Anal. Chem. 1982, 5 4 , 1065-1071
Ion Interaction Chromatography of Organic Anions on a Poly(styrene-divinylbenzene) Adsorbent in the Presence of Tetraialkylammonium Salts Ziad Iskandaranl and Donald J. Pletrryk" Chemistry Department, University of Iowa, Iowa Cjty, Iowa 52242
The major equlllbrla that contrlbute to the retention of an organic analyte anlon on PRP-1 (a nonpolar poly(styrenedlvlnylbenrene) adsorbent) in the presence of a tetraalkylammonluin cation (R4N+), a coanlon, and OH- are idlentlfled and used to derlve an equation descrlbing the analyto retentlon whlclh was experimentally verified. The significant parameters are structure and concentration of R4N+, mobile phase solvent composition and pH, analyte concentratllon, and type and concentration of coanlon. The ability of a R,,N+ salt to enhance analyte retentlon changes slgnlflcantly as the coanlon lab changed. The analyte retention, referred to as Ion Interactlorr, Is suggested to follow a double layer modell where the R4N+!saR occupies a primary layer at the stationary phase while the analyte anlon and other anions In the systeim compete for tlhe secondary layer. A major equlllbrlum Is the selectivity of one anion over another. Several appllcatlons are lllustratedl.
Adding hydrophobic counterions to a mobile phase to enhance retention and resolution has been widely used in the liquid chromatographic (LC) separation of charged organic species 011 alkyl-modified silica (1-3). Other studies have focused on identifying the interactions that occur in the presence of the counterions, and several retention mechanisms have been proposed (1-12). The ion pair mechanism is one where it is suggested that ion pairs form between analyte ions and the hydrophobic counterions prior to sorption on a hydrophobic alkyl-modified silica stationary phase. Experimental evidence supporting this are isotherm measurements and correlation of retention with counterion concentration. Other workers suggested1 an ion exchange mechanism, where the counterions are first sorbed and these charge sites serve as exchange sites for the analyte ions. Retention of counterion on the stationary phase and correlation of this to analyte retention has been the major supporting evidence for an ion exchange mechanism, It has also been suggested that both occur and the extent to which one is more significant than the other is a function of the conditions employed in the LC separation. If very longchained, bulky, hydrophobic counterions are used, micelle formation and molecular size factors are significant par,meters that influence retention. Because neither the ion pair nor ion exchange mechanism individually explains all the experimental results, retention models that are broader in scope and which attempt to account for all equilibria have been suggested ( 2 , 4 , 5 , 1 0 - 1 4 ) . Thus, Biddlingmeyer et al. ( 2 , 5 )proposed a model that does not require ion pair formation in either phase and is not based on classical ion exchange. This model assumes dynamic equilibrium which is affected by electrostatic, eluophilic, eluphobic, adsorbophilic, and adsorbophobic forces. Although this study was done on a (2-18 bonded phnse the model is very similar to the double layer model proposed by Cantwell and 0003-2700/82/0354-10~15$01.25/0
Puon (13) that accounts for retention of organic ions onto Amberlite XAD-2, a poly(styrene-divinylbenzene) (PSDVB) copolymer which acts as a hydrophobic adsorbent in LC aLpplications. In this model, which has been shown to describe the the retention of both organic cations and anions (13,14), analyte cation (or anion) is sorbed onto the XAD-2 surface as a primary layer and small inorganic counterions such as C1- (or Na+) occupy the diffuse secondary layer. Similarly, in Biddlingmeyer's model ( 3 , 5 )the hydrophobic counterion occupies the primary layer sorbed onto the C-18 stationary phase while the counterions occupy the secondary layer. Sirice the mobile phase also contains the hydrophobic salt a dynamic equilibrium is established between the double layer, the hydrophobic salt and the analyte anion. The overall procesei is called ion-interaction chromatography (IIC). Hydrophobic counterions will also influence retention of organic anions on XAD-2 and -4(15,16).A major advantage of these stationmy phases, which are PSDVB copolymers, over the alkyl modified silica is their stability throughout the entire pH range. Thus, a strongly basic mobile phase can be used to ensure that even very weak organic acid analytes are completely ionized in the presence of the hydrophobic catioins. Because of the nature of the suggested interactions and their dependence on the experimental conditions, chromatography on the XADs in the presence of counterions was also called IIC (15,16). In this report the effect of tetraalkylammonium salts (R41N+ salt), inorganic coanions, mixed solvent, added inert electrolyte, and pH on the retention of anions derived from organic acids, amino acids, and peptides on PRP-1 are considered. PRP-1 is a spherical, uniform, 10-pm particle PSDVB copolymer with a high pore volume and surface area and often provides column efficiencies, depending on the separation, in excess of 20000 plates/m (17).A retention model that fits the experimental results and takes into account the major equilibria that influence the retention of an organic analyte anion on PRP-1 under these experimental conditions is presented.
