ACCLAIM oa - Cromlab

Acclaim 120Acclaim 300Acclaim PA/PA2Acclaim OAAcclaim 120Acclaim PAAcclaim ExplosivesOrdering and Warranty InformationIn the U.S.All Orders Including:• Credit Card Orders (VISA, American Express, MasterCard)• Blanket Orders• Emergency OrdersCall 1-800-Dionex-0 (1-800-346-6390)orFAX 1-408-736-4476Ordering by Credit CardDionex accepts payment by MasterCard, VISA, and AmericanExpress cards with minimum orders of $50.00. Call 1-800-DIONEX-0(1-800-346-6390) for more information.Acclaim OAAcclaim PA/PA2Acclaim 120Blanket OrdersDionex will arrange for blanket orders with a specified shippingtime. Please call 1-800-DIONEX-0 (1-800-346-6390) for furtherinformation.Outside the U.S.Please contact your local Dionex representative.Acclaim 120Acclaim PA/PA2Acclaim OAAcclaim SurfactantShipping and HandlingOrders placed for consumables are typically shipped within twoworking days. Dionex ships FOB Sunnyvale, CA, for all U.S. orders,unless prior arrangements have been made. For UPS, FederalExpress, or any carrier under contract with Dionex, Dionex will prepaythe freight charges and add the charges to the invoice. Unlessotherwise specified, shipments are made by ground transportation.Acclaim 300Acclaim 120Acclaim PA2©2007 by Dionex CorporationAll rights reservedLPN 1668-03Dionex Corporation1228 Titan WayP.O. Box 3603Sunnyvale, CA 94088-3603AQA and MSQ are trademarks of Thermo Electron Corporation.One-A-Day is a registered trademark of Bayer Corporation.NyQuil is a registered trademark of Proctor and Gamble.All other trademarks and registered trademarks are property of Dionex Corporation.- 2 -www.dionex.com

IntroductionINTRODUCTIONPar t I: In t r o d u c t i o nAcclaim Column Families at a GlanceDionex Acclaim columns are silica-based columns designed for high efficiency separations and manufactured using ultra-high purity silica.Product families consists of a broad spectrum of stationary phases in the form of the conventional C8 and C18 phases. polar embedded phases.and several specialty phases, each designed for a specific application or group of applications. All Acclaim columns are designed and manufacturedat Dionex, to tight specifications, to ensure consistent performance.General Purpose HPLC ColumnsAcclaim 120High purity silica for small molecule separationsAcclaim 120 reversed-phase C18 and C8 phases are manufactured using high purity silica with a 120-Å pore diameter,very high surface coverage, low silanol activity, and very low metal content. All the phases are LC-MS compatible withgood capacity ideal for use with high organic mobile phases.CharacteristicsHigh purity spherical silica, LC/MS compatible, monomeric bonding, endcapped, 120 Å pore size, 3 µm, 5 µm.Acclaim 300Wide pore columns for macromolecule applicationsAcclaim 300 columns are ideal for the reversed-phase separation of proteins, peptides, and other biological macromolecules.The unique bonding chemistry results in a high-density, highly uniform phase coverage. The use of a 3-µm silicaparticle results in fast, high-resolution separations.CharacteristicsHigh purity spherical silica, LC/MS compatible, monomeric bonding, endcapped, 300 Å pore size, 3 µm, 5 µm.Acclaim PAPolar-embedded Reversed-phase columns for separating a wide range of analytesAcclaim PolarAdvantage (PA) columns feature a patented bonding chemistry that incorporates a polar sulfonamide groupnear the surface of the silica particle. This unique chemistry provides enhanced hydrolytic stability and allows a singlecolumn to resolve both polar and nonpolar analytes. The Acclaim PA is compatible with 100% aqueous mobile phases andexhibits high reversed-phase capacity.CharacteristicsHigh purity spherical silica, LC/MS compatible within guidelines, endcapped, 120 Å pore size, 3 µm, 5 µm.Acclaim PA2Polar-embedded columns with enhanced hydrolytic stabilityAcclaim PolarAdvantage II (PA2) columns feature a patented bonding chemistry that provides enhanced hydrolytic stabilityfrom pH 1.5–10 and allows a single column to resolve both polar and nonpolar analytes. The Acclaim PA2 is compatiblewith 100% aqueous mobile phases and exhibits high reversed-phase capacity, with selectivity complementary toconventional C18 columns such as the Acclaim 120 C18.CharacteristicsHigh purity spherical silica, LC/MS compatible, endcapped, 120 Å pore size, 3 µm, 5 µm.- 4 -www.dionex.com

IntroductionAcclaim General Purpose Column SpecificationsDionex phase Acclaim 120 C18 Acclaim 120 C8 Acclaim 300 C18 Acclaim PA Acclaim PA2Bonded phase C18 C8 C8 Sulfamido C16 Amide C18USP type L1 L7 L1 na naEndcapped Yes Yes Yes Yes YesSubstrate Ultrapure silica Ultrapure silica Ultrapure silica Ultrapure silica Ultrapure silicaParticle shape Spherical Spherical Spherical Spherical SphericalParticle size 3 and 5 µm 3 and 5 µm 3 µm 3 and 5 µm 3 and 5 µmMetal impurity (ppm)

IntroductionINTRODUCTIONSelecting an Acclaim ColumnThere are four questions to consider when choosing an HPLCcolumn:1. What are the goals of the separation?• Speed of analysis• Critical peaks requiring resolution• Solvent consumption/minimal waste• Sensitivity requirements• Operating costs• Other criteria2. What are the best silica characteristics for this separation?This is explained in the section below.3. What is the most appropriate column format? seepage 8 for a summary of the impact of column dimensionson the separation4. What is an appropriate stationary phase chemistry?see pages 4–5 for a summary of the Acclaim family ofcolumns and Part 2 for detailed information.Choosing the Best Column ChemistryWhen a chromatographer designs a separation, selectivityof the stationary phase is the first of many factors that must beconsidered. Selectivity is the result of the differing interactionsbetween each analyte and the stationary phase. Determining thecolumn with optimal selectivity is the essential starting point. Thisdetermination depends on the nature of the desired separation.The easiest first step in choosing a column is to identifywhether there is a column designed specifically for your separation.This can be readily established if the column is namedaccording to its application, as in the case of some of the DionexAcclaim columns. Thus, if you are separating surfactants, theAcclaim Surfactant column is the best first choice; likewiseAcclaim Explosives column are used for EPA Method 8330, andAcclaim OA is for organic acid analysis.If there is no specialty column for your application, thenyou will need some understanding of chemistry of your sampleto choose the best column chemistry. For those cases where thenature of the sample is completely unknown, the best first choiceis usually C18 because of its excellent peak efficiency, low silanolactivity, and ability to separate many organic molecules. The C8phase is a good choice if you want less retention of the sample onthe column. If you need highly aqueous mobile phase conditions,or different selectivity, then the polar embedded phases of theAcclaim Polar Advantage are the best option.For samples that are well characterized, various tests are usefulto characterize the stationary phases and to compare differentcolumns. The tests include studies of effects such as hydrophobicinteractions, molecular shape, and ion-exchange interactionsbetween the stationary phase and the sample analytes. A good,purely reversed-phase interaction will exhibit strong retention ofstrongly hydrophobic molecules and this will be the only retentionmechanism. As the density of the bonded phase decreases,other retention mechanisms will come into play, such as ionexchange interactions between the sample and residual silanolgroups on the surface of the silica gel, or sieving effects for similarsized molecules of different shapes. Thus, these studies can beused to choose a column with specific characteristics, or to find anew column with characteristics similar to an old favorite.HydrophobicityThe strength of the hydrophobic interaction betweenan analyte and the bonded silica is usually characterized bymeasuring the selectivity of ethylbenzene relative to toluene.Because of their high-density bonding, Acclaim C18 and C8columns have near maximum hydrophobicity compared withother C18 and C8 columns, and therefore almost pure reversedphasebehavior.Ion-Exchange ActivityIon-exchange activity is usually present where low surfacecoverage of the bonded phase, or insufficient endcapping is present.Since this effect is generally not manufactured into reversedphase stationary phases on purpose, ion-exchange activity ofreversed phase columns is not reproducible and separations relianton this effect are not reliable. Selectivity between phenol, anilineand pyridine is affected by the degree of their ion-exchangeactivity with residual surface silanol groups at pH 7, becauseunder these conditions the two bases are protonated but phenolis neutral. As illustrated in Acclaim Selectivity figure, overall theAcclaim columns exhibit low ion-exchange activity under neutralconditions and the general purpose C8 and C18 show particularlylow ion-exchange activity, emphasizing their high surface coverageof bonded phase.Molecular ShapeWhen hydrophobic interaction alone is insufficient to producethe required separation, it is often convenient to have secondaryeffects that will have an impact. Triphenylene and o-terphenylare both tricyclic hydrocarbons of similar size but with differentshapes. The type of bonding chemistry of the stationary phase andthe bonding density will both influence how these compoundsinteract with a stationary phase. These compounds are unresolvedon the C8 phase, similarly resolved on the 120-Å or 300-Å C18phases but as shown in the figure above, shape differences have amuch larger effect with the polar embedded phases than they dowith C18 phases.Hydrogen Bonding and Dipole-dipole InteractionsIn addition to hydrophobic retention, and ion exchange ifthe stationary phase has exposed charged sites, phenol, anilineand pyridine are also retained by dipole-dipole interactions andhydrogen bonding. These secondary effects can be exploited toimprove a separation by choosing a stationary phase that hasstronger interactions of this type, such as the polar embeddedphases.- 6 -www.dionex.com

IntroductionINTRODUCTIONUnderstanding More about theBonded PhaseDionex offers highest quality silica-based reversed phase packingmaterials. The type of bonding, carbon load and endcappingare all factors that contribute to the superior bonded phase.Type of BondingFunctional groups are attached to the silica substrate either viaa single attachment (monomeric bonding), or via multiple attachments(multidentate bonding). Monomeric bonded phases providehigher column efficiencies, than polymeric phases, but polymericphases are very stable under pH extremes.Carbon Load and EndcappingThe Percent (%) carbon is a rough guide to the capacity of thecolumn. Given the same type of silica the higher carbon load is anindicator of surface coverage. Phases with higher carbon loads aremore strongly hydrophobic, resulting in higher capacity, longerretention times, and often better resolution.When the functional groups are attached to the silica particle,not all silanol groups on the surface of the silica particle are covered.Free silanol groups will interact with polar analytes, alteringthe retention times and often causing peak tailing for organicbases. To minimize these secondary interactions, the free silanolgroups are endcapped. All Acclaim column packings are endcapped.Choosing the Optimum Column FormatColumn efficiency, which is measured in theoretical plates (N),is mostly a function of the column length and particle size. Acclaimcolumns are available in many formats, therefore it is important tocarefully select the best one for a particular application.Column LengthShorter columns provide faster run times and longer columnsprovide better resolution. If all else is constant, peaks are betterseparated on longer columns. However, the pressure, mobilephase consumption and time of analysis all increase in proportionto column length, thereby raising the cost per analysis. Detectionlimits are better on shorter columns.Column LengthColumn Length SelectionApplication10–75 mm Fast analysis. Works best with 3-µm particles size100–150 mm Standard separations. Works well with 3- or 5-µm particle sizes>250 mm High resolution applications. Works best with 5-µm particle sizeColumn DiameterSelect a column diameter according to your requirements forsample size, solvent consumption and sensitivity. Column diameterimpacts sensitivity (narrow columns provide better sensitivity),loading capacity (wider is better for larger sample volumes and forpurifying samples), solvent consumption (narrow columns use lesssolvent) and backpressure (narrow columns exhibit higher backpressureso you will need to decrease the flowrate). Scale the flowrate by the square of the column diameter to preserve backpressureand retention time. To gain the full benefits of smaller columndiameters, your HPLC instrumentation needs to be optimized forthe flow rate.Column DiameterColumn Diameter SelectionApplication0.075 to 1.0 mm I.D High sensitivity or limited volume samples.Use with LC/MS instrumentation2.1 mm I.D High sensitivity or limited volume samples.Use with low dead-volume instrumentation3.0 mm I.D Lower solvent consumption. Use with standard instrumentation4.0–4.6 mm I.D Standard formats. Use with standard instrumentationQuick Tips when Choosing Column FormatOptimize the analysis time and chromatographic efficiencyby evaluating the trade-offs between the particle diameterand column lengths.• As a general rule, the maximum sample load, solventconsumption, and detection limits are proportionalto the square of the column diameter. Scale the flowrate by the square of the column diameter to preservebackpressure and retention time: a 2.1-mm columnrequires 21% of the flow rate of a 4.6-mm column.• Given the same mass of sample, the peak area scalesaccording to the inverse square of column diameter;for example, a 1.0-ng sample injected on a 2.1-mmcolumn would yield 4.8 times more area than on a4.6-mm column.• A 3-µm, 4.6 × 150-mm column delivers similar peakwidths as a 5-µm, 4.6 × 250-mm column in 60% of thetime.• 50-mm columns sometimes provide adequate resolutionwhile saving time and solvent.• LC-MS applications can increase throughput usingshorter column or larger particle size than required forUV detection.• When using gradient elution, to preserve the relativeelution times, scale the gradient time proportionally tothe column length.• When run time is more important than sharp peaks,optimize the resolution by shortening the columninstead of lengthening the gradient.• By default, a 5-µm, 150-mm-long column is always agood starting point.- 8 -www.dionex.com

IntroductionMobile Phase ConsiderationsThe effect of mobile phase composition on column lifetimeshould be part of the consideration for designing a method.Use the highest practical quality of water, solvent, and buffercomponents; HPLC-grade material has low UV absorbance andis submicron-filtered by the manufacturer. To prevent fouling ofyour Acclaim column, use mobile phases that have been filteredthrough a 0.5-µm or smaller filter.Both alkaline conditions and strong acid conditions willdegrade silica-based columns over time. The silica substrate resistsacids, but is soluble in alkali. The upper pH limit is determinedby the rate of dissolution of the silica gel, which is also affectedby buffer ions and organic co-solvents. When organic solventsare added to buffers of inorganic anions such as phosphate, thebuffer becomes more alkaline; when organic solvents are addedto buffers of organic bases, such as TRIS, the buffer becomes lessalkalineAcid conditions hydrolyze the bonded layer, rather than thesilica itself, and the rate of this effect is proportional to the hydrogenion concentration. The rate of hydrolysis is slower toward themiddle of the pH range. Fortunately, organic modifiers usuallyprovide protection against degradation.INTRODUCTIONwww.dionex.com - 9 -

IntroductionINTRODUCTIONAcclaim Columns Dedicated to HighQualityReliability and DurabilityAt Dionex we know that quality and reliability are essential toa successful analysis. Our columns are thoroughly tested individually,so that chromatographers can have full confidence in them.Manufacturing starts with an ultrapure silica substrate, using onlycarefully selected lots with narrow ranges of physical parameters.By design, the bonding processes are clean and repeatable with nounexpected changes in performance. Each batch of bonded silicareceives a full suite of validation tests appropriate to its intendeduse. The bonded silica is packed in precision-polished 316 stainlesssteel hardware using highly reliable processes. Each packedcolumn is tested to ensure the same great performance every time.The quality assurance reports for silica lot validation and columnperformance explain the test protocols, list the specifications, andshow the actual chromatograms.Base AsymmetryAU1.51.00.50Acclaim Columns Base Performance: AmitriptylineDCBA1234Amitriptyline Performance:# Acclaim Type EP Asym. N/mD 300 Å, 3 µm, C18 1.02 138KC 120 Å, 3 µm, C8 1.20 106KB 120 Å, 3 µm, PA C16 1.33 105KA 120 Å, 3 µm, C18 1.21 123KPeaks:1. Uracil2. Propranolol3. Toluene4. Doxepin55. Amitriptyline0 Minutes1220385Performance IndicatorsAcclaim bonded-silica columns have been designed to meetthe high quality standard needed in laboratories today. The innovativesurface chemistries deliver exceptional peak efficienciesfor a broad range of analytes. To ensure optimal performance, allDionex Acclaim products are thoroughly characterized using anumber of performance indicators, including surface coverage ofthe bonded phase, metal contamination, steric selectivity, columnpolarity, column hydrophobicity, and low silanol activity forbases. The specialty columns are also application-tested for theirspecific analysis, to ensure that each lot of bonded silica provideshigh-performance separations. The following charts and chromatogramsexplain how to interpret each of these tests.Good surface coverage of the silica gel with the bonded phase, and exhaustiveendcapping ensure that Acclaim columns provide symmetrical peaksand superior performance for the separation of basic drugs. Amitriptylineis a well-known example of a basic pharmaceutical that interacts stronglywith residual silanol groups on the silica gel, to produce asymmetricalpeaks.Acid AsymmetryPeak Asymmetry of AcidsPolarity IndexPolarity= [ K BBA · K’ phenol /K’ 2 toluene] × 100100806040201314202852535980AU01 231 23 4246Polar-EmbeddedLeading Brand E5 (As. = 3.49)5 (As. = 1.03)AcclaimPolarAdvantage64 6 8 10MinutesMobile Phase: 40/60 v/v 50 mM phosphate buffer,pH 3.25/CH 3 CNFlow Rate: 1.0 mL/minInj. Volume: 5 µLTemperature: 30 °CDetection: 254 nmPeaks: 1. Uracil2. Pyridine3. Phenol4. N,N-Dimethylaniline5. p-Butylbenzoic acid6. Toluene176540Acclaim C18Luna(2) C18Acclaim PASymmetryShield C18Discovery RP-Amide C16ProntoSIL EPS C18Acclaim PA2ZORBAX Bonus-RP20152Many polar-embedded columns perform well for a limited group of compounds.Acclaim PolarAdvantage columns use state of the art methodsof synthesis to eliminate unwanted secondary interactions. The result isoutstanding performance for hydrophilic and hydrophobic acidic, basic,and neutral analytes.The polarity index chart above ranks commercially available stationaryphases according to their polarity relative to one another, under the sameconditions. The more similar the polarity index of two stationary phases,the more similar their separation of polar compounds should be. If onecolumn does not provide adequate resolution, choosing a column with adifferent polarity index may deliver a better separation.- 10 -www.dionex.com

IntroductionMetal Contamination400300Column: Acclaim 300 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: 50/50 v/v water/methanolTemperature: 30 °CFlow Rate: 1.0 mL/minRA5.75Evaluation of Metal Contamination1Inj. Volume: 10 µLDetection: 214 nmSamples: 1. 4,4'-bipyridyl 250 ng/µL2. 2,2'-bipyridyl 10002Competitor A, 5 µmSteric SelectivityComparison of Steric Selectivity forAcclaim 120 C18 and Acclaim PAAcclaim 120 C182 3 42 34Competitor 1AU11Competitor 212 3 4Columns: As indicatedMobile Phase: 90% methanolTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLPeaks: 1. Toluene2. Phenanthrene3. o-Terphenyl4. TriphenyleneINTRODUCTION200mAU10002.121.445.154.481.29Competitor B, 5 µmCompetitor C, 3 µmCompetitor D, 5 µmCompetitor E, 3 µmAcclaim 300 C18, 3 µm–500 1 2 3 4 5 6MinutesNote: Ratio of asymmetries (RA) is quoted as the asymmetry of Peak 2 divided by Peak 1.Metal contamination can interfere with the separation of many differentsubstances, resulting in peak tailing and concomitant poor peakresolution. 2,2’-bipyridyl is a strong chelating agent, and will revealany contamination by metal ions. The figure above shows that Acclaimoutperforms five competitor columns in this test.17590AU0 2 4 6 8 10Minutes12, 34Competitor 3Competitor 21 2 3 4112 3 4Competitor 12 3 Acclaim PolarAdvantage40 2 4 6 8 10 12MinutesPeaks:1. Toluene 2. Phenanthrene 3. o–Terphenyl 4. TriphenyleneMe17037Column: 5 µmDimensions: 150 × 4.6 mmMobile Phase: 90/10 v/v MeOH/D.I. H 2OTemperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: UV, 254 nmDepending on the bonding chemistry, various C18 columns can havedifferent selectivity to molecular shape. Acclaim may be just what youneed for your unique separation challenge.19309www.dionex.com - 11 -

