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Gradient Elution in Ion Chromatography

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Gradient Elution in Ion Chromatography j;, " , DIONEX Technical Note TN 19 January 1987 Gradient Elution In Ion Chromatography.. Anion Exchange With ConductivityDetection Gradientelution is a powerful techniquein ion chromatography.By varying the concentrationof the eluant,ions with widely differing affinitiesfor the separatorresin can be elutedin one run. However,just as in otherforms of chromatography,gradient elutionplaces certain restrictionson the choiceof eluant. In particular, it is importantto useeluants that produce a minimumshift in baselineconductivity during the run, as well as a fast equilibration timefrom one run to the next. In this TechnicalNote, guidelineswill bepresented to help develop successfulanalyses using gradient elution. In addition, a simplegradient program whichproduces a good separationof many commoninorganic and organic anionswill be described.Although this discussionwill focus on anion e.xchange,most of the conceptsare applicableto cation exchangechromatography. How is gradient elution accomplished? Concentrationgradients avoid theseproblems by alwayskeeping the fonn of the resin the same. Eluantsused in anion exchangecontain an anionic compoundin high concentrationwhich competes With gradientelution, changingthe concentrationof with the analyteions for siteson the resin. Gradient the eluantresults in changesin background elution is bestaccomplished by increasingthe conductivity. By using chemicaleluant suppression concentrati?nof the eluant .aniond~g the run..~. e. to reducethe highestbackground conductivity to less by perfOrmInga concentratIongradient. In addItIon, than 10 ~S, baselinechanges during a run can be a pH gradi.entcan be used,.in ,whi<?h a fix;ed limited to only a few ~S. This is acc°!llplished concentratIonof a weak acId ISmIXed WIth an using an appropriateeluant and an Amon increasing concentration of a strong base such as MicroMembrane Suppressor. In addition, baseline sodium hy~oxide.. pH gradients are essen,tially . changesof up to 20 ~S can be compensatedby a concentratIon gradients, as the purpose of mcreasmg technique described later in this technical note. the pH during an anion exchangerun is to increase the concentrationof the dissociatedform of the weak TIT acid eluant. " hat, .JpeshI 0 if eI uant scan be use.d? Othertypes of gradientsare more difficult to use. Thereare two typesof eluantswhich can be For example, a composition gradient can be suppressedto produce background conductivities of performed by changing from a weakly retained only a few ~S. One type is the salt of a weak acid eluant ion to a much more strongly retained ion. The weakest acid in aqueous solution capable of There is a wide difference in affmity for the resin forming a salt is water itself, with the salt being a between weakly and strongly retained analytes, such strong base such as sodium hydroxide. It is an as monovalentacetate and trivalent citrate. To elute excellentchoice as an eluantfor gradientelution asit the more stronglyretained citrate, ions from the is convertedin the suppressorto water regardlessof strongereluant must completelydisplace the weakly its concentration.There are other saltsof weak acids retainedeluant ions from the resin. Dependingon which can be suppressedto producelow the ratio of column ion exchange capacity to eluant background conductivities. These include borate concentration, it may take a long time for the resin to and many phenatessuch as p-cyanophenate. In be convertedfrom the weak eluantto the strong general,the saltsof weak acidswith pKa'sgreater eluantform. (This effect is similar to solvent than 7 are acceptable.As pKa decreases,the extent demixingin reversedphase HPLC.) Also, to repeat of dissociationfollowing suppressionincreases, the run, the resin fonn must be convertedback to the resultingin increasedbackground conductivity and weakeluant. This can only be doneby pumping a increasedbaseline shift during gradientelution. The high concentrationof the weak eluant,thus other type of eluantwhich can be usedincludes increasingthe equilibrationtime betweenruns. thoseanions which are anionic at high pH but are Dionex Corporation Sunnyvale,CA (408) 737-8522 Dionex CanadaLtd Diooex(UK) Ltd. Diooex GmbH Dionex S.RL. Diooex S.A. Dionex B.V. P.O.Box 3603 Itasca,lL (312) 773-6850 Etobicoke,Ontario Carnberley D-6270Idstein Roma.Italy Boulogne,France 4817BL Breda Sunnyvale,CA Houston,TX (713) 847-5652 (416) 620-0077 Surrey,England West Gelmany (6) 3792984 (I) 46216666 The Netherlands 94088-3603 Atlanta,GA (404) 432-8100 (276) 691722 (6126)W36 (76) 714800 (408) 737-0700 Marlton, NJ (609) 596-0600 LPN 032834 3/87 . .