j;,,
"
, DIONEX
Technical Note
TN19
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 elutionplacescertain restrictionson the choiceof eluant. In particular, it is
importantto useeluantsthatproducea minimumshift in baselineconductivityduring the run, as well as afast
equilibration timefrom one run to the next. In this TechnicalNote,guidelineswill bepresentedto help develop
successfulanalysesusinggradient elution. In addition,a simplegradientprogram whichproducesa good
separationof manycommoninorganic and organic anionswill be described.Although this discussionwill focus
on anion e.xchange,
mostof the conceptsare applicableto cation exchangechromatography.
How is gradient elution accomplished?
Concentrationgradientsavoid theseproblemsby
alwayskeepingthe fonn of the resin the same.
Eluantsusedin anionexchangecontainan anionic
compoundin high concentrationwhich competes
with the analyteions for siteson the resin. Gradient
elutionis bestaccomplishedby increasingthe
concentrati?nof the eluant.aniond~g the run..~.e.
by perfOrmInga concentratIongradient. In addItIon,
a pH gradi.entcan be used,.in,whi<?h
a fix;ed
concentratIonof a weak acIdISmIXedWIthan
With gradientelution, changingthe concentrationof
the eluantresultsin changesin background
conductivity. By using chemicaleluantsuppression
to reducethe highestbackgroundconductivityto less
than 10 ~S, baselinechangesduring a run canbe
limited to only a few ~S. This is acc°!llplished
usingan appropriateeluantand an Amon
increasing concentration of a strong base such as
sodium hy~oxide.. pH gradients are essen,tially .
concentratIon gradients, as the purpose of mcreasmg
MicroMembrane Suppressor. In addition, baseline
changesof up to 20 ~S can be compensatedby a
technique described later in this technical note.
thepH during an anionexchangerun is to increase
the
of the dissociatedform of the weak
acidconcentration
eluant.
" hat
,
.Jpes0if eIuantscan be use.
d?
TIT
hI
Othertypesof gradientsaremoredifficult to use.
Therearetwo typesof eluantswhich canbe
For example, a composition gradient can be
performed by changing from a weakly retained
eluant ion to a much more strongly retained ion.
There is a wide difference in affmity for the resin
between weakly and strongly retained analytes, such
suppressedto produce background conductivities of
only a few ~S. One type is the salt of a weak acid
The weakest acid in aqueoussolution capable of
forming a salt is water itself, with the salt being a
strong basesuch as sodium hydroxide. It is an
asmonovalentacetateandtrivalent citrate. To elute
the more stronglyretainedcitrate,ions from the
strongereluantmustcompletelydisplacethe weakly
retainedeluantions from the resin. Dependingon
excellentchoiceasan eluantfor gradientelution asit
is convertedin the suppressorto waterregardlessof
its concentration.Thereareothersaltsof weakacids
which canbe suppressed
to producelow
the ratio of column ion exchange capacity to eluant
concentration, it may take a long time for the resin to
background conductivities. These include borate
and many phenatessuch as p-cyanophenate. In
be convertedfrom the weak eluantto the strong
eluantform. (This effect is similar to solvent
demixingin reversedphaseHPLC.) Also, to repeat
the run, the resin fonn must be convertedbackto the
weakeluant. This canonly be doneby pumpinga
high concentrationof the weak eluant,thus
increasingthe equilibrationtime betweenruns.
general,the saltsof weak acidswith pKa'sgreater
than7 areacceptable.As pKa decreases,
the extent
of dissociationfollowing suppressionincreases,
resultingin increasedbackgroundconductivityand
increasedbaselineshift during gradientelution. The
othertype of eluantwhich canbe usedincludes
thoseanionswhich are anionicat high pH but are
DionexCorporation
P.O.Box 3603
Sunnyvale,CA
94088-3603
(408)737-0700
Sunnyvale,CA
Itasca,lL
Houston,TX
Atlanta,GA
Marlton, NJ
(408) 737-8522 DionexCanadaLtd
(312) 773-6850 Etobicoke,Ontario
(713) 847-5652 (416) 620-0077
(404) 432-8100
(609) 596-0600
Diooex(UK) Ltd.
Carnberley
Surrey,England
(276)691722
DiooexGmbH Dionex S.RL.
D-6270Idstein Roma.Italy
West Gelmany (6) 3792984
(6126)W36
Diooex S.A.
