Liquid Chromatography on a Porous Polystyrene-Divinylbenzene Support Separation of Nitro- and Chlorophenols Merlin D. Grieser and Donald J. Pietrzykl University of lowa, Department of Chemistry, l o w a City, l o w a 52242
procedures have been described for simple amino acid mixtures (16), for mono- and disulfonic acids of aromatic and aliphatic hydrocarbons ( 1 7 ) , for potassium terephthalate-benzoate mixtures (18), and for simple flavonoid mixtures (19). Medicinally oriented studies have included the purification of steroids (20, 21), carotenoids (22), biologically important compounds (23-28), and for the removal of toxic drugs in animal and human blood (29, 30). Investigations toward systematizing the broad applications of XAD-2 as an adsorbent are few. However, the adsorption of several carboxylic acids and phenols (,31) and for several water-soluble organic compounds as a function of concentration, temperature, and molecular weight for a homologous series of adsorbates ( 3 ) on XAD-2 was described. Since XAD-2 appears to be a generally useful and powerful adsorbent for organic molecules, a systematic study of its chromatographic properties from the standpoint of Amberlite XAD-2 is a recently synthesized styrene-digeneral eluting conditions is desirable. In this report, data vinylbenzene copolymer which has a large surface area (about 300 m2/gram) and a rigid, porous structure (90 .fi are given which suggest typical eluting conditions, column properties, and an equation for predicting adsorption as a average pore diameter). These properties, plus others function of eluting conditions for weakly acidic organic which have been previously investigated (1-4) and appear molecules. Emphasis is placed on mono- and dichlorophein this report, suggest that the XAD-2 polymer is a very nols since these are usual contaminants in chlorine-treatuseful adsorbent for column chromatography. ed water. Several specific applications employing XAD-2 in liquid chromatography have already been reported. Since XAD-2 EXPERIMENTAL will take up nonpolar solvents, it should be useful as a Reagents. All organic compounds were purchased from Eastsupport in column partition chromatography ( 1 ) . This m a n Kodak Chemical, Matheson Coleman and Bell, and other technique has been used for the separation of metal ion chemical supply houses and used as received. Solvents were t h e mixtures (5, 6). best grade available and were used without further purification. Applications of XAD-2 in column adsorption chromaInorganic salts as sources for buffers and inert electrolyte were analytical reagent grade or better. tography include the removal of colored impurities in Amberlite XAD-2 was purchased from Rohm and Haas Chemisugar and sugar juices (7-9), of ferrichromes in sake and cal Co. as 20 to 60 mesh beads. The resin was washed for 2 to 4 rice koji ( I O ) , of low-molecular weight fractions which days in a Soxhlet extractor with methanol. Subsequently, the cause meat aroma, ( 1 1 , 12) and of color and odor producresin was air-dried prior to drying in a vacuum oven a t 4 to 7 m m ing contaminants in water (13-15). Specific separation Hg at 100 "C for 24 hr. Smaller particle resin was prepared by A porous polystyrene-divinylbenzene resin is investigated as a support for liquid-solid column chromatography. Distribution coefficients for a series of different functional groups as a function of ethanol concentration are reported. The effect of electrolyte concentration is considered. Adsorption of phenol, nitrophenols, and mono-, di-, and trichlorophenols as a function of pH is described. An equation for predicting the distribution coefficients for organic acids as a function of pH is given. Separations of nitro and chlorophenol mixtures are possible by using a stepwise pH elution. p H gradient, or ethanol-water gradient at pH 12. Column parameters including fast flow rates, up to 11 ml/min, are examined. Separations by high-pressure chromatography are possible.
