Anal. Chem. 1985. 57.2247-2253
large solvent peak. By use of a retention gap, solutes can be effectively refocused and held at the head of the column while waiting for the solvent to elute. However, materials leaching out of the valve and the plumbing are quantitatively transferred and focused on the column in splitless mode, rather than being mostly vented when a splitter is used. Mobile phase impurities also focus on the column during the wait, causing additional, unwanted peaks in the chromatogram. Cleaner mobile phase will help, but we have found the valve or the associated plumbing to be the most significant source of impurities. New injection devices and methods will be needed before routine quantitative analysis by capillary SFC-FID is possible. Research in this area should include exploring ways of enriching solute concentrations (or selectively venting the solvent) and verification of quantitative solute transfer using appropriate tracers. As for SPE, additional work will be needed to unambiguously assign peak identities.
ACKNOWLEDGMENT We thank B. A. Charpentier, D. R. Gage, J. K. Howie, R.
2247
J. Jandacek, M. W. McIntosh, and H. W. Wharton for their assistance. Registry No. COz, 124-38-9. LITERATURE CITED (1) Rizzi, G. P.; Taylor, H. M. J. Am. Oil Chem. SOC. 1978, 55, 398-401. (2) Jandacek, R. J. Int. J. Obes. 1984, 8, supp. 1, 13-21. (3) Fallat, R. W.;Glueck, C. J.; Lutmer, R.; Mattson, F. H. Am. J . Clin. Nufr. 1978, 29, 1204-1215. (4) Glueck, C. J.; Jandacek, R.; Hogg, E.;Allen, C.; Baehler, L.; Tewksbury, M. Am. J . Clln. Nufr. 1983, 37, 347-354. (5) Birch, C. G.; Crowe, F. E. J. Am. Oil Chem. SOC. 1876, 53, 581-583.
(6) Novotny, M.; Springston, S.R.; Peadon, P. A,; Fjeldsted, J. C.; Lee, M.
L. Anal. Chem. 1981, 53, 407A-414A. (7) Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56, 619A-828A. (8) Fjeldsted, J. C.; Kong, R. C.; Lee, M. L. J. Chromatogr. 1983, 279, 449-455. (9) Chester, T. L. J. Chromatogr. 1984, 299,424-431. (IO) Van Lenten, F. J.; Rothman, L. D. Anal. Chem. 1976, 4 8 , 1430-1432. (11) Peadon, P. A.; Fjeldsted, J. C.; Lee, M. L.; Springston, S.R.; Novotny, M. Anal. Chem. 1982, 5 4 , 1090-1093. (12) Grob, K., Jr. J. Chromatogr. 1982, 237, 15-23. (13) Chester, T. L.; Innis, D. P., unpublished results, The Procter & Gamble Co., Miami Valley Laboratories, 1984.
RECEIVED for review April 15,1985. Accepted June 19,1985.
Liquid Chromatographic Separation of Inorganic Anions on an Alumina Column Gary L. Schmitt and Donald J. Pietrzyk*
Chemistry Department, University of Iowa, Iowa City, Iowa 52242
An aiumlna column is investigated as a stationary phase anion exchanger for the LC separation of lnorganlc anlonlc analytes. Mobile phase varlables that strongly influence analyte anion retentlon and which can be optlmlred to affect separations of complex anaiyte anion mlxtures are mobile phase pH, lonlc strength, counteranlon type and concentratlon, and, to a lesser extent, solvent composition. Anaiyte anion exchange selectlvitles on alumina are different when compared to selectivities on polystyrene dlvlnylbenzene R4N+ type anion exchangers. An alumlna column provldes excellent effIclency, selectlvlty, and resolution and can be used to separate compiex analyte anlon mixtures, Including trace analytes in the presence of maJorcomponents, wlth favorable analysis times. Welkleflned chromatographic peaks are obtalned and provide the bask for favorable detection ilmits and callbratlon curves over a wide concentration range.