EXPERIMENTAL SECTION Reagents. Amino acids, peptides, organic acids, and R4N+sallts were obtained from Sigma Chemical, Eastman Kodak Chemilcal Co., MCB Manufacturing, Pfaltz and Bauer, Inc., and Aldrich Chemical Co. and used as received. The R4Nt salts purchased were the tetramethyl- (TMABr),tetrapropyl- (TPABr),tetrabutyl(TBABr), tetrapentyl- (TPeABr), and tetrahexylammonium (THxABr) bromide and the tetraethyl- (TEACl), tetraheptyl(THpACl), and trimethylhexadecylammonium (TMHxDACl) chloride. Tetrapentylammonium chloride (TPeACl), fluoride (TPeAF), nitrate (TPeAN03), and formate (TPeAFor) were prepared by passing TPeABr through an Amberlite IRA-400 ion exchange column (Fisher Scientific) charged in the C1-, F-,NO,, or HC02- form. Tetrapentylammonium hydroxide (TPeAOH) was prepared by a reaction of TPeABr with Ag20 followed by centrifugation to remove AgBr. Chromatographic grade CH&N was obtained from MC'B. Water was purified by passing distilled water through a mixed bed ion exchanger, an activated charcoal column, and through 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54,NO. 7, JUNE 1982
2-pm stainless steel filters. Inorganic salts were analytical reagent grade. Instrumentation. A Waters Model 202 LC equipped with a Model 6000 pump, a U6K injector, and either a Waters 254-nm UV, a Waters refractive index, a Tracor Model 970 variable wavelength, or a Wescan Model 213 conductivity detector was used. A 4.1 mm i.d. X 150 mm, 10-pm PRP-1 (PSDVB) column was used. Prepacked columns and slurry packed columns (in this laboratory) prepared from bulk form PRP-1 (Hamilton Co.) were used. Efficiencies in this study were 15 000 to 20000 plates/m for optimum mobile phase conditions. Procedures. The PRP-1 column was conditioned by passing a 0.001 M R4N+ salt solution containing other mobile phase components through the column for 1-2 h at a 1 mL/min flow rate. The R J V salt can be removed by passing at least 20 column volumes of 1:l CH3CN:H20through the column. To charge the PRP-1 column into different R4N+salt forms or anion forms, it is best to first clean the column with 1:l CH3CN:H20and then charge it in the desired R4NfC- form. The column charged in the R4NfC-form was found to be stable from day to day; however, a short conditioning period with the 0.001M R4N+mobile phase was usually employed at the start of each working day. Sample solutions of the organic acids (about 20 mg/mL) and amino acids and peptides (about 1 mg/mL) were prepared by dissolving weighed quantities of the analyte in a known volume of HzO, 95% EtOH, or their mixture. These were prepared in 6-mL Hypovials fitted with Hycar Septa and sealed with aluminum caps (Pierce Chemical) and stored in a refrigerator when not in use. Pressure-Lok series B-110 10-pL or 25-pL syringes (Precision Sampling Corp.) were used to inject 1-4 pL samples. Flow rates were usually 1 mL/min and column inlet pressures ranged from 500 to 1000 psi depending on the mobile phase. Either 254 nm, 208 nm, conductivity,or refractive index was used for detection. Mixed solvents in the mobile phase are expressed as percent by volume. Basic mobile phases were prepared from phosphate salts and NaOH. Ionic strength was controlled when necessary by adding known amounts of inorganic electrolyte. Column temperature (25 "C) was controlled by water jacket. Column void volumes, V,, were determined from retention volumes found for several samples not retained by PRP-1 at the given eluting condition. Depending on these conditions, V, was about 1.1mL. R E S U L T S AND DISCUSSION Consider the system composed of the PRP-1 stationary phase, a mixed solvent with water as the major component, a R4N+cation and its coanion, an inorganic electrolyte, hydroxide ion from the addition of NaOH or a basic buffer, and an anionic analyte. At the equilibrium point the significant equilibria can be the following: (1)dissociation of the species formed between the R4N+cation and all other anions including the analyte anion and their retention on the stationary phase (ion pair model), (2) anion exchange between the surface modified stationary phase and the several different anions present in the solution (ion exchange model), or (3) a set of dynamic equilibria indicating dissociation and exchange which accounts for retention through a double layer at the stationary phase surface (double layer ion interaction model). Because of the experimental conditions used in these studies, several other potential equilibria are insignificant. These include the following. (1)All species derived from Na+ and/or K+, present from added electrolyte, buffer, and/or NaOH, and the several different anions present in the solution are assumed to be completely dissociated. (2) Equilibria involving aggregate formation are assumed to be negligible. (3) Equilibria describing retention of the analyte itself or of added inorganic electrolyte and buffer components are negligible. (4) The organic analyte remains completely dissociated since the mobile phase is basic. (If pH is not controlled then ionization of the analyte must be considered.) Retention of Tetraalkylammonium Salts. Figure 1 shows that retention of R4N+salts on PRP-1 (samples of all
carbon number
9
,
?
16
,
,
I
32
24 I
!
I
-I 0
1
20
% CH3CN
,
.
1
60
40
Figure 1. Retention of R4N+ salts on PRP-1 as a function of (1) CH,CNIH,O concentration and (2) carbon number in the R,N+ salt for (A) 3 7 CH&N:H,O and (B) 4:6 CH,CN:H,O.
