LITERATURE CITED (1) F. H. Rainwater and L. L. Thatcher, Geol. Survey Wafer Supply Pap., 1454, 216-219 (1960). (2) F. B. Hora and P. J. Webber, Analyst (London), 85, 567-69 (1960). (3) “Standard Methods for the Examination of Water and Waste Water”. 14th ed.,American Public Health Association, American Water Association, and Water Pdlutkn Control Federation, New York, N.Y., 1975, pp 420-23. (4) “Standard Methods for the Examination of Water and Waste Water”, 12th ed.,American Public Health Association, American Water Association, and Water Pollution COnW Federatbn, New Yolk, N.Y., 1965, pp 202-205. (5) E. M. Chamot, D. S. Pratt, and H. W. Redfield, J. Am. Chem. SOC.,33, 366-85 (1911). (6) M. J. Taras, Anal. Chem., 22, 1020-22 (1950). (7) A. Macejunas, J. Am. Water Works Assoc., 59, 1190-93 (1967).
.,
(8) C. A. Black, “Methods of Soil Analysis”, Part 2, American Society of Agronomy, Inc., publisher, Madison, Wis., 1965, pp 1217-19. (9) J. H. Wise and M. Volpe, cited In M. Volpe and H. S. Johnston, J. Am. Chem. Soc., 78, 3903-10 (1956). (IO) F. H. Westhelmer and M. S. Kharash, J. Am. Chem. Soc., 68, 1871 (1946). (1 1) F. Snell and C. Snell. “Colorimetric Methods of Analysis”, Vol. 11, 3rd ed., D. Van Nostrand Co., Inc., Prlnceton, N.J., 1949, pp 785-801.
RECEIVED for review October 29, 1976. Accepted February 22,1977. We wish to acknowledge support of the Environmental Protection Agency for one of the authors.
Separation of Organic Acids on Amberlite XAD Copolymers by Reversed Phase High Pressure Liquid Chromatography Donald J. Pietrzyk” and Chi-Hong Chu Chemistry Department, The Unlversity of I o w a , Iowa City, Iowa 52242
Eluting conditions for high pressure llquid chromatographic separation of organic acids on columns packed with 45- to 65-pm particles of rigidly porous Amberlite XAD-2, -4, and -7 copolymers, which act as reversed statlonary phases, are evaluated. XAD-2 and -4 are polystyrene divlnylbenzene copolymers and XAD-7 Is an acrylic ester copolymer. Adds studied Include alkyl and other substituted phenols, substituted benzolc acids, chlorinated phenoxyacetlc acids and their corresponding chlorinated phenols, benzene and naphthalene sulfonic acids, amino acids, dipeptides, and sulfas. The advantages of each type of XAD copolymer are established relative to the broad range of acids studied. Depending on the type of acid, optimum eluting conditlons are based on water+rganlc solvent ratio with or without pH control (the XAD copolymers are stable in acldlc and bask solution) and by the inclusion of electrolyte in the eluting mixture. Establishing the column behavior allows the prediction of optimum stripplng conditions for organic acids. I t is also possible to predict the retention volumes or level of retention from a mlnlmum set of data.
Using organic polymers as the stationary phase in high pressure liquid chromatography (HPLC) has been limited the major reasons being that they often lack the required physical strength, often suffer from slow mass transfer, readily swell and contract, or are not available in a uniform, microparticle size. A group of very rigid, macroporous copolymers, which do not suffer from all of these limitations, are stable to acid and base solutions, and are readily available, are the Amberlite XAD copolymers (1-3). The XAD copolymers should act as reversed stationary phases similar to nonpolar bonded phases, and are unlike silica and diatomaceous earths, which are polar porous stationary phases. Other polymers which have recently been shown to be useful in HPLC are polyvinylalcohol(4) and 2,6-diphenyl-p-phenylene oxide (Tenax GC) (5). XAD copolymers have been widely used in stripping applications for the removal of organics; this has been surveyed previously (6-8). Only a few separations on columns prepared from these copolymers using gravity flow (6) or HPLC (7)have been reported. 860
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
Experimental results are provided which illustrate the advantages and limitations of the XAD copolymers as reversed phase stationary supports. XAD-2 and -4,polystyrenedivinylbenzene copolymers, and XAD-7, an acrylic ester copolymer, were studied. Simultaneously, conditions for the analytical separation of organic acids such as phenols, carboxylic acids, sulfonic acids, amino acids, and sulfas by HPLC are described.