The properties of alumina, like silica, as a stationary phase for adsorption and/or partition chromatography have been studied for a long time. Their ability, like most other metal oxides, to exhibit ion exchange characteristics has also been widely recognized in the past. Alumina has many desirable ion exchange properties. For example, it has a rigid structure, undergoes little swelling or shrinking in water or solutions containing electrolyte and organic modifier, can participate depending on the analyte in ion-sieve and steric effects, exhibits good thermal stability, has good resistance toward strong oxidizing and reducing agents and radioactivity, shows fa-
vorable ion exchange selectivities, and exhibits both anion and cation exchange characteristics (1-4). Even though these are very favorable properties when compared to polymeric type ion exchangers, the development of analytical ion exchange has been mainly with this latter group since their introduction in the 1930s. Probably the major reasons for this lack of acceptance in the past is that alumina by comparison was shown like other metal oxides to provide low ion exchange capacities, low resistance to strong acid and base, and, often, poorly defined peak shapes and zones in chromatographic applications. Alumina was a prominent stationary phase in the early developing stages of modern high-performance liquid chromatography (HPLC) where it was used primarily for normal-phase LC. Even though alumina was shown to be versatile and efficient, and uniform, narrow size, spherical alumina particles were developed, its current role in HPLC is minor and it has been replaced by reverse stationary phases. Applications as an ion exchanger in HPLC, however, should be fruitful for the following reasons: (1) The aforementioned modern micro alumina particles are available. (2) Modern HPLC detection does not require a large loading of analyte samples as in the past. Thus, the requirement for high ion exchange capacity to handle the analyte is no longer a major one; a consequence of the lower exchange capacity means that less mobile phase electrolyte is needed for successful elution of the analyte which can then enhance detector response. (3) Well-packed columns of the modern micro alumina particles should provide efficient ion exchange columns that will yield favorable chromatographic peak shapes. Several examples
0003-2700/85/0357-2247$01.50/00 1985 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
illustrating the potential of metal oxides as ion exchangers in modern LC have already been reported. For example, silica has been used for the cation separation of amines (5) and alkali-metal and alkaline-earth cations (6) while alumina has been used as both an anion and cation exchanger for the separation of carboxylic acids, amines, alkaloids, and proteins (7-1 0). It appears that Schwab and co-workers (ref 11 and references within) carried out the first detailed studies of alumina as an ion exchanger where they examined the inorganic cations and anions on a column of a1 sequently batch equilibration, column, and thin-layer chromatographic experiments with alumina and alumina-impregnated paper all indicated that ion exchange was the major factor in determining retention of inorganic cations or anions on alumina; these studies are reviewed in detail elsewhere (ref 3,4, 11, and 12 and references within). In this paper we have examined the use of an efficiently packed column of modern micro alumina particles as an anion exchanger for the separation of inorganic monovalent anions. Chromatographic variables and their optimization are identified. Since analyte elution order is not the same as that found with typical quaternary ammonium type anion exchangers, which are routinely used in double and single column anion exchange chromatography (ref 13 and 14 and references within), the use of alumina is a valuable addition to these latter strategies in applications requiring the separation and determination of inorganic anions.
EXPERIMENTAL SECTION Reagents. Inorganic and organic salts used for analytes, buffers, and ionic strength salts were analyticalreagent grade when possible or were prepared by titration of the corresponding acid or base with standard NaOH or HCl solution, respectively. Organic solvents were LC quality while LC quality water was prepared by passing distilled water through a Sybron/Barnstead water purification unit. Bulk alumina was obtained from Phase Separations as 5 Fm, spherical particles and slurry packed into 4.1 mm i.d. X 150 mm stainless steel columns. Instrumentation. The LC instrumentation consisted of two Beckman MllOA pumps, a M421 system controller,a Waters U6K injector, and a Beckman M160 UV detector (214 nm) and a Wescan 213A conductivitydetector in series. Column temperature was maintained at 35 OC by a water jacket and a Haake M-FE constant temperature water circulator. A Spectra Physics M4100 computing integrator was used for peak area measurements. Procedures. Aqueous analyte anion solutions were prepared by dissolving weighed quantities of corresponding Na or K salts (1or 5 mg/mL) and were stored in Hypovial (Pierce Chemical) glass containers. Sample aliquots of 2-20 pL were introduced by syringe (Hamilton)or by fixed sample loop injector. Mixed mobile phase solvents are percent by volume. Electrolyte and buffer containing mobile phases were prepared by diluting weighed amounts and/or aliquots of standard solutions of the components to a known volume. Mobile phase pH was adjusted with either standard LiOH, NaOH, or KOH solution; acetate, benzoate, formate, or phthalate buffers were used. All pH values were determined by pH meter combination electrode. Columns were aqueous, slurry packed using a ratio of 2 g of alumina/30 mL of HzO and an initial flow rate of 6 mL/min. This flow rate was used until a stable pressure (Waters M-6000 pump) was obtained. The effluent flow was then restricted to cause the packing pressure to reach 5900 psi and maintained at this level for 5 min. The restriction was removed and the column was allowed to reach its unrestricted flow, This procedure was repeated until the pressure at 6 mL/min (unrestricted) was reproducible. The column was then conditioned overnight with one of the test solution mobile phases (see below) prior to column evaluation. Conditioning during mobile phase changes depended on the mobile phase and usually required several hours (or until a stable base line is obtained) of flow at 1 mL/min. If a commercially available prepacked alumina column (alumina is column packed for normal-phase applications from a nonaqueous solvent mixture) is
used, it is essential to condition the column with large amounts (the minimum equilibration time was not determined) of aqueous mobile phase to ensure hydration of the alumina. Typical inlet pressures at 1mL/min, dependingon mobile phase, were 8o(t2000 psi. The analyte test sample and conditions used to compare the characteristics for different packed alumina columns were as follows: a 2-pL injection of a I-, Br-, and NO, (1mg/mL of each) analyte solution and a 1.00 X 10-1 M NaC1, 1.0 x M HOAc/NaOAc buffer, pH 4.0 mobile phase or a I-, Br- sample and a 1.0 X lo-' M LiOAc buffer, pH 6.5 mobile phase at a 1.0 mL/min flow rate; column efficiencies for these columns were calculated to be 40000 to 75000 plates/m using the peak width at half height method. Capacity factors were calculated in the usual way; column void volume, depending on the column, its age, and mobile phase, was 1.0-1.6 mL.