- convert~ to zwi~rion~ or cationsfoll~wing GradientElution on AS5 suppreSSIon.Ammo acIdssuch as glycme aregood choicesbecause they areprimarily convertedto their cationicform andremoved by the suppressor. Is sodium hydroxide the best choice for an eluant? Becausesodium hydroxide is convertedto waterin i the suppressor,it is the bestchoice for an eluant. ~ As long as the capacityof the suppressoris not Eluant 1: Water little effect on background conductivity. The I Flow Rate: 1 mU min. suppressioncapacity of the AMMS usually exceeds 6 ;:. 100mM NaOHat a flow rateof 1.0mUmin. A i Gradlenl Program: gradient run could begin with a few mM NaOH and I TIm. %1 % 2 end at 100 mM. Examples of gradient elution using F 0 75 25 sodium hydroxide are shown in Figures 1 and 2. In 5 0 100 Figure 1, the HPIC-AS5 column is used. This columncan elute anionswith chargesranging from monovalentfor chloride to hexavalentfor tetrapoly- phosphate.In Figure2, theHPIC-AS5A (5~) I I I I columnis usedto elute a large numberof anionsin . 5 to 15 onerun. The initial eluantis weak enoughto retain MInutes fluoride well out of the void volume and separate severalweakly retainedmonoprotic organic acids, while the final eluant concentrationis capableof eluting triprotic phosphateand citrate. As shownin Figure2, 36 ions elutedin 30 minutesillustrates the powerof gradientelution. Figure 1 GradientSeparation on HPIC-AS5A 23 GradientProgram All anions10 ppmunless noted Minutes: 0 5 15 30 1 F~ 11 . (15. ppm) 19. HP02-3 %1: 100 100 85 57 2. a.Hydroxybutyrate20. SeO32- 19 24 %2: 0 0 15 43 3. Acetate 21. Br~ 4. Glycolate 22. N03~ 13 25 5. Butyrate 23. 50.2- 15 20 27 6. Gluconate 24. Oxalate 12 21 35 36 7. a-Hydroxyvalerate 25. SeO.2- 4 1617 22 30 33 8. Formate(5 ppm) 26. a-Ketoglutarate I / 26 2829 32 9. Valerate 27. Fumarate 123 14 (18 10. Pyruvate 28. Phthalate 8 31 34 11. Monochloroacetate29. Oxalacetate 12. BrO3~ 30. PO.3~ 13. CI~(3ppm) 31. AsO.3- 14. Galacturonate 32. CrO.2- 15. N02 (5ppm) 33. Citrate I I I I I I I 16. Glucuronate 34. I~ocitrat~ 0 5 10 15 20 25 30 17. Dichloroacetate 35. cls-Aconlta.te Minutes 18. Trifluoroacetate 36. trans-Aconltate Figure2 0: I ~~- -- . How should sodium hydroxideeluant beprepare. d? Gradient Elution Using AS6 Carbon dioxide readily dissolves in basic solutions, producing carbonate. Carbonate contamination in sodiumhydroxide eluants can greatlyincrease the Conditions baseline shift during a gradient run. Eluants should beprepared from 50%solutions of sodium Eluant#1: 50 mMMannitol, 2% CH3CN ydroxide asthey containl ower concentrations of Eluant#2: 35mM p-cyanophenol, h , 50 mM NH3, 2% CH3CN carbonatethan the solid. 18 meg-.Qdeionized water Eluant #3: 2% CH3CN should be used and degassed prior to addition of ARt. 20 U I sodium hydroxide. Eluants should be maintained ow a e. "m m n. under an inert atmosphere during use. The Dionex Columns: ATC,AS6, AMMS Eluant Degas Module (pIN 37124) and Eluant Regenerant:10 mM H2SO4, 200 mM H3BO3 ContainerSet (pIN 38752)provide a convenient methodfor ensuringthat the eluantsare properly Range: 30ILs degassedand free from carbonatecontamination. Whenshould p-cyanophenate be used? GradientP[Qgram TIme: 0 3 3.1 7 13 15 p-Cyanophenate is a more powerful eluant than %1" 45 0 0 hydroxide and should be used wi,th higher capacity %2; 7 ~ 5 ~o ~~ ~~ 1 o~ separators. The HPIC-AS6 proVIdes excellent %3: 48 45 30 17 22 0 selectivity of diprotic organic acids. Due to its higher capacity (approximately ten times that of the AS5A), the stronger eluant ion p-cyanophenate is necessaryto elute di- and trivalent ions. An example 50.2- of gradient elution on the HPIC-AS6 using p-cyano- CI- .!:! phenateis shownin Figure 3. In this run, the .!:! ~ ~ concentrationof p-cyanophenatein the eluantis .!:! I ':"~' ~ 5 increasedfrom 2.4 mM at the beginningof the run ~..2 <i.'8 z ~ to 35 mM at theend. &&. "8, %, ~ .~ . .!:! F- 0c Q~ , 0N E.o~ ~ .. 2m~.g &£~ .!:! Can carbonate - bicarbonatebuffers be used u % ~ ~ 5 fi .- - GO for gradient elution? :e 2 0 I 8l: ~ u .c .c ~ Carbonatecontaining eluants are generallynot ! ~ acceptablefor gradientelution. Following suppression,carbonic acid is formed. With a relatively low pKa of 6.2, the baselinedrift is too severe.Although saltsof weak acidswith pKa's I I I I I aslow as5 canbe usedfor isocraticelution, due 0 5 .10 15 20 to their higher backgroundconductivity following Minutes suppression,they are generallynot acceptablefor gradientelution. Figure 3 . ,- How doeschanging the eluantconcentration Canselectivity be changed by affectelution? changingthe gradient? The relationshipbetween elution volume and For ions of different charge,the elution order,and gradientsteepness (i. e. how rapidly the eluant thereforethe selectivity,can be changedby adjusting concentrationis being increased)is describedby the gradient. For example,if two ions with different equation[1] and showngraphically in Figure 4. In chargecoelute, an increasein gradientsteepness will
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