Acclaim 120 C18120 C18Pa r t Tw o: Co l u m n sChapter 1: Acclaim 120 C18High performance reversed-phase columns for theseparation of small moleculesThe Acclaim 120 C18 series columns feature a densely bondedmonolayer of octadecyldimethylsiloxane on a highly pure, spherical,silica substrate with 120 Å pore structure.The Acclaim 120 C18 columns are the classic reversed-phasecolumns. These columns are recommended for general-purposereversed-phase applications where high surface coverage, lowsilanol activity, and excellent efficiency are required. The Acclaim120 C18 columns feature:• Highly efficient, symmetrical peaks for difficult basicand chelating analytes• Ultrapure silica substrate• Optimized surface pretreatment, proprietary highdensitybonding process, and double endcapping• Reliability designed into the manufacturing processand assured by thorough and appropriate testing• High hydrophobicity and low polarity yield highselectivity for hydrophobic substances• LC/MS compatible• Wide range of applications in pharmaceutical,environmental, food testing, and product-qualitytesting for small moleculesBondingUSP codeEndcappingC18 monomericL1Double% C 17.5–18.9Pore sizeCH 3Si CH 2 (CH 2 ) CH 3CH 3O 16Physical Specifications120–140 ÅSurface area 290–320 m 2 /gParticle diameter 2.9–3.2 or 4.2–4.5 µmPerformance SpecificationsLot Polar selectivity ratio 0.12–0.16Base asymmetry0.98–1.50 (5 µm)0.98–1.60 (3 µm)Metal activity ratio 0.90–1.30Steric selectivity ratio 1.58–1.65Column Efficiency (N/m) 90,000 (5 µm)120,000 (3 µm)Asymmetry (EP) 0.98–1.20Retention time8.41–9.29 (5 µm)8.31–9.19 (3 µm)Chromatographic specifications for Acclaim 120 C18 4.6 × 150 mm columns.Please refer to Part 4 for a detailed description of the tests and specifications.Specifications are subject to change at the discretion of Dionex.018-01-062018-01-060018-01-058018-01-056018-01-054018-01-046b018-01-046a018-01-032018-01-020Control Chart for Acclaim 120 C18 5 µm0 1 2 3 4 5 6 7k'Control chart showing k’ (phenanthrene) for nine lots of Acclaim 1205-µm C18. The data show the average and standard deviation for allcolumns shipped from that lot.54321LC-MS of Pesticides23028Column: Acclaim 120 C18, 3 µmDimensions: 2.1 × 100 mmMobile Phase: 70/30% methanol/0.1% formic acidFlow rate 0.25 mL/minDetection: AQA MS, +ESI, 30V, 200 °C,SIM as indicatedm/z mass injectedPeaks: 1. Monuron 199 1 ng2. Altrazine 216.1 1 ng3. Propazine 230 2 ng4. Diuron 233 2.5 ng5. Linuron 249 2 ngRecommended Ranges of OperationTo maintain wetting of the hydrophobic surface, the recommended range fororganic composition is 20–100%. The type 316 stainless steel column hardwareis compatible with common HPLC mobile phases. To prevent corrosion, highconcentrations of halide ions at pH

Acclaim PolarAdvantage IIAU12132Dewetting Test in 100% Aqueous EluentColumns: 5 µm, 4.6 x 150 mmMobile Phase: 2.5 mM methanesulfonic acidTemperature: 30 °CFlow Rate: 1.0 mL/minConventional C18Before stop-flow testAfter stop-flow test3AUInj. Volume: 5 µLDetection: UV, 254 nmPeaks:111. Cytosine2. Uracil3. Thymine22Acclaim PA2Before stop-flow test3After stop-flow test3% Remaining Retention11010090807060Accelerated Stability Test at Low pHCompetitor CCompetitor ACompetitor BAcclaim PA2500 10 20 30 40Time of Purge (Days)Column: See chart, 5 µmDimensions: 4.6 × 150 mmMobile Phase: CH 3 CN/1% TFA*,pH 1 v/v 50/50Temperature: 50 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: UV, 254 nmAnalyte: Toluene*Trifluoroacetic acid201420 2 4 6 8 10MinutesHydrolytic Stability0 2 4 6 8 10MinutesDespite their many advantages, the hydrolytic stability ofpolar embedded phases is often inferior to conventional C18 phasesas a result of lower ligand coverage and the hydrophilic natureof the embedded group. The Acclaim PA2 has been designed toprovide all the advantages of polar embedded phases, but withenhanced stability at both low and high pH.HPLC separations of polar analytes are often run at low pH(PH

Acclaim OAChapter 6: Acclaim OA forOrganic AcidsOptimized and application-tested for the analysis ofhydrophilic organic acidsAcclaim OA organic acid columns are special reversed-phasesilica columns, designed for the separation of aliphatic and aromaticorganic acids at low pH with UV-Visible detection. TheAcclaim OA uses a patented polar-embedded stationary phasethat allows a broad range of operating conditions, including 100%aqueous mobile phases.The Acclaim OA is the recommended column for determiningsmall hydrophilic organic acids, C1 to C7 aliphatic acids, andhydrophilic aromatic acid and is also valuable for the analysis andquality assurance of food and beverage products, pharmaceuticalpreparations, plating baths for semiconductor manufacturing,manufacturing chemicals, and chemical intermediates.The Acclaim OA columns are packed in metal-free PEEK columnbodies, to eliminate unwanted interactions between the analytesand the column body. Benefits of the Acclaim OA columnsinclude:• Use-tested to guarantee consistent organic acidseparations• Compatible with 100% aqueous mobile phases, optimumfor reversed-phase retention of hydrophilic aliphaticorganic acids• Hydrolytic stability at low-pH conditions, optimumfor reversed-phase retention of organic acids• Ideal selectivity for separating a wide spectrum oforganic acids, including hydrophilic aliphatic acids,C1 to C7 aliphatic acids, aromatic acids, and selectedamino acids• Excellent peak shapes for organic acids• High reversed-phase capacity with retention similar tostandard C18 phases• Suitable for analysis of foods, beverages, pharmaceuticals,chemical intermediates, and environmentalsamples• Ideal for quality assurance offering fast run times andminimal sample preparationOARecommended Ranges of OperationpH Range 2.0–8.0Pressure limit 4500 psi (2000 psi above 50%organic solvent content)Organic solventsHydrophilic Organic AcidsHO OHO OO OHOO OHOHOOH OHOHOO OHH OHHOOOHOHO1. Oxalic acid 2. Tartaric acid 3. Formc acid 4. Malic acid 5. iso-Citric acidOHOOO OHOOOHOH OH OHH OHOHO3 CH 3 COHHOOOOHO6. Lactic acid 7. Acetic acid 8. Citric acid 9. Succinic acid 10. Fumaric acidHOOO O O OOH OHOH OH11. cis-Acontic acid 12. trans-Aconitic acidOOH0–100% acetonitrile or methanol19511Surface chemistryUSP codeEndcappingPore sizePhysical SpecificationsProprietarynaYes120–140 ÅSurface area 290–320 m 2 /gParticle diameter 4.2–4.5 µmGuaranteed Performance for Organic AcidsThe performance of the Acclaim OA columns is guaranteedfor the separation of aliphatic and aromatic organic acids at lowpH. These columns undergo extensive testing to ensure columnto-columnreproducibility, and are shipped with quality assurancereports detailing these tests. The Acclaim OA columns are usetestedfor two specific applications—isocratic and gradient.These operating conditions provide a good starting point formethods development.Performance SpecificationsLot Metal activity ratio 0.80–1.50Gradient organic acids Baseline drift

Acclaim OAIsocratic Separation of Hydrophilic Organic AcidsRuggedness Test at pH 2.680.025AU021 3 45 6 7 8 91011120 5 10 15MinutesColumn: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phase: 100 mM Na 2 SO 4 , pH 2.65 (adjustedwith methanesulfonic acid)Temperature: 30 °CFlow Rate: 0.6 mL/minInj. Volume: 5 µLDetection: UV, 210 nmPeaks: 1. Oxalic acid 15 mg/L (ppm)2. Tartaric acid 1203. Formic acid 1804. Malic acid 1205. iso-Citric acid 1206. Lactic acid 1807. Acetic acid 1208. Citric acid 1209. Succinic acid 12010. Fumaric acid 711. cis-Aconitic acid **12. trans-Aconitic acid **** 7 ppm total for cis and trans isomers20011AU021 3 4 85 6 7291011 121013 485 6 7 9 11 122Injection 1Injection 10894 6 8MinutesColumn: Acclaim OA, 5 µmDimensions: 4 × 150 mmMobile Phase: 40 mM Na 2 SO 4 , pH(adjusted with methanesulfonic acid)Temperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: UV, 210 nmPeaks: 1. Oxalic acid 15 mg/L (ppm)2. Tartaric acid 1203. Formic acid 1804. Malic acid 1205. iso-Citric acid 1206. Lactic acid 1807. Acetic acid 1208. Citric acid 1209. Succinic acid 12010. Fumaric acid 311. cis-Aconitic acid**12. trans-Aconitic acid*** *3 ppm total for cis andtrans isomers20012Gradient Separation of C1–C7 Aliphatic Organic Acids0.1800.100AU01234, 5Minutes6789 100 5 10 15 20Column: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phases: A: CH 3CNB: 2.5 mM methanesulfonic acidGradient: -10 0 1 15 20 min.2 2 2 60 60 %A98 98 98 40 40 %BTemperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 30 µLDetection: UV, 210 nm with blank subtractionPeaks: 1. Formic acid 10 mmol/L2. Acetic acid 103. Propionic acid 104. Butyric acid 105. Isobutyric acid 106. Isovaleric acid 107. n-Valeric acid 108. Isocaproic acid 109. n-Caproic acid 1010. Heptanoic acid 10OA23611Ordering Information: Acclaim OAThe standard particle size is nominally 5 µm. Analytical columns are available in 4.0-mm diameters; standard lengths are 150 and 250 mm.Guard cartridges in 4.3 × 10 mm are recommended to protect your analytical columns. Other geometries in PEEK or stainless steel areavailable; inquire with your Dionex sales representative.Please order through your local Dionex office or distributor. Refer to the following part numbers.Acclaim OA Analytical Columns5 µm 4.0 × 150 mm ............................... 0629035 µm 4.0 × 250 mm ............................... 062902Acclaim OA Guard Columns5 µm 4.3 × 10 mm . ...............................062925Biocompatible cartridge holder for2.1 × 10 mm and 4.3 × 10 mm guards ..................059456Guard to analytical column coupler ...................059457Holder and coupler kit. ..............................059526These columns are designed for optimal performance using Dionex UltiMate 3000 and ICS-3000 chromatography instruments.www.dionex.com - 23 -

Acclaim SurfactantChapter 7: Acclaim SurfactantOptimized and application-tested for the analysis ofanionic, nonionic and cationic surfactantsThe Acclaim Surfactant ColumnThe Acclaim Surfactant is a silica-based, reversed phase columnthat features a proprietary bonding chemistry optimizedand application-tested for separating a variety of surfactants. Thisspecialty column is ideal for separating cationic, nonionic, anionicand amphoteric surfactants in a single chromatographic run usingsimple and volatile mobile phases. The Acclaim Surfactant columnis the ideal tool for the analysis of surfactants including linear alkylsulfonates,alkyl sulfates, alkyl quaternary ammonium salts, ethoxylatedquats, Triton-X, PEGs and many more. It allows analysis ofsurfactants in various matrices including consumer products, formulationsfor pharmaceutical products, and environmental samples.mVInorganic Anions, Hydrotropes, Cationic, Nonionic,Amphoteric, and Anionic Surfactants213Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN,(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25% to 85% A in 25 min,then hold 85% A for 10 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: ELS detector45670 5 10 15 20 25 30 35Minutes89101112Peaks:1. Chloride2. Bromide3. Nitrate4. Xylene sulfonate5. Laurylpyridinium chloride6. Lauryldimethylbenzylammoniumchloride7. Triton X-1008. Cetyl betaine9. Decyl sulfate10. Dodecyl sulfate11. C 10 -LAS12. C 11 -LAS13. C 12 -LAS14. C 13 -LAS131422793Benefits of the Acclaim Surfactant column include:• Ideal selectivity for separating different types ofsurfactants• Excellent peak shapes, especially for cationic surfactants• Improved resolution for ethoxylated surfactants comparedwith other columns• Ability to analyze hydrophilic hydrotropes• Methods compatible with various detection methodsincluding UV, ELSD, suppressed conductivity,and more• Broad range of applicationsHydrotrope, Cationic, Nonionic, Amphotericand Anionic SurfactantsmVµSSeparation of Commonly Used Cationic SurfactantsELS Detector123Suppressed Conductivity1 234455Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN(B) 20 mM Acetic acidGradient: 20– 50% A in 15 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELS detector orSuppressed CD (CMMS III)Peaks: 1. Lauryl pyridinium chloride2. Lauryldimethylbenzylammonium chloride3. Octylphenoxyethoxyethyldimethylbenzyl ammoniumchloride4. Cetyltrimethylammoniumchloride5. Cetylpyridinium chlorideSURFACTANTCH 3CH 3SO 3 NaSodium Xylene Sulfonate+N Cl–Laurylpyridinium Chloride0 510 15Minutes21086CH 3 –ClCH 2 N +CH 3Lauryldimethylbenzyl Ammonium ChlorideCetyl BetaineSodium Dodecyl SulfateOO-S-ONaOMe+NMeC 8 H 17(OCH 2 CH 2 ) n OHn ~ 10Triton X-100OOO-S-ONaO–OSodium Decyl SulfateHCH 3(CH 2 ) n C (CH 2 ) m CH 3SO Na m + n = 103Sodium Dodecylbenzene Sulfonates22792BondingUSP codePore sizePhysical SpecificationsProprietaryna120–140 ÅSurface area 300 m 2 /gParticle diameters 4.2–4.5 µmRecommended Ranges of OperationThe type 316 stainless steel column hardware is compatible with common HPLCmobile phases. To prevent corrosion, high concentrations of halide ions atpH

Acclaim SurfactantGuaranteed Performance for SurfactantsThe exceptional performance of Acclaim Surfactant columnsfor the separation of anionic, cationic and non-ionic surfactantsis confirmed with every new lot of bonded silica that is manufactured.In addition each column is individually tested with a simplemixture of anionic, cationic and non-ionic probes, to ensure thatevery column is packed consistently and well.Performance SpecificationsColumn Efficiency (N/m) 76,500Asymmetry (EP) 0.95–1.32Retention time 7.50–8.60Example chromatographic specifications for Acclaim Surfactant4.6 × 150 mm. Please refer to Part 4 for a detailed description of thetests and specifications. Specifications are subject to change at thediscretion of Dionex.900LOT VALIDATION #6 #1, SFC_P01 (isocratic)Vlittle or no modifications, because both detectors share the samemobile phase requirements. Conductivity detection of cationic surfactantscan also be used. Because of the versatility of this column,methods using all these detectors have been developed and can befound in this catalog, in the Acclaim product information bulletin,or on the Dionex website.AbundanceAnalysis of POE (1) Lauryl Sulfate (LC-ESI-MS)12:712:812:912:512:612:412:212:312:012:1 (alkyl chain: n)14:2Column: Acclaim Surfactant, 5 µmDimension: 4.6 × 150 mmMobile Phase: CH 3 CN/0.1 M NH 4 OAc,pH 5.4 v/v 60/40Temperature: 25 °CFlow Rate: 1 mL/min (0.2 mL/min to MS)Inj. Volume: 25 µLDetection: MS (ESI negative)Sample: POE (1) lauryl sulfate (100 ppm)14:014:1n ~ 1OSodium POE (1) Lauryl SulfateO(OCH 2 CH 2 ) n - O-S-O Na1 23 450050 10 20 30 40Minutes22795-100min0.0 10.0 20.0 30.0Chromatogram of a group of surfactants, taken from the certificate forlot #013-05-002. Dionex monitors and controls the selectivity of cationic,nonionic and anionic surfactants using the recommended standard conditionsfor this column.Detector CompatibilityAlthough UV detection is the most commonly used of HPLCdetectors, not all surfactants possess chromophores. This problemcan be overcome by the use of a universal detector, such asevaporative light-scattering detection (ELSD) or refractive index(RI) detection. ELSD is compatible with gradient methods and isfar more sensitive than RI. In addition, methods developed withELSD can be easily transferred to LC-ESI-MS applications withAUDimensions: 4.6 × 150 mmMobile Phases: Acetonitrile/100 mMNH4OAc, pH 5.4,v/v 70/30 for Acclaim SurfactantAcclaim SurfactantC11-LASv/v 50/50 for Acclaim PAC10-LASTemperature: 30 °CC12-LASFlow Rate: 1 mL/minInj. Volume: 5 µLC13-LAS Detection: UV, 225 nmC11-LASC10-LASC12-LASAcclaim PAHC13-LASCH 3 (CH 2 ) n C (CH 2 ) m CH 30 5Analyis of Sodium Dodecylbenzene Sulfonate (LAS)10 15 20 25MinutesSO 3 Nam + n =7-10Sodium Dodecylbenzene Sulfonate21078SURFACTANTOrdering Information: Acclaim SurfactantStandard particle size is nominally 5-mm. Analytical columns are stocked in 4.6–mm-diameter widths and a couple of lengths. Guard cartridgesin 4.3-mm sizes, packed with 5-mm particles are recommended to protect the analytical columns. Other geometries can be made toorder. Enquire with your Dionex sales representative.Please order through your local Dionex office or distributor. Refer to the following part numbers.Acclaim Surfactant Analytical Columns5 µm 4.6 × 150 mm ............................... 0632015 µm 4.6 × 250 mm ............................... 063203Acclaim Surfactant Guard Columns5 µm 4.3 × 10 mm (pack of 2) . .....................063215Biocompatible guard cartridge holder ................059456Guard for analytical column coupler. ..................059457Acclaim holder and coupler kit .......................059526These columns are designed for optimal performance using Dionex UltiMate 3000 and ICS-3000 chromatography instruments.www.dionex.com - 25 -