RESULTS AND DISCUSSION Alumina, like other hydrous oxides, has a very complex surface which is dependent on pretreatment and chemical environment. Three viewpoints have been suggested to account for retention of inorganic ions on alumina (ref 3 , 4 , 7 , 8,11,12, and 15-18 and references within). One view considers the key interactions between the analyte and the alumina surface to be due to hydrolytic and precipitation processes. A second focuses on adsorption of an analyte ion and its analyte counter ion and travel of this neutral species over the alumina surface. The third, which embraces an ion exchange concept, is that the analyte ion is retained via the formation of an electrical double layer a t the alumina surface. It is suggested that a surface charge appears due to dissociation at surface =AlOH groups and detachment of either hydrogen or hydroxide ions. As a result of the charge site, ions of opposite charge are attracted from the bulk of the solution with the resulting formation of two charged planes at the interface. While the first two types of interactions may contribute to retention of inorganic analyte ions on alumina (their contribution will be dependent on the chemical environment), the major factor in an aqueous environment is one of ion exchange ( 3 , 4 ,7,11,15,17-19). In simplest terms this can be represented by dissociation equilibria, eq 1and 2, which
+A1-0-H +A1-0-H
2
+Al+ 0-H-
+ +A1-0-
(1)
Hf
(2)
yield positive and negative surfaces, respectively. Anion and cation exchange can then take place at these charge sites as shown in eq 3 and 4, respectively, where X is the analyte ion.
+Al+ 0-H-
+ X- e +Al+X- + OH+ X+ +A1-O-Xt + H+
(3)
+Al-O-H+ (4) The presence of a hydroxylated alumina surface is consistent with several key observations. (1)Alumina is hygroscopic and its water content markedly influences its adsorptive strength (16). (2) The heat of water adsorption indicates that water bonding to alumina is not uniform (16,20). (3) Infrared studies clearly identify the presence of hydroxyl groups (16, 21), whose number and nature are dependent on heat treatment of the alumina. Thus, alumina's amphoteric character and conversion into an anion and cation exchanger via hydration and subsequent treatment with acid or base (buffers) can be viewed as (3, 4 , 7, 11, 17, 18) Alumina
T
H20
+ H20 I
j
-
+ -$l-~-~I-~-
t'
HYy -AI-p-i-&
;1'-
P
H
-@?l-QHCl
C'
a -@-Al-b-
(5)
Y t H20
tl
moH
where anion exchange takes place between added analyte anions and the alumina surface hydroxyl anions while cation exchange occurs between added analyte cations and H+ pro-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
vided by dissociation of the alumina hydroxyl groups. The isoelectric pH for alumina, which is dependent on buffer components and the type and treatment of the alumina, can vary from pH 3.5 (citrate) to 9.2 (carbonate), while its ion exchange capacity, which approaches 2 mequiv/g, is also very dependent on the analyte ion, the pH, and the type and pretreatment of the alumina (1-4, 7, 11, 15, 17-19, 22, 23). Anion exchange capacity increases as the acidic pH decreases while cation exchange capacity increases as the basic pH increases. In general, the changeover from an anion exchanger to a cation exchanger is a gradual one and occurs in the vicinity of the isoelectric pH. Rates of ion exchange, which are also pH, environment, analyte, and type of alumina dependent are favorable and it is suggested that alumina under appropriate pH conditions functions as a monofunctional cation exchanger and a polyfunctional anion exchanger (22). While our initial studies have focused on cation and anion exchange properties of alumina (24),only the latter are discussed here. If it is assumed that inorganic analyte anion retention on alumina follows anion exchange as shown in eq 3, then the major mobile phase variables affecting the retention should be those that typically influence anion retention on a weak base anion exchanger. Thus, the retention should be dependent on ionization of the exchange site, anion exchange selectivities between the analyte anion and any other counteranion present, counteranion concentration, and analyte anion concentration. It should also be possible to equate these variables to the capacity factor, k’, for the retention of the analyte anion as was done when using a weak acid cation exchanger for the separation of cations (6). With analyte anion concentration small and the number of exchange sites large, the retention will be independent of analyte concentration and it will then be determined by the other three factors. pH. Mobile phase pH influences analyte anion retention on alumina in several ways. (1)Since the exchange sites are weakly basic, a pH below its isoelectric pH is required to generate anion exchange sites. (2) Because of the complex nature of the alumina surface, the transition between an anionic and cationic charged surface is a gradual one. Thus, anion exchange capacity increases gradually as the pH decreases from the vicinity of the isoelectric pH to a more acidic condition. (3) Increasing the anion exchange capacity via pH increases analyte anion retention for a given column. (4) The mobile phase pH can affect analyte anion retention in three ways. First, addition of buffers to adjust the pH provides competing counteranions which will influence analyte retention due to mass action and the exchange selectivity exhibited by the counteranion. Second, retention of analytes derived from weak acids will be favored by pH conditions which ensure their ionization. Third, the type of buffer used influences the isoelectric pH (7-10, 23). Third, the type of buffer used influences the isoelectric pH (7-10, 23). Figure 1 shows that as mobile phase pH decreases below the isoelectric pH anion retention, expressed as capacity factor, k’, increases. This trend is consistent with alumina acting as a weak base anion exchanger. Retention below pH 4 was not determined even though anion exchange capacity is still increasing because (1)analyte retention is already very high at this pH (a high level of retention requires a stronger eluent for elution which will severely influence detection limits) and (2) alumina’s stability is poor below pH 2.0. Figure 2 illustrates the effect of pH on the resolution of mixtures of monovalent inorganic anions. At the higher mobile phase pH, Figure 2A, the seven-component mixture is resolved into five distinct peaks. Lowering the mobile phase pH to 4.05,Figure 2B, which increases the number of anion exchange sites on the alumina, completely resolves the mixture into seven quantitatively useful, well-defined, efficient chroma-
2249
oquroua 0.100 Y NoC104, 0.0010 M acddm buffor Spharlrorb A5Y. 150 x 4.1 rnrn flow: 1.0 rnl/rnln.: UV (214 nrn); 35.0°C
15.0’
10.0
lS.0
A
!