EXPERIMENTAL Reagents. All organic compounds were obtained commercially and used as received or distilled or recrystallized. Solvents and salta were analytical reagent grade and were used without further purification. Amberlite XAD copolymers were obtained from Rohm and Haas Chemical Company and Mallinckrodt Chemical Works as 20 to 60 mesh (500 Irm average) particles. These copolymers were cleaned by a Soxhlet extraction procedure described previously (8) and subsequently crushed in a blender (HzO-alcohol slurry) or in a ball mill. Column Preparation. A narrow, particle size range was obtained by dry sieving in U.S. Standard Screens or slurry sieving (HzO-alcohol) with a screen-filtering system from Cistron Corporation. The 45-65 Hm range was placed in methanol, stirred, and allowed to settle. Particles that did not settle after 15 min were discarded; this procedure was repeated severaltimes allowing the particles to air dry before addition of the MeOH. If the fines are not completely removed, an excessive pressure drop will occur in the column. After air drying, the columns were packed by a dry-packing technique. (Recently, slurry packing was shown to be feasible (9)J Since the XAD copolymers swell, particularly XAD-7 (8,9),the crushed particles should not be thoroughly dried or baked; activation is not necessary. Initial conditioning of the column is completed by passing alcohol through the column. Thereafter, different eluting mixtures can be used; however, the polarity change should be gradual and time should be allowed for the column to recondition according to the new eluting condition. For example,if a 10% EtOH-90% HzOeluting mixture was to be used, 10 to 20 mL of 80% EtOH would be passed through the column, followed by 60%, 40%, 20%, and finally 10% EtOH. A very slow gradient change could also be used. The pressure drop of the XAD-7 column was observed to be very dependent on polarity of the mobil phase, more so than for the XAD-2 or -4 column, and is probably due to the fact that XAD-7 swells more readily (8, 9). XAD-7 columns with low pressure drops were best prepared by dry packing with XAD-7
Table I. Separation of Phenol Derivatives on XAD-2 Mixture
VR, mL"
Eluting agentb
mg Taken
mg Found
0.100 0.128
0.128 0.084 0,132
2. 2,4-dinitrophenol p-nitrophenol m-nitrophenol
3.8 8.1 13.3
0.075 0.121 0.091
0.075 0.128 0.096
3. p-nitrophenol 2,6-dichlorophenol m-nitrophenol 2,5-dichlorophenol 2,4-dichlorophenol 3,5-dichlorophenol 4. o-isopropylphenol 2,6-diisopropylphenol 2,6-di-tert-butylphenol
1.9 6.9 12.1 19.2 25.4 34.5 1.3 2.6 6.8 2.5 6.2 1.5 7.1
0.1 M NaOH into 30 mL of 0.1 M pH 10.1 phosphate buffer at constant 10% EtOH 0.2 M NaOH into 30 mL of 0.1 M pH 7.8 phosphate buffer at constant 10% EtOH 0.1 M NaOH into 40 mL of 0.05 M pH 8.95 carbonate buffer at constant 10% EtOH
0.136
phenol p-chlorophenol
3.7 7.6 16.5
0.175 0.158 0.158 0.193 0.172 0.236 0.045 0.060 0.045 0.045 0.450 0.450 0.04 5
0.165 0.159 0.160 0,190 0.161 0.230 0.049 0.063 0.043 0.043 0.439 0.438 0.034
1. o-chlorophenol
5 . 2-methyl-6-tert-butylphenol
2,6-di-tert-butylphenol 6. o-isopropylphenol 2,6-di-tert-butylphenol
66% CH,CN-34% H,O 66% CH,CN-34% H,O 66% CH,CN-34% H,O
Separations 1 to 3 employed a gradient whereby a 30 x 0.19 cm, 45 to 65 pm XAD-2 using a flow rate of 1 mL/min. the first solution was continuously pumped into the second solution maintained at a fixed volume. Separations 4 to 6 employed isocratic elution. particles as they just become free flowing in the MeOH-air drying procedure. Stainless steel tubing and end fittings from Waters Associates were used for all columns. Column Experiments. A Waters Model-202 equipped with a Model-6000 pump, 254-nm UV detector, 8-MLcell, and a Model U6K sample injection system was used. Sampleswere introduced by Pressure Lok Series B-110 10-pL and 25-wL syringes from Precision Sampling Corporation. In general, known stock samples of 5 to 10 mg/mL were prepared in EtOH, HzO,or their mixtures in 6-mL Hypovials sealed by Hycar Septa with aluminum caps (Pierce Chemical); 1 to 5 pL were removed by the syringe and introduced into the chromatograph. Pressures and flow rates were in the range 300 to 700 psi and 0.5 to 1.0 mL/min. Mixed solvents are water-organic solvent mixtures expressed as % by volume. The pH values (apparent pH) for mixed solvents containing buffer were determined by a pH meter. All buffer concentrations are 0.1 M unless stated differently. Retention data are expressed as the net retention volume, VN (mL),or the measured retention volume, V, (mL). The former is calculated by
v, = v, - v, where V , (mL) is the column void volume, which was determined by eluting a compound that was known to have no retention at the given eluting condition. The capacity factor, k', was calculated for isocratic elution by
Distribution coefficients, KD,were determined by procedures previously outlined (8). Gradients were exponential or linear as described previously (6, 7). Quantitative data were obtained by peak height calibration or by peak isolation, dilution to known volume, and Beer's law calibration through UV absorption (Beckman Model DBG).