DionexB.V.
Boulogne,France 4817BL Breda
(I) 46216666
The Netherlands
(76) 714800
LPN 032834 3/87
I
.
.-
convert~ to zwi~rion~ or cationsfoll~wing
suppreSSIon.
Ammo acIdssuchasglycme aregood
choicesbecausethey areprimarily convertedto their
cationicform andremovedby the suppressor.
GradientElutionon AS5
Is sodium hydroxide the best choice
for an eluant?
Becausesodiumhydroxideis convertedto waterin
the suppressor,it is the bestchoicefor an eluant.
As long asthe capacityof the suppressoris not
i
~
Eluant 1: Water
little effect on background conductivity. The
suppressioncapacity of the AMMS usually exceeds
I
6 ;:.
gradient run could begin with a few mM NaOH and
end at 100 mM. Examples of gradient elution using
I
Flow Rate: 1 mU min.
i
100mM NaOHat a flow rateof 1.0mUmin. A
Gradlenl Program:
TIm. %1
F
sodium hydroxide are shown in Figures 1 and 2. In
%2
0
75
25
5
0
100
Figure 1, the HPIC-AS5 column is used. This
columncanelute anionswith chargesrangingfrom
monovalentfor chloride to hexavalentfor tetrapoly-
phosphate.
In Figure2, theHPIC-AS5A(5~)
I
columnis usedto elute a largenumberof anionsin
onerun. The initial eluantis weakenoughto retain
fluoride well out of the void volume and separate
severalweakly retainedmonoproticorganicacids,
while the final eluantconcentrationis capableof
eluting triprotic phosphateandcitrate. As shownin
Figure2, 36 ions elutedin 30 minutesillustratesthe
powerof gradientelution.
I
.
5
I
to
MInutes
I
15
Figure 1
GradientSeparationon HPIC-AS5A
23
GradientProgram
Minutes:
11
19
13
15
20
12
21
1617
22
4
I
123
I
0
8
I
5
14
/
%1:
%2:
24
25
26 2829
(18
I
10
I
15
Minutes
0
5
15
30
1
100
0
100
0
85
15
57
43
2. a.Hydroxybutyrate20. SeO32-
27
30
33
35 36
32
31
I
20
All anions10 ppmunlessnoted
3.
4.
5.
6.
7.
8.
.
F (15
. ppm)
19 HP023
Acetate
Glycolate
Butyrate
Gluconate
a-Hydroxyvalerate
Formate
(5ppm)
21.
22.
23.
24.
25.
26.
~
Br~
N03~
50.2Oxalate
SeO.2a-Ketoglutarate
9. Valerate
27. Fumarate
10. Pyruvate
28. Phthalate
11. Monochloroacetate29. Oxalacetate
34
I
25
.
I
30
12. BrO3~
13. CI~(3ppm)
14. Galacturonate
15. N02 (5ppm)
16. Glucuronate
17. Dichloroacetate
18. Trifluoroacetate
30.
31.
32.
33.
PO.3~
AsO.3CrO.2Citrate
34. I~ocitrat~
35. cls-Aconlta.te
36. trans-Aconltate
Figure2
0:
~~-
--
.
How should sodium hydroxideeluant
b
d?
eprepare.
Gradient Elution Using AS6
Carbon dioxide readily dissolves in basic solutions,
producing carbonate. Carbonate contamination
in
sodiumhydroxideeluantscan greatlyincreasethe
baseline shift during
a gradient
run.
Eluants
beprepared
from 50%solutionsof sodium
Conditions
should
Eluant#1:
hydroxide, asthey containlower concentrations of
carbonatethan the solid. 18 meg-.Qdeionizedwater
50 mMMannitol,2%CH3CN
Eluant
#2: 35mMp-cyanophenol,
50 mM NH3, 2% CH3CN
Eluant #3:
2% CH3CN
should
used and degassed
prior to
of
sodium be
hydroxide.
Eluants should
beaddition
maintained
ARt.
20
under an inert atmosphere during use. The Dionex
Eluant Degas Module (pIN 37124) and Eluant
Columns: ATC,AS6,AMMS
Regenerant:
10mMH2SO4,
200mMH3BO3
ContainerSet (pIN 38752)provide a convenient
methodfor ensuringthat the eluantsareproperly
degassedandfree from carbonatecontamination.