/1 % CH3CN
0
-4
I6
-4 log
-3
DPeAfl
-2 in mobile phase
Figure 2. Amount of TPeAF retained/PRP-1 column calculated from breakthrough volumes as a function of (1)TPeAF concentration in the mobile phase (1:4CH,CN:H,O) and (2)CH3CN/H20concentration (1.00 X lo-, M TPeAF).
R4N+ salts were introduced as Br- salts except TEAC1, THpAC1, and TMHxDACl into a mobile phase free of electrolyte and buffer) follows two general trends. First, as the percent of CH&N in the mobile phase decreases retention increases in a regular manner. Second, for any given mobile phase condition where there is retention, retention increases in a regular manner as the carbon number in the R4N+salt increases. If electrolyte is added to the mobile phase, retention of the R4N+ salts on the PRP-1 increases. Thus, for 0.0010-0.10 M NaBr or NaOH it was shown that log k'increased in a regular manner as the ionic strength of the mobile phase was increased. This trend has been observed previously when using other types of PSDVB stationary phases and charged analytes (13-16). Retention of the R4N+salts was further demonstrated via a determination of breakthrough volumes; a summary of these data is shown in Figure 2. In these experiments the mobile phase containing TPeAF in the CH3CN-H20 mixture was continuously passed through the column at constant flow rate until the TPeAF appeared in the column effluent. The breakthrough profile appeared as a very sharp rise in the detector response which rapidly reaches a maximum value. The breakthrough volume was taken as the inflection point of this sigmoid-shaped curve. Breakthroughs were determined a t 1.0 X lom3M TPeAF as a function of CH3CN:H20 concentration and at 1:4 CH3CN:H20as a function of TPeAF concentration. As the percent CH&N increases the break-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
10167
Table I. ]Effect of Added Electrolytes on the Retention of Organic Analyte Anions in the hesence of TPeABr capacity factor, k' analyte a p-NH,BA rn-NH,BA rn-OHBA p-OHBA o-phthalic acid rn-phthalic acid p-phthalic acid p-NH,BSA p-OHBSA sulfaguanidine sulfacetamide sulfanilamide
NaF
3.97 8.04 11.6 3.40 11.9 32.5 13.7 14.5 13.7 4.06 16.2 3.55
Na,SO,
IrJa,PO,
NaOOCH
Na citrate
NaCl
NaBr
NaNO,
3.61 7.56 11.4 3.12 13.9 33.9 13.2 13.4 13.3 3.57 15.3 3.4
3.44 7.13 9.92 2.67 11.6 31.1 12.6 13.3 12.5 3.57 15.3 3.07
2.80 5.40 5.97 2.08 7.41 16.4 7.01 8.98 6.72 3.77 9.63 2.50
2.79 5.60 4..10 L51 3.97 11.5 5.37
2.49 4.67 5.29 2.14 5.90 12.9 5.94 6.65 5.35 3.99 7.19 2.44
1.27 1.91 1.40 0.87 1.29 2.68 1.73 2.45 1.40 4.37 2.25 1.38
1.33 1.59 1.22 0.80 1.16 2.04 1.37 1.77 1.15 4.69
11.1
5.06 4.26 12.7 2.81
1.81
1.39
a BA = benzoic acid; BSA = benzenesulfonic acid. A 5:95 CH,CN:I -0, 1.00 X lo-, M TPeABr, 1.00 X 10- M, pH 11.00 (phosphate buffer), mobile phase containing the electrolyte so that the ionic strength is 1.00 x lo-' M. The flow rate was 1.00 mL/min, V,,= 1.30 mL, and detection 'was at 254 nm.