RESULTS AND DISCUSSION XAD Columns. The XAD copolymers are inexpensive and commercially available as average 40-mesh spherical particles. Many fines are produced during size reduction and are not completely removed by sieving since they tend to aggregate. Hence, settling techniques must be used to remove these fines prior t o packing the column. Although the XAD copolymers undergo a small amount of swelling (largest for XAD-7), the initial eluting agent should
be an organic solvent, such as MeOH, EtOH, or CHBCN rather than water. Organic impurities are removed but, more importantly, a better packed column is obtained since the copolymers are more easily wetted by organic solvents, particularly XAD-2 and -4. For XAD-7 it is best to dry pack the column with preswollen particles. (Slurry packing techniques were not explored in detail.) Although the XAD copolymers are macroporous, diffusion limitations in column experiments do not appear t o be a significant limitation. I t is possible that this property would be more significant when attempting to separate large molecular weight molecules; details of the effect of size of the molecules being separated are not yet available (9, IO). The XAD particles used in the columns are random and irregular in size (45 t o 65 pm). These factors plus the properties of diffusion and swelling contribute to an efficiency in which H values of 0.3 to 0.6 cm are typical; pressures depending on ability to remove fines should be in the range of 300 to 1000 psi at flow rates of 0.5 to 1.5 mL/min. Faster flow rates are possible while still maintaining symmetrical elution peaks. However, broadening and an increase in H does occur. Although efficiencies are lower than those found for more uniform, smaller type commercially available packings, resolution is still favorable for a wide variety of applications. Separations. In tackling a specific separation problem by HPLC, the optimum eluting conditions can be rapidly predicted from information that describes the properties of the column packing materials, the eluting power for a series of eluting systems, the structural properties of the compound that influences ita retention, and the resulta of several typical examples. In this report the significance of these factors in the separation of mixtures of organic acids on XAD copolymers is described. Phenol Derivatives. The acidity of phenol derivatives covers a wide range and depends on the type and number of substituents within the molecule. Based on the batch K D - ~ H graphs previously reported (6,8), pH should be an excellent variable to affect the separation of phenol derivatives. Eluting conditions were predicted for several separations and the results are shown in Table I in separations 1 to 3. The optimum eluting condition is achieved by using a gradient in which the pH increases a t a fixed alcohol concentration. Separations were also possible by using a gradient whereby ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
881
Table 11. Retention Volumes and Capacity Factors for Benzoic Acid Derivatives as a Function of pH in 50% Ethanol V N mL, ( k ' ) b
Compound Benzoic acid H P-"2
m-NH, 0-NH, P-NO, m-NO2 p-OH m-OH p-c1 p-Br Phenoxyacetic acid
p-c1
0-c1
PH PKaa
3.9
4.9
6.6
9.4
9.4c
4.17 2.41, 4.85 3.12, 4.74 2.11, 4.95 3.43 3.49 4.57 4.08 3.98 3.97
7.8(6.5) 1.8(1.5) 1.8( 1 . 5 ) 5.3(2.7) 13.4(11.2) 11.0(9.2) 1.7(1.4) l.g(l.6)
...
4.3( 3.6) l.O(O.8) LO( 0.8) 3.2(2.7) 2.9( 2.4) 2.7( 2.3) O.g(O.8) 0.9(0.8) 9.8( 8.2) 13.2(11.0)
O.a(O.7) 0.5(0.4) 0.3(0.3) l.O(O.9) 0.5(0.4) 0.5(0.4) 0.4(0.3) 0.3(0.3) 1.3(1.1) 1.5(1.3)
0.3(0.3) 0.2(0.2) 0.2(0.2) 0.3(0.3) 0.4(0.3) 0.4(0.3) O.l(O.2) 0.2(0.2) 0.5(0.4) 0.6(0.5)
l.l(l.0) 0.3(0.3) 0.3(0.3) 0.8(0.7) 3.0(2.3) 2.7(2.3) 0.3(0.3) 0.4(0.3) 5.6(4.7) 9.6(8 . 0 )
2.95 2.92 2.73 2.88
11.2(9.8) 9.0(7.5) 23.3(19.4) 42.8
2.2(1.8) 1.9(1 . 6 ) 3.8(3.2) 6.0(5.0)
0.7(0.6) 0.6(0.5) l.l(O.9) 1.8(1,5)
0.6(0.5) 0.5(0.4) O.g(O.8) 1.6(1.3)
10.2(8.4) 5.4(4.5) 35.6(29.)
...
2,4-diCl 2,4,5-triCl a Aqueous pK, values, A 45 to 65 Mm, 0.71 g, 45 x 0.236 cm XAD-2 column at a flow rate of 0.5 mL/min, V, = 1.2 mL, and the buffer at 0.1 M (carbonate for pH of 9.4 and 6.6 and phosphate for pH of 4.9 and 3.9) in 50% EtOH. The Values are for pH of 9.4 in 10%EtOH. values in parentheses are capacity factors.
...
the organic solvent concentration increases and the pH is held constant. Other mixtures of phenol derivatives separated were p-OH, p-H, rn-C1, and p-CH3 and p-OH, p-NOz, rn-CH3,and o-CH~;0.02 M NaOH in 10% EtOH was used in both cases. Resolution of phenol mixtures using the same eluting condition were better on an XAD-2 column than on a XAD-7 column. For example, for phenol derivatives with smaller V R values than phenol, their sorption is generally greater on XAD-7 in comparison to XAD-2. The opposite is observed for those derivatives that have larger VR values than phenol (8).