Acclaim ExplosivesChapter 8: Acclaim ExplosivesE1 and E2 ColumnsFor the separation of the 14 explosives targeted byEPA SW-846 Method 8330The novel and unique chemistries of these columns providesuperior resolution to other commercially available columns. TheAcclaim Explosives columns provide a "Total Solution" to EPAMethod 8330; two columns each of which provides baseline resolutionof all 14 explosives. The Acclaim Explosives E1 providesselectivity similar to conventional C18 columns. The Acclaim E2column provides selectivity that is complementary to that obtainedusing the Acclaim E1 and is recommended for the confirmatoryanalysis. With their complementary selectivities, the E1 and E2provide both primary and confirmatory columns.EXPLOSIVESAcclaim Explosives ColumnsThe Acclaim Explosives E1 and E2 are specialty reversedphase columns that have been optimized and application testedfor the separation of the 14 explosives targeted by EPA SW-846Method 8330, “Nitroaromatics and Ntramines by HPLC.”Fourteen Target Explosives Listed in EPA Method 8330CH 3NO 2O 2 N NO 2NO 2 N N N NO 2NNO 2NO 2TNTHMXNO 2 N2-NT 4-NT 3-NTNO 2NRDXNNO 2NO 2 NO 2NO 2NH 2CH 3NO 2CH 3CH 3NO 2CH 3CH 3NO 2O 2 N NO 2NO 2NO 22,6-DNT 2,4-DNT1,3-DNB NBCH 3CH 3 NO 2O 2 NO 2 N NO 2NO 2 NH 2O 2 N NO 22-A-4,6-DNT4-A-2,6-DNT 1,3,5-TNBH 3 C NO 2NO 2 N NO 2NO 2Tetryl22770AUBaseline Separation of 14 Target Explosive CompoundsListed in EPA SW-846 Method 8330Peaks: 1. HMX2. RDX3. 1,3,5-Trinitrobenzene4. 1,3-Dinitrobenzene5. Nitrobenzene6. Tetryl7. 2,4,6-Trinitrotoluene1122334545 768 9 10 1112 1314Acclaim Explosives E10 9 18 273645MinutesBenefits of the Acclaim Explosives E1 and E2 columns76 10• Superior resolution for all 14 explosives listed in EPAMethod 8330• Complementary selectivity between Acclaim E1 andE2, for a complete solution for explosives analysis• 5-µm, 120 Å high purity silica gel for high-resolutionseparations, with good mechanical stability• Unique selectivity of the Acclaim E2 allows for simultaneousseparation of some nitrate ester explosives118. 4-Amino-2,6-Dinitrotoluene9. 2-Amino-4,6-Dinitrotoluene10. 2,6-Dinitrotoluene11. 2,4-Dinitrotoluene12. 2-Nitrotoluene13. 4-Nitrotoluene14. 3-Nitrotoluene81291314Acclaim Explosives E222787- 26 -www.dionex.com

Acclaim ExplosivesSeparation of 18 Explosives Showing the Unique Selectivityof the Acclaim Explosives E2 ColumnAU1Column: Acclaim Explosives E2, 5 µm Peaks: (25 ppm each)Dimensions: 4.6 × 250 mm1. 2,4-Di-Amino-6-NitrotolueneMobile Phase: 48/52 v/v MeOH/H 2 O2. HMXTemperature: 30 ºC3. EGDNFlow Rate: 1 mL/min4. RDXInj. Volume: 5 mL5. 1,3,5-TrinitrobenzeneDetection:5UV, 210 nm6. 1,3-Dinitrobenzene7. Nitrobenzene8. 2,4,6-Trinitrotoluene9. Tetryl10. 2,6-Dinitrotoluene11. 2,4-Dinitrotoluene12. 2-Nitrotoluene13. 4-Nitrotoluene14. 3-Nitrotoluene215. 4-Amino-2,6-Dinitrotoluene416. 2-Amino-4,6-Dinitrotoluene6 717. DMDNB818. PETN9 1011 12 14 15 1631317 18Recommended Ranges of OperationThese operating conditions follow EPA Method Conditions.Acclaim Explosives E1 Acclaim Explosives E2Mobile phase MeOH:H 2 O 43:57 MeOH:H 2 O 48:52Temperature 31 ± 1 °C 29 ± 1 °CFlow rate 1.0 mL/min 1.0 mL/minGuaranteed Performance for EPA Method 8330The exceptional performance of both Acclaim Explosives columnsfor EPA Method 8330 is guaranteed. Both products undergoextensive testing to ensure column-to-column reproducibility andare use-tested for this specific application. Both columns performreproducibly under their optimized conditions, although separationconditions for the Acclaim Explosives E2 can be readilychanged to accommodate inclusion of additional explosives compounds.0 10 20 304050Minutes22782Physical SpecificationsAcclaim Explosives E1 Acclaim Explosives E2Bonding Proprietary ProprietaryPore size 120–140 Å 120–140 ÅSurface area 300 m 2 /g 300 m 2 /gParticle diameters 4.2–4.5-µm 4.2–4.5µmPerformance SpecificationsAcclaim Explosives E1Efficiency (N) 20,000 20,000Asymmetry 0.95–1.32 0.95–1.32Retention time 6.96–7.54 7.76–8.64Acclaim Explosives E2Example chromatographic specifications for Acclaim E1 and E24.6 × 250 mm. Please refer to Part 4 for a detailed description of thetests and specifications. Specifications are subject to change at thediscretion of DionexOrdering Information: Acclaim Explosives ColumnsStandard particle size is nominally 5-µm. analytical columns are stocked in 4.6–mm-diameter widths and 250-mm length. Guard cartridgesin 4.3-mm sizes, packed with 5-µm particles are recommended to protect the analytical columns. Other geometries can be made to order.Enquire with your Dionex sales representative.Please order through your local Dionex office or distributor. Refer to the following part numbers.Acclaim Explosives Analytical ColumnsAcclaim Explosives Guard Columns5 µm 4.6 × 250 mm E1 ............................ 064305 5 µm 4.3 × 10 mm E1 (pack of 2) . ..................0643035 µm 4.6 × 250 mm E2 ............................ 064309 5 µm 4.3 × 10 mm E2 (pack of 2) . ..................0643075 µm E1 and E2 kit ............................... 064312 Biocompatible guard cartridge holder ................059456Guard for analytical column coupler. ..................059457Acclaim holder and coupler kit .......................059526These columns are designed for optimal performance using Dionex UltiMate 3000 and ICS-3000 chromatography instruments.EXPLOSIVESwww.dionex.com - 27 -

ApplicationsPa r t Th r e e: ApplicationsContentsAPPLICATIONSPharmaceuticalIsocratic Resolution of Antihistamines and TheirImpurities on Acclaim 120 Å, C18............................ 29Separation of Four Benzodiazepine Drugson Acclaim 120 C18..................................................... 29Separation of Five Antidepressant onAcclaim PA................................................................... 29Spectinomycin and Lincomycin on Acclaim PAwith Electrochemical Detection................................. 29Beta Blockers on Acclaim PA2.................................. 29Venlafaxine and Related Substances........................ 29Assay for Pramoxine in Topical Anesthetic Ointmenton Acclaim 120 Å, C18 with SPE Cleanup onOnGuard II RP.............................................................. 30Determination of Four Active Ingredients in aMultisymptom Cold Remedy Using Acclaim PA. 30Separation of Basic Drugs on Acclaim 120 Å, C18....30Baseline Separation of Catecholamines & Serotoninat Physiological Concentrations on Acclaim120 Å, C18 with Electrochemical Detection............ 30Gradient Resolution of Sulfa Drugs onAcclaim PA................................................................... 30Catecholoamines in Urine on Acclaim PA2............ 30Bromide and Organic Acids in Cough SyrupUsing Acclaim OA....................................................... 31Isocratic Separation of Six Cardiac Anti-Arrhythmic Drugs (Beta-Blockers) on 120 Å C8.... 31Sulfa Drugs on Acclaim PA2..................................... 31Active Ingredients in Zantac 75 Acid-ReducerMedication..................................................................... 31Loratidine and Pseudoephedrine on Acclaim PA. 31Propafenone and Related Substances...................... 31Antidepressants on Acclaim PA2............................. 32Active ingredients in Cough Syrup.......................... 32Barbiturates on Acclaim 120 C18.............................. 32Budesonide and Related Substances........................ 32Hydrocortisone in Skin Ointment............................ 32Phenytoin and Related Substances........................... 32Active Ingredients in Sunscreen Lotion onAcclaim 120 Å, C18 Using ASE Extraction............. 33Glucocorticosteroids in Serum on Acclaim 120 C18.33Nonsteroidal Anti-inflammatory Drugs.................. 33Fexofenadine & Pseudoephedrine on Acclaim PA...33EnvironmentalLC/MS Detection of Herbicides onAcclaim 120 C18........................................................... 34LC Separation of Nitrosamines in EPA 8270Using Acclaim PA........................................................ 34Benzidines by EPA Method 605 on Acclaim120 C18 with Electrochemical Detection................. 34Phenol Standards by EPA Method 604 onAcclaim PA................................................................... 34Separation of Phthalate Esters in EPAMethod 606 Using Acclaim PA................................. 34Brodifacoum in Rodent Bait...................................... 34Thirteen Carbonyl-DNPH Derivatives forCARB Method 1004 on Acclaim 120 Å, C18........... 35Carbamate Insecticides in EPA 531.1 onAcclaim 120 C8 with MS Detection.......................... 35Improved Separation of 13 Carbonyl-DNPHDerivatives by CARB Method 1004 UsingCoupled Acclaim PA and Acclaim C18Columns......................................................................... 35Polynuclear Aromatic Hydrocarbon Standardsby EPA Method 8310 on Acclaim PA..................... 35LC Separation of Benzidines in EPA 8270 UsingAcclaim PA................................................................... 36Sensitive Detection of Triazine Herbicides onAcclaim 120 C18 with MS Detection........................ 36EPA Method 8330 Using Acclaim Explosives E2.. 36EPA Method 8330 Using Acclaim Explosives E1.. 36Four Antibiotics in Animal Feed onAcclaim 120 C18........................................................... 36Carbamate Insecticide Standards in EPA 531.1 onAcclaim PA................................................................... 36Food and BeverageFat-Soluble Vitamin Standards onAcclaim 120 C18........................................................... 37Amino Acids in Vitamin Premix on Acclaim OA.. 37Organic Acids in Orange Juice on Acclaim OA..... 37Organic Acids in White Wine on Acclaim OA...... 37Carotenoids in Serum on Acclaim PA..................... 37Pigments in Turmeric on Acclaim PA2................... 37Assay of Aspartame and Its Impurities on ThreeFormats of Acclaim 120 C18 Columns..................... 38Assay for Water-Soluble Vitamins in VitaminTablets Using Acclaim PA.......................................... 38Nitrofuran Antibiotic Residues in Animal Feed.... 38Fat-Soluble Vitamins and Carotenoids inVitamin Tablets............................................................ 38Determination of Quinine in Tonic Water.............. 39Determination of Ingredients of an Energy Drink. 39Hops Extract on Acclaim 120 C18............................ 39Sudan Dyes in Paprika............................................... 39Water-Soluble Vitamins Using Acclaim PA2......... 39Parabens on Acclaim PA2.......................................... 39Four Aflatoxin Standards on Acclaim 120 C18...... 40Fat-Soluble Vitamin Standards UsingAcclaim PA................................................................... 40Glutamate in Beer by Automated Precolumn OPADerivatization............................................................... 40Determination of Soft Drink Ingredients................ 40Tetracycline Antibiotic Residues in Pork................ 40Fat-Soluble Vitamins and Carotenoids in DryPet Food......................................................................... 40Antioxidants in Edible Oils by AOAC 983.15 onAcclaim 120 C18........................................................... 41Sulfonamide Anibiotic Residues in Milk................. 41Bitter Principles in Beer on Acclaim 120 C18......... 41ChemicalSeparation of Cationic Surfactants Using ELSD.... 42Separation of Ethoxylated Quats.............................. 42Analysis of ZONYL FSO Fluorosurfactant............. 42Analysis of a Shampoo............................................... 42Analysis of a Fabric Softener..................................... 42Analysis of a Mouthwash........................................... 42Separation Hydrotropes, Anionic, Cations, andAmphoteric Surfactiants............................................. 43Analysis of Sodium Lauryl Sulfate UsingLC-ESI-MS..................................................................... 43Analysis of PEG monoethyl ether (MW-550)......... 43Analysis of Triton X-100 Using LC-ESI-MS............ 43Analysis of a Laundry Washing Detergent............ 43Analysis of a Dishwashing Liquid........................... 43Analysis of Ammonium Laureth Sulfate UsingConductivity Detection............................................... 44Separation of Inorganic Anion, Hydrotropes,Cations, Nonionic, Amphoteric and AnionicSurfactiants.................................................................... 44Analusis of Toilet Bowl Cleaner............................... 44Analysis of a Strongly Hydrophilic Hydrotrope... 44Analysis of Quats Using LC-ESI-MS........................ 44Analysis of NEODOL 25-7......................................... 44Analysis of PEG-200 Distearate................................. 45Separation of Cationic Surfactants withSuppressed Conductivity Detection......................... 45Separation of Quaternary ImidazoliniumCompounds................................................................... 45Separation of Different Polyethylene Glycols........ 45POE(30) Ammonium Lauryl Sulfate........................ 45Sudan Dyes on Acclaim 120 C18.............................. 45BioscienceTryptic Peptide Map of Cytochrome con Acclaim 300 C18..................................................... 46Capillary LC for Peptide Mapping UsingAcclaim 300 C18.........................................................46Separation of Protein Standards by Micro-LC onAcclaim 300 C18........................................................... 46Tryptic Peptide Map of Bovine Serum Albuminon Acclaim 300 C18..................................................... 46Cytochrome c from Three Species onAcclaim 300 C18........................................................... 47Protein Mixture by Reversed-Phase HPLC onAcclaim 300 C18........................................................... 47Peptide Test Mix on Acclaim 300 and PA2 withFormic Acid................................................................... 47Comparison of Acid Additives for PeptideAnalysis......................................................................... 47Natural ProductsAlkaloids in Goldenseal Root onAcclaim 120 C18 with ASE Extraction..................... 48Isoflavones in Red Clover on Acclaim 120 C18with ASE Extraction.................................................... 48Flavonoids in Green and Black Teas UsingAcclaim 120 C18........................................................... 48Organic AcidsResolution of Benzene Polycarboxylic Acids onAcclaim OA................................................................... 49Acrylic Acid Oligomers on Acclaim OA................. 49Separation of Hydroxybenzoic Acids onAcclaim OA................................................................... 49- 28 -www.dionex.com

Pharmaceutical ApplicationsPharmaceutical12mAU01Isocratic Resolution of Antihistaminesand Their Impurities on Acclaim 120 C1820 2 4 6 8Minutes341012 14Column: Acclaim 120 C18, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 50 mM sodium acetate(B) MethanolIsocratic: (A) 20%, (B) 80%Temperature: 25 °CDetection: UV, 249 nmPeaks: 1. Thenyldiamine HCI2. Phenothiazine3. Promethazine HCI4. Pyrrobutamine phosphate175100.18AU12345 60 5 10MinutesBeta Blockers on Acclaim PA278Column: Acclaim PA2, 5 µmDimensions: 4.6 × 150 mmMobile Phase: MeOH/0.2% NH 4 OH, pH10, v/v, 60/40Temperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: UV, 210 nmPeaks: (40 ppm each)1. Maleate2. Labetalol3. Metaraminol4. Atenolol155. Acebutolol6. Metoprolol7. Timolol8. Oxprenolol21134Separation of Four Benzodiazepine Drugs on Acclaim 120 C18200mAU01230 1 2 3 4 5 6 7 8 9 10Minutes4Column: Acclaim 120 C18, 5 µmDimensions: 4.6 × 150 mmMobile Phase: 60% acetonitrileFlow Rate: 1.0 mL/minDetection: UV, 235 nmPeaks: 1. Oxazepam2. Clonazepam3. Temazepam4. Diazepam17511350mAU0Separation of Five Antidepressants on Acclaim PA120 1 2 3 4 5 6 7 8Minutes345Column: Acclaim PA, 5 µmDimensions: 4.6 × 150 mmMobile Phase: 80/20 v/v MeOH/30 mM phosphate, pH 6.0Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 220 nmPeaks: 1. Protriptyline 502. Nortriptyline 253. Doxepin 504. Imipramine 405. Amitriptyline 5019299700nC0–1000Spectinomycin and Lincomycin on Acclaim PAwith Electrochemical Detection51013215 20Minutes42553035Column: Acclaim PA, 3 µmDimensions: 2.1 × 150 mmMobile Phase (A) 10 mM acetic acid,3.3 g/L pentanesulfonic acid,pH 4.0 with NaOH(B) 10 mM acetic acid,0.55 g/L pentanesulfonic acid,pH 4.0 with NaOH(C) AcetonitrileGradient: Time % A % B % C0.0 70 30 010.0 70 30 010.1 0 100 020.0 0 100 023.0 0 75 2537.0 0 75 25Flow Rate: 0.30 mL/minTemperature: 40 °CInj. Volume: 10 µLDetection: Postcolumn addition of0.5 M NaOH at 0.12 mL/min;pulsed amperometric detectionPeaks: 1. Benzyl alcohol2. Spectinomycin anomer 13. Spectinomycin anomer 24. Spectinomycin5. Lincomycin800mAU01234Venlafaxine and Related Substances5 6 70 10 20 30 40 50MinutesData courtesy of: V. Bhate of Analytical Solutions89 10 11Column: Acclaim 120 C18, 5 µm,4.6 × 250 mmPump: Summit P680AMobile Phase: (A) 10% acetonitrile; 90% water;1.0 mL/L triethylamine,adj. to pH 3.5 with phosphoric acidMobile Phase: (B) 75% acetonitrile; 25% water;1.0 mL/L triethylamine,adj. to pH 3.5 with phosphoric acidFlow: 1.0 mL/minGradient Times: 0 35 40 45 47 55%A: 95 25 10 10 95 95%B: 5 75 90 90 5 5Temperature: TCC100 thermostat at 35 °CInjection: ASI-100 autosampler; 20 µLDetector: UVD 340U; UV at 226 nmSample: “Resolution” test mixPeaks: 1. Impurity 1 10 µg/mL2. Impurity A 103. FL II 104. Venlafaxine 10005. Impurity III 106. Impurity C 107. Impurity B1 108. PMPA 109. Impurity IV 1010. FL I 1011. Impurity V 10APPLICATIONS2038422660www.dionex.com - 29 -

