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colu
mns A c c l a i m C 3 0 C o l u m n
Passion. Power. Productivity.
Acclaim® C30 columns provide unique selectivity and superior separations of hydrophobic, structurally related analytes.
• Highshapeselectivity
• Uniqueselectivitycomplementarytootherreversed-phasecolumns
• Compatibilitywithhighlyaqueousmobilephases
• High-qualitycolumnswithlowcolumnbleed,highefficiency,andruggedpacking
Columns for Separating Hydrophobic Structurally Related Isomers
TheAcclaimC30isdesignedtoprovidehighshapeselectivityforseparatinghydrophobicstructuralrelatedisomersanduniqueselectivitycomplementarytootherreversed-phasecolumns(e.g.,C18).Itisbasedoncova-lentmodificationofC30alkylsilaneonhigh-purity,spherical,poroussilicagel.ThecombinationofadvancedsurfacemodificationtechnologyandcarefulmatchingofC30alkylchainwiththeporesizeofthesilicasubstrateprovidesthefollowingbenefits:
10 12 14 16 18 20 22 240
100
pA
MinutesCorona® ultra™ detector
Standards in Isopropanol
Saponified Chicken Fat
1 2
3 4
5
6 7
8
9 10
11
12
13
14 Peaks: 1. SDA 2. EPA 3. ALA 4. GLA 5. DHA 6. Arach. 7. LLA 8. DPA 9. 9E, 14Z-CLA 10. ETA 11. Adrenic 12. Oleic 13. EDA 14. Erucic
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High Shape Selectivity TheAcclaimC30columnprovides
significantlygreatershapeselectiv-itycomparedtoC18columns,makingitsuitedtoseparatinghydrophobicstructuralisomers,suchascarotenoids,tocopherol,etc.Shapeselectivitycanbecharacterizedbyelutionorderofthreepolycyclicaromatichydrocar-bons(PAHs)inacetonitrile:benzo[a]pyrene(BaP),1,2:3,4:5,6:7,8-tetra-benzonaphthalene(TBN,alternatename,dibenzo[g,p]chrysene),andphenanthro[3,4-c]phenanthrene(PhPh)describedinNISTSRM869a.Asmallerα(TBN/BaP)(=kʹTBN/kʹBaP)valueindicates
highershapeselectivity.AsshowninFigure1,theAcclaimC30columnexhibitsthehighestshapeselectivityofallAcclaimreversed-phasecolumns.
Unique Selectivity Complementary to Other Reversed-Phase Columns
Selectivityisthekeyforsuccess-fulseparation.WhileC18columnsaremostcommonlyusedforsmallmoleculeseparations,otherreversed-phasecolumnswithdifferentselectivityareoftenneededforoptimalresult.Comparedtoothertypesofreversed-phasecolumns—includingC18,polar-embedded,andphenylphases—theAcclaimC30demonstratesdifferentselectivityandprovidesacomple-mentarytoolformethoddevelopment(Figure2).
100% Aqueous CompatibilityToseparatehydrophilicanalytes,
suchasorganicacidsandwater-solublevitaminsonareversed-phasecolumn,ahighlyaqueousmobilephaseisoftenrequiredtoachieveadequateretention.However,mostC18columnswilldewetundersuchcondition,resultinginunreliableresults.DuetoarigidandhighlyorderedC30alkylchain,alongwiththeuseofawiderporesilicasubstrate,AcclaimC30columnsexhibit100%aqueouscompatibility—nolossofretentionwasobservedafter50stop-flowcycles(seeFigure3).
Figure 1. Shape selectivity comparison. Note: lower (TBN/BaP) suggests higher shape selectivity.
Figure 2. The unique selectivity of the Acclaim column family.
