589) for gradient applications is highly recommended for minimum baseline change when performing eluent step changes or gradients. Use the LC5 or LC3 for temperature control. The GM-3 Gradient Mixer should be used for gradient analysis on systems other than the GP4/GP5/IP/IP5 and the DX-3 HPLC Pump. NOTE: Use of an EG4 (P/N 539) or EG5 (P/N 585) with an EGC II KOH cartridge (P/N 589) for gradient applications is highly recommended for minimum baseline change when performing eluent step changes or gradients. Use the LC5 or LC3 for temperature control.

9 IonPac AS-HC Manual Document No Page 8 of 45 CONFIGURATION -mm 4-mm Detectors AD/AD5 Cell AD/AD5 Cell (-mm, 7.5 µl, P/N 443) (-mm, 9 µl, P/N 49393) VDM- Cell (3-mm,. µl, P/N 43) VDM- Cell (-mm, µl) P/N 433 CD, CD5, CD5A, ED4, ED5, or ED5A Conductivity Cell with DS3 P/N 443 or Conductivity Cell with shield P/N 443 CD, CD5, CD5A, ED4, ED5, or ED5A Conductivity Cell with DS3 P/N 443 or with shield P/N 443 CDM-/CDM-3 Cell P/N 477 CDM-/CDM-3 Cell P/N 477 Replace the TS- with the TS- (P/N 437) on the CDM- or the CDM-3. The TS- has been optimized for -mm operation. Do not use the TS- or the TS- with the ED4/ED5 or the CD/CD5. Either the TS- with the TS- can be used with the CDM- or the CDM-3. Do not use the TS- or the TS- with the ED4/ED5 or the CD/CD5. DIONEX Back Pressure Regulator 75 psi rating (P/N 397, 448) or Tubing (see Table 3) Ensure 5-75 psi back pressure. DIONEX Back Pressure Regulator 75 psi rating (P/N 397, 448) or Tubing (see Table 3) Ensure 5-75 psi back pressure. Color Dionex P/N I.D. inch I.D. cm Table 3 Tubing Back Pressure Volume ml/ft Back pressure Psi/ft. at ml/min Back pressure Psi/ft. at.5 ml/min Back pressure Psi/cm at ml/min Green Orange Blue Black Red Yellow

10 IonPac AS-HC Manual Document No Page 9 of 45 SECTION 3 - INSTALLATION 3. System Void Volume When using -mm columns, it is particularly important to minimize system void volume. The system void volume should be scaled down to at least /4 of the system volume in a standard 4-mm system. For best performance, all of the tubing installed between the injection valve and detector should be.5" (P/N 44) i.d. PEEK tubing.." i.d. PEEK tubing (P/N 4) or." Tefzel tubing (see, Dionex Product Selection Guide ) may be used but peak efficiency will be compromised which may also result in decreased peak resolution. Minimize the lengths of all connecting tubing and remove all unnecessary switching valves and couplers. If you need assistance in properly configuring your system contact the Dionex North America Technical Call Center at -8-DIONEX- ( ) or the nearest Dionex Office (see, Dionex Worldwide Offices ). 3. System Requirements 3.. System Requirements for -mm Operation The IonPac AS-HC -mm Guard and Analytical Columns are designed to run on Dionex Ion Chromatographs equipped with suppressed conductivity detection. Isocratic analyses at flow rates of.5 ml/min or greater can be performed on a pump with standard (/8" pistons) pump heads. For isocratic analyses at flow rates below.5 ml/min and gradient analyses, a microbore pump (/" pistons) must be employed. 3.. System Requirements for 4-mm Operation The IonPac AS-HC 4-mm Guard and Analytical Columns are designed to run on any Dionex Ion Chromatograph equipped with suppressed conductivity detection. Gradient methods and methods requiring solvent containing eluents should be performed on a system having a pump with a standard pump heads (/8" pistons). Isocratic analysis can also be performed on a pump with standard bore pump heads (/8" pistons). 3.3 Installing the CR-ATC Trap Column for Use with EGC II KOH Cartridge For IonPac AS8 applications using the EG4 or EG5 with EGC II KOH cartridge, a CR-ATC Continuously Regenerated Trap Column (P/N 477) should be installed at the EGC eluent outlet to remove trace level anionic contaminants from the carrier deionized water. See the CR-TC Product Manual (Document No. 39) for instructions. As an alternative, the ATC-HC Trap Column (P/N 594) should be installed between the pump outlet and the inlet of the EluGen Cartridge in the EG4 or EG5 Module to remove anionic contaminants from the carrier deionized water. The ATC-HC is for use with EGC II KOH cartridge in the EG4 and EG5 Eluent Generators. See the ATC-HC Product Manual (Document No. 397) for instructions. If the lower capacity ATC-3 Trap Column (P/N 59 and 59) is used, it should be installed between the gradient pump and the injection valve to remove anionic contaminants from the eluent. The ATC-3 column is used when performing sodium hydroxide gradient anion exchange applications using hand-prepared bottled eluents. See the ATC-3 Product Manual (Document No. 397) for instructions. The ATC-HC (P/N 594) and ATC-3 Trap Columns will require off-line regeneration. To use the ATC-HC or ATC-3 Anion Trap Columns, refer to the Product Manuals. 3.4 The Sample Concentrator For 4-mm concentrator work, use the IonPac AG-HC Guard Column when a single piston pump such as the DQP or DXP pump is used for sample delivery. Use the Low Pressure Trace Anion Concentrator Column (TAC-LP, P/N 4) or TAC-ULP (P/ N 4) when the sample is delivered with a syringe or with an autosampler. For -mm concentrator work, use the IonPac AG- HC Guard Column when a single piston pump such as the DQP or DXP pump is used for sample delivery. The function of the TAC- LP, the TAC-ULP (P/N 4), or the AG-HC Guard Column in these applications is to strip ions from a measured volume

