Anal. Chem. 7992, 84, 2283-2287
2283
Ion Chromatographic Separation of Inorganic Anions and Carboxylic Acids on a Mixed-Mode Stationary Phase Raaidah Saari-Nordhaus' and James M. Anderson, Jr. Alltech Associates, Inc., 2051 Waukegan Road, Deerfield, Illinois 60015
Applicatlons of a mlxedmode stationary phase for ionchromatographlc separatlon of lnorgank anions and carboxyik acids are deocrlbed. The statlonary phase b siilca-based, bonded with ilgands containing both reversed-phase and ionexchange functions in a fixed 1:l ratlo. Inorganic anions such as chkrkle, nitrtte, bromide,nltrate, phosphate, phorpMte, seientte, and sulfate and carboxylic aclds such as acetk, la&, fonnlc, propknlc,butyric, and ioobutyrlc can be analyzed on this stationary phase. The separatlon of these anaiytes is affected by several factors such as the type of alkyl group on the support, eluant pH and lonlc strength, and organic modlfkr concentration in the eluant. By careful selection of eluant pH and Ionic strength, the simultaneous determination of inorganic anions and carboxylic acids can be achleved.
INTRO DUCT10N Mixed-mode stationary phases or multifunctional stationary phases have been used widely in liquid chromatography for the separation of biologicalsamples. Pinkerton and Hagestaml describe a packing material that has both hydrophilic and hydrophobic character, designed to allow direct injection of high protein containing samples for drug analysis with minimal protein interference. Hartwick and co-workers24 describe mixed-mode stationary phases that are composed of bonded silanespossessing both ion-exchangeand hydrophobic functionalities. These phases have been shown to result in improved separations of various biomolecules, such as oligonucleotides, oligodeoxyribonucleotides, and other nucleic acid constituents. The use of a mixed-mode stationary phase has also been reported to result in improved peak shape6and eliminate the use of ion-pairing reagent for ion-pair separations.6 This paper examines the use of a mixed-mode stationary phase for ion chromatographic separation of inorganic anions and carboxylic acids. In ion chromatography (IC), anions are separated by ion-exchange mechanism while carboxylic acids are separated by ion-exclusion technique. The two separation mechanisms are essentiallyopposite of one another, where strongly ionized inorganic anions are more strongly retained than weakly ionized carboxylic acids on an anion exchanger, whereas the reverse is true for ion-exclusion. Therefore, two separation modes or column switching methods798 are required to separate mixtures of inorganic anions and carboxylic acids. This unique stationary phase, which contains a single ligand incorporating both reversed-phase and anion-exchange functions in a fixed 1:lratio allows the
* Author to whom correspondence should be addreeaed.
(1) Pinkerton, T. C.; Hagestam, H. Anal. Chern. 1985,57,508. (2) Crowther,J.; Fasio, S.;Hartwick, R. J.Chrornatogr. 1983,282,619. (3) Floyd, T.;Yu,L.;Hartwick, R. Chromatographia 1986,21, 402. (4) Floyd, T.;Crother, J.; Hartwick, R. LC Mag. 1986,3, 508. (5) h a q , H. J.; Gutierrez, J. J. Liq. Chrornatogr. 1988, 11, 2851. (6) Alltech associates Product data sheet, bulletin no. 168, 1989. (7) Jones, W.R.; Jandik, P.; Swartz, M. T. J. Chrornatogr. 1989,473, 171. (8) Dunn, M.H. LCGC 1989, 7,138. 0003-2700/92/0364-2283$03.00/0
separation of both inorganic anions and carboxylic acids using one column. By adjusting the pH and ionic strength of the eluant, a simultaneous separation of anions and carboxylic acids can be achieved. EXPERIMENTAL SECTION Instrumentation. The ion chromatograph used in this work was an Alltech (Deerfield, IL) metal-free ion chromatography system. It consists of a Model 325 HPLC pump, a Rheodyne 9125 injection valve (100-rL sample loop), a Model 320 conductivity detector, and a Model 330 column heater. The temperature of the column heater and the conductivity detector cell was maintained at 35 O C . All data was recorded on a Spectra Physics (Santa Clara, CA) SP 4400 Chromjet integrator. Columns. Separations were carried out on the Alltech (Deerfield, IL)Mixed-Mode RP-C18/Anion column (250 mm X 4.6 mm). The column is packed with a multifunctionalsupport that consists of a high purity, 100 A, 7-wm spherical silica substrate which is bonded with a single ligand containing both reversedphase (Cl8) and anionic (dialkyl amine) functionalitiesin a fiied 1:l ratio. The material is not end capped. Other columns used for comparison purposes were Mixed-Mode RP-C4/Anion, and RP-CS/Anion columns. All columns were 250 mm X 4.6-mm i.d. and obtained from Alltech Associates Inc. (Deerfield, IL). Reagents. Standards and eluantswere prepared from reagentgrade chemicalsobtained from Aldrich ChemicalCo. (Milwaukee, WI). Water and organic solvent were HPLC grade, obtained from Alltech associates (Deerfield, IL). The eluant used for this study was an aqueous solution of phthalic acid. The pH of the eluant was adjusted with lithium hydroxide. Acetonitrile was used as the organic modifier. Column Precondition. The anion exchanger of the new mixed-mode columns are in the iodide form. Since iodide is strongly retained on the column, newly packed columns are preconditioned by passing approximately 300 mL of 8 mM phthalic acid, pH 4.5, to elute iodide off the column.