I-
0.0
- =
-_
=
4
I
I
3.5
4.5
4.0
1
I
5.5
5.0
6.0
7.0
6.5
PH oquoour \ 0.100 M NoCI, 0.0010 M aaotoh buffor x 4.1 mrn Sphorlrorb ASY5l,0 Flow: 1.0 rnL/mln.; UV (214 nrn); Js.O°C
6.0
k 4.0
2.0
B
0.0
4.0
4.5
5h
5.0
PH Flgure 1. Effect of pH on the retention of anions on alumina.
pH = 4.05
pH=5.60
a+ 0.100 M Naa. 0.0010 M acdute buffrr sphw*orb Asy; 10 rnL/tnin.; 2u nm
-
B -
0
1
5
10
mL
0
I
I
I
I
1
5
10
15
20
25
mL
Figure 2. Effect of pH on the resolution of a mixture of inorganic anions on alumina.
tographic peaks. This separation also points out one of several major differences between alumina and polystyrene divinylbenzene strong base anion exchangers of the tetraalkylammonium (R4N+)type which are currently being used in single and double column ion chromatography of inorganic analyte anions (13, 14). For alumina, which is a weak base anion exchanger, mobile phase pH can be used to alter its anion exchange capacity. Reducing the pH increases the capacity, increases analyte anion retention, and, when optimized, can increase resolution. For strong base anion exchangers modest pH changes have no effect on their exchange capacities. Anion Exchange Selectivity. The retention order for several inorganic analyte anions is indicated in Figure 1 and remains constant over the pH range studied. However, as the pH increases toward the isoelectric pH and the number of
2250
* ANALYTICAL CHEMISTRY, VOL.
57, NO. 12, OCTOBER 1985
aquaour 0.0010 M acatata buffar, pH = 5.60 lonlc rtnngth: Varlad wlth NaC104 Sphorlrorb ASY, 150 x 4.1 mm; 35.OoC Row: 1.0 mL/mln.; UV (214 nm) a I-
15.0
aq. 0.0010 M awtah buffw, pH = 5.80
jl war varld urlng W O 4
.).
a
b Br-
f
k'
U=0200M
b
INOS-
Sph.rlrorb A5Y 1.0 mL/mh; W ~ nm U
10.0
5.0
-
0.0
0 0.0
5.0
10.0
15:o
20.0
IONIC STRENGTH (U)X~O-~U Flgure 3. Effect of Ionic strength on the retention of Inorganic anions
on alumina. anion exchange sites drops accordingly, the difference in anion selectivity progressively diminishes. The improved selectivity at a lower pH is also illustrated by comparing resolution at pH 4.05 in Figure 2B to Figure 2A where the pH is 5.60. A second major difference between alumina and a polystyrene divinylbenzene strong base R4N+type anion exchanger is that the anion exchange selectivity on alumina is markedly different than the selectivity on the R4N+exchanger. For alumina and a mobile phase of pH 4 the anion retention order based on the data in Figures 1and 2 and subsequent studies, and which is in general agreement with previous alumina thin layer and column studies (1-4, 11, 12), is
F- > SO-: > Cr,0:- > HC02- > benzoate > C102- > Br03- > C1- > NO2- > NO3- > Br- > C103- > SCN- > I- > C104- > C2H30{ ( 6 ) while for the R4N+type anion exchanger ( 3 , 2 5 )the order is
S042-, C104- > I- > SCN-, C 1 0 > ~ NO3- > Cr042-> Br- > NO2- > Br03- > C1- > HC02- > C2H302-> F(7) Because several selectivity differences in eq 6 and 7 are small, the observed elution order will depend on the mobile phase conditions, particularly pH, since several are anions of weak acids. Fluoride ion is strongly retained on alumina. Repeated experiments with F- indicated that it was either not eluted or not detected even when using a strong eluting mobile phase. The observed halide analyte elution order on alumina, eq 6, is consistent with Al-halide complex formation constants (26) and supports the suggestion that the analyte anion interaction is with the A1 within the alumina structure. Multivalent inorganic analyte anion retention, which is also pH dependent, is much higher than monovalent anion retention. For example,when using the mobile phases in Figure 2, estimated 502- retention times would be over 1-2 h, respectively. Thus, successful elution of multivalent anions in reasonable analysis times requires either a stronger eluting mobile phase, a higher mobile phase pH to reduce the number of anion exchange sites, or a combination of these two eluent modifications. Ionic Strength-Counteranion. As ionic strength increases analyte anion retention decreases; this is illustrated in Figure 3 where retention of several monovalent anions is shown as a function of ionic strength using NaC104as a major source of ionic strength. A dilute M) acetate buffer was
..