11 IonPac AS-HC Manual Document No Page of 45 of a relatively clean aqueous sample matrix. This process concentrates all anionic analyte species onto the TAC-LP, the TAC- ULP, or the AG-HC leading to a lowering of detection limits by -5 orders of magnitude. The unique advantage to the analytical chemist of the TAC-LP, the TAC-ULP, or the AG-HC in these applications is the capability of performing routine trace analyses of sample matrix ions at µg/l levels without extensive and laborious sample pretreatment. For a detailed discussion of anion concentration techniques, refer to Section 3, Operation, of the Low Pressure Trace Anion Concentrator (TAC-LP) and TAC-ULP Column Product Manual (Document No. 3497). CAUTION IonPac Trace Anion Concentrator (TAC-) Column (P/N 43) is not optimized for use with hydroxide eluents and should not be used for concentrator work with the IonPac AS-HC. Use the TAC-LP, TAC-ULP, or the AG-HC 4-mm or AG -mm guards. 3.5 The Injection Loop Table 4 Smallest Injectable Volumes (µl) Valve Type Dionex BF Valve (8 µl Internal Volume) ( cm Loop) Dionex MicroInject Valve (.5 µl Internal Volume) (4 cm Loop) Rheodyne Microinjection Valve Model 9 (.8 µl Internal Volume) ( cm Loop) Using." ID Tefzel Tubing Using.7" ID Tefzel Tubing Using." ID PEEK Tubing Using.5" ID PEEK Tubing The -mm System Injection Loop, - 5 µl For most applications on a -mm analytical system, a - 5 µl injection loop is sufficient. Generally, you should not inject more than nanomoles of any one analyte onto a -mm analytical column. Injecting larger number of moles of a sample can result in overloading the column which can affect the detection linearity. For low concentrations of analytes, larger injection loops can be used to increase sensitivity. The AS-HC -mm requires a microbore HPLC system configuration. Install an injection loop one-fourth or less (<5 µl) of the loop volume used with a 4-mm analytical system (Section, Comparison of -mm and 4-mm Ion Chromatography Systems ) The 4-mm System Injection Loop, - 5 µl For most applications on a 4-mm analytical system, a - 5 µl injection loop is sufficient. Generally, you should not inject more than 4 nanomoles of any one analyte onto the 4-mm analytical column. Injecting larger number of moles of a sample can result in overloading the column which can affect the detection linearity. For low concentrations of analytes, larger injection loops can be used to increase sensitivity.

