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

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

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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

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 this equation,elution volume is usedinstead of separatethe two, causingthe ion of higher chargeto retentiontime; the two arerelated by the eluantflow elutefIrSt. In theory,one could obtain the optimum rate. conditionsfor a gradientelution from a plot suchas Vr - Vrn A that shownin Figure 4. In practice,this is not log = -log R + log Const [1] straightforwardbecause equation [1] assumesthat a V rn A + E singlelinear gradientis usedwith the initial eluant concentrationequal to zero. Fastergradient runs can V r is the retentionvolume, V rn is the mobile phase be obtainedby startingthe eluantconcentration at a volume (column void volume), A is the analyteion's highervalue. Also, the bestcombination of speed charge,E is the eluantion's charge,R is the gradient and selectivityis often the result of combining ramp steepnessin mM of eluantconcentration per severaldifferent gradients,often with isocratic mL of eluant pumped, and log Const is the Y sections and step changes. With more complicated intercept of the log/log plot This equation predicts gradient programs, the retention equation becomes that an increasein gradientsteepness will decrease unwieldy. the retentiontime of divalent ions more than monovalentions. An advantagemonovalent eluants Equation[1] can be easily usedto predict trends. haveover divalent (or other polyvalent)eluants is This is demonstratedin Figure 5. In all three that a given changein eluantconcentration produces chromatograms,the initial eluantconcentration is a greaterchange in analyteretention time. Compared 2.5 mM p-cyanophenate.This concentrationis to a monovalent eluant, during a gradient run, the increased between 2 and 7 min. to 18.5 mM in (a) concentration of a divalent eluant must be changed (where sulfate and nitrate co-elute) to 22.5 mM by a much larger amountto elute ions of widely in (b), resultingin baselineseparation. In (c), differing retention. the eluantconcentration is increasedas in (a) to 18.5mM, but in 5 min. insteadof 9 min. The effect is similar in that the divalent anionselute RetentionYs. Ramp Slope earlier,but becauseof the long period holding at 18.5mM, the separationbetween the divalent anions Theoretical, for monovalent eluant is not compressedas in (b), but is insteadsimilar to (a). The chromatogramin Figure 3 of anionson the HPIC-AS6 was developedwith this methodand usesa combinationof stepchanges, linear gradients, and isocraticsections.

v - v How canbaseline slope be minimized? Log~ Vm When eluantsother than sodiumhydoxide are used, it is often necessaryto minimize the baselineslope which resultsas the eluantconcentration is increased.In anionexchange, this can be accomplishedchemically by addingmannitol to the weakeluant and boric acid to the regenerant. Although the reactionbetween boric acid and LogR mannitolis not well understood,they do combineto Figure 4 producean acid considerablystronger than either of

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the ~o by the~selves. As a ~eutralpoly~cohol, Effectof GradientRamp on ElutionAS6, mannl,tolhas li!tle e!fe;cton el.thersepara,tion ,or p-CyanophenateEluant detection. Bonc acId m the dilute sulfunc acId regenerantis neutral,and can diffuse through the " cationexchange membranes of the AMMS. The .:.. two then combine in the suppressor, producing a (1)1%6'0- CI)6 ~

conductivespecies. By decreasingthe mannitol :~ CoO concentrationin approximatelyinverse proportion to :~ .~ 8 :i :EI .~ the increasein eluantconcentration, the baseline 8 ~ r7,..;: 8 ~ changescan be counteracted This methodis demonstratedin Figure 6, in which mannitolwas usedto balancethe baselinein (b), but not in (a). Anothermethod of baselinecompemsation is computerb3;Seline .s~b~cti?n. For ~s method,a 0 5 10 i 5 10 0 5 10 blank run WIth no mjectlon IS stored m the mY computor'smemory and subtractedpoint by point 22.5 - - - -

longfrom as succeding the baseline runs. profIle This is method reproducible.works well as 18.5 - ~ - r 7- - ~-/

How are contaminantsin the eluant 2.5 removed? (a) (b) (c) . . .. Figure 5 A problemencountered m gradIentelutIon ISthe presenceof extraneouspeaks in the chromatogram causedby traceimpurities in the eluant. In anion Balancingthe GradientBaseline with Mannitol exchange,the most commonimpurities arechloride, and BoricAcid AS6,p-Cyanophenate Eluant sulfate,and carbonate.In the early part of a gradient run, while the eluantis weak, theseions are concentratedat the front of the separator.As the eluantstrength increases, they are eventuallyeluted, and appearas interferingpeaks at the expected retentiontimes. Carbonatecan be minimized by usingproperly deionizedwater and low carbonate sodiumhydroxide. The effect of contaminantscan be minimized by placing a small column,called the Anion Trap Column (A TC), in front of the injection valve. The A TC is filled with high capacity,low efficiency anion exchangeresin. It successfully preventstrace anionic contaminants from reaching the separatorcolumn during the early part of the run. Later in the run, the eluantstrength may be high enoughto elute the contaminants.The low efficiency of the resin spreadsout the contaminant I I I I I I peaks so that they do not interfere with the 0 10 zo 0 10 zo chromatography.A consequenceof the useof an a. b. ATC is that if a compositiongradient is used,the time neededto convertthe form of the resin in the A TC must be considered. Figure 6