Pharmaceutical ApplicationsAU10 1 2 3Separation of Basic Drugs on Acclaim 120 C1823 4CBA4 5 6 7 8 9 10 11 12 13.2Minutes5Column: Acclaim 120 C18, 5 µmDimensions: 4.6 × 150 mmMobile Phase: 80/20 methanol/30 mM phosphate, pH 6Temperature : 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 220 nmAmitriptyline Mass:Trace A: 1200 ngTrace B: 400 ng (normalized peak height)Trace C: 94 ng (normalized peak height)Peaks: 1. Uracil2. Propranolol3. Toluene4. Doxepin5. AmitriptylineNote: The high performance of this column ismaintained as the sample is diluted.18357180mAU0Gradient Resolution of Sulfa Drugs on Acclaim PA123 450 1 2 3 4 5 6 7 8 9 10 11 12 13Minutes6789Column: Acclaim PA, 3 µmDimensions 4.6 × 150 mmMobile Phase: (A) 90/10 methanol/20 mM phosphate, pH 2.7(B) 10/90 methanol/20 mM phosphate, pH 2.7Inj. Volume: 10 µLGradient: (A) 15–30% at 5 min, to 75%at 10 min, to 85% at 12 minTemperature: 30 °CFlow Rate: 1 mL/minDetection: UV, 254 nmPeaks: 1. Sulfanilic acid 6 µg2. Sulfanilamide 43. Sulfadiazine 104. Sulfathiazole 105. Sulfamerazine 106. Sulfamethazine 107. Sulfamethoxazole 148. Sulfisoxazole 129. Sulfadimethoxine 18193002.86nA1Baseline Separation of Catecholamines and Serotoninat Physiological Concentrations on Acclaim 120 C18with Electrochemical Detection12 340 5 10 15 20 25 30 35 40 45 50Minutes5Column: Acclaim 120 C18, 5 µmDimensions: 4.6 × 150 mmMobile Phase: 57 mM citric acid,43 mM sodium acetate,0.1 mM EDTA,1 mM sodium octanesulfonate,10% methanolFlow Rate: 1 mL/minDetection: dc amperometry,Glassy Carbon at 700 mV,Ag/AgCl Referemce electrodePeaks: 1. Norepinephrine 10 pmol2. Epinephrine 103. Dihydroxybenzylamine 104. Dopamine 105. Serotonin 2018358300mAUSample Prep: 1. Disperse 0.5 g ointment in 8.5 mL MeOH2. Add 0.5 mL conc. NH 4 OH and make to 10 mL with MeOH3. Apply 1.0 mL to a conditioned OnGuard II RP 1-cc cartridge and expel with 1 mL air4. Elute twice with 2.0 mL of 2% acetic acid in acetonitrile and expel with 1 mL air.5. Combine and make to 5 mL0Assay for Pramoxine in Topical Anesthetic Ointmenton Acclaim 120 C18 with SPE Cleanup on OnGuard II RPPramoxine0 Minutes7Column: Acclaim 120 C18, 3 µmDimensions: 4.6 × 50 mmMobile Phase: (A) MeOH(B) 10 mM H 3 PO 4 , adjustedto pH 3.1 with NH 4 OHGradient: 25%A until 0.7 min, ramp to 85% Aat 6 min, end at 7 minFlow Rate: 1.0 mL/minTemperature: 30 °CDetection: UV, 226 nmInj. Volume: 5 µL20396APPLICATIONSDetermination of Four Active Ingredients in aMultisymptom Cold Remedy Using Acclaim PA5002Column: Acclaim PA, 3 µmDimensions: 4.6 × 150 mmInjection: 10 µLmAU1Mobile Phase: (A) Acetonitrile(B) 10 mM phosphoric acidadjusted to pH 3.1 with34ammonium hydroxideTemperature: 30 °CDetection: UV, 210 nm–100Gradient : Time % A % B Flow0 Minutes 170 8 92 1.02 8 9210 20 8017 50 50Peak # Compound Label Concentration % Recovery1 Pseudoephedrine HCl 2.0 mg/mL 962 Acetaminophen 33 1003 Doxylamine succinate 0.41 904 Dextromethorphan HBr 1.0 95203622.4nA1 32 4 5Catecholamines in Urine on Acclaim PA26789 100.20 5 10 15 20 25 30MinutesColumn: Acclaim PA2, 3 µmDimensions: 2.1 × 150 mmMobile Phase: Buffer (11.98 g citric acid,3.53 g NaOAc, 37.2 mg EDTA,10 mL of 0.1 M methanesulfonic acidin 1 L D.I. water)/MeOH v/v 90/10Temperature: 30 °CFlow Rate: 0.2 mL/minInj. Volume: 2 µLDetection: DC amperometry [GC electrode,800 mV]Peaks: (1 nM each)1. 4-Hydroxy-3-methoxymandelic acid2. 4-Hydroxy-3-methoxyphenylglycol3. Norepinephrine4. Epinephrine5. 3,4-digydroxybenzlamine6. Normetanephrine7. Metanephrine8. Dopamine9. 4-Hydroxy-3-methoxyphenylacetic acid10. 5-Hydroxyindoleacetic acid20466- 30 -www.dionex.com

Pharmaceutical Applications0.10AU0Bromide and Organic Acids in Cough SyrupUsing the Acclaim OA120 5 10 15Minutes3Column: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phase: 0.1 M Na 2 SO 4 , pH 2.68(adjusted with methanesulfonicacid)Temperature: 30 °CFlow Rate: 0.6 mL/minInj. Volume: 5 µLDetection: UV, 210 nmSample Prep: OnGuard II PSample: NyQuil ®Peaks: 1. Bromide (Dextromethorphan HBr)2. Citrate (Inactive ingredient)3. Succinate (Doxylamine succinate)NyQuil is a registered trademarkof Proctor and Gamble.200250.60AU12 3 4 56 7Sulfa Drugs on Acclaim PA20 5 10 15 20Minutes89Column: Acclaim PA2, 5 µmDimensions: 4.6 × 150 mmMobile Phase: CH 3 CN/0.1 M NH 4 OAc,pH 5.4, v/v, 25/75Temperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: UV, 265 nmPeaks: (50 ppm each)1. Sulfanilamide2. Sulfathiazole3. Sulfamerazine4. Sulfamethazine5. Sulfisoxazole6. Sulfapyridazine7. Sulfamethoxazole8. Sulfadimethoxine9. Sulfaquinoxaline sodium2113225mAU01Loratidine and Pseudo-ephedrine in aTime-Release Tablet on Acclaim PA257 nm–100 1 2 3 4MinutesSample Preparation:– Finely grind a weighed tablet.– Weigh an aliquot containing1 mg loratidine (120 mg) into a dry100 mL flask.– Add 10 mL methanol and disperse.– Add 3 mL of 1N HCl with mixing.– Dilute to 100 mL with water.– Filter through 0.45 µm membrane filter.2Column: Acclaim PA C16, 5 µm, 4.6 × 50 mmPump: Summit P680A DGP-6Mobile Phases: (A) 15:85:0.050 acetonitrile:water:TFA(B) 40:60:0.050 acetonitrile:water:TFAFlow: 1.25 mL/minTemperature: 35 °CGradient: Times 0.00 0.40 0.41 4.00%A 100 100 0 0%B 0 0 100 100Injection: Summit ASI-100, 10 µLDetector: Summit UVD 340U diode array,UV at 206, 240 and 257 nmPeaks: 1. Pseudo-ephedrine 240 µg/mL2. Loratidine 1021785600mAU0BA1Propafenone and Related Substances2 3 4 5 60 10 20 30 40MinutesSample Preparation:Weigh accurately about 230 mg of sample in100 ml standard volumetric flask. Add 60 ml ofmethanol and sonicate for 20–25 min and diluteup to the mark with methanol. Centrifuge theabove solution for 2–3 min and further dilute1 ml of clear supernatant solution to 25 ml withdiluent. Diluent is mobile phases A:B 40:60.Data courtesy of: V. Bhate of Analytical SolutionsColumn: Acclaim PA C16, 5 µm,4.6 × 150 mmPump: Summit P680A LPGMobile Phase: (A) 0.010 M ammonium acetate, adj.to pH 2.4 with phosphoric acid(B) MethanolFlow: 1.0 mL/minGradient Times: 0 5 10 15 30 33%A: 80 50 40 30 5 80%B: 20 50 60 70 95 20Temperature: TCC100 thermostat at 25 °CInjection: ASI-100 autosampler; 20 µLDetector: UVD 340U; UV at 249 nmSample: (A) “Resolution” test mix(B) FormulationPeaks: 1. Propafenone 10 µg/mL2. Epoxyefenone 223. Chlorohydrine 824. Tertiary amine 445. HBAH 346. Dimer 34226630.25AU0Isocratic Separation of Six Cardiac Antiarrhythmic Drugs(Beta-Blockers) on Acclaim 120 C81236 7 8540 10MinutesColumn: Acclaim 120 C8, 3 µmDimensions: 4.6 × 150 mmMobile Phase: 51/49 w/w MeOH/25 mM phosphate, pH 7.0Flow Rate: 1.0 mL/minTemperature: 40 °CInj. Volume: 5 µLDetection: UV, 214 nmPeaks: 1. Maleic acid –2. Acebutolol 50 µg/mL3. Metoprolol 504. Timolol 1005. Oxprenolol 506, 7. Labetaloldiasteromers 508. Propranolol 20203970.6AU0.0Active Ingredient in Zantac ® 75 Acid-Reducer Medication20 5 1015MinutesReference: Ho C, Huang HM, Hsu SY, Shaw CY,Chang BL, Drug Dev. Ind. Pharm. 1999 25:379-385.Zantac is a registered trademark ofWarner-Lambert Co.1Column: Acclaim 120 C18, 5 µm,4.6 × 150 mmPump: GP50 microMobile Phase: 40 mM KH 2 PO 4 :acetonitrile:methanol:triethylamine345:20:35:0.7 v:v:v:v isocraticFlow: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetector: AD20 UV/VIS at 320 nmSample Prep.: One tablet dissolved in100 mL water; filteredPeaks: 1. Ranitidine2. Ranitidine S-oxide (impurity)21535APPLICATIONSwww.dionex.com - 31 -

Pharmaceutical Applications00–24 mAU FS12 3 4 5 6 9 100–400 mAU FSBudesonide and Related Substances0 5 10 15 20MinutesReference: Hou S, Hindle M, Byron PR;J. Pharm. Biomed. Analy. 2001 24:371-80.78Column: Acclaim 300 C18, 3 µm,4.6 x 150 mmPump: Summit P580 HPG/4Mobile Phase: (A) Acetonitrile:ethanol 15:1(B) 0.1% phosphoric acidIsocratic 66% BFlow: 1.0 mL/minTemperature: 30 °CInjection: ASI-100 autosampler, 15 µLDetector: UVD 340U; UV at 240 nmSample: Budesonide, 500 µg/mLafter three daysPeaks: 7, 8. Budesonide epimers, 99%215230.14AU0.00Hydrocortisone in Skin Ointment120 246Minutes3Column: Acclaim 120 C8, 3 µm,4.6 × 150 mmPump: Dionex GP50 microboreMobile Phase: Acetonitrile:water 49:51Flow: 1.0 mL/minTemperature: 30 °CInjection: 1 µLDetector: AD-20; UV at 245nmSample Prep.:– Disperse 100 mg of ointment in7.0 mL denatured ethanol– Mix in 3.0 mL hexane then 1.0 mL water– Centrifuge to separate emulsion– Filter ethanol layerPeaks: 1. Hydrocortisone 1.05% (label 1.0%)2. Methyl paraben 0.32%3. Propyl paraben 0.21%2152660mAU01Barbiturates on Acclaim 120 C180.0 1.5 3.0 4.5 6.0 7.5Minutes2345Column: Acclaim 120 C18 5µm, 4.6 x 50 mmPump: Summit P580 HPG/4Mobile Phase: (A) Acetonitrile:methanol 4:1 v:v(B) 0.1% phosphoric acidGradient: Time 0.0 5.0 7.5%A 20 45 45%B 80 55 55Flow: 1.0 mL/minTemperature: 30 °CInjection: ASI-100 autosampler, 3 µLDetector: UVD 340U, UV at 210 nmSample: 25 µg/mL each in methanolPeaks: 1. Barbital2. Phenobarbital3. Butabarbital4. Amobarbital5. Secobarbital215210.8AU012Active Ingredients in Cough Syrup30 5 10 15Minutes4Column: Acclaim PA2, 5 µmDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/50 mM phosphate buffer,pH 1.9 from 10/90 to40/60 (v/v) in 15 minTemperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 20 µLDetection: UV, 210 nmPeaks: 1. Doxylamine2. Pseudo-ephedrine3. Acetaminophen4. Dextromethorphan211360.3AU001Antidepressants on Acclaim PA22Column: Acclaim PA2, 5µmDimensions: 4.6 × 150 mmMobile Phase: MeOH/10 mM triethylamine,pH 10.8, v/v 75/25Temperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: UV, 254 nmPeaks: Concentration Asymmetry(µg/mL) (EP)1. Uracil 10 1.082. Doxepin 100 1.1433. Imipramine 80 1.1544. Amitriptyline 100 1.115 101520MinutesPhenytoin and Related Substances100Column: Acclaim 120, C18 5 µm,14.6 × 150 mmPump: Summit P680A2 Mobile Phase: 20% methanol; 30% acetonitrile;mAU 3 50% 0.05 M NH 4 H 2 PO 4 adj. to pH 2.5Flow: 1.5 mL/minB4 6Temperature: TCC100 thermostat at 60 °C7 8 Injection: ASI-100 autosampler; 25 µL0A5Detector: UVD 340U; UV at 220 nm0 5 10 15 20Samples: (A) “Low load” test mixMinutes(B) “Resolution” test mixData courtesy of: V. Bhate of Analytical SolutionsPeaks: 1. Impurity A 1 µg/mL2. Impurity B 13. Benzoic acid 94. Benzaldehyde 95. Phenytoin sodium 1000 (B) or 3 (A)6. Benzoin 97. Benzophenone 18. Benzil 92046522664APPLICATIONS- 32 -www.dionex.com

Pharmaceutical ApplicationsGlucocorticosteroids in Serum on Acclaim 120 C18Nonsteroidal Anti-inflamatory Drugs on Acclaim PA2 C18100mAU0BA1 2 345 6 70 5 10 15 20 25MinutesReference: McWhinney B C, Ward G, HickmanP E; Clin. Chem, 1996, 42:979-981.Column: Acclaim 120 C18 5 µm,4.6 × 250 mmPump: Summit P580 HPG/4Mobile Phase: Methanol:tetrahydrofuran:water3:25:72 v:v:v, isocraticFlow: 1.0 mL/minTemperature: 30 °CInjection: ASI-100 autosampler, 60 µLDetector: UVD 340U; UV at 240 nmSamples: (A) Extract of Bovine Serumextracted with ethyl acetate fromserum alkalized with NaOH(B) Six steroid standards,5 µg/mL in mobile phasePeaks: 1. Prednisone2. Prednisolone3. Cortisol4. Fludrocortisone (I.S.)5. Methylprednisolone6. 11-Deoxycortisol7. Dexamethasone21532Column: Acclaim PA2 C18, 5 µm,4.6 × 150 mmPump: Summit P 680A DGP-6Mobile Phase: Acetonitrile:0.1% phosphoric acid,55:45 v/v; isocratic90mAU0254.5335.4200 nm 39812Flow: 1.0 mL/minTemperature: 30 °CInjection: Summit ASI-100, 20 µLDetector: Summit UVD340U diode array,265nm, and spectra 200–400 nmPeaks: 1. Ketoprofen 10 µg/mL2. Naproxen 103. Ibuprofen 50200 nm 3980 2 4 6 8 10 12Minutes21557229.8271.4262.0208.5219.9263.2200 nm 39831,800mAU 10Active Ingredients in Sunscreen Lotion onAcclaim 120 C18 Using ASE Extraction0 Minutes222Extraction Conditions:ASE 200 with 11 mL extraction cell50/50 MeOH/CH 2 Cl 2Column: Acclaim 120 C18Mobile Phase: Isocratic 85/14/0.75methanol/water/HOAcInj. Volume: 20 µLDetection: UV, 310 nmPeaks: 1. Benzophenone-32. Octyl methoxycinnamate33. Octyl salicylate20398400mAU0Fexofenadine and Pseudoephedrine on Acclaim PA*UV218 nm10 0.5 1.0 1.5 2.0 2.5 3.0 3.5Minutes*2Column: Acclaim PA C16, 5 µm, 4.6 × 50 mmPump: Summit P 680A DGP-6Mobile Phases: A. 15:85:0.047 acetonitrile:water:TFAB. 44:66:0.050 acetonitrile:water:TFAFlow: 1.25 mL/minTemperature: 35 °CGradient Times: 0.00 0.80 0.81 3.50%A 100 100 0 0%B 0 0 100 100Injection: Summit ASI100, 20 µLDetector: Summit UVD340U diode array,UV at 207, 218 and 257 nmPeaks: 1. Pseudo-ephedrine 200 µg/mL2. Fexofenadine 20*. Artifacts21559APPLICATIONSwww.dionex.com - 33 -

Environmental ApplicationsEnvironmental50Brodifacoum in Rodent BaitColumn: Acclaim 120 C18, 5 µm,4.6 × 150 mmPump: Summit P580 HPG/4Mobile Phase: (A) Acetonitrile(B) 0.10% conc. phosphoric acid in watermAU1Isocratic A:B 82.5:17.52Flow: 1.20 mL/minTemperature: 30 °C0Injection: Summit ASI-100, 10 µLDetector:0 Minutes10Summit UVD 340U diode array,265 nm, and spectra 200–400 nmSample Prep.:- Mutually saturate ethyl acetate and waterfor use in the next step- Shake 1.0g of finely ground bait with 10.0 mLethyl acetate, 5.0 mL water and 0.10 mL acetic acid- Centrifuge to separate the emulsion- Evaporate 3.0 mL of ethyl acetate fraction to dryness- Reconstitute in 0.60 mL acetonitrile- FilterPeaks: 1. Brodifacoum isomer 12. Brodifacoum isomer 2215220.10AU012Separation of Phthalate Esters inEPA Method 606 Using Acclaim PA3 45 60 5 10 15MinutesColumn: Acclaim PA, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) D.I. H 2 O(B) MeOHGradient: Time (min) % B0 2510 10015 100Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: UV, 254 nmPeaks: (100 µg/mL each)1. Dimethyl phthalate2. Diethyl phthalate3. Bis(2-ethylhexyl) phthalate4. Di-n-butyl phthalate5. Di-n-octyl phthalate6. Butyl benzyl phthalate20107LC Separation of Nitrosamines in EPA 8270 Using Acclaim PAAU12 3 4 5 60 10 20 30Minutes78 9Column: Acclaim PA, 5 µmDimensions: 4.6 × 250 mmMobile Phase: (A) D.I. H 2 O(B) AcetonitrileGradient: Time (min) % B0 15 125 9030 90Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 15 µLDetection: UV, 230 nmPeaks: (20 µg/mL)1. N-Nitroso-dimethylamine2. N-Nitroso-morpholine3. N-Nitroso-methylethylamine4. N-Nitroso-pyrrolidine5. N-Nitroso-diethylamine6. N-Nitroso-piperidine7. N-Nitroso-dipropylamine8. N-Nitroso-dibutylamine9. N-Nitroso-diphenylamineNote: This gradient will also resolve benzidines.20109a0.45AU0Phenol Standards by EPA Method 604 on Acclaim PA13967102 4 5 80 5 10 15 20MinutesNote: The lower pH provided by methanesulfonic acid(compared to acetic acid in the standard method) givesoptimum separation for the Acclaim PA column.11Column: Acclaim PA, 3 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 2.5 mM MSA in H 2 O(B) MethanolGradient: Time (min) % B0 604.5 6010.5 9520 95Temperature: 25 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 285 nmPeaks:(50– 250 µg/mL each)1. Phenol2. 2-Chlorophenol3. 4-Nitrophenol4. 2-Nitrophenol5. 2,4-Dimethylphenol6. 4-Chloro-3-methylphenol7. 2,4-Dichlorophenol8. 2,4-Dinitrophenol9. 2-Methyl-4,6-dichlorophenol10. 2,4,6-Trichlorophenol11. Pentachlorophenol20106APPLICATIONS054321LC/MS Detection of Herbicides on Acclaim 120 C18Column: Acclaim 120 C18, 3 µmDimensions: 2.1 × 100 mm0.0010Mobile Phase: 70/30% methanol/10.1% formic acidFlow Rate: 0.25 mL/minDetection: AQA MS, +ESI, 30 V, 200 °C,SIM as indicatedµA2Peaks: m/z mass injected1. Monuron 199 1 ng32. Atrazine 216.1 13. Propazine 230 24. Diuron 233 2.515. Linuron 249 2–0.00012 3 4 5 0 2.5 5.0MinutesMinutes7.5 10.017922Benzidines by EPA Method 605 on Acclaim 120 C18with Electrochemical DetectionColumn: Acclaim 120 C18, 3 µmDimensions: 2.1 × 100 mmMobile Phase: 50/50 acetonitrile/100 mM NaOAc, pH 4.7Flow Rate: 0.17 mL/minInj. Volume: 1 µLDetection: Glassy carbon electrode, 800 mV(1 µg/mL)Peaks 1. Benzidine2. 3.3'-Dimethylbenzidine3. 3.3'-DichlorobenzidineNote: Scaling down the column size and flowrate produces a superproportional increase insensitivity in the electrochemical detector.20400- 34 -www.dionex.com