12 IonPac AS-HC Manual Document No Page of The IonPac AG-HC Guard Column An IonPac AG-HC Guard Column is normally used with the IonPac AS-HC Analytical Column. Retention times will increase by approximately % when a guard column is placed in-line prior to the analytical column. A guard is placed prior to the analytical column to prevent sample contaminants from eluting onto the analytical column. It is easier to clean or replace a guard column than it is an analytical column. Replacing the AG-HC Guard Column at the first sign of peak efficiency loss or decreased retention time will prolong the life of the AS-HC Analytical Column. 3.7 Eluent Storage IonPac AS-HC columns are designed to be used with hydroxide eluent systems. Storage under a helium atmosphere ensures contamination free operation and proper pump performance (nitrogen can be used if eluents do not contain solvents). 3.8 Anion Self-Regenerating Suppressor Requirements An Anion Self-Regenerating Suppressor should be used for applications that require suppressed conductivity detection. It is compatible with solvent containing eluents and aqueous ionic eluents of all concentrations with which the systems and columns are compatible. Aqueous ionic eluents can be used in all ASRS -ULTRA modes of operation. NOTE Solvent containing eluents should be used in the AutoSuppression External Water Mode. If you are installing an IonPac AS-HC 4-mm Analytical Column, use an ASRS ULTRA II (4-mm, P/N 5). If you are installing an IonPac AS-HC -mm Analytical Column, use an ASRS ULTRA II (-mm, P/N 5). For detailed information on the operation of the Anion Self-Regenerating Suppressor, see Document No. 395, the Product Manual for the Anion Self-Regenerating Suppressor ULTRA II, the ASRS ULTRA II. 3.9 Anion MicroMembrane Suppressor Requirements An Anion MicroMembrane Suppressor (AMMS III) may be used instead of an ASRS ULTRA II for applications that require suppressed conductivity detection. Use an AMMS III (P/N 575) with the IonPac AS-HC 4-mm Analytical Column. It is compatible with all solvents and concentrations with which the systems and columns are compatible. For -mm operation, use the AMMS III (-mm) (P/N 575). For detailed information on the operation of the Anion MicroMembrane Suppressor, see Document No. 377, the "Product Manual for the Anion MicroMembrane Suppressor III, the AMMS III". 3. Using Displacement Chemical Regeneration (DCR) with the Chemical Suppression Mode Dionex recommends using the Displacement Chemical Regeneration (DCR) Mode for chemical suppression using sulfuric acid and the Anion MicroMembrane Suppressor (AMMS III). See the DCR kit manual, Document P/N 34, for details. SAFETY Use proper safety precautions in handling acids and bases. 3. Using AutoRegen with the ASRS ULTRA II or the AMMS III in the Chemical Suppression Mode To save regenerant preparation time and reduce regenerant consumption and waste, Dionex recommends using an AutoRegen Accessory (P/N 39594). For more detailed information on the use of the AutoRegen Accessory see the AutoRegen Accessory manual (Document No. 3853). For more detailed information on the use of AutoRegen Regenerant Cartridges, see the Product Manual for the AutoRegen Regenerant Cartridge Refills (Document No. 385).

13 IonPac AS-HC Manual Document No Page of Detector Requirements See Section, Comparison of Ion Chromatography Systems, for -mm and 4-mm system detector, cell and thermal stabilizer requirements. 3.3 Using the EG4 or EG5 with AS-HC Please refer to the EG4 manual, Document No. 3373, for information on the operation of the EG4. For the EG5, please refer to the EG5 Product Manual, Document No. 398, for information on the operation of the EG5.

14 IonPac AS-HC Manual Document No Page 3 of 45 SECTION 4 - OPERATION 4. General Operating Conditions Sample Volume: Column: Eluent: Eluent Flow Rate: SRS Suppressor: or MMS Suppressor: -mm:.5 µl Loop +.8 µl Injection valve dead volume 4-mm: µl Loop +.8 µl Injection valve dead volume -mm: AS-HC -mm Analytical Column + AG-HC -mm Guard Column 4-mm: AS-HC 4-mm Analytical Column + AG-HC 4-mm Guard Column 3 mm NaOH (for test chromatogram) -mm:.38 ml/min 4-mm:.5 ml/min Anion Self-Regenerating Suppressor ULTRA II, ASRS ULTRA II (- or 4-mm) AutoSuppression Recycle Mode for aqueous gradients AutoSuppression External Water Mode for eluents with solvent Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity: < 3 µs Storage Solution: mm Sodium Borate 4. IonPac AS-HC Operation Precautions CAUTIONS Filter and Degas Eluents Filter Samples Eluent ph between and 4 Sample ph between and 4.75 ml/min Maximum Flow Rate for -mm Columns 3. ml/min Maximum Flow Rate for 4-mm Columns Maximum Operating Pressure = 4, psi (7.57 MPa) 4.3 Chemical Purity Requirements Obtaining reliable, consistent and accurate results requires eluents that are free of ionic impurities. Chemicals, solvents and deionized water used to prepare eluents must be of the highest purity available. Low trace impurities and low particle levels in eluents also help to protect your ion exchange columns and system components. Dionex cannot guarantee proper column performance when the quality of the chemicals, solvents and water used to prepare eluents has been compromised Inorganic Chemicals Reagent Grade inorganic chemicals should always be used to prepare ionic eluents. Whenever possible, inorganic chemicals that meet or surpass the latest American Chemical Society standard for purity should be used. These inorganic chemicals will detail the purity by having an actual lot analysis on each label Deionized Water The deionized water used to prepare eluents should be Type I Reagent Grade Water with a specific resistance of 8. megohmcm. The deionized water should be free of ionized impurities, organics, microorganisms and particulate matter larger than. µm. Bottled HPLC-Grade Water (with the exception of Burdick & Jackson) should not be used since most bottled water contains an unacceptable level of ionic impurities.