IAuthor to whom reprint requests should be sent. (1) D. J. Pietrzyk, Taianta, 16, 169 ( 1 9 6 9 ) . (2) A. D. Wilks and D. J . Pietrzyk. Anal. Chem.. 44, 6 7 6 ( 1 9 7 2 ) . ( 3 ) R. L. Gustafson, R . L. Albright, J. Heisler, J. A. Lirio, and 0. T. Reid, Jr., Ind. Eng. Chem., Prod. Res. Develop., 7 , 107 ( 1 9 6 8 ) (4) J. Paleos, J . Colioid Interface Sci., 31, 7 ( 1 9 6 9 ) (5) J. S. Fritz, R. T. Frazee. and G. L. Latwesen. Talanta, 1 7 , 8 5 7 (1970) ( 6 ) J . S. Fritz and D. R. Beuerman, Ana/. Chem., 44, 6 9 2 ( 1 9 7 2 ) . ( 7 ) K. J. Parker and J. C. Williams, Proc. Tech. Sess. Cane Sugar Refining Res., 117, ( 1 9 6 8 ) . (8) G . Assalini, Ind. Sac. Ita/., 61, 69 ( 1 9 6 8 ) . (9) J. Houssiau, R . Waegeneers, and J. Gurny. Surc. Eelgelsugar Ind. Absfr., 90, 3 1 0 ( 1 9 7 1 ) . (10) H. Narahara. Nippon Jozo KyokaiZasshi, 65, 3 4 0 ( 1 9 7 0 ) . (11) L. L. Zaika. A. E. Wasserman. C . A. Monk, Jr.. and J. Salay, J. FoodSci., 33, 5 3 ( 1 9 6 8 ) . (12) L. L. 2aika.J. Agr. FoodChem., 17, 8 9 3 ( 1 9 6 9 ) . (13) G. A. Segar, Effiuent Water Treat. J . , 9. 4 3 3 ( 1 9 6 9 ) . (14) A. K. Burnham, G . V. Calder. J . S. Fritz, G . A. Junk, H. J. Svec, and R . Willis, Ana/. Chem.. 44, 139 ( 1 9 7 2 ) . (15) T. F. Walser, Proc. S. Water Resour. Poliut. Contr. Conf.. 1 6 , 81 (1967).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
L. L. Zaika, J. Chromatogr.. 49. 222 ( 1 9 7 0 ) . M. W. Scoggins and J. W. Miller, Anal. Chem.. 40, 1155 ( 1 9 6 8 ) M. W. Scoggins.Anai. Chem., 44, 1285 (1972) M. Hori. EuiI. Chem. SOC.Jap. 42. 2333 ( 1 9 6 9 ) . C. H. L. Shackleton, J. Sjovall. and 0. Wisen, Clin. Chin;. Acta. 2 7 , 354 ( 1 9 7 0 ) . H. L. Bradlow, Steroids. 11, 265 ( 1 9 6 8 ) . T. Kamikubo and H. Narahara, Vitamins, 37. 225 ( 1 9 6 8 ) J. M. Fujimoto and V. 6 . Haarstad, J. Pharmacoi. Exp. Ther.. 165, 45 ( 1 9 6 9 ) . J. E. Stambaugh. L. G. Feo, and R . W. Manthei. Life S c i , 6, 1811 (1967). V. R . Mattox and W. Vrieze. Fed. Amer. SOC. Exp. Biology Fed. Proc., 2 6 , -2, Abstract 9 4 5 ( 1 9 6 7 ) , P. W. Hollowayand G . Popjak, Eiochem. J.. 104, 5 7 ( 1 9 6 7 ) . G. Popjak, J. Edmond, K. Clifford, and V. Williams, J. Bioi. Chem.. 244, 1897 ( 1 9 6 9 ) . W. Scheider and J. K. Fuiler. Eiochem. Eiophys. Acta, 221, 3 7 6 (1970). J. L. Rosenbaum, S. Winsten, M. S., Kramer, J. Moros, and R . Raja, Trans. Amer. SOC.Artif. Intern. Organs, 1 6 , 134 (1970) J. L. Rosenbaum. M. S. Kramer. R. Raja, and C. Boreyko, New EnglandJ. Med., 28. 874 ( 1 9 7 1 ) . J. S. Fritz and A. Tateda. Anai. Chem.. 40, 2115 (1968)
crushing t h e XAD-2 suspended in water in a blender. This was followed by air-drying, sieving into fractions (U.S. Standard sieves), washing the fractions in a Soxhlet extractor (methanol), air-drying, and finally vacuum-drying. All vacuum-dried resins were stored in glass stoppered weighing bottles. Procedures. D i s t r i b u t i o n Coefficients. Distribution coefficients, KD, were measured with the vacuum dried 20 t o 60 mesh XAD-2 resin. One gram of weighed resin was transferred into a 125-ml ground glass stoppered Erlenmeyer flask. Aliquots of solvent, buffer, electrolyte, a n d stock solution (compound of interest in ethanol) were added such t h a t the desired concentrations of each were obtained. A ratio of 50 mllgram of resin a n d a solution concentration of t h e compound of interest a t 7.0 X 10-3!V was maintained unless otherwise stated. (This concentration level was shown t o he on t h e linear portion of t h e sorption isotherm.) Standards were prepared in t h e same way except t h a t t h e resin was omitted. After a 48-hr equilibration a t 25 f 1 "C, suitable aliquots were taken from the unknown a n d standard flasks and the concentration of t h e solute in each was determined spectrophotometrically with a Beckman Model DB-G spectrophotometer. Values for Kr, were calculated by