Since most phenols tend to be weak acids, a strong eluting condition is achieved under conditions whereby the phenols are predominately in their salt form (basic conditions). Sorption of the phenols in the neutral form (acid solution) is very high (6,8) and this condition is not a suitable eluting condition. However, an acidic solution would be preferred if phenols are being stripped and concentrated as a group from the unknown sample. As the number of substituents increases in the phenol derivative, the sorption of the salt form increases and added salt becomes a more significant factor in influencing the elution volume (8). Hence, the pH as well as the concentration of the buffer will effect the resolution. For example, in Figure 1,where four chlorinated phenols are separated, the VRvalues, particularly for the 2,4- and 2,4,6-chlorinated derivatives, are dependent on salt concentration; in the absence of 0.1 M NaCl in Figure 1, the VR values are 1.9, 3.2, 6.0, and 23 mL, respectively. A difference in sorption was found for 0-,m-, and p-substituted phenols, but the order does not appear to be uniform. For example, on XAD-2 and XAD-7 columns, VR values change in the order p-C1> m-C1> 0-C1 and o-CH3 > p-CH3 > m-CH3. A basic solution is not suitable for elution of certain phenol derivatives because of their instability under these conditions. Examples are the alkylated phenols some of which are used as antibacterials while others are antioxidants. Separation with good resolution is still possible by using an isocratic or gradient elution employing an organic solvent-water mixture. A broad range of eluting power can be achieved by selecting different organic solvents (see Figure 3 in reference 8). Separations 4 to 6 in Table I illustrate the separation of several base-sensitive phenols with a CH3CN-HzO eluting mixture. 862
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
ml Figure 1. Separation of chlorinated phenols. 0.71 g, 45 X 0.236 cm, 45 to 65 pm XAD-2 using 0.02 M NaOH, 0.1 M NaCl in 10% EtOH at 1 mL/min. a. @chlorophenol, 1.8 pg; b. pchlorophenol, 4.0 pg; c. 2,4-dichlorophenol, 4.0 pg; d. 2,4,5-trichlorophenoI, 6.3 pg
For an XAD-2,45 to 65 pm, 45 X 0.236 cm column and 66% CH3CN a t 0.5 mL/min, the retention order ( V R )was found to be 2,6-di-tert-butylphenol (>>lo) > 2,6-diisopropylphenol (7.2) > 2-methyl-6-tert-butylphenol(6.5) > o-isopropylphenol (3.2) > phenol (2.2). Since these compounds are weaker acids than phenol (due to the presence of ortho substituents), it can be assumed that they are present as the neutral form and sorption of the neutral form is being compared. Thus, an increase in the number of o-alkyl groups and in the hydrophobic nature of the group results in larger VR values. Benzoic Acid Derivatives. Table I1 lists retention volumes and capacity factors for benzoic acid derivatives in 50% ethanol as a function of apparent pH. Since the benzoic acids are weak acids with pK, values of approximately 4, the pH range covers conditionswhereby the acids are essentially 100% salt form (pH 9.4) to approximately 1:l salt to neutral form (pH 3.9). At more acidic pH's, dissociation is repressed and sorption is greater. Reducing the EtOH concentration at a fixed pH increases the sorption. This was studied at a pH where salt form sorption would occur. Only the data a t 10% EtOH and pH 9.4 are shown in Table 11. The position and type of substituent strongly influences the sorption (8). For example, the greatest change in V , or
Table 111. Retention Volumes and Capacity Factors for Phenoxyacetic Acid Derivatives on XAD-2 as a Function of Ethanol at pH 9.4 VN, mL, (k’)’ % EtOH
Compound
10%
30%
50%
Phenoxyacetic acid
ml
Figure 2. Separation of benzoic acid derivatives. 0.71 g, 45 X 0.236 crn, 45 to 65 prn, XAD-2 using 0.1 M NaHC0, in 10% EtOH at 0.5 rnL/rnin. a. phydroxybenzoic acid, 1 Fg; b. benzoic acid, 10 wg; c. pnitrobenzoic acid, 3 wg; d. pchiorobenzoic acid, 10 hg; e. p bromobenzoic acid, 10 wg
k’ with EtOH concentration or pH is noted for the benzoic acid derivatives that have the largest VN values; namely, nitroand halogen-substituted compounds even though the derivatives are completely in the salt form at these conditions. The retention order for each series of ortho, meta, and para substituted benzoic acids, like the phenols, is not the same. For example, on XAD-2 and -7, VN values for benzoic acid derivatives change in the order o-NH2 > m-NH2 L p-NH2 (same for -OH) while p N 0 2 > rn-NOz. Contributing to these differences is the acidic or basic properties of the substituents. Also, as described here and elsewhere (6-8, I I ) , the order may differ as the experimental conditions (pH, organic solventwater ratio, or type of organic solvent) are altered. Figure 2 illustrates a typical chromatogram for the separation of a mixture of benzoic acid derivatives on XAD-2 using isocratic elution. Depending on the mixture, other organic solvents, pH values, or gradients can be used to effect the separation. For example, a mixture of p-, m-, and oaminobenzoic acids were separated using pH 5.5 (phosphate buffer) and 10% ethanol. If benzoic acid esters were present, these will have larger VNvalues and their elution would require a higher concentration of organic solvent or one that has a strong eluting power (8);buffers at modest concentration levels will have little influence on the elution of the esters. Resolved peaks are also obtained for separations on a XAD-7 column, however, the peaks tend to be broader than found on XAD-2. For example, a mixture of p-OH, o-OH, and p-Br benzoic acids was easily separated (baseline) on XAD-7 using 0.1 M NaHC03 in 10% EtOH. Phenoxyacetic Acids. Retention of phenoxyacetic acid derivatives (PA) are influenced by pH even though they are stronger acids than benzoic acids. Hence, potential eluting or stripping conditions are controllable by adjusting pH, type and concentration of organic solvent, salt concentration (8), and by using either isocratic or gradient elution. The study was limited to chlorine derivatives of PA since these are of environmental interest. Because the pK, values are constant for the chlorine-substituted PA derivatives, the differences in sorption are due to the number and position of the chlorine substituents rather than the pK,. Batch KD-pH plots can be calculated (6-8). These curves were used to predict eluting conditions to illustrate the effect of pH at constant EtOH concentration and the effect of EtOH concentration at constant pH on VN. These data are included in Tables I1 and 111. Increasing the pH (converting the PA derivative to the salt form) causes a decrease in the V, value; similarly, increasing the EtOH concentration a t fixed pH (or in the absence of pH control) decreases the VN value. Figure 3(a) illustrates a typical chromatogram for the quantitative separation of a mixture of chlorinated PA derivatives on XAD-2 using a fixed pH, EtOH-H20 eluting
10.2(8.4) 5.4(4.5) 35.6(29)
p-c1 041 2,4-diC1 2,4,5-triCl
2.1(1.8) 0.6(0.5)
1.3(1.1) 0.5(0.4) 4.8(4.0) O.g(O.8) 13.2(11) 1.6(1.3)
a 0.71 g, 45 X 0.236 cm, 45 to 65 p m XAD-2 using pH 9.4 (0.1 M carbonate buffer) at various EtOH concentrations; V,,,= 1.2 mL.