15 IonPac AS-HC Manual Document No Page 4 of Solvents Solvents can be added to the ionic eluents used with IonPac AS-HC columns to modify the ion exchange process or improve sample solubility. The solvents used must be free of ionic impurities. However, since most manufacturers of solvents do not test for ionic impurities, it is important that the highest grade of solvents available be used. Currently, several manufacturers are making ultrahigh purity solvents that are compatible for HPLC and spectrophotometric applications. These ultrahigh purity solvents will usually ensure that your chromatography is not affected by ionic impurities in the solvent. Currently at Dionex, we have obtained consistent results using High Purity Solvents manufactured by Burdick and Jackson and Optima Solvents by Fisher Scientific. When using a solvent in an ionic eluent, column generated back pressures will depend on the solvent used, concentration of the solvent, the ionic strength of the eluent and the flow rate used. The column back pressure will vary as the composition of water-methanol and water-acetonitrile mixture varies. The practical back pressure limit for the IonPac AS-HC columns is 4, psi (7.57 MPa). The AS-HC can withstand common HPLC solvents in a concentration range of - %. Solvents and water should be premixed in concentrations which allow proper mixing by the gradient pump and to minimize outgassing. Ensure that all of the inorganic chemicals are soluble in the highest solvent concentration to be used during the analysis. Table 5 HPLC Solvents for Use with IonPac AS-HC Columns Solvent Maximum Operating Concentration Acetonitrile % Methanol % -Propanol % Tetrahydrofuran %* *Higher concentrations may only be used for limited duration applications such as column clean-up at pressures < psi. CAUTION The Anion Self-Regenerating Anion Suppressor (ASRS ULTRA II) must be operated in the AutoSuppression External Water Mode when using eluents containing solvents. 4.4 Making Eluents that Contain Solvents When mixing solvents with water remember to mix solvent with water on a volume to volume basis. If a procedure requires an eluent of 9% acetonitrile, prepare the eluent by adding 9 ml of acetonitrile to an eluent reservoir. Then add ml of deionized water or eluent concentrate to the acetonitrile in the reservoir. Using this procedure to mix solvents with water will ensure that a consistent true volume/volume eluent is obtained. Premixing water with solvent will minimize the possibility of outgassing. NOTE When purging or degassing eluents containing solvents, do not purge or degas the eluent excessively since it is possible that a volatile solvent can be "boiled" off from the solution. Always degas and store all eluents in glass or plastic eluent bottles pressurized with helium. Only helium can be used to purge and degas ionic eluents containing solvents, since nitrogen is soluble in solvent containing eluents. Acetonitrile (ACN) hydrolyzes to ammonia and acetate when left exposed to basic solutions. To prevent eluent contamination from acetonitrile hydrolysis, always add acetonitrile to basic aqueous eluents by proportioning the acetonitrile into the basic eluent with the gradient pump. Keep the acetonitrile in a separate eluent bottle containing only acetonitrile and water.

16 IonPac AS-HC Manual Document No Page 5 of 45 WARNING Never add the acetonitrile directly to the basic carbonate or hydroxide eluent bottle. 4.5 Regenerant Preparation for the AMMS III The Anion MicroMembrane Suppressor III (AMMS III) requires the use of a regenerant solution. If you are using the AMMS III instead of the Anion Self-Regenerating Suppressor ULTRA II (ASRS ULTRA II) see Document No. 377, the Product Manual for the Anion MicroMembrane Suppressor III, the AMMS III. 4. Sample Concentration The Low Pressure Trace Anion Concentrator, TAC-LP (P/N 4), the TAC-ULP (P/N 4), or the IonPac AG-HC Guard Column can be used for trace anion concentration work required in high purity water analysis. The function of a concentrator column in these applications is to strip ions from a measured volume of a relatively clean aqueous sample matrix. This process concentrates the desired analyte species onto the concentrator column, lowering detection limits by -5 orders of magnitude. The concentrator column is used in lieu of the sample loop. Pump the sample onto the concentrator column in the OPPOSITE direction of the eluent flow. When using concentration techniques, do not overload the concentrator column by concentrating an excessive amount of sample. Concentrating an excessive amount of sample can result in inaccurate results being obtained. It is possible during the concentration step for the polyvalent anions such as phosphate and sulfate to elute the weakly retained anions such as fluoride and acetate off the concentrator column. For more detailed information on sample concentration techniques for high sensitivity work refer to Section 3, Operation, of the Low Pressure Trace Anion Concentrator (TAC-LP and TAC-ULP) Column Product Manual (Document No. 3497). These techniques can also be applied to the AG-HC.