Environmental ApplicationsAUImproved Separation of 13 Carbonyl-DNPHDerivatives by CARB Method 1004 Using CoupledAcclaim PA and Acclaim 120 C18 ColumnsColumns: Acclaim PA, 5 µm, 4.6 × 150 mm,coupled with Acclaim 120 C18, 5 µm,4.6 × 150 mmMobile Phase: (A) H 2 O(B) CH 3 CNGradient: Time (min) % B0 5420 540.0330 9035 9012435Temperature: 30 °CFlow Rate: 1.5 mL/minInj. Volume: 10 µLDetection: UV, 360 nmPeaks:678 910(0.3 µg/mL each)DNPH derivatives of1. Formaldehyde2. Acetaldehyde3. Acrolein4. Acetone5. Propionaldehyde6. Crotonaldehyde7. Methacrolein8. Butyraldehyde9. Methylethylketone10. Benzaldehyde11. Valeraldehyde12. m-Tolualdehyde13. Hexaldehyde11 12 13Background Information:LC/MS of 12 insecticides, 1 ng each on column.Overlay of 12 simultaneous single-ion chromatograms.Column: Acclaim 120 C8, 5 µm, 2.1 × 100 mmMobile Phase: (A) MeOH(B) 20 mM NH 4 HCO 2 , pH 3.4Gradient:Time %A %B Flow0 12 88 0.251 12 8815 70 3017 70 3018 100 0Inj. Volume: 1–50 µLSample: 100 ppm stock diluted1000× in Eluent B300,000Counts 1Carbamate Insecticides in EPA 531.1on Acclaim 120 C8 with MS Detection2345Detection: MSQ MS +ESIMS Settings: Probe 400 °CNeedle 2.0 kVCone voltage set per ionPeaks:m/z1. Aldicarb sulfoxide 2072. Aldicarb sulfone 2403. Oxamyl 2374. Methomyl 1635. 3-Hydroxy carbofuran 2206. Aldicarb 2087. Unknown 2088. Propoxur 1689. Carbofuran 22210. Carbaryl 14511. Unknown 16912. Methiocarb 16989101206110 4 8 12 16 20 24 28 32 35Minutes7Notes: (1) No conventional C18 column can produce equivalent resolution.0(2) CARB = California Air Resource Board 4.4 Minutes17.9201122039970mAU00Polynuclear Aromatic Hydrocarbon Standards byEPA Method 8310 on Acclaim PA123456789101113121516145 10 15 20 25 30 35 40 45MinutesColumn: Acclaim PA, 5 µmDimensions: 4.6 × 250 mmMobile phase: (A) CH 3 CN(B) D.I. H 2 OGradient: Hold A/B (50/50) for 5 min,A/B (50/50) to A/B (85/15)in 35 min,Hold A/B (85/15) for 5 minTemperature: 25 °CFlow Rate: 2 mL/minInj. Volume: 10 µLDetection: UV, 254 nmPeaks: 1. Naphthalene2. Acenaphthylene3. Acenaphthene4. Fluorene5. Phenanthrene6. Anthracene7. Fluoranthene8. Pyrene9. Benzo[a] anthracene10. Chysene11. Benzo[b] fluoranthene12. Benzo[k] fluoranthene13. Benzo[a] pyrene14. Dibenz[a,h] anthracene15. Benzo[g,h,i] perylene16. Indeno[1,2,3-cd] pyrene19305mAU–2Thirteen Carbonyl-DNPH Derivatives byCARB Method 1004 on Acclaim 120 C18Column: Acclaim 120 C18, 5 µmDimensions: 2.1 × 250 mmMobile Phase: 40% acetonitrile at 0–7 min,gradient to 100% acetonitrile at 20 minFlow Rate: 0.25 mL/minInj. Volume: 10 µLDetection: UV, 360 nmPeaks (DNPH and 13 carbonyl derivatives) 0.3 µg/mL:1. 2,4-DNPH2. Formaldehyde-DNPH163. Acetaldehyde-DNPH124. Acrolein-DNPH5. Acetone-DNPH6. Propionaldehyde-DNPH7. Crotonaldehyde-DNPH8. Methacrolein-DNPH9. 2-Butanone-DNPH10. Butyraldehyde-DNPH11. Benzaldehyde-DNPH12. Valeraldehyde-DNPH13. m-Tolualdehyde-DNPH14. Hexaldehyde-DNPHNote: CARB = California AirResource Board3 485 7 96 1011 12 14130 Minutes2020111APPLICATIONSwww.dionex.com - 35 -

Environmental Applications0.5 Column: Acclaim PA, 5 µm9Dimensions: 4.6 × 150 mmMobile Phase: (A) D. I. H 2O(B) CH 3CNAU21345Carbamate Insecticide Standardsin EPA 531.1 on Acclaim PA86 7100 10 20 30MinutesGradient: Time (min) % B0 1030 70Temperature: 25 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 210 nmPeaks: (100 µg/mL each)1. Aldicarb sulfoxide2. Aldicarb sulfone3. Oxamyl4. Methomyl5. 3-Hydroxycarbofuran6. Aldicarb7. Propoxur8. Carbofuran9. Carbaryl10. Methiocarb201052,500,000CountsSensitive Detection of Triazine Herbicides onAcclaim 120 C18 with MS Detection1 234502.9 18.6Minutes67Background Information: Seven herbicides at 1 ngon column. Seven simultaneous single-ionchromatograms optimized for positive identification.Column: Acclaim 120 C18, 5 µmDimensions: 2.1 × 100 mmMobile Phase: 35% Acetonitrile65% Ammonium acetate, pH 5Temperature: 30 °CFlow Rate: 0.25 mL/minInj. Volume: 10 µLDetection: MSQ MSESI: PositiveProbe: 400 °CNeedle: 2.5 kVCone Voltage: Set per ionPeaks m/z1. Simazine 2022. Atrazine 2163. Prometon 2264. Ametryn 2285. Propazine 2306. Prometryn 2427. Terbutryn 24220401LC Separation of Benzidines in EPA 8270 Using Acclaim PAColumn: Acclaim PA, 5 µmDimensions: 4.6 × 250 mmMobile Phase: (A) D.I. H 2 O3(B) AcetonitrileGradient: Time (min) % B20 15 11AU25 9030 90Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 15 µLDetection: UV, 230 nmPeaks: (20 µg/mL)0 10 20 301. BenzidineMinutesNote: This gradient will also resolve N-nitrosamines2. 3.3'-Dimethylbenzidine3. 3.3'-Dichlorobenzidine20109b0.25AU0Four Antibiotics in Animal Feed on Acclaim C18120 2 4 6 8 10 12 15Minutes34Column: Acclaim 120 C18, 5µm,4.6 × 150 mmPump: GP50 microboreMobile Phase A: 20 mM formic acidMobile Phase B: AcetontirileGradient Times: 0 15%A: 85 50%B: 15 50Flow: 1.00 mL/minTemperature: 30 °CInjection: 5 µLDetector: UV, 280 nmPeaks: (100 µg/mL each)1. Sulfamerazine2. Oxytetracycline3. Chloramphenicol4. Sulfadimethoxine22662APPLICATIONSAUEPA Method 8330 Using Acclaim Explosives E1 Column1234567 118 9101213140 9 18 27 36 45MinutesColumn: Acclaim Explosives E1, 5 mm,4.6 × 250 mmMobile Phase: 43/57 v/v MeOH/H 2 OTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 254 nmPeaks:1. HMX2. RDX3. 1,3,5-Trinitrobenzene4. 1,3-Dinitrobenzene5. Nitrobenzene6. Tetryl7. 2,4,6-Trinitrotoluene8. 4-Amino-2,6-Dinitrotoluene9. 2-Amino-4,6-Dinitrotoluene10. 2,6-Dinitrotoluene11. 2,4-Dinitrotoluene12. 2-Nitrotoluene13. 4-Nitrotoluene14. 3-Nitrotoluene22784AUEPA Method 8330 Using Acclaim Explosives E2 Column1 234560 8 16 24 32 40Minutes97 81410 12 1311Column: Acclaim Explosives E2, 5 mm,4.6 × 250 mmMobile Phase: 50/50 v/v MeOH/H 2 OTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 254 nmPeaks:(EPA 8330 mix, 50 ppm each)1. HMX2. RDX3. 1,3,5-Trinitrobenzene4. 1,3-Dinitrobenzene5. Nitrobenzene6. 2,4,6-Trinitrotoluene7. Tetryl8. 2,6-Dinitrotoluene9. 2,4-Dinitrotoluene10. 2-Nitrotoluene11. 4-Nitrotoluene12. 3-Nitrotoluene13. 4-Amino-2,6-Dinitrotoluene14. 2-Amino-4,6-Dinitrotoluene22779- 36 -www.dionex.com

Food and Beverage ApplicationsFood and Beverage50mAU0Carotenoids in Serum on Acclaim PA10 5 10 15 20 25Minutes234SpectrumlibrarymatchColumn: Acclaim PA C16, 3 µm,4.6 × 150 mmPump: Summit P580 HPG/4Mobile Phase: (A) Water(B) 54:44:2methanol:acetonitrile:isopropanolGradient: Time 0 8.0 8.1 25%A 5 5 0 0%B 95 95 100 100Flow: 1.25 mL/minTemperature: 30 °CInjection: Summit ASI-100, 25 µLDetector: Summit UVD 340U diode array,450 nm and spectra 200–595 nmSample: Extract of bovine serum, fortified withlycopene equivalent to 300 ng/mLPeaks: 1. Unidentified carotenoid2. trans-lycopene3. trans-β-carotene4. cis-β-carotene21524Pigments in Turmeric on Acclaim PA2901 Column:Pump:Acclaim PA2, 3 µm, 4.6 × 50 mmSummit P680 DGPMobile Phase: (A) 10 mM phosphoric acidmAU(B) Methanol32Isocratic: 78% BFlow: 1.50 mL/minTemperature: Summit TCC-100, 30 °C0Injection: Summit ASI-100, 10 µLDetector: Summit UVD340U diode array,0 1 2 3 4428 nm, and spectra 200–595 nmMinutesPeaks: 1. Curcumin2. DemethoxycurcuminSample Preparation:3. Bis-demethoxycurcumin– Extraction solvent: 70% ethanol, 29.5% water,0.5% phosphoric acid– Extract 100 mg of ground turmeric with 20 mLof solvent in a capped vial at 65 °C for 15 min– Centrifuge– Dilute 10× in 75% methanol– Filter2265910mAU–10Fat-Soluble Vitamin Standards on Acclaim 120 C1810 223454 6 8Minutes610Column: Acclaim 120 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: 95/5 v/v acetonitrile/methanolFlow Rate: 2.0 mL/minTemp.: 30 °CInj. Volume: 5 µLDetection: UV, 280 nmPeaks: 1. All-trans retinol (Vitamin A) 0.12 mg/mL2. δ-Tocopherol (impurity) -3. Ergocalciferol (Vitamin D 2 ) 0.124. Cholecalciferol (Vitamin D 3 ) 0.065. β-Tocopherol (impurity) -6. α-Tocopherol (Vitamin E) 1.317834600mAU–50Amino Acids in Vitamin Premix on Acclaim OA213485 6 70 2 4 6 8 10 12 14MinutesColumn: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phase: 40 mM Na 2 SO 4 adjusted to pH 3.05with methanesulfonic acidFlow Rate: 0.60 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 210 nmPeaks: 1. Lysine2. Glutamine3. Valine4. Ascorbic acid5. Isoleucine6. Leucine7. Unknown8. Unknown204120.07AU0Organic Acids in Orange Juice on Acclaim OA1230 5 10 15Minutes4Column: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phase: 0.1 M Na 2 SO 4 , pH 2.68(adjusted with methanesulfonic acid)Temperature: 30 °CFlow Rate: 0.6 mL/minInj. Volume: 5 µLDetection: UV, 210 nmSample Prep.: OnGuard II P,diluted 2× with D.I. H 2 OPeaks:1. Malic acid2. Ascorbic acid (Vitamin C)3. Citric acid4. Fumaric acid200230.10AU0Organic Acids in White Wine on Acclaim OA1234 50 5 10 15MinutesColumn: Acclaim OA, 5 µmDimensions: 4 × 250 mmMobile Phase: 0.1 M Na 2SO 4, pH 2.68 (adjustedwith methanesulfonic acid)Temperature: 30 °CFlow Rate: 0.6 mL/minInj. Volume: 5 µLDetection: UV, 210 nmSample prep: OnGuard II PPeaks: 1. Tartaric acid2. Malic acid3. Lactic acid4. Citric acid5. Succinic acid20024APPLICATIONSwww.dionex.com - 37 -

Food and Beverage ApplicationsAssay for Water-Soluble Vitamins inVitamin Tablets Using Acclaim PA015 6 7 8 Peaks: 1. Thiamine210 nm2. Ascorbic acid246 nm3. Pyridoxine265 nm285 nm4. NiacinamideColumn: Acclaim PA, 3 µm1000Dimensions: 4.6 × 150 mm2Mobile Phase: Phosphate pH 3.4,gradient 0–20% CH 3 CNDetection: UVD 340 at listed wavelengthsSample: One-A-Day ® brand "Active Formula"Sample Prep.: 1. Grind a 1500 mg tabletmAU2. Extract 150 mg with 100 mL of4pH 3.4 buffer3. Sonicate 10 min4. Filter5. Inject 20 µL3–2005. Pantothenic acid0 Minutes176. Folic acid7. Vitamin B 128. RiboflavinOne-A-Day is a registered trademark of Bayer Corp.19350Nitrofuran Antibiotic Residues in Animal Feed on Acclaim 120 C1835mAU012 30 2 4 6 8 10 12MinutesReference:R.J. McCracken, D.G. Kennedy;J. Chromatogr. A, 1997, 771, 349–354.420 mg/kg5 mg/kgControlColumn: Acclaim 120 C18, 5 µm,4.6 × 150 mmPump: Summit P680A DGP-6Mobile Phase: Acetonitrile:10 mM ammonium acetatepH 5.0, 20:80 v/v; isocraticFlow: 1.0 mL/minTemperature: 30 °CInjection: Summit ASI100, 20 µLDetector: Summit UVD340U diode array,365 nm, and spectra 200–500 nmSample Preparation:1. 3.0 g guinea pig feed in a 50 mL centrifuge tube2. Add 9 mL water and let stand 5 min.3. Add 21 mL methanol:acetonitrile 1:1and extract for 30 min.4. Pass through cleanup cartridge containing1.7g of neutral alumina; discard first 1.7 mL,retain next 3.5 mLPeaks: 1. Nitrofurazone2. Nitrofurantoin3. Furazolidone4. Furaltadone215582,500,000CountsSensitive Detection of Triazine Herbicides onAcclaim 120 C18 with MS Detection1 234502.9 18.6Minutes67Background Information: Seven herbicides at 1 ngon column. Seven simultaneous single-ionchromatograms optimized for positive identification.Column: Acclaim 120 C18, 5 µmDimensions: 2.1 × 100 mmMobile Phase: 35% Acetonitrile65% Ammonium acetate, pH 5Temperature: 30 °CFlow Rate: 0.25 mL/minInj. Volume: 10 µLDetection: MSQ MSESI: PositiveProbe: 400 °CNeedle: 2.5 kVCone Voltage: Set per ionPeaks m/z1. Simazine 2022. Atrazine 2163. Prometon 2264. Ametryn 2285. Propazine 2306. Prometryn 2427. Terbutryn 24220401300mAU0Fat-Soluble Vitamins and Carotenoids in a Vitamin Tablet12 3473 nm325 nm285 nm0 5 10 15 20MinutesSample Preparation:– Finely grind a weighed tablet.– Weigh an aliquot of about 1/10 tablet (150 mg)into a screw-cap vial.– Partition between 1.0 mL water and 5.0 mLethyl acetate with vigorous shaking.– Warm to 60 °C for 10 min.– Shake vigorously again for 0.5 min.– Centrifuge to separate phases.4567Column: Acclaim PA C16, 3 µm,4.6 × 150 mmPump: Summit P680A DGP-6Mobile Phase: (A) Water(B) 54:44:2 methanol:acetonitrile:isopropanolGradient: Times 0 8.0 8.1 25%A 5 5 0 0%B 95 95 100 100Flow: 1.25 mL/minTemperature: 30 °CInjection: Summit ASI-100, 15 µLDetector: Summit UVD 340U diode array,473, 325 and 285 nm,and spectra 200–595 nmPeaks: 1. trans-Retinol acetate2. trans-Lutein3. α-Tocopherol acetate4. trans-Lycopene5. cis-Lycopene6. trans-β-Carotene7. cis-β-Carotene21786APPLICATIONS- 38 -www.dionex.com

Food and Beverage ApplicationsParabens on Acclaim PA20.15Column:Dimensions:Acclaim PA2, 5 µm4.6 × 150 mm1Mobile Phase: CH 3 CN/H 2 O v/v 60/402Temperature: 30 °CFlow Rate: 1.0 mL/minAU3Inj. Volume: 2 µL4Detection: UV, 254 nmPeaks: ( 0.1 mg/mL each)1. Methylparaben2. Ethylparaben3. Propylparaben04. Butylparaben0 2 4 6 8 10Minutes20464450mAU01230 10 20 30Minutes*Obtained from Am. Soc. Brewing ChemistsHop Extract on Acclaim 120 C184Column: Acclaim 120 C18, 5µm, 4.6 × 150 mmPump: Summit P680A DGP-6Mobile Phase: Methanol, water, phosphoric acid;85:17:0.25 (v/v/v)Flow: 1.2 mL/minTemperature: TCC-100 thermostat, 35 °CInjection: ASI100 autosampler, 25 µLDetector: UVD-340U, UV 314 nm;spectra 200–400 nmStandard*: ICE-2 certified hop extract,986 µg/mL in acidified methanolPeaks: 1. Cohumulone 142 µg/mL2. Humulone + adhumulone 3443. Colupulone 1274. Lupulone + adlupulone 11821997Water-Soluble Vitamins Using Acclaim PA2Determination of Quinine in Tonic Water Using Acclaim PA0.3047 8AU235 6 9100 2 4 6 8 10 12 13MinutesColumn: Acclaim PA2, 5 µmDimensions: 4.6 150 mmMobile Phase: (A) CH 3 CN(B) 25 mM phosphate buffer, pH 3.31Gradient: Hold B for 5 minB to A/B (40/60) in 7 minthen to A/B (75/25) in 1 minHold A/B (75/25) for 4 minTemperature: 30 °CFlow Rate: 1.0 mL/minInj. Volume: 50 µLDetection: UV, 210 nmPeaks: 1. Thiamine 2.5 µg/mL2. Ascorbic acid 603. Pyridoxine 2.54. Niacinamide 205. Pantothenic acid 106. Vitamin B12 1.257. Folic acid 58. Riboflavin 59. Biotin 12.5202810.7AU0.0A. Tonic Water1B. Quinine standard,100 µg/mL20 51015MinutesReference: U.S. Pharmacopeia, 22nd Ed.,1990, 1209.34Column: Acclaim PA C16, 5 µm,4.6 × 150 mmPump: GP50 microboreMobile Phase: (A) 896 mL water,100 mL acetonitrile,1.40 mL methanesulfonic acid,0.80 mL acetic acid,2.00 mL diethylamine(B) 70:30 v:v acetonitrile:waterGradient: Time 0.0 10.5 10.6 15.0%A 100 100 50 50%B 0 0 50 50Flow: 1.0 mL/minTemperature: 30 °CInjection: 25 µLDetector: AD20, UV 235 nmSample Prep.: FilterPeaks: 1. Cinchonidine2. Quinine3. Dihydroquinine4. Benzoic acid21528Determination of Energy Drink Ingredients with Acclaim PA2600mAU0210nm246nm265nm285nm123 546780 4 8 12 16MinutesSample Prep:Peaks:Column: Acclaim PA2, 3 µm, 4.6 × 150 mmPump: Summit P680 DGP6Mobile Phases: (A) Acetonitrile(B) 30 mM phosphate buffer, pH 3.28(C) 30 mM phosphate buffer, pH 2.54Gradient: Times 0.0 14.0 16.0%A 0 40 40%B 100 0 0%C 0 60 60Flow: 1.0 mL/minTemperature: 30 °CInjection: ASI-100 autosampler, 5 µLDetector: UVD 340U, UV 210, 246, 265,285 nm; spectra 200–400 nmFilter1. Ascorbic acid2. Pyridoxine3. Niacinamide4. Pantothenic acid5. Caffeine6. Riboflavin7. Sorbic acid8. Benzoic acid21784300mAU0BA1 2 3 40 2 4 6 8 10 12MinutesSample Preparation:– Finely grind dried peppers– Take an aliquot of about 400 mg– Partition between 2.0 mL water and 6.0 mLethyl acetate with vigorous shaking for 2 min– Centrifuge to separate phases– Evaporate 1.0 mL of ethyl acetate phase andreconstitute in 0.33 mL ethanol5Sudan Dyes in PaprikaColumn: Acclaim 120 C18, 5 µm,4.6 × 150 mmPump: Summit P680 DGPMobile Phase: (A) Water(B) 1:1 tetrahydrofuran:methanolGradient: Times 0 7.0 7.1 13%A 20 8 0 0%B 80 92 100 100Flow: 1.25 mL/minTemperature: 30 °CInjection: Summit ASI-100, 15 µLDetector: Summit UVD340U diode array,500 nm, and spectra 200–595 nmSamples: (A) Control paprika without artificialcoloring(B) Paprika spiked with approx.25 mg/kg of each dyePeaks: 1. Sudan I2. Sudan II3. Sudan III4. Sudan IV5. Oil Red O (2 peaks)(Natural pigments 8-12 min.)21787APPLICATIONSwww.dionex.com - 39 -