K,
=
(volume solution)(weight of solute o n t h e resin) (1) (weight of resin)(weight of solute in solution) Initial experiments demonstrated t h a t miximum Kl1 values were reached in 30 min for 100 t o 200 mesh XAD-2 (20% ethanol). For 20 to 60 mesh resin (10% ethanol), a very gradual increase in K D was observed for equilibration times over 4 hr. T o ensure complete equilibration, a contact time of 48 hr was used for the KD measurements. T h e K , , values were also reproducible for different lots of XAD-2 resin. Separations. Smaller particle size resin was used for separations in 0.9- a n d 1.5-cm diameter columns which were obtained from Pharmacia Fine Chemicals. Columns were packed by the slurry method and flow rates were controlled by either t h e AutoAnalyzer Proportioning p u m p or Buchler Polystaltic p u m p . Gradients were established by delivery of one eluting mixture from a reservoir t o a mixing chamber which contained a second eluting mixture ( 3 2 ) . This continually stirred solution was subsequently delivered to t h e column. T h e gradient apparatus and colu m n were connected in such a way t h a t one p u m p controlled the flow in t h e entire system. When a pH gradient was used, the change in p H in t h e mixing chamber as a function of time was followed continuously with ;a recording p H meter. Initially, the weak eluting agent was passed through t h e colu m n until achieving a stable base line in the chromatogram. S u b sequently. pumping was stopped, and a n aliquot of the mixture (usually 5 t o 40 111) was added t o the column by micropipet or syringe. T h e volume of weak eluting agent in t h e mixing chamber was adjusted, and t h e strong eluting agent reservoir was connected t o the system. Pumping was resumed, a n d column effluent was monitored in a flow-through cell (Hellma Cells. No-l'i5QS) with a Beckman Model DB-G spectrophotometer. For quantitative measurements, each hand was collected and determined spectrophotometrically.
RESULTS AND DISCUSSION The adsorptive forces present when using XAD-2 as a chromatographic adsorbent are primarily of the van der Waals type (1-4). Thus, in the adsorption process, the portion of the solute which has little affinity for water (hydrophobic portion) i s preferentially adsorbed on the hydrophobic polystyrene surface of the resin while the hydrophilic section of the molecule will remain oriented in the aqueous phase. Alteration in the hydrophobic/ hydrophilic balance within the solute or within the solvent mixture in comparison to the resin will affect the adsorption of the solute. Although the beads are highly porous, there is evidence which suggests that penetration of the solute into the interstices is minor and, therefore, major adsorption is on the outer bead surface ( 3 ) .Consequently, ideal elution behavior would be predicted, and it is only necessary to establish the optimum eluting conditions. (32) L. R . Snyder, Chrornalogr, R e v . , 7,1 (1965)
Table I. Distribution Coefficients for Selected Compounds as a Function of Ethanol Concentration Ethanol,
Compound Benzene Benzoic acid Methyl benzoate Chlorobenzene p - N itroaniline Acetophenone Phenol p-Nitrophenol p-Chlorophenol Picric acid p-Chlorophenoxyacetic acid o-Chlorophenoxyacetic acid Pyridine p-Toluenesulfonic acid
YO
10
25
50
75
100
329 120 1240 824 118 403 42 65 179 13 175
134 44 259
0.2 34 27 21 3.7 14
0 0
0 0
19 1.6
114
5.4
2.6
0 36 68 10 69 6.7 0.1
4.6 6.8
1.0
0
0.1
1.4 1.2
0 0 0 0
0
0 0
7.8
0.6
5.8 0 0
0 0
0 0
0 0