II
1
5
I
ml
IO
I
’
15
20
ll
II
Figure 3. Separation of chlorinated phenoxyacetic acids and phenols. 0.71 g, 45 X 0.236 crn. 45-65 wm, XAD-2, (a) 0.1 M NaHCO, in 10% EtOH at 0.5 rnL/rnin. (b) 0.1 M NaOH in 2 0 % EtOH pumped into 20 rnL 0.1 M NaHCO, in 2 0 % EtOH at 0.5 mL/rnin. (c) 0.1 M NaHCO, in 2 0 % CH3CN at 0.6 rnL/rnin. a. echlorophenoxyacetic acid, b. pchlorophenoxyacetic acid, c. 2,4-dichlorophenoxyacetic acid, d. 2,4,5-trichlorophenoxyaceticacid, e. ochiorophenol, f. pchlorophenol, g. 2,4-dichlorophenoi, h. 2,4,5-trichiorophenoi
mixture. When 80% EtOH was used, resolution was not as good and the peaks tended to be broader than those obtained in the presence of pH control. If CH&N is used in place of EtOH, the separation is possible at a lower CH3CN percentage. ANALYTICAL CHEMISTRY, VOL. 49, NO. 6,MAY 1977
a
863
b
al
Flgure 4. Separation of sulfonic acids. (a) 16 X 0.48 cm, 45 to 65 pm XAD-2 using a gradient of 5 % EtOH into 50 mL of 3 % EtOH at 0.7 mL/min. (b) Same as (a), 5 % EtOH only. (c) 0.71 g, 45 X 0.236 cm, 45 to 65 pm XAD-2 using 0.1 M NaCl in 2 0 % MeOH at 0.4 mL/min. (d) 0.84 g, 45 X 0.236 cm, 45 to 65 pm XAD-4 using 0.1 M NaCl in 40% MeOH at 0.4 mL/min. a. phydroxybenzenesulfonic acid, b. benzenesulfonic acid, c. pmethoxybenzenesulfonic acid, d. pnitrobenzenesulfonic acid, e. 2,4dinitrobenzenesulfonicacid, f. 2,4,6-trinitrobenzenesulfonic acid, g. 2-naphthalenesulfonic acid
Hydrolysis of chlorinated PAS will yield the corresponding chlorinated phenols plus other products. Since the chlorinated phenols plus other products will have larger VN values than the corresponding PA derivatives, separation of their mixtures should be possible. This is illustrated in Figure 3(b) and 3(c) for the separation of 0- and p-chlorinated phenol and PA derivatives and 2,4-di- and 2,4,5-trichlorinatedphenol and PA derivatives. These examples which employ isocratic or gradient elution, also demonstrate that separation time and resolution is subject to pH control, and type and concentration of organic solvent. Addition of salt will also increase V, values particularly for the more highly retained compounds. Chlorinated dibenzo-p-dioxanes are very toxic by-products that are often found in chlorinated PAS. Since these compounds do not have acidic sites, their VN values are large and they would appear at very large retention volumes relative to the acids and phenols shown in Figure 3. To elute the chlorinated dioxanes conveniently, it would be necessary to increase the concentration of the organic solvent or use an organic solvent of higher eluting power (8). Separation of mixtures of other chlorinated phenoxyacetic, propionic, and butyric acids and esters and chlorinated dioxanes will be described elsewhere. Sulfonic Acids. Sulfonic acids, unlike the phenols and carboxylic acids, are strong acids and should be nearly fully ionized in aqueous solution. Hence, pH should have little or no effect on retention. Being very polar, sorption should be low even a t low organic solvent concentration. The influence of the type and number of substituents and the presence of salt or strong acid have a significant effect on the level of retention of the sulfonic acid derivative (8). Thus, useful eluting mixtures are based on controlling these latter variables. Batch KD values in EtOH-water mixtures and VN values in MeOH-water with and without added salt have been re864
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
ported before (8). In general, MeOH is preferred over EtOH in column experiments on XAD-2 since sorption is greater in the MeOH-containing mixtures. Although there is significant difference in VN values for the substituted benzenesulfonic acids (BSA), resolution of complex mixtures is poor even if a gradient is used. Typical separations employing gradients are illustrated in Figure 4(a) and (b). By slight changes in the gradient, the resolution in different parts of the separation can be improved (compare Figure 4(a) to 4(b)). The best resolution is obtained by adding salt to the eluting mixture. Not only are the bands sharpened, but the differences in VN values are magnified (8). This is illustrated in Figure 4(c). If 2-naphthalene sulfonic acid was in the mixture in Figure 4(c) it would appear at a much larger retention volume. VN values for BSA derivatives were compared in 20% MeOH containing 0.1 M NaC1, LiC1, or NaNO,. Only slight differences were noted for those derivatives that were retained the most; in these cases NaN03 tended to yield the higher VN value. A more detailed and careful study would have to be completed before making any general conclusion regarding effects of different salts. Sorption of organic acids as well as many other compounds is significantly higher on XAD-4 relative to XAD-2 (6, 8). There are many cases where it would be an advantage to choose a support of higher adsorbent power. For example, the compounds in the mixture may be suspected of having low sorption; hence, a stronger adsorbent, would be suitable. If the mixture contained compounds covering a wide range of sorption, the stronger adsorbent would permit the use of a wider range of eluting systems. Also, the stronger adsorbent might be preferred in stripping applications. The sulfonic acids are not highly retained on XAD-2 and can be used to illustrate the stronger adsorbing properties of XAD-4. Furthermore, the experiments illustrate that XAD-4
Table IV. Retention Volumes for Benzenesulfonic Acid Derivatives on XAD-4 as a Function of Methanol Concentration at Constant Salt Concentration mLb % MeOH 20%