-___
through time decreases and the amount of TPeAF retained by the PRP-1 decreases. From the flow rate and TPeAF concentration the number of millimoles of TPeAF per column can be calculated. This also represents the potential number of charged sites per column for the given mobile phaiae condition. As shown in Figure 2 the log millimoles of TPeAF decreases linearly as the percent CH3CN increases while a t a fixed CI13CN-H20 ratio (Figure 2) the log millimoles of TPeAF per column increases linearly as the log TPeA,F concentration in the mobile phase increases. The column capacity in Figure 2 is expressed per column since the weight of stationary phase in the 15-cm column is not known. From the column dimensions and packing experience with PRP-1 columns, the amount of stationary phase is estimated to be about 0.9-1.0 g per column. Thus, depending 011 the CH3CN:H20ratio the capacity or number of charge sites ranges from about 0.008 to 0.018 mmol/g. In contrast, conventional anion exchangers have capacities of about 3.5 mmol/g. Dissociation of R4N+ Salts. Conductances of 1:3 CH3CN:H20solutions of TPeAF, TPeAFor, and TPeAE3r were determined as a function of R4N+ salt concentration. Repeated measurements indicated that the percent ioniization of the three R4N+salts was >go%. Although the differences are small it also appeared that the percent ionization isi in the order TPeAF > TPeAFor > TPeABr. Conductanci,= measurements with TPeAOH solutions also indicated that its percent ionization is >go%. The percent ionization will depend on thie CH,@NH20ratio. In the experiments reported here the CIH3CN:W20ratio covers only a relatively small range and thus the percent ionization of the R4N+salts is not only very large but it also remains nearly constant. Retention of Analyte Anions. Figure 3 illustrates that the retention of several organic anionic analytes increases on PRP-1 as tbe number of carbons in the R chain of the R4N+ salts increases. In these experiments the mobile phase was 1:9 CH3CN:H20 (Figure 3A) or 100% H20 (Figure 313); the R4N+ salts were 1.0 x M (all were Rr- salts except for TEAC1) and the pH was 11.0 (phosphate buffer with controlled ioniic strength using NaCl) to ensure that all analytes were in the anionic form. Retention data for the R4N+salts (taken from Figure 1)are also shown in Figure 3. It is seen that the rise in retention of the analyte anion corresponds to the rise in retention of the R4N+ aialt. In Figure 3A, the sharp rise occurs when R = propyl while in Figure 3B it occurs when R = ethyl. This difference is due to the stronger eluent (1:9 CH3CN:H20)used in Figure 3A. The correlation of increased analyte retention with increalsed retention of R4N+salts has been observed on
10-
5-
0
4
8
I2 16 carbon number
20
Flgure 3. Retention of several organic analyte anions on PRP-1 as a function of R,N+ carbon number. The mobile phase conditions in (14) are 1:9 CH,CN:H,O, 1.00 X lo-, M R,N+ salt, pH 11.0 (phosphate buffer), p = 0.100 M at a flow rate of 1.00 mL/min; V , = 1.30 mL. (B) is the same except the solvent is 100% H20. The dotted lines aire R,N+ salt retention as a function of R,N+ salt carbon number (from Figure 1).
other PSDVB stationary phases (15,16). The significance of the retention maxima in Figure 3B is not readily apparent. Since the R4N+ salt retention occurs at a higher carbon number in the stronger eluent (Figure 3A), it may be that the analyte retention, which is shifted in the same direction, has not yet reached its retention maximum. Measurements of k' values for the analytes in Figure 3 at higher carbon numbeirs appeared to indicate that some of the analytes would pans through a maximum. The data in Figures 1-3 suggested to us that the TPeA,+ salt was an optimum R4N+salt for additional studies. The reasons for this are the following. (1)The effect of the TPeA,+ salt on retention of the organic analyte is large. (2) Since retention is enhanced other, mobile phase variables affecting retention can be altered significantly and their effect can ble readily ascertained. (3) If R4N+salts containing longer alkyl chains were used, aggregate and/or micelle formation effects could become significant. Effect of R4N+Coanion. Only a few studies (14-16) have considered that the type of anions in solution or accompanying the R4N+salt will influence retention of the organic anioniic analytes. Table I, which lists the k'values for the retention of several different weak organic acids and ampholytes on
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
Table 11. Effect of TPeA' in Different Anionic Forms on the Retention of Organic Analyte Anions on PRP-1 analyte'
F-
p-NH,BA p-OHBA o-phthalicacid p-NH,BSA p-OHBSA sulfanilamide sulfaguanidine sulfacetamide
3.13 22.6 20.7 3.86 25.9 2.12 0.81 3.77
a
Table 111. Retention of TPeAX Salts on PRP-1 analyte
capacity factor, h ' b Cl- formate NO;
Br-
3.01 19.7 16.3 3.73 22.8 2.03 0.80 3.61
2.53 15.0 12.6 3.10 17.3 2.02 0.79 3.07
3.04 20.0 18.6 3.82 23.7 2.20 0.69 3.75
2.78 16.6 21.5 3.49 20.9 2.00 0.70 3.43
BA = benzoic acid; BSA = benzenesulfonic acid.
A
M TPeA+C- where C is F-, 1 : 3 CH,CN:H,O, 1.00 x M NaOH mobile W ,formate, NO,-, or Br-, 1.00 x phase. The flow rate was 1.00 mL/min, V, = 1.30 d, and detection was 254 nm.