Range: 30ILs
ow
a e.
U
"m
Whenshouldp-cyanophenatebe used?
I
m n.
GradientP[Qgram
TIme:
p-Cyanophenate
is a more powerful eluant than
hydroxide and should be used wi,th higher capacity
0
%1"
separators. The HPIC-AS6 proVIdes excellent
3
45
3.1
0
%2;
7
%3:
48
7
13
15
0
~5
~o
~~
~~
45
30
17
22
1 o~
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 elutedi- and trivalent ions. An example
of gradient
elution
on the HPIC-AS6
50.2-
using p-cyano-
CI-
phenateis shownin Figure 3. In this run, the
concentrationof p-cyanophenate
in the eluantis
increasedfrom 2.4 mM at the beginningof the run
.!:!
~
.!:!
I
.!:!
':"~'
<i.'8
~..2
to 35mM at theend.
&&.
-
Can carbonate bicarbonate
buffersbeused
"8,
c
Fu
~
for gradient elution?
~
.~
.o~
N
%
~
5
z
%, ~
0 Q , 0
2m~.g
:e .-2
~
.
.!:!
E ~ ..
&£~
~
~
0
5
8l:
aslow as5 canbeusedfor isocraticelution,due
to their higherbackgroundconductivity following
suppression,
they aregenerallynot acceptablefor
gradientelution.
I
~
.c
Carbonatecontainingeluantsaregenerallynot
acceptablefor gradientelution. Following
suppression,
carbonicacid is formed. With a
relatively low pKa of 6.2, the baselinedrift is too
severe.Although saltsof weak acidswith pKa's
fi
.!:!
GO
u
.c
~
!
~
I
I
I
I
I
0
5
.10
15
20
Minutes
Figure 3
I
.
,-
How doeschangingtheeluantconcentration
affectelution?
Canselectivitybechangedby
changingthegradient?
The relationshipbetweenelution volume and
gradientsteepness
(i. e. how rapidly the eluant
concentrationis being increased)is describedby
equation[1] and showngraphicallyin Figure 4. In
this equation,elution volume is usedinsteadof
retentiontime; the two arerelatedby the eluantflow
rate.
Vr - Vrn
A
log
= -log
R + log Const
[1]
V rn
A +E
V r is the retentionvolume,V rnis the mobile phase
volume (columnvoid volume),A is the analyteion's
charge,E is the eluantion's charge,R is the gradient
ramp steepness
in mM of eluantconcentrationper
For ions of different charge,the elution order,and
thereforethe selectivity,canbe changedby adjusting
the gradient. For example,if two ions with different
chargecoelute,an increasein gradientsteepness
will
separatethe two, causingthe ion of higherchargeto
elutefIrSt. In theory,one could obtain the optimum
conditionsfor a gradientelution from a plot suchas
that shownin Figure4. In practice,this is not
straightforwardbecauseequation[1] assumesthat a
singlelinear gradientis usedwith the initial eluant
concentrationequalto zero. Fastergradientrunscan
be obtainedby startingthe eluantconcentrationat a
highervalue. Also, the bestcombinationof speed
andselectivityis often the result of combining
severaldifferent gradients,often with isocratic
mL of eluant pumped, and log Const is the Y
intercept of the log/log plot This equation predicts
sections and step changes. With more complicated
gradient programs, the retention equation becomes
that an increasein gradientsteepness
will decrease
the retentiontime of divalent ions more than
monovalentions. An advantagemonovalenteluants
haveover divalent (or otherpolyvalent)eluantsis
that a given changein eluantconcentrationproduces
a greaterchangein analyteretentiontime. Compared
unwieldy.
Equation[1] canbe easilyusedto predict trends.
This is demonstratedin Figure 5. In all three
chromatograms,
the initial eluantconcentrationis
2.5 mM p-cyanophenate.This concentrationis
to a monovalent eluant, during a gradient run, the
concentration of a divalent eluant must be changed
increasedbetween 2 and 7 min. to 18.5 mM in (a)
(where sulfate and nitrate co-elute) to 22.5 mM
by a muchlarger amountto elute ions of widely
differing retention.
in (b), resultingin baselineseparation.In (c),
the eluantconcentrationis increasedasin (a) to
18.5mM, but in 5 min. insteadof 9 min. The
effect is similar in that the divalent anionselute
earlier,but becauseof the long period holding at
18.5mM, the separationbetweenthe divalentanions
is not compressedasin (b), but is insteadsimilar to
(a). The chromatogramin Figure 3 of anionson the
HPIC-AS6wasdevelopedwith this methodand
usesa combinationof stepchanges,linear gradients,
andisocraticsections.