17 IonPac AS-HC Manual Document No Page of 45 SECTION 5 - EXAMPLE APPLICATIONS 5. Recommendations for Optimum System Performance The chromatograms in this section were obtained using columns that reproduced the Production test Chromatogram (see Section 5., Production Test Chromatogram ) on optimized Ion Chromatographs (see Section 3, Installation ). Different systems will differ slightly in performance due to slight differences in column sets, system void volumes, liquid sweep-out times of different components and laboratory temperatures. The IonPac AS-HC is designed to perform analyses of large numbers of anions of varying valencies through gradient elution. In any type of gradient elution system it is important to use eluents that produce a minimum shift in baseline conductivity during the run, as well as a fast equilibration time from one run to the next. Because sodium hydroxide is converted to water in the suppressor, it is the best choice for an eluent. As long as the capacity of the suppressor is not exceeded, the eluent hydroxide concentration has little effect on background conductivity. For example, a gradient run could begin at a few mm NaOH and end at mm NaOH, with only a resulting to 3 µs total baseline change. Ensure that your system is properly configured. It is very important that applications run on -mm columns utilize the proper pump configuration (see Section, Comparison of -mm and 4-mm Ion Chromatography Systems ) and have all system void volumes minimized. Fluctuations in operating temperature can affect the retention time and resolution of analytes and should be controlled. Ensure that adequate equilibration time is allowed between runs. If downward shift in baseline is observed during the isocratic section of the chromatogram, increase the equilibration time. Ensure that all of the eluents have been made from high purity reagents and deionized water. All water used in the preparation of eluents should be degassed, deionized water. For chemical purity requirements see Section 4.3, Chemical Purity Requirements. The addition of chromate to the sample will help stabilize organic acids. If your sample or standard contains organic acids, adding chromate (about mg/l) will help stabilize them from bacterial degradation at room temperature. The sample chromatograms in Sections 5.3, Gradient Elution of a Large Number of Inorganic and Organic Acid Anions, (see Figure 3, Separation of Mono-, Di-, and Trivalent Anions in One Sample Run ). Install a CR-ATC Continuously Regenerated Trap Column (P/N 477), or an ATC-HC (P/N 594), or an Anion Trap Column, ATC-3 (4-mm) or the ATC-3 (-mm) in the system. See Section 3. of the Product Manual for the Anion Trap Column to minimize the baseline shift and to improve retention time reproducibility of analytes when doing gradient chromatography and to keep baseline shift to a minimum. (Refer to the ATC column cleanup protocol in Section.., A Contaminated Trap Column. ) Use a guard column to protect the analytical column. If column performance deteriorates and it is determined that the guard and analytical columns has been fouled, refer to the column cleanup protocols in, Column Care. You can increase the sensitivity of your system by using sample concentration techniques (see Section 3.3, The Sample Concentrator ). NOTE Carbon dioxide readily dissolves in dilute basic solutions, forming carbonate. Carbonate contamination of eluents can effect the retention times of the anions being analyzed. Eluents should be maintained under an inert helium atmosphere to avoid carbonate contamination.

18 IonPac AS-HC Manual Document No Page 7 of Eluent Preparation Sodium Hydroxide Eluent Concentration Weight Method When formulating eluents from 5% sodium hydroxide, Dionex recommends weighing out the required amount of 5% sodium hydroxide. Use the assayed concentration value from the sodium hydroxide bottle. Volume Method Example: To make L of 3 mm NaOH use.4 g of 5% sodium hydroxide: (as used in Section 5.3, Production Test Chromatogram ) For 3 mm:.3 mole/l x 4. g/mole =.4 g diluted to L 5% Although it is more difficult to make precise carbonate-free eluents for gradient analysis volumetrically, you may choose to use the following formula to determine the correct volume of 5% sodium hydroxide to be diluted. g = dvr Where: g = weight of sodium hydroxide required (g) d = density of the concentrated solution (g/ml) v = volume of the 5% sodium hydroxide required (ml) r = % purity of the concentrated solution Sodium Hydroxide Eluents Example: To make L of mm NaOH use.5 ml of 5% sodium hydroxide: (as used in Section 5., Production Test Chromatogram ) For mm:.3 mole/l x 4. g/mole =.57 ml diluted to L 5% x.53* g/ml *This density applies to 5% NaOH. If the concentration of the NaOH solution is significantly different from 5%, the upper (weight method) calculation should be used instead. Dilute the amount of 5% (w/w) NaOH Reagent specified in Table, Dilution of 5% (w/w) NaOH to Make Standard AS-HC Eluents with degassed, deionized water (8. megohm-cm) to a final volume of, ml using a volumetric flask. Avoid the introduction of carbon dioxide from the air into the aliquot of 5% (w/w) NaOH bottle or the deionized water being used to make the eluent. Do not shake the 5% (w/w) NaOH bottle or pipette the required aliquot from the top of the solution where sodium carbonate may have formed. Table Dilution of 5% (w/w) NaOH to Make Standard AS-HC Eluents 5% (w/w) Concentration of NaOH NaOH Eluent g (ml) (mm).8 (.53).4 (.) 5.4 (.57) 3 8. (5.3). (.5) 4. (.5) 5