1
5
1 0 0
mL
1
1
I
I
I
5
10
15
20
29
mL
Figure 4. Effectof ionic strength on the resolution of a mixture of
inorganic anions on alumlna.
used so that its contribution to ionic strength and as a source of a second counteranion (OAc-) was significant only at low NaC104concentrations. The effect of ionic strength on analyte anion retention cannot be separated from the effect of the selectivity exhibited by the counteranion where both its concentration and choice of the counteranion will determine how the retention changes. Increasing counteranion concentration, which occurs when the concentration of the ionic strength salt is increased, will decrease analyte anion retention according to mass action effects. This result is consistent with anion exchange as shown in eq 3. Also, analyte anion retention will be different at a given ionic strength according to the selectivity of the counteranions being compared. Thus, the anion elution order (anion selectivity) for alumina given in eq 6 also indicates the eluting power of the anions when used as the counteranion in the mobile phase. Although F- is one of the strongest eluent counteranions, its use is not recommended because its interaction with alumina is too strong and leads to permanent changes in the alumina column. Hydroxide ion is also a strong eluent counteranion because its presence, if concentrated enough, causes the alumina to shift from an anion to a cation exchanger. The effect of ionic strength on chromatographic separation is illustrated in Figure 4. In Figure 4A, where the ionic strength is 0.20 M, five distinct peaks are obtained for the separation of the fivecomponent mixture. If the ionic strength is reduced, for example to 0.050 M as shown in Figure 4B, a base line resolution of the five-component mixture is obtained with only a modest increase in analysis time. Figure 4 also shows that excellent chromatographic peak shapes are obtained over a wide range of ionic strength. Comparison of Figure 1A and Figure 1B indicates that the counteranion has a significant effect on retention which is typical of anion exchange. At a given pH the mobile phase ionic strengths are the same in Figure 1A and Figure 1B and the mobile phases differ only in the type of counteranion present. Thus, Cl- (Figure 1B) is a much stronger eluent counteranion than C10, (Figure 1A) by a factor of 3-4. This is in contrast to the polystyrene divinylbenzene R4Nf type anion exchangers where C104- not C1- is the much stronger eluent counteranion (25). If NaC104 was used in Figure 2 in place of NaCl as the source of the counteranion at the two pH conditions, analyte retention times for the NaC104mobile phase would be 3 to 4 times larger. Since multivalent anions are highly retained, a stronger eluting mobile phase can be prepared by using salts of multivalent anions, for example, a SOf salt, as the source of ionic strength and/or counteranion. Preliminary studies indicate
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
2251
I n 0
I
I
I
I
0
5
10
Is
mL
200
5
10
ml
Is
0
5
10
Ij
(s
ml
Figure 5. Effect of CH,CN:H,O composition on the resolution of a mixture of inorganic anions on alumina. that acidic phosphate mobile phases are compatible with the alumina while an oxalate mobile phase is not, presumably because of hydrolytic tendencies between the alumina surface and oxalate anions. Analyte Concentration. Analyte anion retention was shown to be independent of analyte concentration a t low column loadings. In these studies, NO, and I- were injected in 2.0-pL aliquots. The amount injected, calculated as the anion, covered the range from 10 to 2000 ng of anion. An aqueous 0.10 M NaC104,0.0010 M acetate buffer (pH 5.60) mobile phase was used. No attempt was made to determine the upper column loading limit or the point where retention is affected by concentration. However, subsequent calibration curve studies indicated that retention was independent of concentration a t higher analyte concentrations, particularly if a lower mobile phase pH is used. Alumina's anion exchange capacity changes with pH since it is a weak base anion exchanger. Thus, the maximum loading permitted for retention to be independent of concentration will depend on mobile phase pH and will increase as pH decreases. For example, if the pH is raised to 6.50, it was found that for I-, Br-, or C1as the analyte, the upper limit for retention-analyte concentration independence using an aqueous 0.010 M Li acetate mobile phase was about 500-1000 ng of anion. Since anion exchange capacities for alumina approach about 2 mequiv/g at a favorable pH, the independent analyte retention-concentration relationship is favored by a low pH mobile phase condition. A general guideline used with polystyrene divinylbenzene R4N+ type anion exchangers is that analyte loadings should be below 1% of the exchange capacity in order to ensure a favorable analyte retention-concentration relationship. Mobile Phase Solvent Composition. The effect of organic modifier (MeOH and CH3CNwere studied) on inorganic analyte retention was studied up to 70% by volume of organic solvent. The mobile phase conditions were the same as those used in Figure 1B except for the addition of organic modifier to the solventmixture. As the CH3CN concentrationincreases, retention for benzoate, Br03-, NOz-, NO3-, Br-, and I- decreases slightly except for benzoate which undergoes a large decrease in retention while retention for CIOz-rises slightly. Even though changes are small, reversals in selectivity were found over the solvent range studied. For example, at 1:l CH3CN-Hz0, NO3- elutes before Br- and benzoate elutes before Br03- and C10, when compared to 100% HzO as the mobile phase solvent. This is useful chromatographically as shown in Figure 5. Figure 5A shows the elution order in 100% H20. When the CH3CN Concentration is increased to 30%, Figure 5B, Br- and NO3- are coeluted and benzoate appears between Br03- and ClOZ-. A t 50% CH3CN, NO3- appears before Br- and benzoate is eluted between NOz- and Br03(see Figure 5C). Chromatographic peak shape and plate
rnL
rnL
Figure 8. Separation of inorganic anions on alumina.