Food and Beverage ApplicationsSulfonamide Antibiotic Residues in Milk on Acclaim 120 C18Antioxidants in Edible Oils by AOAC 983.15 on Acclaim 120 C184012 3 4 5C6 7 8mAUB*0A5 10 15 20 25MinutesColumn: Acclaim 120 C18, 5 µm,4.6 × 250 mmPump: Summit P580 HPG/4Mobile Phase: (A) 30 mM acetic acidadjusted to pH 4.2 with NaOH(B) MethanolGradient Times: 0.0 20 25%A 88 60 60%B 12 40 40Flow: 1.5 mL/minTemperature: 35 °CInjection: ASI-100 autosampler, 100 µLDetector: UVD 340U, UV at 265 nmwith spectral confirmationSample Prep.: AOAC 993.32; refer to procedurefor important notesSamples: (A) Spiked extract of skim milk,1.0 µg/g each(B) Milk extract(C) Sulfonamide standards,0.20 µg/mL in waterPeaks: 1. Sulfadiazine (SDZ)2. Sulfathiazole (STZ)3. Sulfapyridine (SPD)4. Sulfamerazine (SMR)5. Sulfamethazine (SMZ)6. Sulfachloropyridazine (SCP)7. Sulfadimethoxine (SDM)8. Sulfaquinoxzline (SQX)*. TheobromineNote: Most interferences are organic acids thatmay be moved out of the way by smalladjustements to the pH of the mobile phase.21533Background Information: Nine antioxidants forAOAC 983.15 spiked into wheat germ oil.Column: Acclaim 120 C18, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 0.75% HOAc in H 2 O(B) 50/50/0.5 MeOH/acetonitrile/HOAcInj. Volume: 10 µLDetection: UV, 280 nm; diode array confirmationGradient:Time % A % B Flow0.0 30 70 2.09.6 0 100 2.011.4 0 100 2.012.0 0 100 4.017.0 0 100 4.01502713 456mAU01 232.5 Minutes4567Peaks: 1. Propyl gallate2. THBP3. TBHQ4. NDGA5. BHA6. Ionox-1007. Octyl gallate8. BHT9. Dodecyl gallate98Note: Ethoxyquin (not shown) has been resolved under the same conditions.89Extract of SpikedWheat Germ OilExtract ofWheat Germ OilStandards, 100 ng each1520404Bitter Principles in Beer on Acclaim 120 C18550 StandardsmAU ICS-H1ICS-R1ICS-T2ICS-I206.5 Märzen-style beer1 2mAU300 10 20 30MinutesColumn: Acclaim 120 C18 5µm,4.6 × 150 mmPump: Summit P680A DGP-6Mobile Phase: 75% methanol, 24% water,1% phosphoric acid (v/v/v)Flow: 1.2 mL/minTemperature: TCC-100 thermostat, 35 °CInjection: ASI100 autosampler, 25 µLDetector: UVD-340U, UV 270 nm;spectra 200–400 nmStandards*:ICS-I2: isohumulones 205 µg/mLICS-T2: tetrahydroisohumulones 135ICS-R1: rho-isohumulones 200ICS-H1: hexahydroisohumulones 210Peaks: 1. Isocohumulone2. Mixed isohumulone congeners3. IsoadhumuloneSample Preparation:Extraction per ASBC- 10 mL beer + 1 mL 3N HCl + 50 µL 1-octanol +20 mL iso-octane- Shake vigorously- Centrifuge to separate phases- Bitterness Units = 50 × A 275For HPLC- Evaporate dry and reconstitute in mobile phase*Obtained from Am. Soc. Brewing Chemists22027APPLICATIONSwww.dionex.com - 41 -

Ch e m i c a l ApplicationsSurfactantsmVSeparation of Cationic Surfactants Using ELSD1230 10 2030Minutes45Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–85% A in 30 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: ELSDPeaks:1. Lauryl pyridinium chloride2. Lauryldimethylbenzyl ammoniumchloride3. Octylphenoxyethoxyethyldimethylbenzyl ammonium chloride4. Cetyltrimethylammonium chloride5. Cetylpyridinium chloride21082mVUV, 225 nmELSDAnalysis of a ShampooLaureth Sulfate0 5 10 15 20 25 30 35 40 45MinutesColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–80% A in 30 min,then hold at 80% A for 15 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSD, UV21083Analysis of a MouthwashAnalysis of a Fabric SoftenermV123Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–80% A in 30 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 20 µL (direct injection)Detection: ELSDPeaks: 1. Sodium sacchrin and benzoate2. Domiphen bromide3. Cetylpyridinium chloridemVColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) THF(C) 0.1 M NH 4 OAc, pH 5.4Gradient: 40% A/10% B/50% C to 75% A/10% B/75% C in 20 min,then hold for 10 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 20 µLDetection: ELSDPeaks: Methyl bis[ethyl(tallowate)]-2-hydroxyethyl ammonium methylsulfate (500×)010 2030Minutes21120010 2030Minutes21121Analysis of ZONYL FSO FluorosurfactantSeparation of Ethoxylated QuatsmVColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile,(B) H 2 OGradient: 40–70% A in 30 min,then hold at 70% A for 5 minFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSDR f CH 2 CH 2 O(CH 2 CH 2 O) x HmVColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/0.2 M NH 4 OAc,pH 5.4 v/v 40/60Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: ELSDSample: Ethoquad 18/25 (500×)R f = F(CF 2 CF 2 ) yx = 0 to about 15y = 1 to about 7Zonyl FSO fluorosurfactantAPPLICATIONS0 10 20 30 35Minutes210810 5 10 15 20 25 30 35 40Minutes21116- 42 -www.dionex.com

Ch e m i c a l ApplicationsSurfactantsAnalysis of Sodium Lauryl Sulfate Using LC-ESI-MSAnalysis of PEG Monoethyl Ether (MW-550)AbundanceColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/0.1 M NH 4 OAc,pH 5.4 v/v 75/25Temperature: 25 °CFlow Rate: 1 mL/min(0.2 mL/min to mass spectrometer)Inj. Volume: 25 µLDetection: MS (ESI negative, probe 300 °C,cone 20 V needle 2.5 KV,SIM 265.2, span 0.3 m/z)Peak: Sodium lauryl sulfate (1 ppm)mVColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) H 2 OGradient: 5–20% A in 25 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSD0 5 10 15 20Minutes211260 2 4 6 810 12 14 16 18 20 22 25Minutes21118AbundanceAnalysis of Triton X-100 Using LC-ESI-MS0 4 8 12 16 20 24MinutesColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/50 mMNH 4 OAc, pH 5.4 v/v 45/55Temperature: 25 °CFlow Rate: 1 mL/min (0.2 mL/minto mass spectrometer)Inj. Volume: 25 µLDetection: MS (ESI positive, probe 300 °C,cone 20 V, needle 2.5 kV, SIM 312.2,356.2, 400.4, 444.4, 488.4, 532.4,576.5, 620.6, 664.6, 708.6, 752.5,796.6, 840.7, 884.7, 928.7, 972.7,1016.8, 1060.7, 1104.7, span 0.5 m/z)Peak: Triton X-100 (100 ppm)C 8 H 17(OC 2 HCH 2 ) n OHmVSeparation of Hydrotrope, Anionic, Cationic,and Amphoteric SurfactantsColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN,(B) 0.16 M NH 4 OAc, pH 5.4Gradient: 25% to 85% A in 30 min,then hold 85% A for 10 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 25 µL10Detection: ELS detector12 3 45 6 789111213Peaks:1. Xylene sulfonate2. Laurylpyridinium chloride3. Lauryldimethylbenzyl ammonium chloride4. Octylphenoxyethoxyethyl dimethylbenzylammonium chloride5. Myristamidopropyl betaine6. Cetyltrimethyl ammonium chloride7. Cetylpyridinium chloride8. Cetyl betaine9. Decyl sulfate10. Dodecyl sulfate11. C 10 -LAS12. C 11 -LAS13. C 12 -LAS14. C 13 -LAS14n ~ 10Triton X-1000 5 10 15 20 25 30 35Minutes2112822794UV, 225 nmDodecylbenzene sulfonateELSDAnalysis of a Laundry Washing DetergentDodecylbenzene sulfonateand Laureth sulfateColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–80% A in 30 min,then hold at 80% A for 15 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: ELSD, UVSample Prep: Dilute 10× with 70% acetonitrile,then filtered through 0.2-µmmembraneUV, 225 nmDodecylbenzenesulfonateELSDDodecylbenzene sulfonateand laureth sulfateAnalysis of a Dishwashing LiquidColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–80% A in 30 min,then hold at 80% A for 10 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSD, UVSample Prep: Dilute 10× with 70% acetonitrile,then filtered through 0.2-µmmembraneAPPLICATIONS0 5 10 15 20 25 30 35 40 45Minutes0 5 10 15 20 25 30 35 40Minutes2108421085www.dionex.com - 43 -

Ch e m i c a l ApplicationsSurfactantsAUAnalysis of a Strongly Hydrophilic HydrotropeAcclaim SurfactantConventional C180 5 10 15MinutesDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/0.1 M NH 4 OAc,pH 5.4 v/v 30/70Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV, 225 nmSample: Sodium xylene sulfonate (1000 ppm)210744µS0Analysis of Ammonium Laureth SulfateUsing Conductivity Detection0 10 2030MinutesColumn: Acclaim Surfactant, 5 µmDimension: 4.6 × 150 mmMobile Phase: Acetonitrile/0.5 M H 3 BO 4and 20 mM NaOH v/v 55/45Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: Suppressed CD (AMMS III)Sample: Ammonium laureth sulfate21076Analysis of NEODOL 25-7Analysis of Quats Using LC-ESI-MSmVR—(OCH 2 CH 2 ) n OHR = C 12 to C 15n ~ 7NEODOL 25-7Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) H 2 OGradient: 40–65% A in 30 min,then hold at 65% A for 5 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSDAbundanceC15C16C17C18Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: Acetonitrile/50 mM NH 4 OAc,pH 5.4 v/v 60/40Temperature: 25 °CFlow Rate: 1 mL/min (0.2 mL/min tomass spectrometer)Injection Vol.: 25 µLDetection: MS (ESI positive, probe 300 °C,cone 30 V, needle 2.5 kV, SIM 332.4,360.4, 374.4, 388.5, span 0.5 m/z)Peak: Benzyl dimethyl hydrogenatedtallow ammonium chloride (10 ppm)0 10 20 30 35Minutes210790 5 10 15Minutes22127APPLICATIONSmV121Analysis of Toilet Bowl Cleaner0.1 M NH 4 OAc0 5 10 15 20 25 30Minutes56 7 8430.02 M NH 4 OAcSeparation improved by changingmobile phase ionic strength54 6 738Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN(B) NH 4 OAc, pH 5.4Gradient: 25% to 85% A in 25 min,hold for 5 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELS detectorSample: Toilet bowl cleaner(20× dilution)Peaks: 1. Chloride2. PEG3–7. Cationic surfactants8. Nonionic surfactant22797mVInorganic Anions, Hydrotropes, Cationic, Nonionic,Amphoteric, and Anionic Surfactants213Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN,(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25% to 85% A in 25 min,then hold 85% A for 10 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: ELS detector45670 5 10 15 20 25 30 35Minutes89101112Peaks:1. Chloride2. Bromide3. Nitrate4. Xylene sulfonate5. Laurylpyridinium chloride6. Lauryldimethylbenzylammoniumchloride7. Triton X-1008. Cetyl betaine9. Decyl sulfate10. Dodecyl sulfate11. C 10 -LAS12. C 11 -LAS13. C 12 -LAS14. C 13 -LAS131422793- 44 -www.dionex.com

Ch e m i c a l ApplicationsSurfactantsSeparation of Different Polyethylene GlycolsSeparation of Quaternary Imidazolinium CompoundsmVPEG-300PEG-400Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) H 2 OGradient: 3–15% A in 20 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSDmV2-Hydroxyethylbenzylcoco imidazoliniumchlorideDiethyl heptadecylimidazoliniumethylsulfateColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–85% A in 30 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: ELSDPEG-6000 5 10 15 20Minutes0 5 10 15 20 25 30Minutes2107721117µS01Separation of Cationic Surfactants withSuppressed Conductivity Detection25345Minutes10Column: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) Acetonitrile(B) 20 mM acetic acidGradient: 20–50% A in 15 minTemperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: Suppressed CD (AMMS III)Peaks: 1. Lauryl pyridinium chloride2. Lauryldimethylbenzylammonium chloride3. Octylphenoxyethoxyethyldimethylbenzyl ammonium chloride4. Cetyltrimethylammonium chloride5. Cetylpyridinium chloride1521086mVAnalysis of PEG-200 Distearate0 5 10 15 20MinutesOOColumn: Acclaim Surfactant, 5 µmDimensions: 4.6 × 150 mmMobile Phase: (A) CH 3 CN,(B) 0.1 M NH 4 OAc, pH 5.4Gradient: 25–85% A in 10 min,then hold 85% A for 10 minTemperature: 30 °CFlow Rate: 1.5 mL/minInj. Volume: 20 µLDetection: ELS detectorSample: PEG-200 DistearateOOOO22796POE(30) Ammonium Lauryl SulfateDyes15µSColumn: Acclaim PA2, 5 µmDimension: 4.6 × 150 mmMobile phase: Acetonitrile/25 mM borate buffer v/v 40/60Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 20 µL (1000 ppm)Detection: Suppressed conductivity detection(AMMS III)Sample: POE(30) Ammonium lauryl sulfate40mAUSudan Dyes on Acclaim 120 C18132 4Column: Acclaim 120 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: 50/50/0.5 MeOH/acetonitrile/HOAcFlow Rate: 1.0 mL/minInj. Volume: 20 µLDetection: UV, 280 nmPeaks: 1. Sudan I2. Sudan I, impurity3. Sudan II4. Sudan III00 10 2025Minutes211370 1 2 3 4 5 6Minutes20411APPLICATIONSwww.dionex.com - 45 -

Bioscience ApplicationsProteins and PeptidesAUTryptic Peptide Map of Cytochrome c on Acclaim 300 C181 Minutes30Column: Acclaim 300 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 95/5/0.1 H 2 O/acetonitrile/TFA(B) 5/95/0.1 H 2 O/acetonitrile/TFAFlow Rate: 1.0 mL/minGradient: 15% B to 50% B in 35 minInj. Volume: 40 µLDetection: UV, 214 nm20405100mAU–10010Separation of Protein Standards byMicro LC on Acclaim 300 C18132420 30Minutes40Column: Acclaim 300 C18, 3 µmDimensions: 300-µm i.d. × 15 cmMobile Phase: (A) Water. 0.05% TFA(B) 8/2 CH 3 CN/ water,0.04 % TFAGradient: 15–60% B in 30 minFlow Rate: 4 µL/minDetection: UV, 214 nmPeaks: 1. Ribonuclease 25 ng2. Insulin 253. Cytochrome c 254. Myoglobin 2520409AUTryptic Peptide Map of BovineSerum Albumin on Acclaim 300 C18Column: Acclaim 300 C18, 3 µmDimensions: 4.6 × 50 mmMobile Phase: (A) 95/5/0.1 H 2 O/acetonitrile/TFA(B) 5/95/0.1 H 2 O/acetonitrile/TFAFlow Rate: 1.0 mL/minGradient: 5% B to 40% B in 35 minInj. Volume: 40 µLDetection: UV, 214 nmCapillary LC for Peptide Mapping Using Acclaim 300 C1822Column: Acclaim 300 C18, 3 µmDimensions: 75 µm i.d. × 15 cmMobile Phase: (A) 95/5 (v/v) water/acetonitrile, 0.05% TFA(B) 20/80 (v/v) water/acetonitrile, 0.04% TFAGradient: 5–60% B in 60 minSample: Tryptic digest of BSA,1 pmol/µLDetection: UV, 214 nmmAU2 Minutes3020406–215 20 25 30 35 40 45 50 55 60 65 70Minutes20408APPLICATIONS- 46 -www.dionex.com