RetentionYs.RampSlope
Theoretical,
for monovalent eluant
v -v
Log~
How canbaselineslopebeminimized?
Vm
LogR
Figure4
Wheneluantsotherthan sodiumhydoxideareused,
it is often necessaryto minimize the baselineslope
which resultsasthe eluantconcentrationis
increased.In anionexchange,this canbe
accomplishedchemicallyby addingmannitolto the
weakeluantandboric acid to the regenerant.
Although the reactionbetweenboric acid and
mannitolis not well understood,they do combineto
producean acid considerablystrongerthaneitherof
--
,
.
.
the ~o by the~selves. As a ~eutralpoly~cohol,
mannl,tolhasli!tle e!fe;cton el.thersepara,tion
,or
detection. Bonc acIdm the dilute sulfunc acId
Effectof GradientRampon ElutionAS6,
p-Cyanophenate
Eluant
regenerant
is neutral,andcandiffusethroughthe
"
6'0(1)1%
cation
exchangemembranesof the AMMS. The
two then combine in the suppressor, producing a
conductivespecies.By decreasingthe mannitol
concentrationin approximatelyinverseproportionto
the increasein eluantconcentration,the baseline
changescanbe counteracted This methodis
demonstrated
in Figure 6, in which mannitolwas
usedto balancethe baselinein (b), but not in (a).
Anothermethodof baselinecompemsationis
computerb3;Seline
.s~b~cti?n. For ~s method,a
blank run WIth no mjectlon IS stored m the
computor'smemoryand subtractedpoint by point
longasthebaselineprofIleis reproducible.
from
succeding
runs.
This
method
works
well
as
How are contaminantsin the eluant
mY
0
22.5
18.5
5
~-
-
-
.
..
:E
8 ~
I .~
r7,..;:
7
10 i
-
r
CoO
8 :i
.~
8 ~
5
10 0
-
-
5
10
~
-
-/
2.5
(a)
.
.:..
~
:~
:~
removed?
A problemencounteredm gradIentelutIonISthe
presenceof extraneouspeaksin the chromatogram
causedby traceimpuritiesin the eluant. In anion
exchange,the mostcommonimpurities arechloride,
sulfate,andcarbonate.In the early part of a gradient
run, while the eluantis weak,theseions are
concentratedat the front of the separator.As the
eluantstrengthincreases,they areeventuallyeluted,
andappearasinterferingpeaksat the expected
retentiontimes. Carbonatecan be minimizedby
usingproperlydeionizedwater and low carbonate
sodiumhydroxide. The effect of contaminantscan
be minimizedby placing a small column,calledthe
Anion Trap Column (ATC), in front of the injection
valve. The A TC is filled with high capacity,low
efficiency anionexchangeresin. It successfully
preventstraceanioniccontaminantsfrom reaching
the separatorcolumn during the early part of the run.
Later in the run, the eluantstrengthmay be high
enoughto elute the contaminants.The low
efficiencyof the resin spreadsout the contaminant
6
CI)
(b)
(c)
Figure 5
Balancingthe GradientBaselinewith Mannitol
and BoricAcid AS6,p-Cyanophenate
Eluant
I
I
I
I
I
I
peaks so that they do not interfere with the
0
10
zo
0
10
zo
chromatography.A consequence
of the useof an
ATC is that if a compositiongradientis used,the
time neededto convertthe form of the resin in the
A TC mustbe considered.
a.
Figure 6
b.
.An alternateapproachis to usea higherefficiency
trap column which is regeneratedat the end of each
gradientrun with a high concentrationeluant,
removingany accumulatedionic contaminants.The
HPIC-AG8 guardcolumn canperform asa trap
columnbecauseof its high affmity for carbonate,the
primary contaminantin sodiumhydroxidee1uants.
An exampleof the useof an AG8 asa trap columnis
the separationof anionsin coffee shownin Figure 7.