19 IonPac AS-HC Manual Document No Page 8 of Production Test Chromatograms Isocratic elution of common anions on the IonPac AS-HC Analytical Column has been optimized utilizing a hydroxide eluent. By using this eluent, common inorganic anions can be used to test the performance of the AS-HC Column. The IonPac AS- HC Analytical Column should always be used with the IonPac AG-HC Guard Column. To guarantee that all IonPac AS- HC Analytical Columns meet high quality and reproducible performance specification standards, all columns undergo the following production control test. An operating temperature of 3 C is used to ensure reproducible resolution and retention. Note that the AG-HC Guard is packed with a microporous resin of proportionally lower capacity and contributes approximately % increase in retention times when a guard column is placed in-line prior to the analytical column. Sample Volume: -mm:.5 µl Loop +.8 µl Injection valve dead volume 4-mm: µl Loop +.8 µl Injection valve dead volume Column: See chromatogram Eluent: 3 mm NaOH Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression Recycle Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity: < 3 µs Storage Solution: mm Sodium Borate Analyte mg/l (ppm). Fluoride.. Chloride Nitrite. 4. Sulfate. 5. Bromide.. Nitrate. 7. Phosphate 3. AS-HC 4-mm Analytical Column Only AS-HC -mm Analytical Column Only μs 7 μs AS-HC 4-mm & AG-HC 4-mm Analytical & Guard Columns AS-HC 4-mm & AG-HC -mm Analytical & Guard Columns μs μs Figure IonPac AS-HC Production Test Chromatograms 5

20 IonPac AS-HC Manual Document No Page 9 of Test Chromatograms at Room Temperature Isocratic elution of seven common anions on the IonPac AS-HC Analytical Column has been optimized at 3 C. However, the column can be operated at room temperature. Notice that at room temperature ( C) the divalent and trivalent ions have shorter retention times with 3 mm NaOH. The optimum eluent for room temperature operation is 5 mm NaOH. Sample Volume: Column: Eluent: Eluent Flow Rate: Operating Temperature: SRS Suppressor: or MMS Suppressor: -mm:.5 µl Loop +.8 µl Injection valve dead volume 4-mm: µl Loop +.8 µl Injection valve dead volume IonPac AS-HC Analytical and AG-HC Guard See chromatogram.38 ml/min (-mm),.5 ml/min (4-mm) Room temperature ( C) Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression Recycle Mode Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity: < 3 µs Storage Solution: mm Sodium Borate 3 mm NaOH Analyte mg/l (ppm). Fluoride.. Chloride Nitrite. 4. Sulfate. 5. Bromide.. Nitrate. 7. Phosphate 3. μs mm NaOH μs 7 5 Figure IonPac AS-HC Test Chromatograms at Room Temperature

21 IonPac AS-HC Manual Document No Page of Gradient Elution of a Large Number of Inorganic Anions and Organic Acid Anions Using NaOH Gradient A large number of inorganic anions and organic acid anions can be separated on the IonPac AS-HC using gradient elution. The sodium hydroxide gradient is optimized in order to elute mono-, di-, and trivalent organic acid anions in a single run. The starting eluent in the beginning of the gradient has a low concentration allowing fluoride to elute after the void volume and also separates several weakly retained monovalent organic acids. The hydroxide concentration in the later part of the gradient elutes polyvalent ions such as trivalent phosphate, citrate, and cis- and trans-aconitate. See Section 5., "Eluent Preparation", for eluent preparation instructions. Figure 3 demonstrates the effect of temperature on the selectivity of the AS-HC. Operation at 3 C improves the separation of monovalents such as fluoride and lactate or formate and butyrate. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume: -mm:.5 µl 4-mm: µl Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: See Chromatogram SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression Recycle Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: psi (5.5 MPa) Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 7 min 7. 8 Analysis mm NaOH, Inject Inject Valve to Load Position 5. 8 Isocratic analysis Gradient analysis Analyte mg/l(ppm). Quinate. Fluoride 3 3. Lactate 4. Acetate 5. Glycolate. Propionate 7. Formate 8. Butyrate 9. Methylsulfonate. Pyruvate. Chlorite. Valerate 3 Galacturonate 4. Monochloroacetate 5. Bromate. Chloride 5 7. Nitrite Analyte mg/l(ppm) 8. Sorbate 9. Trifluoroacetate. Bromide. Nitrate. Glutarate 3. Carbonate 4. Succinate 5 5. Malate 5. Malonate 5 7. Tartrate 5 8. Maleate 5 9. Sulfate 5 3. Oxalate 5 3. Fumarate 5 3. Ketomalonate 33. Tungstate Analyte mg/l(ppm) 34. Phosphate 35. Phthalate 3. Citrate 37. Isocitrate 38. Chromate 39. cis-aconitate 4. trans-aconitate

22 IonPac AS-HC Manual Document No Page of 45 at Room Temperature ( C) S µs 3 4, ,,4,5 8,9 3,, at 3 C,7 9 S µs 3 7 4, , , , Figure 3 Gradient Elution of a Large Number of Inorganic Anions and Organic Anions using NaOH Gradient