PRP-1 in the presence of TPeABr and several different types of added inorganic electrolytes, demonstrates that not only is the coanion influence significant but the effect appears to follow a regular pattern. Although there are small variations for individual analytes, the effect of the added anion in reducing the analyte retention follows the order Br-
> NO3- > C1- > citrate > formate > Po43-> SO:- > F-
(1)
I t should be noted that if TPeABr is omitted from the mobile phase in Table I, all analyte samples would have little or no retention on PRP-1. Also, the mobile phase was basic ensuring that the analytes are anionic (all ampholytes are monovalent anions while several of the diprotic acids are divalent anions) and the electrolyte was added to yield mobile phases that were 0.10 M ionic strength. The mobile phase used in Table I contains several different anions. In addition to the added anion all mobile phases contain Br- (from TPeABr), OH- (pH ll),and HP0?--P043(buffer). This complex anion mixture contributes to the variation noted in the elution for the different analytes. Furthermore, since Br- is a strong eluent anion its presence will dominate the weaker eluting power exhibited by anions below Br- in the elution order (eq 1). Thus, the actual difference in anion eluting power should be significantly larger than that indicated by Table I. Experiments similar to those in Table I were repeated in such a way that the effects of the competing anions could be systemically minimized. In the first experiment the R4N+salt was TPeAFor and the basic p H was achieved by using only NaOH. Since the formate is a weaker eluent anion, only electrolytes were added that provided equal or stronger eluent power, namely, formate ion, Cl-, and NO3-. The effect of the added anion in reducing the analyte retention follows the expected order NOs- > C1- >> formate. Furthermore, the elution range in the Br--P043--free solution is greater than that obtained in their presence (Table I). The full effect of the anion on the analyte retention is shown in Table I1 where each mobile phase contained only OH- and the TPeA+ salt of the anion being examined. Experiments with TPeAOH suggested that its influence was less than that of F-. In Table I1 enhancement in retention is greatly increased since the competing effect of additional anions is absent. It should also be noted that a 1:3 CH3CNH20mobile phase is used which itself decreases retention relative to the conditions used in Table I. Confirmation of the elution order and further demonstration that the TPeA+C- salts are retained by the PRP-1 were demonstrated by measurement of k' values for analytical samples of the TPeA+C- salts. These data are shown in Table
k''
TPeAOH TPeAF TPeAFor
0.27b 2.81 3.05
analyte TPeACl TPeABr TPeANO,
k'' 3.27 3.75 4.44
A 3:7 CH,CN:H,O mobile phase. The flow rate was 1.00 mL/min and V , = 1.17 mL. Conductivity detection was used while a refractive index detector was used for all others. I11 where the mobile phase was a mixed solvent free of electrolyte. The k'retention order of NO; > Br- > C1- > formate > F- > OH- is consistent with the elution order exhibited by these anions toward the retention of organic anionic analytes. Retention Model. Since the conductance data indicate that the R4N+salts are highly dissociated under the conditions of the LC experiments it can be assumed that ion pair formation between R4N+ and the analyte anion is negligible. Furthermore, retention of the R4N+ salt on PRP-1 and its dependence on the type of coanion present suggest that selectivity of one anion over another is a major influence on analyte retention. Taking into account that certain equilibria, as outlined earlier, are negligible and experimental conditions can be devised to minimize or eliminate certain other competing equilibria, an expression can be derived which focuses on those equilibrium constants and experimental variables which significantly influence the retention of an organic analyte on PRP-1 in the presence of a R4N+salt. Certain competing anion equilibria are avoided in the following way. (1)The basic pH is achieved with NaOH rather than by a buffer, thus, eliminating the presence of buffer anions. Since OH- ion is similar or weaker than F- (see eq 1)in eluting power its competing effect will be very small. (2) The R4N+salt and the inorganic salt used, when required to provide different ionic strengths, contained the same coanion. Thus, the only anions present in the mobile phase are OH-, the coanion from the R4N+and ionic strength salt, and the analyte anion. The equilibrium and equilibrium constant, K(Q,,)~ describing the retention of a R4N+ salt by PRP-1 is given by Q+,
+ C-, + A, ==(QCA),
(2) (3)
where Q+ is the R4N+ species, C- is its coanion, A, is the stationary phase, and m and s are the mobile and stationary phase, respectively. Similarly, for retention of the analyte anion, X-, the expressions are Qfm
+ X-, + A, == (QXA),
(4)
where K(QXA)B is the constant for QX retention. Since the anions can exchange the exchange equilibrium and the exchange constant, KQX,are given by (QCA),
+ X-,
== (QXA),
+ C-,
(6)
The sorption capacity, KO,for the stationary phase is a measure of the number of sites that can be occupied in the retention process (moles sorbed per gram of sorbent). Thus,
.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
o p-H00C02H
0.20-
b 0-phthalic ocld
t
0.15Ilk’
020b/
0.10-
0 051 1
0 030Lb8001
1
’
0 16ixr
002
I
0 005 ionic strength (NoCI). M
I 004
0 010
Flgure 4. Retention of organic analyte anions on PRP-1 as H function of lonlc strength (NaCI). The mobile phase conditlons are 1 3 M TPeACI, 1.00 X M NaOH, and added CH,CN:H,O, 1.00 X NaCl at a flow rate of 1.00 mL/min, V , = 1.30 mL.
I 006 Onolyte , rng
I069
I 1
I 008
010
Figure 5. Retention of organic analyte anions on PRP-1 as a function of analyte concentration. The mobile phase conditions are 1:3 CH,CN:H,O, 1.00 X I O 3 M TPeACI, 1.00 X I O 3 M NaOH, 1.00 X iiO-2 M NaCl at a flow rate of 1.00 mL/min, V , = 1.30 mL.
writing a mass balance that accounts for all occupied and free sites gives
KO= (A),
+ (QXA)EtOtal + (QCA),
(8)
where (Q.XA)EtOtnl represents the contribution of (QXA), from the retention of QX and the exchange of X- with (QCA),. combining eq 2-8 with elimination of (A), and (QCIA), and solving for (QX.A), gives
-4
-3
log ionic strength (TPeABr)
-2
I
Figure 6. Retention of organic analyte anions as a function of TPeABr concentration. The mobile phase conditions are 1:3 CH,CN:H,O, 1.OO X IOm3 M NaOH at a flow rate of 1.00 mL/min, V , = 1.30 mL.