For this chromatogram,carbonateandother
contaminantson the trap column wereremovedwith
a strongborateeluant The sampleis theninjected
beforethe sodiumhydroxideeluanthasremovedall
of the boratefrom the trap column. The small
amountof borateleft forms a complexwith quinate,
increasingits retentionfrom coe1utionwith acetateto
elution betweenglycolateandformate.
How are gradients used with MPIC?
Mobile PhaseIon Chromatography(MPIC) is a
form of reversedphaseion pair chromatographyin
which chemicallysuppressed
conductivitydetection
is used. As in reversedphaseHPLC, gradient
elution is accomplishedby varying the percentage
of organicsolventin the eluantduring the run.
Although the ionic strengthof the eluantmay remain
the samethroughoutthe run, the backgroundconductivity candecreasedue to the changingdielectricconstant Thesebaselinechangescanbe compensated
eitherby chemicalmeansor by computerbaseline
subtraction.Often, :MPICis usedto eluteions
which arevery stronglyretained. When this is the
case,the baselineconductivitychangecanbe minimizedwithout affectingthe separationby adding
very weakly retainedions directly to the stronger
eluant (higher percentageof organic solvent). Using
gradient :MPIC, the separation of five alkyl sulfonates and sulfates is shown in Figure 8. The AMMS:MPIC suppressor(pIN 37106) should be used.
Anionsin Coffee,AS5A
Gradient Proaram
%1:
100
100
85
Conditions
55
0
0
%2:
0
0
15
45
40
40
%3:
0
0
0
0
60
~
0
0
100
100
0
0
Thenext injectionwas alwaynwoo42 nin. atE. tIE begiming of tIE ~viol8
.
G
e.~
..
0
~.c
-
GG
U
Eluant
'a
-
mM H3BO3
0
FI
run.
1-.
U>0-
#3:
:.
G
'(j
.
.c
a.
-G -.
G
0
.c
Co
c
~3
a
I
0
Figure7
I
5
I
10
I
15
Mlnut..
II
20
25
200
'
ow Rate: 1.0 mL/mln
-G.
.
C.OG
NaOH
Eluant
..
-:
-
mM
0
G
u
0.75
mM NaOH
-.
0
#1:
Eluant #2: 200
G
G
..
100
~I
30
.
,
,
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
~
~
:§
uU)
!
recommendedas a good startingpoint The HPIC-
:s
c:
u
AS5A (51.1)separatorwith sodium hydroxide eluant
provides the optimum combination of efficiency,
3~
selectivity,and speedwithout an unacceptable
baselineslope. If fewer ions than the 36 shownin
~c! ~
!
~~ ~ ~
~
!
!
~
I.
Figure 2 need to be separated,the gradient steepness
can be increased to reduce the run time. Remember
that changing the gradient will affect the elution
~ u
! ~
I~
0
=:!!
orderof ions of different charge. In particular,
increasing the gradient ramp slope will causesulfate
N
andotherdivalentions to elute earlierthannitrate.
-I
0
When the considerations discussedin this technical
note are followed, gradient elution can be used to
solvemany chemicalanalysisproblemsthat are
difficult to solveusing isocraticelution.
Ion chromatograph:
Trap column:
Dionex series4OOOi
or IC with GPM
ATC (pIN 37151)or HPIC-
Guardcolumn:
AG8 (pIN 37023)
HPIC-AG5A (pIN 37134)
Separatorcolumn:
HPIC-AS5A (PIN 37131)
Eluant #1:
0.75 roM sodium hydroxide
Eluant #2:
200 roM sodium hydroxide
Eluantflow rate:
1.0mL/mm
Suppressor:
AnIon MicroMembrane,
1
'--
0
~
8
112 16
Minutes
Columns: IIPIC-NSI
Eluant 1:
Eluant 2:
Eluant 3:
Eluant 4:
Flow Rate: 1.0 mUmln.
water
CH3CN
0.5 II H3B03, 10 mil TIIAOH, 40 mil mannitol
0.5 II H3B03. 10 mil TIIAOH, 50% CH3CN
Time(mln)
0
7.5
10
15
15.1
1%
2%
3%
4%
74
8
4
14
17
7
47
57
24
20
9
55
16
74
8
4
12
16
14
14
Figure 8
.
.
AMMS (pIN 38019)
Suppressor: AIIII5-IIPIC
Regenerant:
.
25mN H2SO4
Regenerant:
25 InN H2SO4
Regenerantflow rate:
10mL/min