C
d
d
CI-
d
I: 1 : 1 : 100
D
d
Flgure 7. Separation of a halide mixture on alumina. aquroua 0.100 M NoCI, 0.0010 M buffrr pH 4.U 1.0 mL/mIn. Sphrrlaorb UY UV ( 214 nm ) 10 or 9800 ng of rach
!\
mL mL mL Figure 8. Separation of N03--N02- mixture on alumina.
number are not significantly affected by the change in mobile phase solvent composition and well-defined, quantitatively useful chromatographic peaks are obtained even at 70% organic solvent. Separations. Optimization of the elution variables depends on the type of analytes in the mixture and the desired analysis time, resolution, and detection limits. Figures 6-8 illustrate this optimization by focusing on the separation of several multicomponent mixtures where the analytes are at equal or trace concentrations. For the separation of simpler mixtures, the aforementioned elution variables, namely, pH, ionic strength, type of counteranion, and, to a lesser extent, solvent composition, can be adjusted accordingly using Figures 1-5 as a guide. In Figure 6, nine- and eight-component analyte anion mixtures are separated on the alumina column. The sample
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
injection volume was 10 pL and the weight of each analyte in this aliquot was 0.2-2 mg calculated as the anion. A mobile phase pH approaching the alumina isoelectric point pH was used. This reduces the alumina anion exchange capacity and decreases analyte retention, and therefore a weaker eluent in terms of ionic strength and counteranion is required. The NaOAc buffer not only satisfies these requirements but also provides a counteranion that does not interfere in inorganic anion detection. Also, the reduced mobile phase electrolyte concentration relative to Figure 2 provides a better detection limit when using a conductivity detector. Similar separations are possible with other eluents. For example, Clod-, I-, ClO,, Br-, NOz-, C1-, and Br03- were separated with a 0.0050 M benzoate, pH 6.62, aqueous mobile phase. This mobile phase provides the stronger eluent benzoate counteranion and thus its concentration can be reduced, will permit detection of acetate anion, and provides a modestly lower detection limit by conductance because of reduced electrolyte concentration. On the average the retention times for the analytes using the benzoate mobile phase are about 1.5 times greater than those shown in Figure 6. A phthalate eluent can also be used; however, for complex analyte mixtures the benzoate mobile phase provides better resolution because of a more favorable selectivity. Even a t a mobile phase pH of 7.0 resolution, depending on the analyte mixture, is still possible. For example, in a pH 7.01,O.OlOM Li acetate buffer, ClO;, I-, Br-, C1-, C102-, and formate are base line resolved in less than 14 min. If the pH is lowered, anion retention is higher due to increased anion exchange capacity and resolution can be improved. For example at a pH 4.14 (0.0010 M NaCZH3Oz buffer), I- and SCN- are base line resolved, which is not the case in Figure 6, and the NOf-NO, base line separation is increased by over 2 min. Because of increased retention, mobile phase electrolyte concentration was also increased to reduce analysis time; in this example the mobile phase also contained 0.10 M NaCl. Since alumina's ion exchange capacity is modestly high, which allows both high analyte loadings and favorable resolution, the separation of trace analytes is possible. Examples illustrating this are shown in Figures 7 and 8. Figure 7A shows the base line separation of equal amounts of the halides. Resolution is so favorable that eluent strength can be modestly increased to reduce retention time while still maintaining a base line resolution. Analyte weights, calculated as the anion, were injected as 2-pL aliquots. Figure 7B-D, illustrates that a base line separation is still obtained even though each of two of the halides are only 1% of the mixture while the third halide makes up 98% of the mixture. Chromatographicpeaks are still sharp, well-defined,and quantitatively useful. Also, base line separation should still be possible at even larger ratios since the major component is still not overloading the available exchange sites or is tailing into adjacent halide peaks. This is particularly striking in Figure 7B since the first eluted component, I-, is also the major component. Also, noteworthy is that this separation is of considerable practical importance because typical ion chromatographic procedures for the halides, which use polystyrene divinylbenzeneR4N+type ion exchangers ( 1 3 , 1 4 ) ,give the reverse elution order (Figure 7). Not only is I- more retained on the R4N+exchanger but also a stronger eluent relative to Cl- and Br- is required to successfully elute I- in a reasonable analysis time. Figure 8 illustrates the separation of N03--NOz- mixtures at a 1:l ratio and combinations where one of the two components is about 0.1 % of the sample. The mobile phase pH was lowered to provide more anion exchange sites and prevent an overload. Since this also increases analyte anion retention, the mobile phase eluent strength was increased by using a higher concentration of an electrolyte (NaCl)that also provides