RESULTS AND DISCUSSION The stationary phase used in this study is a silica-based support covalently bonded with ligands containing both reversed-phase and anion-exchange (dialkylamine) functionalities in a fixed 1:lratio. The structure of the phase is shown in Figure 1. The reversed-phase functionality has an alkyl chain length of 18,8,or 4 carbons. The fiied ratio of 1anionexchange functionalityto each 1reversed-phase functionality assures batch to batch reproducibility of the support. The silica substrate has a particle size of 7 pm and a pore size of 100 A. This stationary phase is stable in organic solvents; however, because of the silica substrate, the elutant pH is limited within the pH range of 2-8. Both functionalities on the mixed-mode stationary phase, reversed-phase and anionexchange, will affect the separation of the inorganic anions and carboxylic acids. Effect of an Alkyl Group. The effect of an alkyl group on the analyte retention on the mixed-mode column was studied by injecting 20 ppm chloride and 100 ppm each of acetic acid and propionic acid. The Mixed-Mode RP Cl8/ Anion, RP CB/Anion, and RP C4lAnion columns were used. An aqueous solution of phthalic acid (a common eluant used Q l S S 2 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992 30
Si - o - Si - (cH,),- N - R
\
/
+
20 4
fi -0'
+
+ + +
L 10
R = C4, C8 or C18
Chloride Nitrite Bromide Nltrate Sulfate
Fburel. Structureof mixed-mdereversedghaseandanWxchange stationary phase. 0
3 4 5 pH of 4mM Phthalic acld
2
6
Flgurr 3. Effect of pH on the retention time of Inorganic anions.
1 \ 0
Chloride Acetic Propionic
+ 4
+
0
0
10
+
20
Acetic Propionic Isobutyric Butyric Formic
Number of carbon on the alkyl group
Figure 2. Relatlonshlp between anlon retention time and the carbon numbers on the alkyl group.
for the separation of anions on a silica-based anion-exchange column with conductivity detectiong was used as the eluant. The phthalic acid concentration was 4 mM adjusted to pH 4.0 with lithium hydroxide. The relationship between retention time, expressed as capacity factor, k', and the chain length of the alkyl group is shown in Figure 2. As the number of carbons on the alkyl group increases, the retention time decreases. The decrease in the retention time is more drastic for chloride than for acetic or propionicacids. We hypothesize that the increased hydrophobicity of the stationary phase with increased alkyl chain length is responsiblefor this trend. Referring to Figure 1,the secondary amine ion exchanger is immediately adjacent to a hydrophobic alkyl group. This hydrophobic region limits access to the ion exchanger by the hydrophillic chloride anion used in this study. As this hydrophobicity of the alkyl group increases (Le. the chain length increases), this shielding effect is more pronounced, reducing the inorganic anion's access to the ion exchanger with a resultant decrease in retention. Unlike inorganic anions, carboxylic acids are retained by both ion-exchangeand reversed-phasemechanisms.1° As the hydrophobicity of the alkyl group increases, the reversedphase interaction between the acid and the support increases. This should result in an increase in the retention by reversed phase as the chain length increases. However, the shielding of the ion-exchangerby the alkyl group increases as the chain length increases. This acts to decrease retention by ion exchange. These two effects off-set one another, resulting in a slight decrease in retention for acetic and propionic acids as shown in Figure 2. Since propionic acid is more hydrophobic than acetic acid, the repellent effect by the ion exchanger is less pronounced and the reversed-phase effect is more pronounced. The combination of these two effects result in a smaller change in the propionic acid retention than in the acetic acid retention as the chain length increases. Further studies were performed using only the Mixed-Mode RP C18/Anion column. (9)Gjerde,D. T.;Fritz, J. S. Ion Chromatography;Huthig: New York, 1987; Chapter 7, p 133. (10) Walker, T.A.; Ho, T. V.; Akbari, N. In Advances in Zon ChroCentury International, matography; Jandik, P., Cassidy, R. M., Me.; Inc.: MA,1990; Vol. 2, p 271.