Resin-Solvent Properties. The compatibility of XAD-2 with water and other organic solvents has been investigated and reported elsewhere (1-4). Choices of potential eluting solvents and mixtures were partially based on these observations. One fact should be emphasized, since it affects the application of XAD-2 in column experiments. If the XAD-2 is completely dehydrated, the dried resin will not be wetted by water. It must first be wetted by an organic solvent (methanol or ethanol is convenient) and subsequently water is continually added until the solvent system is completely aqueous. Because of this property, distribution coefficients, KD, were not measured in pure water. Dried XAD-2 was used for the KD measurements SO that the KD values could be calculated in reference to a reproducible resin state. Furthermore, the organic solvent in the KD studies was always added first. Thus, floating of the resin is prevented and the rate a t which equilibrium is reached is rapid. Distribution Coefficients. In order to achieve the optimum conditions for the adsorption and elution of organic molecules on XAD-2 columns, KD values were determined for a variety of experimental conditions. The batch technique was used for all K D measurements. Table I lists KD data in ethanol-water mixtures for simple representatives of a wide variety of functional groups. Several conclusions regarding the sorption performance on XAD-2 can be made from these data. All of the compounds are retained by the resin to some extent a t low-ethanol concentration. As the ethanol concentration increases (hydrophobic/hydrophilic balance increases), the degree of retention decreases. The same retention behavior would be predicted upon considering the solubilities of these compounds in water and ethanol or in their mixtures. The wide variation in the KD values for the different compounds a t a given ethanol-water composition indicates that the resin does not have the same preference for all organic molecules. Those molecules with potential charged sites (acidic or basic groups) have the least retention. However, retention does not necessarily follow the order of acidity or basicity. (It might have been anticipated that retention would decrease at a given solvent composition as the acidity increases.) For example, the stronger acids benzoic acid and p-nitrophenol both have larger retentions than phenol itself. Although the potential charged site influences the retention, retention is also influenced by the side groups or remaining portion of the molecule. For example, p-chlorophenol and p-chlorophenoxyacetic acid have essentially the same K D values ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
1349
Table I I. Distribution Coefficients for Nitro- and Chlorophenols as a Function of pH in 10% Ethanol PH
o-Nitrophenol (7.23)=
rn-Nitrophenol (8.40)
p-N itrophenol (7.16)
2,4-Dinitrophenol (4.09)
Picric acid (0.71)
4.0 6.0 8.0 10.0
647 507 104 6.8
112 104 76 10.3
56 25 1.8
167 5.9 2.4 2.3
7.1 5.9 5.2 3.6
Phenol (9.98)
PH 1.3 2.2 6.8 8.4 9.2 11.9 2,4-Dichlorophenol (7.89)
ii
o-Chlorophenol (8.48)
42.2
rn-Chlorophenol (9.02)
p-Chlorophenol (9.38)
158 255 254 157
108
197
131 3.8
34.3
0.5 2.5-Dichlorophenol (7.50)
2.9 2,6-Dichlorophenol (6.79)
3.1
3,5-Dichlorophenoi (8.18)
PH
Ku
PH
KD
PH
KD
PH
2.1 5.9 6.8 7.9 11.1 12.0
842 815 701 278 7.4 6.1
2.1 5.9 6.8 7.9 11.1 12.0
902 871 766 321 6.9 5.8
2.1 6.3 7.0 8.8 9.3 12.0
842 630 383 28 14 4.1
2.1 7.0 7.9 10.1 11.1 12.0
KD
1090 1050 646 45 13 7.3
2,4,6-TrichlorophenoI PH
KD
5.9 6.8 9.1 11.1 12.0
21 10 833 43 12.6 11.7
3,4,5-TrichlorophenoI PH 2.1 7.0 7.9 10.1 11.1 12.1
KD 3400 2610 1330 55.3 22.0 14.2
K a values are given in parentheses.