Bioscience ApplicationsProteins and Peptides350mAU0Cytochrome c from Three Species on Acclaim 300 C1821–1000 2 4 6 8 10 12Minutes3Column: Acclaim 300 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 95/5/0.1 v/v water/acetonitrile/TFA(B) 95/5/0.1 v/v acetonitrile/water/TFAGradient: Time (min) % A % B–5 80 200 80 2015 45 5520 45 55Flow Rate: 1.0 mL/minInj. Volume: 10 µL (1 µg/µL)Detection: UV, 214 nmPeaks: 1. Yeast cytochrome c2. Equine cytochrome c3. Bovine cytochrome c19104Protein Mixture by Reversed-Phase HPLC on Acclaim 300 C181,000mAU14325–506 8 10 12 14 16Minutes67Column: Acclaim 300 C18, 3 µmDimensions: 4.6 × 150 mmMobile Phase: (A) 5 mM MSA in 95/5 H 2 O/acetonitrile(B) 5 mM MSA in 5/95 H 2 O/acetonitrileInj. Volume: 25 µLDetection: UV, 214 nmGradient: Time % A % B Flow0 100 0 1.03 100 018 0 10023 0 100Peaks: 1. RNAse A2. Impurity in carbonic anyhdrase3. Impurity in lysozyme4. Lysozyme5. Impurity in carbonic anhydrase6. Myoglobin7. Carbonic anhydrase20407Comparison of Acid Additives for Peptide Analysis on Acclaim 3002AU0ABC2 4 6 8 10 12MinutesColumn: Acclaim 300 C18, 3 µm,4.6 × 150 mmPump: Summit P680AMobile Phases: A: 70% acetonitrile v/vB: WaterC: Acid stock solutionGradient Times: –8 0 16%A 7 7 75%B 68 68 0%C 25 25 25Flow: 1.0 mL/minTemperature: TCC100 thermostat at 30 °CInjection: ASI-100 autosampler; 5 µLDetector: UVD 340U; UV at 214 nmAcid Additive: A. Phosphoric Acid, 0.15%B. Trifluoroacetic Acid, 0.1%C. Formic Acid, 0.1%Sample: Sigma H2016 Peptide MixPeaks: 1. Gly-Tyr2. Val-Tyr-Val3. Met-Enkephalin4. Angiotensin-II5. Leu-Enkephalin230641.2AUPeptide Test Mix on Acclaim 300 and PA2 with Formic Acid0AB2 4 6 8 10 12MinutesColumns: (A) Acclaim 300 C18, 3 µm,4.6 × 150 mm(B) Acclaim PA2, 3 µm,4.6 × 150 mmPump: Summit P680AMobile Phases: (A) 70% acetonitrile v/v(B) Water(C) 0.4% formic acid v/vGradient Times: –8 0 16%A 7 7 75%B 68 68 0%C 25 25 25Flow: 1.0 mL/minTemperature: TCC100 thermostat at 30 °CInjection: ASI-100 autosampler; 5 µLDetector: UVD 340U; UV at 214 nmSample: Sigma H2016 Peptide MixPeaks: 1. Gly-Tyr2. Val-Tyr-Val3. Met-Enkephalin4. Angiotensin-II5. Leu-Enkephalin23066APPLICATIONSwww.dionex.com - 47 -

Lot Qualification TestsPa r t Fo u r: AppendixAppendix 1: Lot Qualification Testsfor Acclaim-Bonded SilicaColumn Performance Test, Acclaim C18, C8, PA, PA2Polar SelectivityThis test protocol is part of the column performance test:column pressure, retention time, efficiency, and asymmetry forphenanthrene are specified for each column. This parameter alsois used to monitor batch-to-batch reproducibility as measured bythe selectivity of a polar analyte (dimethylphthalate) relative to anonpolar analyte (phenanthrene).Mobile Phase: 70/30 v/v acetonitrile/waterFlow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 254 nm, 6-mm path lengthPeaks: 1. Uracil 0.015 mg/mL2. Dimethylphthalate 0.075 µL/mL3. Phenanthrene 0.015 mg/mLMetal Activity2,2’-Bipyridyl chelates with metals whereas 4,4’-bipyridyldoes not form chelation complexes. If metals are present, the 2,2’isomer elutes with a high asymmetry factor (tailing). The peakasymmetry ratio for the two analytes should be approximately 1.0if no metal contamination is present.Mobile Phase: 50/50 v/v methanol/waterFlow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 254 nmPeaks: 1. 4,4’-Bipyridyl 50 ng/µL2. 2,2’-Bipyridyl 200 ng/µLBase AsymmetryBasic compounds exhibit poor peak shape when the silica surfaceis incompletely covered during the bonding process, or whenthe silica substrate is inadequately prepared. This test monitorssurface coverage with an application-based analysis.Mobile Phase: 80/20 v/v methanol/30 mM phosphate, pH 6.0Flow Rate: 1.0 mL/minTemperature: 30 °CIn. Volume: 5 µLDetection: UV, 220 nmPeaks: 1. Uracil 0.04 mg/mL2. Propranolol 0.06 mg/mL3. Toluene 0.08 µL/mL4. Amitriptyline 0.24 mg/mLHydrophobic Steric SelectivityThis parameter is used to monitor batch-to-batch bondingreproducibility as measured by the relative selectivity of two differentlyshaped aromatic analytes: o-terphenyl and triphenylene. Thistest is used to characterize all the C18 and polar-embedded phases.Mobile Phase: 90/10 v/v methanol/waterFlow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 254 nmPeaks: 1. Toluene 2 µl/mL2. Phenanthrene 0.03 mg/mL3. o-terphenyl 0.14 mg/mL4. Triphenylene 0.03 mg/mLAPPENDIXAcid AsymmetryThis test is used to characterize polar-embedded phases.Acidic compounds exhibit poor peak shape when the bondedphase has basic sites due to degradation or contamination.Mobile Phase: 35/65 v/v acetonitrile/0.1% TFA in waterFlow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume 5 µLDetection: UV, 240 nmPeaks: 1. 4-Hydroxybenzoic acid 50 ng/µL2. Benzoic acid 70 ng/µL3. Ethyl 4-hydroxybenzoate 50 ng/µLSome application-specific tests are used for lot validation. Readabout these tests in the chapters for Acclaim 300 and Acclaim OA.- 50 -www.dionex.com

Lot Qualification TestsColumn Performance Test, Acclaim SurfactantIsocratic Surfactant SelectivityEach column is tested under these conditions to guarantee thatthe column is well manufactured. Column pressure, efficiency,asymmetry and retention time of propylbenzene are specifiedfor each column. This test also is used to monitor batch-to-batchreproducibility as measured by the relative retention of an anionicsurfactant to a neutral marker.Mobile Phase: 50:50 (v/v) acetonitrile : 0.1M NH 4 OAc, pH 5.2Flow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 220 nmPeaks: 1. Uracil 0.10 mg/mL2. 2-(4-octylphenoxy)ethoxyethyldimethylbenzylammonium chloride 0.10 mg/mL3. Sodium p-Toluenesulfonate 0.20 mg/mL4. Propylbenzene 0.40 mg/mLColumn Performance Test, Acclaim Explosives E1 and E2Each column is tested under these conditions to guarantee thatthe column is well manufactured. Column pressure, efficiency,asymmetry, and retention time of naphthalene are specified foreach column.Mobile Phase: 70/30 v/v acetonitrile/waterFlow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV, 254 nmPeaks: 1. Uracil 0.05 mg/mL2. Naphthalene 0.5 mg/mLGradient Surfactant ResolutionAn application-based test for the resolution of anionic, nonionicand cationic surfactants.Mobile Phase A: AcetonitrileMobile Phase B: 0.10 M Ammonium acetate, pH 5.4Time %A %B0.0 25 7525.0 85 1530.0 85 15Flow Rate: 1.0 mL/minTemperature: 30 °CInj. Volume: 20 µLDetection: Evaporative Light ScatteringAnalytes: 1. Laurylpyridinium chloride 0.10 mg/mL2. Lauryldimethylbenzylammonium chloride 0.10 mg/mL3. Triton X-100 0.40 mg/mL4. Sodium decylsulfate 0.20 mg/mL5. Sodium dodecylsulfate 0.10 mg/mLAPPENDIXwww.dionex.com - 51 -

Topical IndexAppendix 2:Topical IndexAcclaim 120 C18 4-5, 7, 10-11, 1213, 29-41, 48, 50120 C8 4-5, 7, 10, 14-15,31, 35, 50300 C18 4-5, 7, 10, 16-17,32, 46-47, 50PA 4-5, 7, 10-11,18-20, 29-31,33-40, 50PA2 4-5, 7, 10, 20-21,30-33, 39, 47, 50PolarAdvantage,see Acclaim PAand PA2OA 4-6, 22-23, 31,37, 40, 49Surfactant 4-6, 24-25,42-45, 51Explosives 4-6, 26-27,36, 51Acid Aromatic 22, 49Asymmetry 10, 19, 21Asymmetry test50Hydrophilic 22Organic 4, 6, 22-23, 37Alkaline Buffers 9, 21Amitriptyline 10, 29, 50AntibioticsAsymmetry 12, 14, 16, 19,21,22, 24, 27Acid 10, 19, 21, 22, 50Base 10, 19, 21, 50Base Asymmetry 10, 19, 21Asymmetry test12, 50Basic Buffers 9, 21Drugs 14, 29-33Beverages 22, 37-41Bipyridyl 11, 50Buffers 9Capillary 13, 15, 17, 19CARB Method 1004 35Carbon Load 8Column Diameter 8Format 6, 8Length 8Corrosion 12, 14, 16, 18,20, 24Cytochrome c 16, 46-47Detection limit 8De-wetting 18, 20Efficiency 7, 12, 14, 16,19, 21, 22, 25,27, 50ELSD 24, 42-45, 51Endcapping 5, 8, 12, 14, 16,18, 20Environmental 34-36EPA Methods531 36604 34605 34606 348270 34, 368310 358330 26-27, 36Evaporative Light Scattering Detection(ELSD) 24, 42-45, 51Explosives 5, 6, 26-27, 36Flow rate 8Food and Beverages 37-41Guard cartridge 13, 15, 17, 19,21, 23, 25, 27Hydrolytic stability 4, 9, 18, 20-21Hydrophilic 18, 20HydrophobicSteric selectivity test11, 12, 50Hydrophobicity 6Ion Exchange 6k’ 12, 14Loading Capacity 8Lot Qualification (see lotvalidation)Lot Validation 10, 12, 14, 16,19, 21, 22, 25,27, 50-51LC-MS 34, 36, 43Compatibility13, 15, 17, 19Metal Activity test 12, 14, 16, 19,21, 50Contamination11Methanesulfonic acid 23Mobile Phase 9Natural products 48Nitramine 26, 36Nitroaromatic 26, 36Normal-phase 18Octadecyldimethylsilane 12, 16Octyldimethylsilane 14Ordering information inside frontcover,13, 15, 17, 19,21, 23, 25, 27Organic acids 22-24, 31, 37,39PA 4-5, 7, 10-11,18-20, 29-31,33-40, 50PA2 4-5, 7, 10,20-21, 30-33,39, 47, 50Particle Size 5, 7PEEK 22Peptide 4, 16, 46-47pH 4, 9, 12, 14,16, 18, 20, 22,24, 50-51Pharmaceutical 29-33Phenanthrene 50Polar Embedded 4-5, 18-21Selectivity Test50PolarAdvantage, see PA and PA2Polarity index 10, 20Pore Size 5, 7Pressure 7, 8Protein 4, 16-17,46-47Resolution 22Reversed-phase 6Selectivity 5, 6-7, 12, 18,20, 22, 24, 26Base 7Ethylbenzene/toluene7Peptide 16Polar 50Shape 6-7Steric 50Tripheylene/o-terphenyl6-7, 50Silanol 6, 8, 10, 12,14, 18Silica Substrate 5Suppressed Conductivity Detection44, 45Surface Area 5, 7, 12, 14,16, 18, 20, 22,24, 27Surfactants 6, 24-25,42-45, 51Tryptic digest 16, 46USP Code 5, 12, 14, 16Validation(see lotValidation)Vitamins 37-40Warrantyinside frontcoverWetting 12, 14, 18, 20,APPENDIX- 52 -www.dionex.com

Part Number IndexAppendix 3:Part Number IndexPart No. Acclaim Type Size Page Part No. Acclaim Type Size Page Part No. Acclaim Type Size Page059122 120, 3 µm, C8 2.1 × 50 mm 15059123 120, 3 µm, C8 2.1 × 100 mm 15059124 120, 3 µm, C8 2.1 × 150 mm 15059125 120, 3 µm, C8 4.6 × 50 mm 15029126 120, 3 µm, C8 4.6 × 100 mm 15059127 120, 3 µm, C8 4.6 × 150 mm 15059128 120, 3 µm, C18 2.1 × 50 mm 13059129 120, 3 µm, C18 2.1 × 100 mm 13059130 120, 3 µm, C18 2.1 × 150 mm 13059131 120, 3 µm, C18 4.6 × 50 mm 13059132 120, 3 µm, C18 4.6 × 100 mm 13059133 120, 3 µm, C18 4.6 × 150 mm 13059134 120, 5 µm, C8 2.1 × 50 mm 15059135 120, 5 µm, C8 2.1 × 100 mm 15059136 120, 5 µm, C8 2.1 × 150 mm 15059137 120, 5 µm, C8 2.1 × 250 mm 15059138 120, 5 µm, C8 4.6 × 50 mm 15059139 120, 5 µm, C8 4.6 × 100 mm 15059140 120, 5 µm, C8 4.6 × 150 mm 15059141 120, 5 µm, C8 4.6 × 250 mm 15059142 120, 5 µm, C18 2.1 × 50 mm 13059143 120, 5 µm, C18 2.1 × 100 mm 13059144 120, 5 µm, C18 2.1 × 150 mm 13059145 120, 5 µm, C18 2.1 × 250 mm 13059146 120, 5 µm, C18 4.6 × 50 mm 13059147 120, 5 µm, C18 4.6 × 100 mm 13059148 120, 5 µm, C18 4.6 × 150 mm 13059149 120, 5 µm, C18 4.6 × 250 mm 13059446 120, 5 µm, C18 4.3 × 10 mm 13059447 120, 5 µm, C18 2 × 10 mm 13059448 120, 5 µm, C8 4.3 × 10 mm 15059449 120, 5 µm, C8 2 × 10 mm 15059456 Guard Cartridge holder 13, 15, 17,19,23, 25, 27059457 Guard to analyical coupler 13, 15, 17,19,23, 25, 27059526 Guard holder & coupler kit 13, 15, 17,19,21, 23, 25, 27060263 300, 3 µm, C18 2.1 × 50 mm 17060264 300, 3 µm, C18 2.1 × 150 mm 17060265 300, 3 µm, C18 4.6 × 50 mm 17060266 300, 3 µm, C18 4.6 × 150 mm 17060393 300, 3 µm, C18 4.3 × 10 mm 17060395 300, 3 µm, C18 2 × 10 mm 17061316 PA, 3 µm, C16 2.1 × 100 mm 19061317 PA, 3 µm, C16 2.1 × 150 mm 19061318 PA, 3 µm, C16 4.6 × 150 mm 19061319 PA, 5 µm, C16 4.6 × 50 mm 19061320 PA, 5 µm, C16 4.6 × 150 mm 19061321 PA, 5 µm, C16 4.6 × 250 mm 19061331 PA, 5 µm, C16 2 × 10 mm 19061332 PA, 5 µm, C16 4.3 × 10 mm 19062902 OA, 5 µm 4.0 × 250 mm 23062903 OA, 5 µm 4.0 × 150 mm 23062925 OA, 5 µm Guard 4.3 × 10 mm 23063187 PA2, 3-µm 2.1 × 150 mm 21063189 PA2, 3-µm 4.6 × 50 mm 21063191 PA2, 3-µm 4.6 × 150 mm 21063193 PA2, 5 µm Guard2.0 × 10 mm (pack of 2) 21063195 PA2, 5 µm Guard4.3 × 100 mm (pack of 2) 21063197 PA2, 5-µm 4.6 × 150 mm 21063199 PA2, 5-µm 4.6 × 250 mm 21063201 Surfactant 5 µm 4.6 × 150 mm 25063203 Surfactant5 µm 4.6 × 250 mm 25063215 Surfactant 5 µm Guard4.3 × 10 mm (pack of 2) 25064305 Expolsives 4.6 × 250 mm E1 27064303 Expolsives 5 µm Guard4.3 × 10 mm E1 (pack of 2) 27064307 Expolsives 5 µm Guard4.3 × 10 mm E2 (pack of 2) 27064309 Expolsives 5 µm 4.6 × 250 mm E2 27064312 Expolsives 5 µm E1 and E2 kit 27161450 120, 5 µm, C18 1.0 × 50 mm 13161451 120, 5 µm, C18 1.0 × 150 mm 13161452 120, 5 µm, C18 1.0 × 250 mm 13161453 120, 5 µm, C18 300 µm × 50 mm 13161454 120, 5 µm, C18 300 µm × 50 mm 13161455 120, 5 µm, C18 300 µm × 250 mm 13161456 120, 5 µm, C18 75 µm × 50 mm 13161457 120, 5 µm, C18 75 µm × 150 mm 13161458 120, 5 µm, C18 75 µm × 250 mm 13162204 120, 3 µm, C8 1.0 × 50 mm 15162205 120, 3 µm, C8 1.0 × 150 mm 15162206 120, 3 µm, C8 300 µm × 50 mm 15162207 120, 3 µm, C8 300 µm × 150 mm 15162208 120, 3 µm, C8 75 µm × 50 mm 15162209 120, 3 µm, C8 75 µm × 150 mm 15162210 120, 5 µm, C8 1.0 × 50 mm 15162211 120, 5 µm, C8 1.0 × 150 mm 15162212 120, 5 µm, C8 1.0 × 250 mm 15162213 120, 5 µm, C8 300 µm × 50 mm 15162214 120, 5 µm, C8 300 µm × 150 mm 15162215 120, 5 µm, C8 300 µm × 250 mm 15162216 120, 5 µm, C8 75 µm × 50 mm 15162217 120, 5 µm, C8 75 µm × 150 mm 15162218 120, 5 µm, C8 75 µm × 250 mm 15162219 300, 3 µm, C18 1.0 × 50 mm 17162220 300, 3 µm, C18 1.0 × 150 mm 17162221 300, 3 µm, C18 300 µm × 50 mm 17162222 300, 3 µm, C18 300 µm × 150 mm 17162223 300, 3 µm, C18 75 µm × 50 mm 17162224 300, 3 µm, C18 75 µm × 150 mm 17162234 120, 3 µm, C18 1.0 × 50 mm 13162235 120, 3 µm, C18 1.0 × 150 mm 13162236 120, 3 µm, C18 300 µm × 50 mm 13162237 120, 3 µm, C18 300 µm × 150 mm 13162238 120, 3 µm, C18 75 µm × 50 mm 13162239 120, 3 µm, C18 75 µm × 150 mm 13162240 PA, 3 µm, C16 1.0 × 50 mm 19162241 PA, 3 µm, C16 1.0 × 150 mm 19162242 PA, 3 µm, C16 300 µm × 50 mm 19162243 PA, 3 µm, C16 300 µm × 150 mm 19162244 PA, 3 µm, C16 75 µm × 50 mm 19162245 PA, 3 µm, C16 75 µm × 150 mm 19162246 PA, 5 µm, C16 1.0 × 50 mm 19162247 PA, 5 µm, C16 1.0 × 150 mm 19162248 PA, 5 µm, C16 1.0 × 250 mm 19162249 PA, 5 µm, C16 300 µm × 50 mm 19162250 PA, 5 µm, C16 300 µm × 150 mm 19162251 PA, 5 µm, C16 300 µm × 250 mm 19162252 PA, 5 µm, C16 75 µm × 50 mm 19162253 PA, 5 µm, C16 75 µm × 150 mm 19162254 PA, 5 µm, C16 75 µm × 50 mm 19162261 120, 5 µm, C8 1.0 × 5 mm 15162262 120, 5 µm, C8 1.0 × 15 mm 15162263 120, 5 µm, C8 800 µm × 5 mm 15162264 120, 5 µm, C8 500 µm × 5 mm 15162265 120, 5 µm, C8 500 µm × 15 mm 15162266 120, 5 µm, C8 300 µm × 5 mm 15162302 PA, 5 µm, C16 300 µm × 5 mm 19162304 120, 5 µm, C8 300 µm × 1.0 mm 15162306 120, 5 µm, C18 300 µm × 1.0 mm 13162310 PA, 5 µm, C16 300 µm × 1.0 mm 19162321 120, 5 µm, C18 1.0 × 5 mm 13162322 120, 5 µm, C18 1.0 × 15 mm 13162323 120, 5 µm, C18 800 µm × 5 mm 13162324 120, 5 µm, C18 500 µm × 5 mm 13162325 120, 5 µm, C18 500 µm × 15 mm 13162326 120, 5 µm, C18 300 µm × 5 mm 13162333 PA, 5 µm, C16 1.0 × 5 mm 19162334 PA, 5 µm, C16 1.0 × 15 mm 19162335 PA, 5 µm, C16 800 µm × 5 mm 19162336 PA, 5 µm, C16 500 µm × 5 mm 19132337 PA, 5 µm, C16 500 µm × 15 mm 19APPENDIXwww.dionex.com - 53 -