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1.0
1.5
2.0
2.5
3.0
AcclaimC30
Acclaim120 C18
AcclaimPA
AcclaimPA2
AcclaimPhenyl-1
0.99 2.10 1.79 1.94 1.85
α (T
BN/B
aP)
Column Name
Columns: (4.6 × 150 mm)Mobile Phase: Acetonitrile/water 85:15 v/vTemperature: 25 °CFlow Rate: 2 mL/minInj. Volume: 5 µLDetection: UV (254 nm)Sample: NIST SRM 869a standard mix 1. Benzo[a]pyrene (BaP)
2. Phenanthro[3,4-c]phenanthrene (PhPh)
3. Dibenzo[g,p]chrysene (TBN)
α(T
BN/B
aP) =
kT́B
N/ kB́a
P
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1 2 3
Acclaim Phenyl
Acclaim Polar Advantage
Acclaim Polar Advantage II
Acclaim 120 C18
Minutes0 6 12 9 3
AU
0
12
3
12
3
12 3
Acclaim C30 2
3, 1 Columns: (4.6 × 150 mm)Mobile Phase: Acetonitrile/water 85:15 v/vTemperature: 25 °CFlow Rate: 2 mL/minInj. Volume: 5 µLDetection: UV (254 nm)Sample: NIST SRM 869a standard mix 1. Benzo[a]pyrene (BaP)
2. Phenanthro[3,4-c]phenanthrene (PhPh)
3. Dibenzo[g,p]chrysene (TBN)
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4
6
8
10
12
0 10 20 30 40 50
Retn
etion
of T
hym
ine (
min
)
Number of Stop-Flow Cycle
De-wetting Test Column: Acclaim C30, 5 µm (4.6 × 150 mm)Mobile Phase: 25 mM Ammonium acetate, pH 5Temperature: 30 °CFlow Rate: 1 mL/minInj. Volume: 5 µLDetection: UV (254 nm)Sample: 0.1 mg/mLProbe: Thymine
Stop-Flow Protocol:1. Test the column under above condition2. Stop the pump for 5 min (pump pressure drops to 0)3. Resume the flow and condition the column under above condition for 10 min4. Inject the sample and run the test under the same conditions.5. Repeat 2 through 4
Figure 3. Aqueous compatibility of the Acclaim C30 column.
3
High Quality of Column ManufacturingLowcolumnbleedisessentialfor
gooddetectionlimits,especiallyformassspectrometer(MS)andchargedaerosoldetectors(CAD).Thepropri-etarybondingprocessensurestheC30bondedphasemaintainsgoodhydrolyticstabilitywithextremelylowcolumnbleed.Ourinnovativepackingmethodprovideshighcolumnefficiencyandro-bustcolumnpackingformostchalleng-ingapplications.EachAcclaimcolumnismanufacturedtostrictspecificationstoensurecolumn-to-columnreproduc-ibility.Inaddition,eachcolumnisindividuallytestedandshippedwithaqualificationassurancereport.
Wide Range of ApplicationsTheAcclaimC30columnisa
reversed-phaseHPLCcolumn.LikeC18columns,theAcclaimC30canbeusedinabroadrangeofapplicationsincludingfoodandbeverage,chemical,environmental,pharmaceutical,academia,etc.TheAcclaim30alsooffersseveraluniquefeaturesthatsetitapartfromC18columns.First,itexhibitshighershapeselectivitysuitedtoseparationofhydrophobic,long-chain,structuralisomers(e.g.,carotenoids,steroids,etc).Second,itisfullycompatiblewithvariousaqueousbuffers,resultinginabroaderapplica-tionrange(e.g.,water-solublevitamins,organicacids,etc)andmoreflexibilityinmethoddevelopment.Third,itcanbeaviablealternativetonormal-phasecolumnsforlipidanalysis.Therefore,theAcclaimC30beusedasageneral-purposecolumnthatcomplementsC18columns,andalsoserveasaspecialtycolumnwhenaC18columnfailstoprovidesatisfactoryresults.
CarotenoidsCarotenoidsareoccurnaturally
inthechloroplastsandchromoplastsofplants,andsomefungiandbacte-ria.Theyservetwokeyrolesinplantsandalgae:1)theyabsorblightenergyforuseinphotosynthesis,and2)theyprotectchlorophyllfromphotodamage.Inhumans,fourcarotenoids(beta-car-
Figure 5. Glucocorticosteroids.
Figure 4. Carotenoids in vegetables.
otene,alpha-carotene,gamma-carotene,andbeta-cryptoxanthin)havevitaminAactivity,andcanalsoactasantioxidants.AsshowninFigure4,sixcommoncarotenoidsandchlorophyllaresepa-ratedontheAcclaimC30columnwithexcellentselectivityandresolution.
GlucocorticosteriodsGlucocorticosteroidsareagroupof
naturallyoccurringandsynthetichor-monesthatmoderateinflammationandotherstressresponses.Thesecompounds
areincludedontheWorldAnti-DopingAgency’slistofsubstancesprohibitedincompetition.Separationofthesesubstancesischallengingduetotheirstructuralsimilarity.ThemethodofMcWhinneyiscommonlyusedinclini-callaboratoriesformonitoringofthesesubstances.WhiletheoriginalmethodusesaC18column,theAcclaimC30offersimprovedresolutionandthrough-putwiththesameelutionorderusedtheliteraturemethod.Figure5showsbaselineseparationofeightglucocortico-steroidsusingtheAcclaimC30column.