Table V. Retention Volumes for Several Amino Acids, Peptides, and Sulfas
3.68
mL PHa 5.70 7.40
tYr Phe trP glY-Phe Phe-glY gly-gly-phe
0.8
0.8
1.5 3.0 2.2 1.7 2.7
1.3
tyrb pheb trpb gly-pheb phe-glyb sulfamerazineC sulfathiazoleC sulfamethazineC
0.2
0.2
0.6
0.6
3.2
2.3 0.2 0.3
VN,
V,,
Compounda
10%
p-NH,-BSA p-OH-BSA p-H-BSA p-OCH,-BSA p-CH,-BSA p-NO,-BSA 2,4-diN02-BSA 2,4,6-triN02-BSA 1,5-NDSA 2,6-NDSA 2,7-NDSA
1.5
1.2(0.6)c 1.4(0.6) 3.8(1.3) 8.8(2.1)
1.7
40% 0.6
0.7 1.4 2.2 2.7
4.7 8.4 13.1 1.7(0.6) 2.0(0.7) 3.5(0.9)
1.0 1.1 1.8
0.6 0.6
0.6
BSA = benzenesulfonic acid and NDSA = naphthalene0.84 g, 45 X 0.236 cm, 45-65 pm, disulfonic acid. XAD-4 using 0.1 M NaCl in various MeOH concentrations VN values in parentheses at 0.5 mL/min; V, = 0.9 mL. are at the same eluting conditions on a XAD-2 column. a
0.7 1.3 2.7
10.35 0.7 2.2 3.6 2.4 3.8 1.8
1.4 1.3 1.4 2.4 1.4 1.3 % Organic solvent 10 15 20 30
0.4
0.4 1.7 3.1 5.2
0.3 0.5 2.1 0.1
0.2
0.2 0.3 1.7 0.1
0.2 0.5 0.6 0.6
a A 45 to 6 5 fim, 0.71 g, 45 X 0.236 cm XAD-2 column using 0.5 mL/min, V , = 0.9 mL, and 0.1 M phosphate buffers in 10%EtOH. A 45 to 6 5 pm, 0.32 g, 30 x 0.19 cm XAD-2 column using 0.5 mL/min, V, = 0.7 mL, and no buffer (or salt). Same column as a but using 0.5 mL/min, V , = 0.9 mL, and CH,CN-H,O mixtures containing 0.1 M NaHCO,,
I nb
ml
Compound
ml
Figure 5. Separation of naphthalenedisulfonic acid derivatives. 0.71 g, 45 X 0.236 cm, 45 to 65 pm XAD-2 using 0.1 M NaCl in water at 0.5 mL/min. a. 1,5-naphthalenedisuifonic acid, 2.6 pg; b. 2,6naphthalenedisuifonic acid, 1.6 pg; c. 2,7-naphthalenedisulfonicacid,
3.6 PCLQ
can be successfully utilized in a column. Table IV lists VN values for BSA derivatives on XAD-4 as a function of MeOH concentration a t 0.1 M NaC1. Sorption on XAD-4 is higher than on XAD-2 at comparable conditions. The effect of salt and MeOH concentration are similar to that found for XAD-2. Figure 4(d) illustrates a separation on XAD-4 using the same mixture separated in Figure 4(c) on XAD-2. Since sorption is greater on XAD-4, a higher MeOH concentration is used in the eluting mixture. Resolution appears to be better on the XAD-4 column particularly for compounds d to f. Gradients employing changes in salt or MeOH-H,O ratios as well as EtOH or CH3CN in the eluting mixture are variables which will affect resolution. Table IV contains VN data for three naphthalenedisulfonic acid derivatives (NDSA). The VN value for naphthalenesulfonic acid is >> 14 mL. As expected the introduction of the second ionic group sharply reduces the sorption. Separation of a mixture of the NDSA derivatives is accomplished on XAD-4 using 10% MeOH-0.1 M NaCl. Figure 5 illustrates these separations on XAD-2 where no organic solvent is used in the eluting mixture since sorption is less on XAD-2. A baseline separation was observed for both mixtures in the presence of the salt while, in its absence, a
single peak accounting for both compounds was obtained. The salt has a double effect of (1) sharpening the bands and (2) increasing the difference in the V , values for the NDSA derivatives. Amino Acids-Sulfonamides. Batch KD values were determined for aromatic and nonaromatic amino acids on XAD-2. Even though the KD values were small and the errors large, the measurements clearly established that both types of amino acids are adsorbed by XAD-2. Also, the degree of sorption differed and pH and concentration of organic solvent influenced the sorption. Quantitative data illustrating the sorption of the amino acids were obtained on an XAD-2 column; these data are listed in Table V. Since detection was limited to 254 nm, only the aromatic amino acids were studied. As the alcohol concentration increases, sorption decreases as expected. KD data indicated a minimum in that sorption started to increase at very high alcohol concentration. These conditions were not used in column experiments. If pH is varied, a minimum in VN is observed in the vicinity of the isoelectric point. Similar effects of pH and alcohol concentration on sorption of di- and tripeptides were also found, see Table V. The structure of the dipeptide appears to influence the sorption; however, not enough examples were studied to warrant conclusions. Systematic studies with other dipeptides and more complex peptides will be reported later. If an XAD-4 column is used, sorption is greater and stronger eluting conditions are required; for example, higher EtOH concentration to elute the amino acids. A quantitative separation of tyrosine, phenylalanine, and tryptophan is illustrated in Figure 6. A similar separation and elution order was found for the dipeptide mixture gly-tyr, gly-phe and gly-trp. Column experiments demonstrated that the sulfonamides were retained to a greater extent than the amino acids. Consequently, CH3CN,a stronger eluting solvent than EtOH, was used in mixtures with water. Table V lists VN data for several sulfas using a slightly basic solution to further reduce retention. A typical quantitative separation is shown in Figure ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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a
I
0
4m~
8
Figure 6. Separation of an amino acid mixture. 0.71 g, 45 X 0.236 cm, 45 to 65 pm XAD-2 using 10% EtOH at 0.5 mL/min. a. tyrosine, 13 pg; b. phenylalanine, 26 pg; c. tryptophan, 6 pg
Figure 7. Separation of a tri-sulfa mixture. 0.71 g, 45 X 0.236 cm, 45-65 pm X A D 2 using 0.1 M NaHCO, in 10% CH&N at 0.5 mL/min. a. sulfamerazine, 1.58 pg; b. sulfathiazole, 2.40 pg; c. sulfamethazine, 2.59 hg