0
2
3
4 5 6 pH of 4mM Phthalic ecld
2 7
Flgure 4. Effect of pH on the retention time of carboxyllc acMs.
Effect of pH. Eluant pH influences the retention of inorganic anions and carboxylic acids in several ways. The anion-exchangecapacity of the column itself is dependent on the pH of the eluant. The secondaryamine functional groups (PK,= 10)become increasingly cationic as the pH decreases. Below approximatelypH 9, the support is fully ionized. Under the conditions used in this study (maximum pH 6.5) the support is always 100% ionized and the influence of pH on anion-exchange capacity is negligible. For inorganic anions, the C18 functions do not contribute to retention. In addition, these anions carry the same charge throughout the pH range studied. However, as shown in Figure 3, the retention of inorganic anions increases as the pH decreases. This is explained by the influence of pH on the ionic strength of the phthalate eluant. The pK1 and p& values for phthalic acid are 2.95 and 5.41, respectively. As pH decreases, the charge on the phthalic acid moves from -2 to -1 to neutral with consequent reduction in eluant ionic strength. This reduction in the ionic strength of the eluant is responsible for increased inorganic anion retention with decreasing pH (Figure 3). Note that below pH 4 the phthalate eluant becomes substantially monovalent and is thus incapable of eluting sulfate within a reasonable run time. As described earlier, carboxylic acids are retained by both ion-exchange and reversed-phase mechanisms. In addition, the anionic character of carboxylic acids changes within the pH range studied (3-6.51,influencing the extent of interaction with the support by ion exchange. The carboxylic acids become increasingly neutral when eluant pH is near or below the acid's pK., resulting in less retention by ion exchange. However, the neutral acids are more favorably retained over their anionic counterparts by reversed phase. Thus, as eluant pH decreases, ion-exchange interaction with the support decreases, but reversed-phase interaction with the support increases. This complex combination of retention mechanisms and eluant ionic strength effects produces a markedly different response to changes in eluant pH for carboxylic acids as compared to inorganic anions (Figure 4). Note that
ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992
Table I. Effect of Acetonitrile as Eluant Modifier on the Capacity Factors, k’,of Anions and Carboxylic Acids k’ CH3CNin eluant (76) chloride bromide nitrite nitrate acetic propionic butyric 0 25 50
1.52 3.61 8.09
2.86 5.72 10.68
4.20 8.07 15.32
5.68 9.38 15.32
2.58 3.18
3.72 3.46
9
9
6.13 4.37 2.93
2285
isobutyric 7.31 5.38 3.72
Peaks are not detected due to Door sensitivity. ,-
lo0-
Q
+ L
6-
9
+ +
4-
4
Chloride Formate Bromide Nitrite Nitrate Sulfate
2-
0
Table 11. Nitrate and Propionic Acid Retention Reproducibility on the Mixed-Mode Column. column retention time* (min) serial number nitrate propionic acid 03271UA 05281CA 111510P 11201IP
5.86 6.37 6.95 7.69
6.80 7.22 7.88 8.49
a Eluank 4 mM phthalic acid, pH 4.5. Calculated mean of three injections.