a stronger eluent counteranion. Also, NaCl has little effect on UV detection. Figure 8B shows that 10 ng of NO3- and NOz- are readily base line resolved. When the minor component, NOz-, elutes second, the peak for the major component, NO,, broadens into the NOz- band and the ratio shown is approaching the limit of resolution for the mobile phase conditions used. A weaker eluent will improve the resolution and allow a larger ratio to be separated. For the reverse case (Figure 8C) where the second eluted analyte, NOz-, is the major component, a base line resolution is obtained and favorable resolution is still possible at a higher ratio. The unusually large area for the trace NO3- peak in Figure 8C is from NO3- impurities in the NO2- sample. Calibration-Detection. The column chromatographic data for anion exchange on alumina indicate that reliable, reproducible column efficiency and well-defined peak shapes are readily obtained. Favorable calibration curves of peak area or peak height w. concentrationcovering a wide concentration range are also readily obtained. This was demonstrated by preparation of a calibration curve for NO, using UV (214 nm) detection and the mobile phase conditions listed in Figure 8. A fixed sample loop of 20 pL was used to transfer 1 to 2000 ng of NO3- (calculated as NO3-) into the alumina column. A linear relationship defined by log peak area = 0.94 log NO3+ 3.78 with a correlation coefficient of 0.999 was obtained when log NO3- peak area was plotted vs. log NO3- in nanograms. Mobile phase electrolyte did not interfere since UV detection was used. A favorable linear response can be obtained with a conductivity detector; however, the lower concentration limit for detector response depends on the electrolyte content of the mobile phase and the type of conductance detector used. For example, using the mobile phase conditions listed in Figure 6 , l ng of Cl- (calculated as Cl- and injected as a 2 0 - ~ Laliquot) gave a peak height twice the base line noise when using a Waters M 430 conductivity detector. Indirect photometric detection (27) using an eluent counteranion that can be monitored by absorbance can also be used for analyte detection; for example, a benzoate mobile phase using a wavelength where benzoate absorbs was shown to give an indirect detection of analyte anions. Although not studied, column and membrane electrolyte suppression techniques (13, 14) should be compatible with the alumina column and further reduce detection limits when using the conductance detector. Registry No. I-, 20461-54-5;Br-, 24959-67-9;NO;, 14797-55-8; NO2-, 14797-65-0; C102-, 14998-27-7; BrOs-, 15541-45-4; SCN-, 302-04-5; ClO,, 14866-68-3; C1-, 16887-00-6;Clod-, 14797-73-0; HC02-, 71-47-6; alumina, 1344-28-1; benzoate, 766-76-7; butanesulfonate, 24613-77-2.
LITERATURE CITED Amphlett, C. B. "Inorganic Ion Exchangers"; Elsevier: Amsterdam, 1964; p 84. Clearfleld, A., "Inorganic Ion Exchange Materials"; CRC Press: Boca Raton, FL, 1982. Patterson, R. "An Introduction to Ion Exchange"; Heyden: London, 1970; pp 15, 31, 90. Michai, J. "Inorganic Chromatographic Analysis"; Van Nostrand Reinhold: New York. 1970: D 75. Bidiingmeyer, B. 'A.; Del kios, J. K.; Kolysi, J. Anal. Chem. 1982, 5 4 , 442-447. Smith, R. L.; Pietrzyk, D. J. Anal. Chem. 1984, 56, 610-614. Laurent, C.; Billiet, H. A. H.; deGalan, L. Cbromatographia 1983, 77, 253-258. Laurent. C. J. C. M.: Billiet, H. A. H.; dealan, L. Chromatographla 1983, f 7 , 394-399. Laurent, C. J. C. M.; Billlet, H. A. H.; dealan, L. J . Chromatogr. 1984, 285, 161-170. Laurent, C. J. C. M.; Billiet, H. A. H.; deGalan, L.; Buytenhuys, F. A,; Van Der Maeden, F. P. B. J . Chromatogr. 1984, 287, 45-54. Schwab, G. M.; Ghosh, A. N. Z.Angew. Chem. 1840, 53, 39-40. Lederer, M.; Polcaro, C. J . Chromatogr. 1973, 8 4 , 379-386. Fritz, J. S.; Gjerde, D. T.; Pohlandt. C. "Ion Chromatography"; Huthig Verlag: Heidelberg, 1982. Smith, F. C., Jr.; Chang, R. C. "The Practice of Ion Chromatography"; Wiley: New York, 1983.
Anal. Chem. 1985, 57,2253-2256 (15) Nechaev, E. A.; Volglna, V. A. Russ. J. Phys. Chem. (Eng. Trans/.) 1974, 48, 1364-1367. (16) Snyder, L. R. “Princlples of Adsorptlon Chromatography”; Marcel Dekker: New York, 1968; p 163. (17) Flscher, W.; Kulling, A. 2.Elekfrochem. 1958, 60,680-688. (18) Umland, F. Z . Elekfrochem. 1956, 60,711-721. 1191 Shiao. S. Y.:. Mever. ~. R. E. J. Inoro. Nucl. Chem. 1981. 43, 3301-3307. (20) Cornelius, E. B.; Mulliken, T. H.; Mills, G. A.; Oblad, A. G. J . Phys. Chem. 1955, 59, 809-813. (21) Perl, J. B. J . Phys. Chem. 1985, 69, 220-230. (22) Churms, S. C. J . S.A h . Chem. Inst. 1986, 79, 98-114. (23) Parks, G. A. Chem. Rev. 1965, 65, 177-196. (24) Schmitt, G. L.; Edeimuth, S. H., unpublished results.