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0
80
mAU
Minutes
C
B
A 1
2
3
4 5
6
7
8
260 800
452.4476.6
276.3
nm
Column: Acclaim C30, 5 µm (4.6 × 150 mm)LC System: UltiMate® 3000 RSMobile Phases: A. Acetonitrile B. Methanol/ethyl acetate 1:1 (v/v) C. 200 mM Acetic acid in waterGradient: Time (min) %A %B %C -5 85.0 14.5 0.5 0 85.0 14.5 0.5 2 85.0 14.5 0.5 15 65.0 34.5 0.5 25 65.0 34.5 0.5Flow: 1.50 mL/minTemperature: 30 °CInj. Volume: 8 µLDetector: Diode array; VIS 450 nm, spectra 260–800 nmSamples: A. Maize extract in acetone B. Spinach extract in acetone C. Carrot extract in acetone
Peaks: 1. Lutein 2. Zeaxanthin 3. Chlorophyll-b 4. alpha-Cryptoxanthin 5. beta-Cryptoxanthin 6. Chlorophyll-a 7. alpha-Carotene 8. beta-Carotene
Reference: “HarvetsPlus Handbook for Carotenoid Analysis,” D.B. Rodriguez-Amaya and M. Kimura, International Food Policy Research Institute, 2004.
β-Carotene
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0
100
mAU
Minutes
1 2
3 4
5
6
7
8
Column: Acclaim C30, 3 µm (3.0 × 150 mm)LC System: UltiMate 3000 RSMobile Phase: Methanol/tetrahydrofuran/water 3:25:72 (v/v)Flow: 0.50 mL/minTemperature: 30 °CInj. Volume: 2 µLDetector: Diode array; UV 240 nm
Peaks: 1. Prednisone 50 µg/mL 2. Cortisone 3. Prednisolone 4. Hydrocortisone 5. Dexamethasone 6. 6-Methylprednisolone 7. Corticosterone 8. 11-Deoxyhydrocortisone
Reference: McWhinney B C, Ward G, Hickman P E, Clin. Chem, 1996, 42, 979–981.
4
Figure 7. Analysis of cooking oil: Acclaim C30 vs. C18.
Water-soluble VitaminsWater-solublevitaminsareessential
nutrients.Theyarefoundnaturallyinfoods,andtheyarealsoaddedtofood,andformulatedsupplements.Chemi-cally,theyareaheterogeneousgroupofanions,cations,zwitterions,andneutrals.Duetothevarietyandcom-plexityofmatriceswherevitaminsarefound,theiranalysisisoftenchalleng-ing.TheAcclaimC30columndemon-stratesbothhighhydrophobicretentionandaqueouscompatibility;thisdesir-ablecombinationoffeaturesenablessimplereversed-phaseseparationofthesecomplexanalytes(Figure6).
Triglycerides in Cooking OilsCookingoilsarepurifiedlipids
fromplants,andtheyaretypicallyliquidatroomtemperature.Thesecompoundscontaintriglyceridesasmajorcomponents,andsmallquantitiesoffreefattyacids,mono-,anddiglyc-erides.Thecompositionofcookingoilsishighlycomplexduetothewidevarietyofalkylchainlength,degreeofunsaturation,origin,etc.Whilenormal-phasechromatographyisoftenusedtocharacterizeoilsbytheirhydrophilic-ity,reversed-phasechromatographyprovideshighresolutionforanalyzingmajorandminorcomponents,andobtainingadetailedfingerprint.Duetoitshighshapeselectivity,theAcclaimC30provideshigherresolutionthantheC18columnforoilanalysis(Figure7).SeparationsofseveralcookingoilsareillustratedinFigure8.
Figure 6. Water-soluble vitamins.