1 2 3 4 Phthalic acid concentration (mM),pH 5.4
5
Flgurr 5. Effect of ionic strength on the retention time of inorganic
anions. the retention of carboxylic acids is relatively constant with changing eluant pH due to the offsetting effects of increased reversed-phase interaction at low pH and increased ionexchange interaction at high pH. The notable exception is formic acid, which shows a marked increase in retention as pH drops. One might speculate that formic acid, with very little hydrophobicity to interact with the support by reversed phase, would behave more like an inorganic anion, and this is indeed the case. The retention vs pH profile for formic acid is very similar to that of the monovalentinorganic anions. With a pK, of 3.74, however, one would expect a decrease in retention for formicacid at lower pH as it becomes less anionic. While no data was collected below pH 3, Figure 4 clearly shows that the retention for formic acid is flat or decreasing between pH 3 and 4. Effect of Ionic Strength. Aa the eluant ionic strength is increased, a corresponding decrease in the retention of analytes retained by anion-exchange mechanisms will be observed due to increased competition for the anion-exchange sites. The effect of eluant ionic strength on the separation of inorganic anions and formic acid is illustrated in Figure 5. Aqueous phthalic acid solutions at concentrations of 1-4 mM, adjusted to a pH of 5.4 with lithium hydroxide, was used as the eluant. The retention times decreasewith increasing eluant ionic strength. Identical results are observed for other carboxylic acids. Effect of an Organic Modifier. The effect of an organic modifier on the inorganicanion and carboxylic acid retention was studied by adding up to 50% acetonitrile to the eluant. The eluant was an acetonitrile/water solution of 4 mM phthalic acid, adjusted to an apparent pH of 5.0 with lithium hydroxide. The presence of acetonitrile affects both anions and carboxylic acids retention as shown in Table I. The concentration of the anions were 20 ppm for chloride and 50 ppm each for bromide, nitrite, and nitrate. Carboxylic acids were at a concentration of 200 ppm each. In general, as the concentrationof the acetonitrile increased,the inorganic anion retention increases, whereas the carboxylic acid retention decreases. The presence of acetonitrile affects the phthalate eluant, the inorganic anions,and the carboxylicacids in several ways. The degree of ionization of the phthalic acid in the eluant and the weakly acidic carboxylic acids in the sample
is reduced when acetonitrile is added to the eluant.11 Decreased ionization of the phthalic acid reduces the eluant ionic strength, making it a weaker eluant for separating anions retained by ion-exchange mechanism. As the acetonitrile concentrationincreases, the phthalate eluant becomes weaker, resulting in increased inorganic anion retention. The decreased solubility of the inorganic anions in the less polar eluant may also contribute to the increased retention of the inorganic anions. Carboxylic acids retention decreases as the acetonitrile concentration increases. The decreasedionization of the carboxylic acids in the presence of acetonitrile reduces the retention of the carboxylic acids by ion-exchange mechanism. Addition of acetonitrile also increases the eluant’s reversedphase eluting power. Consequently, the carboxylic acids exhibit a decrease in retention as the acetonitrile concentration is increased. When using an aqueous solution of 4 mM phthalic acid, pH 5.0, with no acetonitrile added, the peaks for the carboxylic acids are negative. When 25 % acetonitrile was added to the eluant, acetic and propionic acids are detected as positive peaks, while butyric and isobutyric acids are detected as negative peaks. In addition, the sensitivity becomes very poor for carboxylic acids when acetonitrile is added to the eluant. At 50% acetonitrile, acetic and propionic acids are not detected by conductivity. This may be due to increased carboxylic acids mobility and/or decreased ionization of the carboxylic acids in organic solvents. When acetonitrile is present, the equivalent conductance of the carboxylic acids is very close to that of the phthalate eluant. Column to Column Reproducibility. The column to column reproducibility was examined using chloride and propionic acid and 4 mM phthalic acid adjusted to pH 4.5 as the eluant. The retention times on the four columns tested are shown in Table 11. The retention times are quite reproducible except for the column with serial number 03271UA, which had been used for 5 months prior to this experiment. The columns with serial number of 111510P and 11201Ip were newly packed. It can be conclude that since the packing contains both reversed-phase and anion-exchangerin a fiied 1:l ratio by the nature of the bonding chemistry, batch to batch and column to column reproducibility is easily achieved. Applications. Examples of the inorganic anion and carboxylic acid separations are shown in Figure 6. When using conductivity detection, the signal is based on the difference (11) Haddad, P.R.;Croft, M.Y.Chromatographia 1986, 21, 648.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992
A
B 12
3.4
10
5
0 I
r
I
15Min.
9
10 20 Min. 0 10 20 30Min. 0 Figure 6. Separatlon of Inorganic anions and carboxylic acids on the mixed-mode RP C18/Anlon column using 4 mM phthalic acid eluant at (A) pH 4.1 and (6)pH 5.5. (A) Peak identlficatlon: (1)Chloride (20 pprn), (2) formate (50 pprn), (3)bromide (40 pprn), (4) nitrite (50 pprn), (5) nitrate (50 ppm), (6) sulfate (70 ppm). Peak identlflcatlon: (1) acetlc acid (100 pprn), (2) propionic acid (100 pprn), (3)butyric acid (100 ppm), (4) isobutyrlc acid (100 ppm); flow rate: 1.0 mL/mln. Detector: conductlvlty, 0.1 ps full scale. Detector polarity Is set at “posltlve” for A and “negative”for B. Injectlon volume: 100 pL. 4
0
5
I
A
I
27
36
45Min.
D
-
1
18
0
-
16Mln.
0
10
20Mln.