7. Using a peak height procedure and seven separate results, a relative standard deviation of 2.1% was found for each component. Calculation of VN. It was shown previously that batch KD values for organic acids and bases could be calculated a t different pH's providing the K D for neutral and salt form sorption and the K, (or Kb) are known (6, 7). A similar equation relating the net retention volume, VN, to pH can be derived or
where VNs and V are net retention volumes for the salt and neutral form. VN values are readily measured but V" values are not since these elution volumes will be very large. If V N values are determined at two intermittent pHs, VN' and V N N can be found by substitution into Equation 3 and solving the two equations simultaneously. These values for VN' and V" can then be used to calculate VN values at other pH conditions. To verify this, VNs and VNNwere calculated for several benzoic and phenoxyacetic acid derivatives in 50% EtOH at pH 9.40and 3.92 and used to calculate the VNvalue a t pH 4.9. The experimental value (see Table 11)was found to be in reasonable agreement with the calculated value. Summary. From data within this report and elsewhere ( 6 4 ,the XAD copolymers are useful in HPLC for several reasons. (1)Multiple eluting conditions for the separation of organic acids and bases are achieved by adjustment of pH, type of organic solvent mixed with water, the ratio of the solvents in the mixture, and the inclusion of salt in the eluting mixture.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
These variables can be controlled by isocratic or gradient elution techniques. (2)XAD copolymers which differ in surface area, porosity, and surface activity are available. Thus, if the interaction between the sample components and the support is weak, a support which provides stronger interactions can be chosen. If the interactions are strong, a less active support can be chosen. (3)Organic acids, bases (7), and nonelectrolytes (9) are retained by the XAD polymers. For acids and bases, KDvalues can be predicted as a function of pH from a minimum of ~ K D values and the K,or Kb (6-8).Similar equations in terms of capacity factor or retention volume (or time) can be derived and used to predict eluting conditions. Further predictions based on the effect of type and number of substituents on sorption are also possible (8). (4)The XAD copolymers can be used in columns prepared with macroparticles (75 to 150 pm) at gravity flow conditions; plate heights of 1 to 2 cm are typical (6). Also, under these conditions, flow rates in excess of 10 mL/min are possible with little sacrifice in plate height or resolution (6). Small particles (50-pm average) can be successfully used in columns and produce plate heights of 0.3 to 0.6 cm. Applied pressures of 300 to 1000 psi at flow rates of 0.5 to 1.5 mL/min are typical. In general, XAD columns allow large sample loading (7, 9). (5) The nonpolar XAD copolymers act as an adsorbent via a reversed phase mode and are unlike other typical adsorbents, such as the polar alumina and silica gel. They are also stable to acidic and basic solutions. Thus,the polar solvents are weak eluting agents and, in general, the eluting power is the reverse of that found for alumina and similar to the order for charcoal. The best eluting conditions, a t least for organic acids (and bases), are water mixed with water-miscible organic solvents with and without pH control. Even though the XAD copolymers are typical reversed phase adsorbents, their use is not restricted to sorption of polar compounds and they can be used to separate organic nonelectrolytes (9). (6) The XAD copolymers are very useful in stripping organic compounds from complex mixtures (6-8). Equally useful is their application in analytical separations and in group separations whereby very complex mixtures are stripped and separated into simpler mixtures directly off the stripping column. The development of the eluting conditions for the analytical separations will also provide information about the optimum condition for stripping, which in the past has been accomplished by trial and error. (7) The XAD copolymers can be used in a partition mode by coating them with a stationary liquid phase. Some progress in this application has been made (12-14). Also, XAD-1, -2, and -4are syntheticaly suited to modifications via the introduction of chemically bound functional groups (9, 15). (8)The XAD copolymers as microparticles can be cast into thin layers with a suitable binder (9). Thus, a preliminary chromatographic result is available.
LITERATURE CITED (1) R. L. Gustafson. R. L. Aibright, J. Heisier, J. A. Lirio, and 0. T. Red, Jr.. Ind. Eng. Chem., Prod. Res. Develop., 7 , 107 (1968). J. Paleos, J . Colloid Interface Sci., SI, 7 (1969). D. J. Pietrzyk, Talanta, 16, 169 (1969). A. Carpenter, S. Siggia, and S. Carter, Anal. Chem., 48, 225 (1976). W. Bertsch, A. Zlatkis, H. M. Lkbich, and H. J. Schneider, J. Chfmfogr., 99, 673 (1974). (6) M. D. Grieser and D. J. Pietrzyk, Anal. Chem., 45, 1348 (1973). (7) C. H. Chu and D. J. Pietrzyk, Anal. Chem.. 48, 330 (1974). (8) D. J. Pietrzyk and C. H. Chu, Anal. Chem., 49, this issue. (9) D. J. Pietrzyk. Unpublished results. (10) R. L. Gustafson and J. Paieos, "Organic Compounds in Aquatic Environments", S. J. Faust and J. V. Hunter, Ed., M. Dekker, New York, 1971, p 213. (11) H. Takahagi and S. Seno, J . Chromatogr., 108, 354 (1975). (12) D. J. Pietrzyk. Talanta, 18, 169 (1969). (13) J. S. Fritz, R. T. Frazer, and G. L. Latwesen, Talanfa, 17, 857 (1970). (14) J. S. Fritz and D. R. Beuerman, Anal. Chem., 44, 692 (1972). (2) (3) (4) (5)
(15) E. M. Mayers and J.