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0
160
Minutes
mAU
200 400
283.6
nm
Folic acid
1
2
3
4
6
5 7
Column: Acclaim C30, 5 µm (4.6 × 150 mm)LC System: UltiMate 3000 RSMobile Phases: A. Acetonitrile B. 7 mM Phosphoric acid + 40 mM sodium sulfate (pH 2.65)Gradient: Time (min) %A %B –3.0 0 100 0.0 0 100 2.0 0 100 10.0 25 75 12.0 25 75Flow Rate: 1.50 mL/minTemperature: 30 °CInj. Volume: 10 µLDetection: Diode array; UV 210 nm, spectra 260–400 nmSamples: Vitamin standards in mobile phase
Peaks: 1. Thiamine 25 µg/mL 2. Ascorbic acid 62 3. Niacinamide 25 4. Pyridoxine 25 5.Pantothenic acid 25 6. Folic acid 6.2 7. Riboflavin 5.0
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30 46 62 54 38 70 Minutes
pA
0
150
A. Acclaim C30
B. Acclaim 120 C18
Columns: A. Acclaim C30, 5 µm (4.6 × 150 mm) B. Acclaim 120 C18, 5 µm (4.6 × 150 mm)Mobile Phase: Acetonitrile (CH3CN)/Iso-propanol (IPA)/ Ammonium acetate buffer (0.1 M, pH 5.0)Gradient: Time (min) CH3CN IPA Buffer -15 90 5 5 0 90 5 5 0.1 90 5 5 60 0 95 5 70 0 95 5Temperature: 40 °CFlow Rate: 1.0 mL/minInj. Volume: 2 µLDetection: Corona ultra (gain = 100 pA; filter = medium; neb. temp. = 25 °C)Sample: Peanut oil (5 mg/mL in iso-propanol)
Figure 8. Analysis of cooking oils.
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0 32 724816 80Minutes
pA
0
200
A
B
C
Column: Acclaim C30, 5 µm (4.6 × 150 mm)Mobile Phase: Acetonitrile (CH3CN)/iso-propanol (IPA)/ ammonium acetate buffer (0.1 M, pH 5.0)Gradient: Time (min) CH3CN IPA Buffer –15 90 5 5 0 90 5 5 0.1 90 5 5 60 0 95 5 80 0 95 5Temperature: 40 °CFlow Rate: 1.0 mL/minInj. Volume: 2 µLDetection: Corona ultra (gain = 100 pA; filter = medium; neb. temp. = 25 °C)Samples: A. Peanut oil B. Olive oil C. Wheat germ oil (5 mg/mL in iso-propanol)
5
Figure 9. Profile of egg lecithin.
Figure 10. Phospholipids in egg lecithin.
Phospholipids in Egg LecithinThisclassoflipidsaremajor
componentsofallcellmembranesastheycanformlipidbilayers.Mostphospholipidscontainadiglyceride—aphosphategroupconnectedtoahydro-philicorganicgroupsuchascholine.Thefirstphospholipididentifiedinbiologicaltissueswaslecithinintheeggyolk.Analysisofphospholipidsischallengingduetoitscomplexity(hydrocarbonlength,degreeofsatura-tionofdiglyceridemoiety,andtypeofthehydrophilicorganicgroup).Figure9showstheprofileoflecithinfromeggyolkandsoybeanobtainedusinganAcclaimC30columnwithaCoronaultra™CAD®detector.Itindicatesthatlecithinfromeggyolkcontainsbothphospholipidsandtriglycerides,withminorimpurities.Toobtaindetailedinformationonphospholipidcomposi-tion,ahigher-resolutionmethodcanbeused(Figure10).
Omega Fatty AcidsOmega-3fattyacidsareafamily
ofunsaturatedfattyacidsthatshareafinalcarbon–carbondoublebondinthen-3position.Omega-3fattyacids,suchasα-linolenicacid(18:3,n-3;ALA),eicosapentaenoicacid(20:5,n-3;EPA),anddocosahexaenoicacid(22:6,n-3;DHA),areimportantinhumannutri-tion.ThebiologicaleffectsofOmega-3arelargelymediatedbytheirinterac-tionswiththeOmega-6fattyacids.SomemedicalresearchsuggeststhatexcessivelevelsofOmega-6relativetoOmega-3fattyacidsmayincreasetheprobabilityofanumberofdiseasesanddepression.AsshowninFigure11,theAcclaimC30columnexhibitsexcellentresolutionpowerforasuiteofcloselyrelatedOmegafattyacids.
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pA
0
60 Triglycerides
Phospholipids
Degradants
Column: Acclaim C30, 5 µm (4.6 × 150 mm)Mobile Phase: Acetonitrile (CH3CN)/iso-propanol (IPA)/ ammonium acetate buffer (0.1 M, pH 5.0)Gradient: Time (min) CH3CN IPA Buffer -15 70 0 30 0 70 0 30 0.1 70 0 30 10 90 0 10 35 10 80 10 50 0 95 5 60 0 95 5Temperature: 40 °CFlow Rate: 1.0 mL/minInj. Volume: 2 µLDetection: Corona ultra (gain = 100 pA; filter = medium; neb. temp. = 25 °C)Sample: Egg lecithin – PL30S (5 mg/mL in iso-propanol)
Figure 11. Omega fatty acids.