Figure 8. Simultaneousseparation of Inorganic anlons and carboxyllc acids using 4 mM phthalic acid, pH 3.0. (A) Standard. Peak Identlflcation: (1)acetic acid (200 pprn), (2)lactic acid (200 pprn), (3) formlc acid (50 ppm), (4) phosphate (30 pprn), (5) butyrlc ac# (200 ppm), (6)lsobutyrlc acid (200 ppm), (7) phosphite (30 ppm), (8) chlorkk (10 pprn). (6) Standard. Peak identlflcatlon: (1) acetic acid (100 ppm), (2) lactic acid (100 ppm), (3)succlnlc acid (100 ppm), (4) formic acid (30 pprn), (5) butyric acid (100 pprn), (6) selenite (20 pprn), (7) phosphite (15 pprn), (8) chloride (10 pprn), (9) nitrate (50 ppm). (C) Cream cheese. Peak identlflcatlon: (1) lactlc acid, (2)phosphate, (3) chloride. (D) Salad dressing. Peak identlflcatlon: (1)acetlc acid, (2) lactlc acid, (3)chloride. Other chromatographic conditions are as In Figure 7.
Table 111. Effect of Eluant pH on Peak Area of Acetic and Propionic Acids peak areaa pH of 4 m M phthalic acid acetic (200ppm) propionic (200ppm) 0
9
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
18 Mln.
Figure 7. Simultaneous separation of inorganic anlons and carboxylic acids using 4 mM phthailcacid, pH 4.5. Peak identlflcatlon: (1)chioride (20 ppm), (2) acetate (200 pprn), (3) proplonlc acid (200 pprn), (4) nitrite (50 pprn), (5)nitrate (50ppm). Flow rate: 1.O mL/mln. Detector: conductivity (positbe polarlty), 0.1 ps full scale. InJectlon volume: 100 pL.
in the equivalent conductance between the eluant anion and the analyte anion. The equivalent conductance for the inorganic anions in this work is constant over the pH range studied and is always higher than the equivalent conductance for phthalate. Therefore, the inorganic anions always appear as positive peaks, independent of eluant pH. The ionization (and the equivalent conductance)of the carboxylicacid anions in the sampleand the phthalate anion in the eluant will depend on eluant pH. When the eluant pH is far below a given carboxylic acid’s pK,, the acid will exist largely in the molecular form and will therefore have a low equivalent conductance. The ionization and equivalent conductance will increase as the eluant pH reaches and exceeds the pK, for the anion in question. At any given eluant pH, if the equivalent conductance of the sample anion is higher than the equivalent
a
+74 870 +59 355 +21803 -19 257 -86 789 -147 118 -183 496 -187 289
+61547 +42 919 +11989 -28 991 -88 431 -146 828 -181 166 -184 822
Peak area reported aa positive (+) or negative (-) peaks.
conductance for the phthalate eluant anion, the sample peak will be positive. If the converse is true, the peak will be negative. The size of the peak will be proportional tb the difference in equivalent conductance of the sample anion and the phthalate eluant anion. The relationship between sensitivity for acetic and propionic acid, expressed in peak area, and the eluant pH is presented in Table 111. In general,higher pH eluants provide better sensitivity for carboxylic acids. The simultaneous separation of inorganic anions and carboxylic acids at an eluant pH of 4.5 is shown in Figure 7. At this pH, inorganic anions appear as positive peaks, while carboxylic acids appear as negative peaks. In order to analyze both inorganic anions and carboxylic acids in one run with
ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992 2287
all positive peaks, the eluant pH must be decreased. Some examples are shown in Figure 8. Cream cheese and salad dressing samples were diluted with deionized water and filtered through syringe filters prior to injection. A variety of inorganic and carboxylic acids can be analyzed using this column. However, since the eluant pH has to be lower than approximately 3.5 to obtain all positive peaks, the phthalate eluant is present predominantly as a monovalent anion and is not able to elute divalent anions. The sensitivity for carboxylic acids is also poor at low eluant pH. One potential solution to these problems is to use indirect UV as the detection method. Phthalate ion absorbs UV light at 254 nm. By setting the detector at this wavelength, anions and
carboxylic acids can be detected aa a reduction in the absorbance. Higher pH eluants can be used with indirect UV detection and allow the separation of strongly retained anions. The sensitivity for carboxylic acids may also be improved. Developing the separation at high pH to elute strongly retained anions followed by reduction of eluant pH by a postcolumn chemical suppression may also solve this problem. Additional studies are required to fully investigate these possibilities.
RECEIVEDfor review February 24, 1992.
24, 1992. Accepted June