23 IonPac AS-HC Manual Document No Page of GRADIENT ELUTION OF A LARGE NUMBER OF INORGANIC ANIONS AND ORGANIC ACID ANIONS USING A NaOH GRADIENT WITHOUT SOLVENT OR WITH SOLVENT Figure 4 illustrates the separation of a large number of inorganic anions and organic acids on the IonPac AS-HC using a sodium hydroxide gradient. A solvent (methanol) gradient is used to optimize the resolution of closely eluting ions such as acetate and glycolate, succinate and malate, as well as malonate and tartrate. An operating temperature of 3 C is used to ensure reproducible resolution and retention times. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume: -mm:.5 µl 4-mm: µl Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: Without Solvent:, psi (5.5 MPa) With Solvent:,7 psi (8. MPa) Gradient Conditions Without Solvent TIME %E %E %E3 %E4 Comments (min) Gradient Conditions With Solvent TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 7 min 7. 8 Analysis mm NaOH, Inject Inject Valve to Load Position 5. 8 End Isocratic analysis, Gradient analysis Equilibration 7. mm NaOH for 7 min 7. 7 Analysis mm NaOH, Inject Inject Valve to Load Position 5. 7 End isocratic analysis, Gradient analysis Analyte mg/l(ppm). Quinate. Fluoride 3 3. Lactate 4. Acetate 5. Glycolate. Propionate 7. Formate 8. Butyrate 9. Methylsulfonate. Pyruvate. Chlorite. Valerate 3 Galacturonate 4. Monochloroacetate Analyte mg/l(ppm) 5. Bromate. Chloride 5 7. Nitrite 8. Sorbate 9. Trifluoroacetate. Bromide. Nitrate. Glutarate 3. Carbonate 4. Succinate 5 5. Malate 5. Malonate 5 7. Tartrate 5 8. Maleate 5 Analyte mg/l(ppm) 9. Sulfate 5 3. Oxalate 5 3. Fumarate 5 3. Ketomalonate 33. Tungstate 34. Phosphate 35. Phthalate 3. Citrate 37. Isocitrate 38. Chromate 39. cis-aconitate 4. trans-aconitate

24 IonPac AS-HC Manual Document No Page 3 of 45 Without Solvent,7 9 µs S 3 7 4, , , , With Solvent µs S 9 34,35 38,39 4 7, , 3, Figure 4 Gradient Analysis of a Large Number of Anions using NaOH without Solvent and with Solvent

25 IonPac AS-HC Manual Document No Page 4 of METHOD OPTIMIZATION FOR FOOD AND BEVERAGE APPLICATIONS USING NaOH GRADIENT WITH A SOLVENT STEP CHANGE OR A SOLVENT GRADIENT Figure 5 illustrates the use of a solvent gradient compared to a step change to optimize peak resolution. These examples illustrate how a solvent gradient or step change can be introduced at different points in the gradient to modify column selectivity for the analytes. Note that in examples A and B, good resolution of lactate/acetate and formate/butyrate can be achieved in the beginning section of the chromatogram without solvent. Resolution of succinate and malate is optimized by the addition of solvent with a linear solvent gradient shown in example B. Sulfate and oxalate resolution is optimized by using a solvent step change, shown in example A. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume: -mm:.5 µl 4-mm: µl Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: Without Solvent:, psi (5.5 MPa) With Solvent:,7 psi (8. MPa) Example A Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject Valve to Load Position. 8 Isocratic analysis. 9 NaOH gradient. 7 8 Methanol step change Example B Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject Valve to Load Position. 8 End isocratic analysis,. 5 5 Methanol/NaOH gradient Start reverse methanol gradient 4. 4 Analyte mg/l(ppm). Quinate. Fluoride 3 3. Lactate 4. Acetate 5. Glycolate. Propionate 7. Formate 8. Butyrate 9. Methylsulfonate. Pyruvate. Chlorite. Valerate 3 Galacturonate 4. Monochloroacetate Analyte mg/l(ppm) 5. Bromate. Chloride 5 7. Nitrite 8. Sorbate 9. Trifluoroacetate. Bromide. Nitrate. Glutarate 3. Carbonate 4. Succinate 5 5. Malate 5. Malonate 5 7. Tartrate 5 8. Maleate 5 Analyte mg/l(ppm) 9. Sulfate 5 3. Oxalate 5 3. Fumarate 5 3. Ketomalonate 33. Tungstate 34. Phosphate 35. Phthalate 3. Citrate 37. Isocitrate 38. Chromate 39. cis-aconitate 4. trans-aconitate

26 IonPac AS-HC Manual Document No Page 5 of 45 Example A µs S 4, , , , ,39 4 Example B S µs 4, , , , ,39 4 Figure 5 Elution of a Large Number of Inorganic Anions and Organic Acid Anions Using NaOH Gradient with Solvent Step Change or Linear Solvent Gradient

27 IonPac AS-HC Manual Document No Page of Analysis of a Beer Sample Figure uses an optimized sodium hydroxide gradient with a methanol step gradient for analysis of a beer sample. The beer sample was diluted :5 with deionized water and treated with an OnGuard RP cartridge. The AS-HC column has a high capacity which allows injection of concentrated samples for determination of trace components. OnGuard RP Sample Pretreatment Procedure. Wash OnGuard RP with ml methanol. Next wash with ml deionized water 3. Discard 3-4 ml diluted sample then collect next -7 ml sample. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume: -mm:.5 µl 4-mm: µl Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: 7 psi (8. MPa) Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject valve to load position. 8 End isocratic analysis. 9 NaOH gradient. 7 8 Methanol step change Analyte. Lactate. Acetate 3. Formate 4. Butyrate 5. Pyruvate. Chloride 7. Bromide 8. Nitrate 9. Succinate. Malate. Carbonate. Fumarate 3. Sulfate 4. Oxalate 5. Phosphate. Citrate 7. Isocitrate 8. cis-aconitate 9. trans-aconitate