Retention of the analyte, X-, on PRP-1 is given by
k
q(QXA),/[X-Im
(10)
where k’iis the capacity factor and q is the ratio of stationary phase volume to mobile phase volume. Combining eq 9 and 10 gives
which cain also be written as 1
1
and indicates that k’is indirectly proportional to the coanion, C-, and ainalyte, X-, concentration and directly to thle R4N+, Q+, concentration. Experiments demonstrating that the retention of organic analytes on PRP-1 follow these relatiionships according; to eq 11’ are shown in Figures 4-6. In Figure 4, TPeACl was used and the analyte retention was determined as a function of NaCl concentration. As the C1- concentration increases, llk’increases as indicated by eq 11’. Also, selectivity for a given pair of analytes appears to decrease. At higher NaCl concentrations than shown in Figure 4 retention decreases rapidly and the relationship is no longer linear. This is consistent with a combined effect due to a high selectivity of C1- over the analyte anion and mass action. Figure 5 shows that l / k ‘increases as analyte concentration increases as indicated by eq 11’. At very high analyte concentrations the equilibrium positions are shifted to favor the
Q+-X- interactions, and its lower retention, over the Q + - C interaction (KQxin the F term in eq 11’). At lower X- concentrations than shown in Figure 5, k’ levels off and approaches the intercept value. It is impossible to carry out an experiment where only Q+ concentration increases; its increase must always be accompanied by an increase in C- by the same amount. Thus, as shown in Figure 6, retention increases as the R4N+salt concentration increases, as indicated by eq 11’. However, retention reaches a maximum a t a point where the accompanying anion (in this case Br-) begins to exert a favoralble competing effect (€3 term in eq 11’) over retention of the analyte anion and its retention begins to drop as the R,N+ salt concentration increases. The choice of the anion as C- will strongly influence the experimental results shown in Figures 4-6. Although not studied in detail, it appears that in Figure 4 both the k’and slope will be affected by the anion chosen for C- while in Figure 5 the slope is retained and only k’is affected. In Figure 6 the choice of C-will affect k’, the slope, and the location of the maximum. This is consistent with the anion order indicated in ey 1. Experimentd verification of eq 11’does not prove that the anion exchange selectivity is a major equilibrium contributing to analyte anion retention since the same equation can be derived where only ion pair formation between R4N+and the different anions is considered. In this case the equilibrium constants in ey 11 are ion pair constants and constants describing retention of these ion pairs. For mobile phase organic solvent-water ratios widely different than used here, it is possible that both ion pair and exchange selectivity are important; both can then be accounted for in the constant terms in eq 11. Since a basic solution was used in the experiments described here, the analyte was always in its anion form. If
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7 JUNE 1982
Figure 7. Retention of three R4N+ salts as a function of ionic strength. The mobile phase conditions are 3:7 CH,CN:H,O containing NaF, NaCI, or NaBr at a flow rate of 1.00 mL/min, V o = 1.55-2.10 mL.
Scheme I primary secondary
this were not the case, eq 11 could be modified to take into account the effect of pH on retention of organic analytes derived from weak acids. The relationship between k'for an analyte ion and mobile phase ionic strength ( p ) according to the Stern-Gouy-Chapman theory of double layer adsorption (13) is given by
where C, and C2 are constants. If a double layer adsorption is involved, eq 12 predicts a linear relationship between (k3-l and pm1I2.Figure 7 shows that the retention of the TPeAX salts conforms to eq 12. Their retention on PRP-1 can therefore be viewed as the R4N+ adsorbed onto the absorbent surface constituting a primary layer and its coanion occupying a diffuse or secondary layer about the stationary phase. All the experimental results suggest that the retention of the analyte anion in the presence of a R4N+ salt on PRP-1 is the result of a complex set of equilibria taking place within a double layer at the stationary phase surface. This ion interaction model is summarized in Scheme I. Although not shown, buffer and added electrolyte anions (OH-, Z-) would contribute to the secondary layer if present in the mobile phase and will be part of the competing equilibria, The final equilibrium state can be reached by either the two routes. In one the analyte is introduced into the mobile phase containing the R4N+salt and equilibrium occurs as this mixture passes over the stationary phase (route A in Scheme I). Alternatively, the stationary phase is first conditioned which establishes the double layer. After the analyte is