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An alternateapproach is to usea higher efficiency How are gradients used with MPIC? trap column which is regeneratedat the end of each gradientrun with a high concentrationeluant, Mobile PhaseIon Chromatography(MPIC) is a removingany accumulatedionic contaminants.The form of reversedphase ion pair chromatographyin HPIC-AG8 guardcolumn can perform as a trap which chemicallysuppressed conductivity detection column becauseof its high affmity for carbonate,the is used. As in reversedphase HPLC, gradient primary contaminantin sodiumhydroxide e1uants. elution is accomplishedby varying the percentage An exampleof the useof an AG8 as a trap column is of organicsolvent in the eluantduring the run. the separationof anionsin coffee shownin Figure 7. For this chromatogram,carbonate and other Although the ionic strengthof the eluantmay remain contaminantson the trap column were removedwith the samethroughout the run, the backgroundconduc- a strongborate eluant The sampleis theninjected tivity can decreasedue to the changingdielectric con- beforethe sodiumhydroxide eluant has removed all stant Thesebaseline changes can be compensated of the boratefrom the trap column. The small eitherby chemicalmeans or by computerbaseline amountof borateleft forms a complexwith quinate, subtraction.Often, :MPICis usedto elute ions increasingits retentionfrom coe1utionwith acetateto which are very stronglyretained. When this is the elution betweenglycolate and formate. case,the baselineconductivity changecan be minimized without affectingthe separationby adding very weakly retainedions directly to the stronger eluant (higher percentageof organic solvent). Using gradient :MPIC, the separation of five alkyl sulfon- ates and sulfates is shown in Figure 8. The AMMS- :MPIC suppressor(pIN 37106) should be used.

Anionsin Coffee,AS5A

Gradient Proaram Conditions

%1: 100 100 85 55 0 0 0 0 100 Eluant #1: 0.75 mM NaOH Eluant #2: 200 mM NaOH %2: 0 0 15 45 40 40 100 100 0 Eluant #3: 200 mM H3BO3 %3: 0 0 0 0 60 ~ 0 0 0 FIow Rat e: 1.0 mL/ mln' Thenext injectionwas alwaynwoo 42 nin. atE. tIE begiming of tIE ~viol8 run.

G -G G -. . 'a :. e.~ G '(j .. 0 - G 0 - . - G ~.c .. . ..- u -:. .ca. GG 1-. C.U - G -G .c0 OGU- - . Co ->0- c ~3 a

I I I I II ~I 0 5 10 15 20 25 30 Mlnut.. Figure 7 .

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Gradient Ion Pairing What conditions does Dionex recommend for gradient elution?

Although many columns and eluants can be used for ! gradient elution, the conditions used in Figure 2 are ~ g recommendedas a good startingpoint The HPIC- ~ :s AS5A (51.1)separator with sodium hydroxide eluant :§ c: provides the optimum combination of efficiency, uU) u 3~ selectivity,and speedwithout an unacceptable ! ! ~ c! ~ baselineslope. If fewer ions than the 36 shownin ~ ~ ~ ~ Figure 2 need to be separated,the gradient steepness ~ ! ~ u can be increased to reduce the run time. Remember ! ~ ! ~ that changing the gradient will affect the elution I. 0 I ~ order of ions of different charge. In particular, =:!! increasing the gradient ramp slope will cause sulfate N and other divalent ions to elute earlierthan nitrate. -I When the considerations discussed in this technical 0 note are followed, gradient elution can be used to solvemany chemicalanalysis problems that are difficult to solveusing isocraticelution. Ion chromatograph: Dionex series4OOOi 1 or IC with GPM

'-- Trap column: ATC (pIN 37151)or HPIC-

0 ~ 8 112 16 AG8 (pIN 37023) Minutes Guardcolumn: HPIC-AG5A (pIN 37134)

Columns: IIPIC-NSI Flow Rate: 1.0 mUmln. Separatorcolumn: HPIC-AS5A (PIN 37131) Eluant 1: water Eluant 2: CH3CN Eluant 3: 0.5 II H3B03, 10 mil TIIAOH, 40 mil mannitol Eluant #1: 0.75 roM sodium hydroxide Eluant 4: 0.5 II H3B03. 10 mil TIIAOH, 50% CH3CN Time (mln) 1% 2% 3% 4% Eluant #2: 200 roM sodium hydroxide 0 74 8 4 14 . 7.5 17 47 24 12 Eluant flow rate: 1.0 mL/mm 10 7 57 20 16 15 9 55 16 14 . . 15.1 74 8 4 14 Suppressor: AnIon MicroMembrane, Suppressor: AIIII5-IIPIC AMMS (pIN 38019) Regenerant: 25mN H2SO4 Regenerant: 25 InN H2SO4 Figure 8 Regenerantflow rate: 10 mL/min