28 IonPac AS-HC Manual Document No Page 7 of 45 Beer Sample (Low Sensitivity) 5 S µs 3 5 Beer Sample (High Sensitivity) µs S Beer Sample Spiked with Butyrate 5 µs S Figure Analysis of a Beer Sample

29 IonPac AS-HC Manual Document No Page 8 of Analysis of Apple Juice Samples Figure 7 uses an optimized sodium hydroxide gradient with a methanol step gradient for analysis of apple juice samples. All three juice samples were diluted : with deionized water and filtered through a.45 mm syringe filter. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume: -mm:.5 µl 4-mm: µl Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: 7 psi (8. MPa) Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject Valve to Load Position. 8 End isocratic analysis. 9 NaOH gradient. 7 8 Methanol step change Analyte. Quinate. Lactate 3. Acetate 4. Propionate 5. Formate Galacturonate 7. Chloride 8. Nitrate 9. Succinate. Malate. Fumarate. Sulfate 3. Oxalate 4. Phosphate 5. Citrate. Isocitrate 7. cis-aconitate

30 IonPac AS-HC Manual Document No Page 9 of Fresh Apple Juice S µs Commercial Apple Juice Sample A µs S Commercial Apple Juice Sample B S µs Figure 7 Analysis of Apple Juice Samples

31 IonPac AS-HC Manual Document No Page 3 of Analysis of Orange Juice Samples Figure 8 uses an optimized sodium hydroxide gradient with a methanol step gradient for analysis of an orange juice sample. Both juice samples were diluted : with deionized water and filtered through a.45 mm syringe filter. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume:.5 µl (-mm), µl (4-mm) Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < 3.5 µs Typical Operating Back Pressure: 7 psi (8. MPa) Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject Valve to Load Position. 8 Isocratic analysis. 9 NaOH gradient. 7 8 Methanol step change Analyte. Quinate. Lactate 3. Acetate 4. Propionate 5. Formate Galacturonate 7. Chloride 8. Glutarate 9. Succinate. Malate. Malonate. Sulfate 3. Oxalate 4. Phosphate 5. Citrate. Isocitrate 7. Trans-Aconitate

32 IonPac AS-HC Manual Document No Page 3 of 45 Commercial Orange Juice Sample 5 µs S Fresh Orange Juice Sample 5 µs S Figure 8 Analysis of Orange Juice Samples

33 IonPac AS-HC Manual Document No Page 3 of Analysis of a Wine Sample Figure 9 uses an optimized sodium hydroxide gradient with a linear methanol gradient for the analysis of red wine. The wine samples were diluted with deionized water and treated with an OnGuard RP cartridge. OnGuard RP Sample Pretreatment Procedure. Wash OnGuard RP with ml methanol. Next wash with ml deionized water 3. Discard 3-4 ml diluted sample then collect next -7 ml sample. Trap Column: ATC-3, (Located between pump and injection valve) Sample Volume:.5 µl (-mm), µl (4-mm) Sample Dilution: Sample A: : Sample B: : Column: IonPac AS-HC Analytical and AG-HC Guard Eluent: E: Deionized water E: 5. mm NaOH E3: mm NaOH E4: % Methanol Eluent Flow Rate:.38 ml/min (-mm),.5 ml/min (4-mm) Operating Temperature: 3 C SRS Suppressor: Anion Self-Regenerating Suppressor, ASRS ULTRA II (-mm or 4-mm) AutoSuppression External Water Mode or MMS Suppressor: Anion MicroMembrane Suppressor, AMMS III (-mm or 4-mm) MMS Regenerant: 5 mn H SO 4 Expected Background Conductivity:. mm NaOH: µs mm NaOH: < µs Typical Operating Back Pressure: 7 psi (8. MPa) Commercial Red Wine Sample A 4 Gradient Conditions TIME %E %E %E3 %E4 Comments (min) Equilibration 8. mm NaOH for 8 min 8. 8 Analysis mm NaOH, Inject Inject Valve to Load Position. 8 Isocratic analysis. 5 5 Methanol/NaOH gradient µs S Commercial Red Wine Sample B Analyte. Lactate. Acetate 3. Formate 4. Pyruvate 5. Galacturonate. Chloride 7. Bromide 8. Nitrate 9. Succinate. Malate. Tartrate. Sulfate 3. Oxalate 4. Phosphate 5. Citrate. Isocitrate 7. cis-aconitate µs S Figure 9 Isocratic Analysis of a Wine Sample