Analyte IndexAppendix 4:Analyte IndexAcebutolol.......................................14, 29, 31Benzyl alcohol............................................ 29Cytochrome-c....................................... 46, 47Fluoranthene.............................................. 35APPENDIXAcenaphthalene......................................... 35Acenaphthene............................................ 35Acesulfame-K............................................. 40Acetaldehyde-DNPH................................ 35Acetaminophen.................................... 30, 32Acetic acid................................................... 23Acetone-DNPH.......................................... 35cis-Aconitic acid......................................... 23trans-Aconitic acid..................................... 23Acrolein-DNPH......................................... 35Acrylic acid................................................. 49Acrylic acid dimer..................................... 49Adhumulone.............................................. 39Adlupulone................................................. 39Aflatoxin B1................................................ 40Aflatoxin B2................................................ 40Aflatoxin G1............................................... 40Aflatoxin G2............................................... 40Aldicarb................................................. 35, 36Aldicarb sulfone................................... 35, 36Aldicarb sulfoxide............................... 35, 36Ametryn...................................................... 362-Amino-4,6-dinitrotoluene.........26, 27, 364-Amino-2,6-dinitrotoluene.........26, 27, 36Amitriptyline......................10, 29, 30, 32, 50Amobarbital................................................ 32Angiotensin-II............................................ 47Aniline........................................................... 7Anthracene.................................................. 35Antibiotics.....................30, 31, 36, 38, 40, 41Antidepressants.................10, 29, 30, 32, 50Antioxidants............................................... 41Ascorbic acid (Vitamin C)............37, 38, 39Aspartame............................................. 38, 40Aspartylphenylalanine....................... 38, 40Aspartyl-phenylalanine methyl ester.38, 40Atenolol................................................. 14, 29Atrazine...........................................13, 34, 36Barbital........................................................ 32Benzaldehyde............................................. 32Benzaldehyde-DNPH............................... 35Benzene 1,2,3-tricarboxylic acid.............. 49Benzene 1,2-dicarboxylic acid.................. 49Benzene 1,3,5-tricarboxylic acid.............. 49Benzene 1,3-dicarboxylic acid.................. 49Benzene 1,4-dicarboxylic acid.................. 49Benzidine.............................................. 34, 36Benzil........................................................... 32Benzo[a]anthracene................................... 35Benzo[a]pyrene.......................................... 35Benzo[b]fluoranthene................................ 35Benzo[g,h,i]perylene................................. 35Benzo[k]fluoranthene................................ 35Benzoic acid........................32, 39, 40, 42, 50Benzoin........................................................ 32Benzophenone............................................ 32Benzophenone-3........................................ 335-Benzyl-3,6-dioxo-2-piperazineacetic acid.................................................... 38Benzyldimethyl hydrogenated tallowammonium chloride.................................. 44Berberine..................................................... 48Biochanin A................................................ 48Biotin...................................................... 38, 392,2’-Bipyridyl........................................ 11, 504,4’-Bipyridyl........................................ 11, 50Bis(2-ethylhexyl)phthalate....................... 34Bisdemethoxycurcumin............................ 37Bovine serum albumin tryptic digest..... 46Brodifacoum............................................... 34Bromide................................................. 31, 44Budesonide................................................. 32Butabarbital................................................ 322-Butanone-DNPH.................................... 354-Butylbenzoic acid............................. 10, 20Butyl paraben............................................. 39Butylated hydroxyanisole (BHA)............ 41Butylated hydroxytoluene (BHT)............ 41Butylbenzylphthalate................................ 34Butyraldehyde-DNPH.............................. 35Butyric acid................................................. 23C10-LAS..........................................25, 43, 44C11-LAS..........................................25, 43, 44C12-LAS..........................................25, 43, 44C13-LAS..........................................25, 43, 44Caffeine...........................................39, 40, 48Caproic acid................................................ 23Carbamate insecticides....................... 35, 36Carbaryl................................................. 35, 36Carbofuran............................................ 35, 36Carbonic anhydrase............................ 46, 47beta-Carotene.................................37, 38, 40cis-beta-Carotene.................................. 37, 38trans-beta-Carotene.......................37, 38, 40Carotenoids....................................37, 38, 40Cetyl betaine...................................24, 43, 44Cetylpyridinium chloride.......24, 42, 43, 45Cetyltrimethylammoniumchloride......................................24, 42, 43, 45Chloramphenicol....................................... 36Chloride...................................................... 44Chlorohydrine............................................ 314-Chloro-3-methylphenol......................... 342-Chlorophenol.......................................... 34Chlortetracycline........................................ 40Cholecalciferol (Vitamin D3)............. 37, 40Chyrsene..................................................... 35Cinchonidine.............................................. 39Citric acid..................................23, 31, 37, 40Clonazepam................................................ 29Cohumulone............................................... 39Colupulone................................................. 39Cortisol (Hydrocortisone).................. 32, 33Crotonaldehyde-DNPH............................ 35Curcumin.................................................... 37Cytochrome-c tryptic digest.............. 16, 46Cytosine................................................ 18, 21Decyl sulfate.............................24, 43, 44, 51Demethoxycurcumin................................. 3711-Deoxycortisol........................................ 33Dexamethasone.......................................... 33Dextromethorphan.............................. 30, 322,4-Diamino-6-nitrotoluene...................... 27Diazepam.................................................... 29Dibenzo[a,h]anthracene............................ 35Di-n-butylphthalate................................... 343,3’-Dichlorobenzidine........................ 34, 362,4-Dichlorophenol.................................... 34Diethyl heptadecyl imidazoliniumethylsulfate................................................. 45Diethylphthalate........................................ 34Dihydroquinine.......................................... 392,5-Dihydroxybenzoic acid...................... 493,5-Dihydroxybenzoic acid...................... 493,4-Dihydroxybenzylamine...................... 30Dimethylaniline...............................7, 10, 203,3’-Dimethylbenzidine...................... 34, 362,3-Dimethyl-2,3-dinitrobutane(DMDNB).................................................... 272,4-Dimethylphenol................................... 34Dimethylphthalate............................... 34, 501,3-Dinitrobenzene........................26, 27, 362,4-Dinitrophenol....................................... 342,4-Dinitrophenylhydrazine (DNPH)..... 352,4-Dinitrotoluene..........................26, 27, 362,6-Dinitrotoluene..........................26, 27, 36Di-n-octylphthalate................................... 34Diuron......................................................... 34Dodecyl gallate.......................................... 41Dodecyl sulfate........................24, 43, 44, 51Dodecylbenzene sulfonate................. 25, 43Domiphen bromide................................... 42Dopamine................................................... 30Doxepin...........................................10, 29, 30Doxycycline................................................ 40Doxylamine.......................................... 30, 32Epicatechin gallate..................................... 48Epichatechin............................................... 48Epigallocatechin......................................... 48Epigallocatechin gallate............................ 48Epinephrine................................................ 30Epoxyefenone............................................. 31Ergocalciferol (Vitamin D2)............... 37, 40Erythorbic acid........................................... 40Ethoquad 18/25......................................... 42Ethyl 4-hydroxybenzoate......................... 50Ethyl benzoate.............................................. 7Ethyl paraben............................................. 39Ethylbenzene................................................ 7Ethyleneglycol dinitrate (EGDN)............ 27Explosives.......................................26, 27, 36Fludrocortisone.......................................... 33Fluorene...................................................... 35Folic acid............................................... 38, 39Formaldehyde-DNPH............................... 35Formic acid................................................. 23Formononotin............................................. 48Fumaric acid......................................... 23, 37Furaltadone................................................. 38Furazolidone.............................................. 38Genistin....................................................... 48Glutamate................................................... 40Glutamine................................................... 37Gly-Tyr........................................................ 47Heptanoic acid........................................... 23Herbicides.......................................13, 34, 36Hexahydroisohumulone........................... 41Hexaldehyde-DNPH................................. 35HMX................................................26, 27, 36Humulone................................................... 39Hydrastine.................................................. 48Hydrocortisone.................................... 32, 334-Hydroxybenxoic acid....................... 49, 503-Hydroxycarbofuran......................... 35, 362-Hydroxyethylbenzyl cocoimidazolinium chloride............................ 455-Hydroxyindoleacetic acid..................... 304-Hydroxy-3-methoxymandelic acid...... 304-Hydroxy-3-methoxyphenylaceticacid............................................................... 30Ibuprofen.................................................... 33Imipramine........................................... 29, 32Indeno[1,2,3-cd]pyrene............................. 35Insulin.......................................................... 46Ionox-100..................................................... 41Isoadhumulone.......................................... 41Isobutyric acid............................................ 23Isocaproic acid............................................ 23Isocitric acid................................................ 23Isocohumulone........................................... 41Isohumulone............................................... 41rho-Isohumulone....................................... 41Isoleucine.................................................... 37Isovaleric acid............................................. 23Ketoprofen.................................................. 33Labetalol..........................................14, 29, 31Lactic acid............................................. 23, 37Laureth sulfate......................................42-44Lauryl sulfate...........................24, 43, 44, 51Lauryldimethylbenzyl ammoniumchloride.......................................24, 42-45, 51Laurylpyridinium chloride.....24, 42-45, 51Leucine........................................................ 37Leu-Enkephalin.......................................... 47Lincomycin................................................. 29Linuron.................................................. 13, 34Loratidine.................................................... 31Lupulone..................................................... 39trans-Lutein........................................... 38, 40- 54 -www.dionex.com

Analyte Indexcis-Lycopene............................................... 38nor-Dehydroguiaretic acid (NDGA)....... 41Propyl paraben..................................... 32, 39t-Butylhydroquinone (TBHQ)................. 41trans-Lycopene..................................... 37, 38Norepinephrine.......................................... 30Protryptyline.............................................. 29Temazepam................................................ 29Lysine.......................................................... 37Normetanephrine...................................... 30Pseudo-ephedrine....................30, 31, 32, 33Terbutryn.................................................... 36Lysozyme.................................................... 47Nortriptyline.............................................. 29Pyrene.......................................................... 35o-Terphenyl.......................................7, 11, 50Maleic acid......................................14, 29, 31Octyl gallate................................................ 41Pyridine.............................................7, 10, 20Tetracycline................................................. 40Malic acid.............................................. 23, 37Octyl methoxycinnamate......................... 33Pyridoxine............................................. 38, 39Tetrahydroisohumulone........................... 41Metanephrine............................................. 30Octyl salicylate........................................... 33Pyrrobutamine phosphate........................ 29Tetryl...............................................26, 27, 36Metaraminol...................................14, 29, 31Met-Enkephalin......................................... 47Methacrolein-DNPH................................. 35Methiocarb............................................ 35, 36Methomyl.............................................. 35, 363-Methoxy-4-hydroxyphenylglycol........ 30Methyl bis(ethyl(tallowate))-2-hydroxyethylammonium methylsulfate.............. 422-Methyl-4,6-dichlorophenol................... 34Methyl paraben.................................... 32, 39Methylethylketone-DNPH....................... 35Methylprednisolone.................................. 33Metoprolol......................................14, 29, 31Monuron............................................... 13, 34Myoglobin............................................. 46, 47Myristamidopropylbetaine...................... 43N,N-Dimethylaniline.......................7, 10, 20Naphthalene......................................... 35, 51Naproxen.................................................... 33NEODOL 25-7............................................ 44Niacinamide......................................... 38, 39Nitrate.................................................... 24, 44Nitrobenzene...............................7,26, 27, 36Nitrofurantoin............................................ 38Nitrofurazone............................................. 382-Nitrophenol............................................. 344-Nitrophenol............................................. 34N-Nitrosodibutylamine............................ 34N-Nitrosodiethylamine............................. 34N-Nitrosodimethylamine......................... 34N-Nitrosodiphenylamine......................... 34N-Nitrosodipropylamine.......................... 34N-Nitrosomethylethylamine.................... 34N-Nitrosomorpholine............................... 34N-Nitrosopiperidine.................................. 34N-nitrosopyrrolidine................................. 342-Nitrotoluene................................26, 27, 363-Nitrotoluene................................26, 27, 36Octylphenoxyethoxyethyl dimethylbenzylammonium chloride................24, 42, 45, 51Oil red O..................................................... 39Ononin......................................................... 48Oxalic acid.................................................. 23Oxamyl.................................................. 35, 36Oxazepam................................................... 29Oxprenolol......................................14, 29, 31Oxytetracycline.................................... 36, 40Pantothenic acid................................... 38, 39PEG monoethyl ether................................ 43PEG-200 Distearate.................................... 45PEG-300....................................................... 44PEG-400....................................................... 44PEG-600....................................................... 44Pentachlorophenol..................................... 34Pentaethyritol tetranitrate (PETN).......... 27Phenanthrene.................................11, 35, 50Phenobarbital............................................. 32Phenol..........................................7, 10, 20, 34Phenols........................................7, 10, 20, 34Phenothiazine............................................. 29Phenytoin.................................................... 32POE(1) Lauryl sulfate................................ 25POE(30) Ammonium lauryl sulfate........ 45Pramoxine................................................... 30Prednisolone............................................... 33Prednisone.................................................. 33Promethazine HCl..................................... 29Prometon..................................................... 36Prometryn................................................... 36Propafenone................................................ 31Propazine........................................13, 34, 36Propionaldehyde-DNPH.......................... 35Propionic acid............................................ 23Propoxur............................................... 35, 36Propranolol...................10, 14, 29, 30, 31, 50Propyl benzene.......................................... 51Quinine........................................................ 39Ranitidine.................................................... 31Ranitidine S-oxide..................................... 31RDX..................................................26, 27, 36trans-Retinol......................................... 37, 40Retinol acetate...................................... 38, 40Riboflavin.............................................. 38, 39Ribonuclease............................................... 46RNAse-A..................................................... 47Saccharin Sodium...................................... 42Secobarbital................................................ 32Serotonin..................................................... 30Simazine...................................................... 36Sissotrin....................................................... 48Sodium lauryl sulfate..............24, 43, 44, 51Sorbic acid................................................... 39Spectinomycin............................................ 29Steroids.................................................. 32, 33Succinic acid...................................23, 31, 37Sudan Orange I.................................... 39, 48Sudan Orange II................................... 39, 48Sudan Orange III................................. 39, 48Sudan Orange IV....................................... 39Sulfachloropyridazine............................... 41Sulfadiazine.......................................... 30, 41Sulfadimethoxine.....................30, 31, 36, 41Sulfamerazine...........................30, 31, 36, 41Sulfamethazine...............................30, 31, 41Sulfamethoxazole...........................30, 31, 41Sulfanilamide....................................... 30, 31Sulfanilic acid............................................. 30Sulfapyridazine.......................................... 31Sulfapyridine.............................................. 41Sulfaquinoxaline.................................. 31, 41Sulfathiazole......................................... 30, 31Sulfisoxazole......................................... 30, 31Tartaric acid................................................ 37Thenyldiamine HCl................................... 29Theobromine.............................................. 41Thiamine..................................................... 38Thymine..........................................18, 21, 39Timolol............................................14, 29, 31alpha-Tocopherol................................. 37, 40alpha-Tocopherol acetate(Vitamin E)............................................ 38, 40beta-Tocopherol......................................... 37delta-Tocopherol........................................ 37m-Tolualdehyde-DNPH............................ 35Toluene............................7, 10, 11, 20, 30, 50Toluene sulfonate...................................... 512,4,6-Trichlorophenol................................ 343,4,5-Trihydroxybenzoic acid.................. 49Trihydroxybenzophenone (THBP)......... 411,3,5-Trinitrobenzene....................26, 27, 362,4,6-Trinitrotoluene......................26, 27, 36Triphenylene....................................7, 11, 50Triton X-100..............................24, 43, 44, 51Uracil.................10, 18, 20, 21, 30, 32, 50, 51Valeraldehyde-DNPH............................... 35Valeric acid................................................. 23Valine........................................................... 37Val-Tyr-Val................................................. 47Venlafaxine................................................. 29Vitamin A acetate................................ 38, 40Vitamin B 12 .......................................... 38, 39Vitamin C........................................37, 38, 39Vitamin D 2 ............................................ 37, 40Vitamin D 3 ............................................ 37, 40Vitamin E.............................................. 37, 40Vitamin E acetate................................. 38, 40Vitamin K 1 .................................................. 40Vitamins................................................ 37–40Xylene sulfonate............................24, 43, 44ZONYL FSO fluorosurfactant.................. 424-Nitrotoluene................................26, 27, 36Propyl gallate............................................. 41APPENDIXwww.dionex.com - 55 -

SystemsAcclaim Columns—Optimal Performance withthe UltiMate 3000 Intelligent LC and ICS-3000Corporate HeadquartersDionex Corporation1228 Titan WayP.O. Box 3603Sunnyvale, CA 94088-3603Tel: (408) 737-0700Fax: (408) 730-9403The UltiMate 3000 IntelligentLC series delivers the full range ofHPLC. Select from a wide rangeof modules to optimize systemsfor all types of applications, fromsensitive nanoflow LC/MS analysesto automated semipreparativepurifications. Whichever configurationyou choose, you will get ahighly integrated solution withoptimum fluidic connections,single-point and intelligent controlthrough Chromeleon ® software,and seamless intermodule communication.The UltiMate 3000Intelligent LC System won:• Gold award for best newproduct from InstrumentBusiness Outlook.The ICS-3000 metal-freesystem is ideal for applicationsrequiring chemically inert (PEEK)chromatography. The ICS-3000 haswon more than five awards:• R&D 100 Award as one of thebest new products of 2006• Two Gold Awards fromInstrument Business Outlook• An IDEA Bronze award forIndustrial design from BusinessWeek's editorial staff.• The Editors' Choice SilverAward as one of the best newproducts at Pittcon 2005Worldwide Sales and ServiceNorth AmericaU.S. (847) 295-7500Canada (905) 844-9650South AmericaBrazil (55) 11 3731 5140EuropeAustria (43) 1 616 51 25Belgium (32) 3 353 4294Denmark (45) 36 36 90 90France (33) 1 39 30 01 10Germany (49) 6126 991 0Italy (39) 02 51 62 1267The Netherlands (31) 20 683 9768Switzerland (41) 62 205 9966U.K. and Ireland (44) 1276 691722Asia PacificAustralia (61) 2 9420 5233China (852) 2428 3282India (91) 22 28475235Japan (81) 6 6885 1213Korea (82) 2 2653 2580Singapore (65) 6289 1190www.dionex.comDionex products are designed,developed, and manufacturedunder an ISO 9001 Quality System.© 2007 Dionex CorporationAQA and MSQ are trademarksof Thermo Electron Corporation.Zantac is a registered trademarksof Glaxo Smith Kline, Inc.All other trademarks andregistered trademarks are theproperty of Dionex Corporation.LPN 1668-03 PDF 07/07Passion. Power. Productivity.- 56 -www.dionex.com