introduced into the mobile phase stream and passes over the stationary phase, a selectivity is established between the analyte anion and any anions that make up the secondary layer (route B in Scheme I). Also contributing to the final state are equilibria that take place between the double layer components and the mobile phase components (including all other competing anions if present). The need for the conditioning step was experimentally verified in two ways. First, the column was conditioned with a mobile phase free of the R4N+ salt, for example 1:3 CH3CN:H20,0.010M NaBr, 0.0010M NaOH. Simultaneously, as an aliquot of an analyte-R,N+ salt mixture was introduced at the sample port, the mobile phase was changed to one that also contained 0.0010 M TPeABr. Thus, the mobile phase-
sample mixture front entered the column head together. No evidence of analyte retention was observed when using several experimental techniques to ensure a simultaneous entry of the R4N+salt-analyte mixture into the column. Even mole ratios of up to 1O:l R4N+sa1t:analyte in the injected sample did not produce analyte retention. Since reducing the flow rate in these experiments had no effect and the retention of the R4N+salts on PRP-1 (see Table 111) were readily determined, the lack of analyte retention was not due to poor kinetics. In the second experiment the column was conditioned with a 1:3 CH,CN:H20, 0.010 M NaBr, 0.0010 M NaOH, 0.0010 M TPeABr mobile phase as a function of time prior to introduction of the analyte. Since the column breakthrough time is known (see Figure 2), location of the TPeABr front in the column can be calculated. Determination of analyte retention as a function of different equilibration times clearly showed that retention followed the uptake (double layer formation) of the TPeABr on the PRP-1 surface and reached its maximum k'value when the TPeABr reached its breakthrough. Although several equilibria are involved, the major one appears to be the step representing the selectivity of one anion over another within the secondary layer. This is represented by the equilibrium constant, KQx,in eq 11 (see eq 6 and 7 ) . In general, the selectivity order observed here (see eq 1) is similar to the selectivity order found on strongly basic anion exchangers (18). Applications. The mobile phase parameters indicated in eq 11 are (1)type and concentration of R4N+salt, (2) choice of coanion, (3) pH, (4)type and concentration of mixed solvent, and ( 5 ) ionic strength. Although these are often interrelated, adjustments of the first three are the most critical. Briefly, the optimum conditions are the following. The combination of R4N+salt and mobile phase solvent mixture should provide a k' > 5 for the retention of the R4N+ salt. The optimum R,N+ salt and concentration (see Figure 6) is where R = pentyl and 1 X to 2 X M, respectively. A CH3CN:H20 mixture is preferred over lower alcohol:H,O mixtures since band broadening is greater in the latter mixtures. The choice of the coanion significantly affects the eluent power of the R4Nt salt (see eq 1). Of the anions studied the coanion, F-, is the weakest eluent anion while Br- is the strongest. Control of the mobile phase pH is important to ensure that analytes, particularly those derived from weak acids, are in their anionic form. Furthermore, selectivity can be changed by using a pH where only certain components of the mixture are ionized. Finally, increasing ionic strength decreases analyte retention, particularly if the electrolyte provides a strong eluent anion. Several separations which illustrate these parameters were carried out. For example, the effect of R4N+salt, its structure, and solvent composition is illustrated in Figure 5, ref 15, where a series of benzenesulfonic acid derivatives were separated. At the optimum condition a near base line separation was obtained in the presence of the R4N+salt while in ita absence no or little resolution is found. Figure 8 illustrates the separation of a mixture of sulfas and aminobenzoic acid derivatives using either a TPeABr or TPeAF mobile phase. For a 1:3 CH3CN:H20mobile phase retention is negligible in the absence of the R4N+salt. Not only is retention high in the presence of the R4N+ salt but resolution is excellent and is significantly improved when using the weaker F eluent over the Br- eluent. Similar results were found when comparing a TPeAF and TPeABr elution of a phenol-benzoic acid mixture where the retention order was phenol = benzoic acid < o-hydroxy- < m-hydroxy- = p hydroxybenzoic acid when the mobile phase was pH 11. In
iavi
Anal. Chem. 1982, 5 4 , 1071-1078 TPeABr
TPeAF
in detail in a subsequent report.
TPeABr
LITERATURE CITED
Figure 8. Effect of eluent anion on the separation of swlfas and aminobenzloic acids on PRP-1 in the presence of TPeA'. The mobile phase conditions a r e 1 3 CH,CN:H,O, 1.00 X M TPeABr or M NaOH at a flow rate of 1.OO mUmin, V o = 1.23 TPeAF, 1.OO X mL.