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(25) Helfferlch, F. “Ion Exchange”; McGrall-HIII: New York, 1962. (26) Sillen, L. 0. “Stability Constants of Metal-Ion Complexes”; The Chemicai Society: London, 1971; Supplement 1, Publication 25. (27) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 54, 462-469.
RECEIVED for review Ami1 9. 1985. AcceDted June 10, 1985. Part of this work was sipported by Grant AM28077 awarded by the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases and was presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, Feb 1985, paper 1104.
Detection Method for Ion Chromatography Based on Double-Beam Laser-Excited Indirect Fluorometry Sun-I1Mho and Edward 5.Yeung* Ames Laboratory-USDOE
and Department of Chemistry, Iowa State University, Ames, Iowa 50011
The concept of Indirect fluorometry for detectlon In Ion chromatography Is presented. A fluorescing eluting Ion Is used to malntaln a constant background slgnal. When the analyte Ions elute, electroneutrallty forces the displacement of an equlvalent amount of the elutlng Ions at the detector. A decrease In slgnal Is then observed. A mass detectablllty of 6.7 ng of chloride Ion Is obtained ( S I N = 3), which corresponds to a concentration detectablllty of 7.5 X IO-’ N. Thls Is posslble due to a novel double-beam arrangement for laserexclted fluorescence, substantlally reduclng the flicker nolse.
Ion chromatography (IC) has become a mature analytical method as a result of research efforts in the last few years (1-3). Significant improvements have been made in the de: velopment of a variety of stationary phases and in new detection methods. Much attention has been given to nonsuppressed ion chromatography, because of the potential for minimizing band broadening. In addition to the standard conductivity detector, several optical methods, including indirect photometric ( 4 ) ,refractive index (5),and direct photometric (6) methods, have been tried to allow the use of standard liquid chromatographic instrumentation. Most of these detectors have very similar limits of detection (LOD), which are in the nanogram range for typical ions. Since these detectors have already been utilized to their full potential, additional improvements in LOD can only come if new detection principles are introduced. In conventional liquid chromatography (LC), the lowest LOD’s have been reported for the fluorescence detector (7). For ions that fluoresce, one can thus expect improved LOD in IC as well. However, the number of ions that do fluoresce is very small, particularly those that are of interest to “real-world”problems. The exceptions are certain rare-earth ions, which phosphoresce in aqueous solutions. The combination of low absorption coefficients and poor emission efficiency leaves the LOD’s for these ions still quite unimpressive. Recently, several indirect detection methods for LC have been demonstrated (4,8). Briefly, the detector responds to some physical property of the chromatographic eluent. So, there is a constant background signal generated at the detector when no analytes are present. When the analyte elutes, it
displaces an equal amount of the eluent at the detector. Even though the detector does not respond to the analyte, the lower eluent concentration at the detector causes a decrease in signal. The analyte can then be monitored as a negative signal, Le., indirectly. So, it should be possible to devise a detection method based on indirect fluorometry. It may then be possible to extend the advantages for fluorescence detection to species that do not themselves fluoresce. To appreciate the potential of indirect measurements, it is necessary to define a figure-of-merit known as the dynamic reserve (DR). This is the ability of a detector to recognize a small change on top of a large background signal. This is quite different from the “dynamic range” concept often used in measurements, which is simply the ratio between the smallest and the largest signal that can be measured, independent of the background level. For example, the absorbance detector for LC can measure a change of AA = 2 X AU when the background absorbance is unity (4,9).The dynamic reserve is simply the ratio of the two, so that DR = 5 X lo3. In other words, the analyte must be at a fractional concentration of at least 1part in 5 X lo3 (of that of the eluent) at the detector before a noticeable decrease in the signal is observed. We note that DR for the absorbance detector cannot be increased by using a larger background absorbance, because noise increases as well (in fact more rapidly) to degrade the minimum detectable AA. So, indirect photometry (4) can only be used at analyte (fractional) concentrations of 1 part in 5 X lo3 or higher. In normal LC situations, this is too high a concentration to be useful. The reason indirect photometry works in IC (4) is because the eluting ion is typically present at a low concentration, e.g., M. So, the analyte need only be at 2 x M to produce a fractional concentration of 1 part in 5 X lo3. The refractive index (RI) detector is also an indirect detector because the solvent provides the major contribution to the signal. One can measure ARI = regardless of the background RI. So, for a solvent-solute RI difference of 0.1, the DR is lo6. This is why RI detectors have been useful in many LC situations. The best DR is obtained with polarimetry (8). When an optically active eluent is used, there will be a background rotation as large at looo (10). By mechanical adjustment to the polarization analyzer, one can suppress this background signal to a level similar to that with an optically inactive eluent. A change of 4 X lo4 deg ( S I N = 3) can still be detected. The DR is then 2.5 X lo7. In ion
0003-2700/85/0357-2253$01.50/00 1985 American Chemical Soclety