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pA
0
50
Phospholipids
Degradants
Column: Acclaim C30, 5 µm (4.6 × 150 mm)Mobile Phase: A. Acetonitrile (CH3CN) B. Iso-propanol (IPA) C. Ammonium acetate buffer (0.1 M, pH 5.0)Gradient: Time (min) CH3CN IPA Buffer –15 60 20 20 0 60 20 20 0.1 60 20 20 150 38 50 12 151 5 90 5 160 5 90 5Temperature: 40 °CFlow Rate: 1.0 mL/minInj. Volume: 5 µLDetection: Corona ultra (gain = 100 pA; filter = medium; neb. temp. = 25 °C)Sample: Egg lecithin – PL100M (5 mg/mL in iso-propanol)
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100
pA
Minutes
A
B
1 2
3 4
5 6 7
8 9
10
11
12
13
14 0
6000
ng
0 9Area
LLA
Column: Acclaim C30, 5 µm (4.6 × 150 mm)LC System: UltiMate 3000 RS, Dual GradientMobile Phases: A. Water:formic acid:mobile phase B 900:3.6:100 (v/v) B. Acetone:acetonitrile:THF:formic acid 675:225:100:4(v/v)Gradient: Time (min) %A %B 0 100 0 1 40 60 13 30 70 22 5 95 24 5 95 29 100 0 32 100 0Flow Rate: 1.00 mL/minTemperature: 30 °CInj. Volume: 2 µLDetection: Corona ultra, nebulizer 15 °C, filter highSamples: A. Standards in isopropanol B. Saponified chicken fat
Peaks: 1. SDA 2. EPA 3. ALA 4. GLA 5. DHA 6. Arach. 7. LLA
8. DPA 9. 9E, 14Z-CLA 10. ETA 11. Adrenic 12. Oleic 13. EDA 14. Erucic
ORDERING INFORMATION
IntheU.S.,call(800)346-6390orcontacttheDionexRegionalOfficenearestyou.OutsidetheU.S.,orderthroughyourlocalDionexofficeordistributor.Referthefollowingpartnumbers:
Acclaim C30 Analytical and Guard Columns Part Number
AcclaimC30,5µmAnalytical,4.6×250mm.......................................................... 075718
AcclaimC30,5µmAnalytical,4.6×150mm.......................................................... 075719
AcclaimC30,3µmAnalytical,4.6×150mm.......................................................... 075723
AcclaimC30,3µmAnalytical,3.0×250mm.......................................................... 075726
AcclaimC30,3µmAnalytical,3.0×150mm.......................................................... 075724
AcclaimC30,3µmAnalytical,2.1×150mm.......................................................... 075725
AcclaimC30,5µm,Guard,4.6×10mm................................................................. 075720
AcclaimC30,5µm,Guard,3.0×10mm................................................................. 075721
AcclaimC30,5µm,Guard,2.1×10mm................................................................. 075722
Accessories Part Number
GuardHolder(V-2).................................................................................................... 069580
GuardHolderkitV-2(HolderV-2andCoupler)....................................................... 069707
GuardCoupler............................................................................................................ 074188
SPECIFICATIONS
Column Chemistry: C
30alkyl
Silica Substrate: Spherical,high-purity Particlesizes: 3and5µm Surfacearea: 200m2/g Poresize: 200Å
Operating pH range: pH2–8
Operating Temperature: Upto60°C
Aqueous compatibility: 0to100%
Solvent compatibility: 0to100%
Operating pressure and flow rate: Seetabletopright
Corona,Acclaim,CAD,andUltiMateareregisteredtrademarks,andultraisatrademarkofDionexCorporation.
North America
U.S./Canada (847) 295-7500 South America
Brazil (55) 11 3731 5140
Europe
Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722
Asia Pacific
Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190Taiwan (886) 2 8751 6655
Dionex Corporation
1228 Titan Way P.O. Box 3603 Sunnyvale, CA 94088-3603 (408) 737-0700 www.dionex.com
LPN 2776 PDF 3/11©2011 Dionex Corporation
Column Dimensions
Particle Size
Part Number
Maximum Pressure
(psi)
Typical Flow Rate (Recommended)
(mL/min)
Maximum Flow Rate (mL/min)
4.6×250mm 5µm 075718 9,000 0.8–1.5 2.0
4.6×150mm 5µm 075719 8,000 0.8–1.5 2.0
4.6×150mm 3µm 075723 8,500 0.8–1.5 1.0
3.0×250mm 3µm 075726 12,000 0.4–0.6 1.0
3.0×150mm 3µm 075724 8,500 0.4–0.6 1.0
2.1×150mm 3µm 075722 8,800 0.2–0.3 0.5
OPERATING PRESSuRE AND FlOw RATE SPECIFICATIONS