S.Fritz, Anal. Chem., 48, 1117 (1976).
RECEIVED for review December 2,1976. Accepted February 23,1977. Part of this work was presented at the ACS Award in Chromatography Symposium at the Centennial American
Chemical Society Meeting, New York, April 4-9, 1976, and at the 12th Midwest Regional ACS Meeting, Kansas City, October 28-29, 1976. This investigation was supported by Grant Number CA 18555, awarded by the National Institute, DHEW.
Acid-Base Properties of Tris(hydroxymethy1)aminomethane (Tris) Buffers in Seawater from 5 to 40 "C Richard W. Ramette,' Charles H. Culberson, and Roger G. Bates* Department of Chemistry, Universiw of Florida, Gainesville, Florida 326 1 1
Buffer solutions composed of tris(hydroxymethyl)aminomethane (lris, Tham) and Tris.H+ have pH values near 8.2 at 25 OC and are useful standards for pH measurements In seawater. By means of emf measurements of cells without liquid Junction, pm, values of equimolal Trts/Trls*HCIbuffer solutlons, based on a scale of hydrogen ton molallty, have been determtned at eight temperatures from 5 to 40 OC In synthetic seawater wlth salinity In the range 30 to 40 parts per thousand (lonlc strength from 0.57 to 0.77 mol kg-'). The pKof Tr1s.H' is higher than that In water by 0.11 to 0.15 unlt and varies llnearly wlth the formal lonlc strength of the seawater medium.
The chemical properties of marine systems are dependent both on equilibrium relationships and on non-equilibrium steady-state processes which are, in turn, intimately dependent on the nature of seawater. This medium is a complex aqueous saline mixture, the chemistry of which can only be described adequately in terms of metal ion speciation, ion pairing, and acid-base processes. Although the total salinity of seawater may vary, the relative concentrations of the major constituents remain remarkably constant. Furthermore, natural seawater has a fairly constant ionic strength, in the range 0.65 to 0.75 mol kg-', corresponding to salinities near 35 parts per thousand (35%0),and the pH near the surface usually falls in the range 7.9 to 8.3. Seawater of this composition thus qualifies as a "constant ionic medium". In media of this sort, activity coefficients tend to be stabilized, as do the liquid-junction potentials encountered in electrochemical measurements with reference electrodes. In earlier publications (I-4),the simplifications afforded by the fixed composition of seawater in respect to acid-base studies and the establishment of a useful pH scale based on hydrogen ion concentrations have been examined. Because of the constant ionic strength of natural seawater, it is possible to base a practical scale of pH on hydrogen ion molality instead of activity. The standard potentials needed to defiie reference solutions based on a scale of pmH (4)can also be used in conjunction with emf measurements to study the dissociation of weak acids and bases in synthetic seawater. The dissociation of ammonium ion in seawater of salinities from 20 to 45% and temperatures from 5 to 40 "C has already been investigated by this method (5). 'On leave 1975-76 from Carleton College, Northfield, Minn.
We have now studied equimolal buffer solutions of tris(hydroxymethy1)aminomethane (Tris, Tham) and Tris hydrochloride in synthetic seawater (sw) a t temperatures from 5 to 40 "C by means of emf measurements of cells of the type Pt;H,(g, 1 atm)ITris(m),TrisHCl(m) in swIAgC1;Ag The molality (m) of each buffer constituent was varied from 0.02 to 0.06 mol/kg of water at nominal salinities of 30,32.5, 35, 37.5, and 40%. The dissociation constant of Tris.H+ and the changes of enthalpy, entropy, and heat capacity for the dissociation process were determined, and values for pmH of equimolal Tris buffers in synthetic seawater have been calculated. The latter are useful for the calibration, on the scale of hydrogen ion molality, of pH equipment for use in seawater.
METHOD As proposed in earlier work (I, 6), we have referred the standard emf, activity coefficients, and equilibrium constants to a standard state in seawater rather than to the hypothetical ideal 1molal aqueous solution. Thus, activity coefficients y* become unity at zero molality of solutes in seawater of a given salinity instead of in the infinitely dilute aqueous solution. As a consequence, the thermodynamic equilibrium constant K for the dissociation of protonated Tris: Tris.H+e Tris + H+
(1) becomes equal to the molality quotient K , in the limit of zero molality of the buffer system in the seawater medium:
where the subscripts T and T H designate Tris and Tris.H+, respectively. Evidence has been presented ( 4 ) that pmH, that is, -log mH, in seawater (a "constant ionic medium") can be determined experimentally from the emf E of cells of type A with an uncertainty amounting to only a few thousandths of a pH unit, when m (the molality of each buffer component) does not exceed 0.04 mol kg-l. Values of E* defined by
E* E E"*- 2 k 1% 'YHC? (A) (3) over the range of seawater salinities from 20 to 45% and temperatures from 5 to 40 "C were determined from measurements of cells of type A in which a part of the NaCl of the synthetic seawater was replaced with HC1 (solution A). In this equation and subsequent relationships, k is written for the Nernst slope, (RTIn 10)/F. In a buffer solution ANALYTICAL CHEMISTRY, VOL. 49,
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