the absence of TPeA+ no retention of the mixture is obtained. If the mobile phase pH is reduced to 7.2, the three hydroxybenzoic acids (diprotic acids which are doubly charged at pH 11)are only singly charged and the retention order with improved resolution becomes p-hydroxybenzoic acid < mhydroxybenzoic acid < benzoic acid < phenol < o-hydroxybenzoic acid. Equation 11 is not restricted only to retention of organic anionic aiialytesi. Since the major equilibrium constant influencing retention represents anion selectivity, it should also be possible to separate inorganic anions on PRP-1 in the presence of R4Nt salts. Not only do the experimental results fit the model but many useful inorganic anion separations are possible, even at, trace levels. These studies will be reported
(1) Hearn, M. 1'. W. "Advances in Chromatography"; Giddings, J. C., Grushka, E., Cazes, J., Eds.; Marcel Dekker, New York, I980 Vol. 18, pp 59-100. (2) Bidiingmeyer, B. A. J . Chromatogr. Scl. 1980, 18, 525-539. (3) Tomlinson, E.; Jefferies, T. M.: Riley. C. M. J . Chromatow. 1978. 159, 315-358. (4) Horvath, C.;Melander, W.; Molnar, I.; Molnar, P. Anal. Chem. 1977, 49 ., 2295-2305 -- - - - - - (5) Bidlingmeyer, B. A.; Deming, S.N.; Price, W. P.; Sachok, B.; Petrusek, M. J . Chromatogr. 1979, 186, 419-434. (6) Knox, J. H.; Jurand, J. J . Chromatogr. 1976, 125, 89-101. (7) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 175, 51-63. (8) Ehmcke, H. U.; Keller, H.; Konig, K. H.; Ullner, H . Fresenius' 2.Anal, Chem. 1978, 294, 251-281. (9) Van De Venne, J. I-. M.; Hendrikk, T. L. H. M.; Deelder, R . S.J . Chromatogr. 1978, 167, 1-16. (IO) Melander, W. R.; Kalghatgi, . . K.; Horvath, C. J . Chromatogr. 1980,
201, 201-224. (11) Hung, C. T.; Taylor, R. B. J . Chromatogr. 1980, 202, 333-345. (12) Tiiiy-Melin, A.; Askemark, Y.; Wahlund, K. G.; Schill, G. Anal. Chelm. 1878. 51. 976-983. (13) Cantwell,'F. F.; Puon, S.Anal. Chem. 1979, 5 1 , 623-632. (14) Rotsch, T. D.; Cahill, W. R., Jr.; Pietrzyk, D. J.; Cantweil, F . F. Can J . Chem. 1981, 5 9 , 2179-2183. (15) Rotsch, T. D.; Pietrzyk, D. J. Anal. Chem. 1980, 5 2 , 1323-1327. (16) Rotsch, T. D.; Pietrzyk, D. J . J . Chromatogr. Scl. 1981, 19, 88-95. (17) Iskandarani, 2.;Pietrzyk, D. J . Anal. Chem. 1981, 5 3 , 489-495. (18) Helfferich, F. "Ion Exchange"; McGraw-Hill: New York, 1962;pp 95.
RECEIVED for review November 30, 1981. Accepted March 17,1982. Part of this; work was presented at the 17th Midwest Regional Meeting of the American Chemical Society, Coluimbia, MO, 1981, and at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, 19132. This investigation was supported by Grant No. CHE 7913203 awarded by The National Science Foundation.
Liquid Chromatographic Separation of Aromatic Hydrocarbons with ChemicalIy Bonded (2 4-Dinit roanilinapropyl)siIica Patrick 1.. Grizzle" and Jane S. Thomson Bartlesvilie Energy Technology Center,
U.S.Department of Energy, P. 0. Box 1398, Bartlesviile, Oklahoma 74003
A detailed comparlson of alumina and chemically bonded (DNAP-silica) sillca-R( NH,), and (2,4dinitroanlllnopropy~)slllca for the compound-class (rlng-numbered) high-performance liquid chromatographlc separation of aromatic hydrocarbons was made wlth 86 model compounds elther expected or known to be present In petroleums, coal Ilqulds, or Shide oils. Model compounds ranged from one-rlng aromatic hydrocarbons i o hlghly condensed polyaromatlc hydrooarbons contalnlngi six aromatic rings. The importance of substltuent and structure effects on separations was determiined by varylng the degree of alkyl and naphthenlc substltutloni on the bask ring structures. Determination of the best system was based upon the relatlve retention strengths, grouping tendencles, arid the observed structure and substltuent effects. The slllca-DNAP and alumlna were determlned to be more sensitive to molecular structure but less sensitive to substituent effects than the slllca-R(NH,),. The retention characteristics of all three systems are strongly dependent upon sterlc effects. On the bask of retentlon strengths and grouping tendencles, the DNAP-slllca Is cconsldered superior to alumlnei and slllca-I%(",),.
Efficient utilization of nonconventional liquid foss8ilfuels This artlcie not subject to
such as heavy petroleums, coal liquids, and shale oils requires detailed chemical compositional data. Due to the complexity of these materials, approaches taken have employed compound-class separations prior to instrumental analysis or characterization. Although the separation schemes are widely varied, they generally involve a preliminary separation ZLCcording to functional groups such as acids, bases, and neutrals (hydrocarbons) followed by subsequent fractionation of the functional-group types into compound classes (1-3). Highperformance liquid chromatographic (HPLC) fractionation of the neutral compounds in liquid fossil fuels into compowid classes has received considerable attention (4-10). However, even with the large effort expended on these HPLC separations, the aromatic-ring number separation of high boiling or residuum hydrocarbons/neutrals from petroleum, coal liquids, and shale oils stdl represents a formidable task. This difficulty reflects an increase in the concentration of compounds containing the hetoroatoms N, 0, and S and the importance of structure and substituent effects on the retention charmteristics of aromatic hydrocarbons. Detailed studies of molecular structure and substituent effects on the retention characteristics of aromatic hydrocarbons on alumina (11-14), silica (15, 16), and various chemically bonded silicas containing -CIB, -NH2, -R(NH,),, -CN, RCN, RQR, and -phenylmercuric acetate have been
U S . Copyright. Published 1982 by the American Chemical Society