2
n
Y
m 0 _I
I
, 2
4
6
8
IO
12
PH Figure 1. Distribution coefficient as a function of pH in 1 0 % ethanol Experimental points (0, 0 , A , a ) , and calculation (----): (A) rn-chlorophenol ( 0 ) ;( 5 )p-chlorophenol ( 0 ) ; (C) o-chlorophenol ( A ) ; ( D ) phenoi (GI).
even though they have different K , values. It is concluded that the orientation of these molecules on the surface of the beads is the same and must be through the chlorophenyl portion. However, the potential charged site must influence the magnitude of KD since the KD for p-chlorobenzene is about four times as large. Obviously, one way to change the retention of the more polar (acidic or basic) compounds is to increase their ionic character through neutralization. The effect of p H on retention will be discussed in more detail later. 1350
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
Effect of Electrolyte and Other Solvents. The effect of electrolyte concentration (KC1) on K D for benzene, methyl benzoate, p-nitrophenol, and picric acid was investigated. Even though this series of compounds represents a wide range of polarity, in general, the change in KD with increasing KC1 concentration waS negligible up to 0.1M KC1, except for benzene; its KD had doubled in comparison to the absence of KC1. Above 0.1M KC1, the KD value for all the compounds rose sharply, and this increase is probably the result of a salting out effect. The effect of solvent mixture on the adsorption of a solute correlates to the polarity of the solvent mixture and solute. For example, in the series methanol, ethanol, and n-propanol mixed with water, a decrease in KD upon increasing the alcohol concentration occurs the earliest for the least polar n-propanol. If a nonpolar solvent is added, for example heptane-ethanol mixtures, KD for polar solutes (phenol and acetophenone) increases only at very high concentrations of heptane. For a nonpolar solute (naphthalene), the KD value gradually decreases as the heptane concentration increases. The tendency, in general, is for adsorption to follow the “like for like rule.” Effect of pH. Since phenolic compounds are weak acids their sorption on XAD-2 should be affected by the pH of the solution. In essence, the adsorption should change sharply a t some point with increasing pH, since the molecule will change from its neutral, poorly dissociated form to the dissociated anionic form. The KD values for a series of nitro- and chlorophenol derivatives in 10% ethanol, 0.1Mbuffer, as a function of pH are listed in Table 11. All of the phenols show similar behavior in that adsorption is high in acid solution (neutral form) and low in basic solution (anion form). Furthermore, as the p H of the solution increases, the change in KD with pH takes on the shape of an acid-base titration curve. Several examples which are typical of all are shown in Figure 1. The major change in KD with p H occurs in a pH region which is similar to the pK, for the phenol derivative. At this point, a small change in pH converts a large percentage of the neutral species, which is highly adsorbed, to the more polar charged form, which has very little adsorption. However, the position of a sharp drop in KD does not cor-
relate directly with K, for the phenols. The degree of adsorption of the neutral form depends on the type of substituents, their number, and their positions, as well as on the K , of the phenol derivative. If the pH is held constant and the ethanol concentration increased, the KD values decrease accordingly. For example, for 2,5-dichlorophenol at p H 12, the KD values in 10 (Table 11), 20, and 40% ethanol are 5.8, 2.9, and 0, respectively. Thus, by increasing the ethanol concentration a t high pH, adsorption on XAD-2 can be eliminated. Eluting conditions for separations can be predicted from this batch distribution data. Even though this is more fruitful than a trial and error process of selecting eluting conditions, it can still be time consuming in that KD values must be measured over a variety of conditions. Since the adsorption of a phenol is extensive when it is in the molecular form and very small when it is in the anionic form, the KD (Equation 1)can be redefined as
where [HA],, [A-IK, [HA],, and [A-1, are the concentrations of the molecular and anionic form on the resin and in the solution, respectively, L’ is the volume of solution, and u: is the weight of resin. Rearrangement and substitution by K, yields
a
b
%%!$+ (1
[H+]/K,
)
(3)
Term a is the KD for the neutral form and is experimentally determined (calculated by Equation 1) a t low p H where adsorption of the molecular form is a t a maximum. Term b is the KD for the adsorption of the anionic form and is experimentally determined (calculated by Equation 1) a t a high pH. Therefore, for a fixed solvent composition, the entire curve of KD as a function of p H can be calculated from the measurement of two KD values. Furthermore, if adsorption of the anionic form is negligible, Equation 3 simplifies to
The agreement between calculation and experiment is shown in Figure 1. Similar agreement was found for all the other phenols. In addition, excellent agreement between calculated and experimental curves was found for benzoic acid and p-chlorophenoxyacetic acid, and it appears that this equation can be used to calculate the KD-pH profile for the adsorption of weak acids on XAD-2. Separations. Several different eluting mixtures for the separation of phenols are suggested by the K D data. The two principal ones are: changing the p H while holding the ethanol-water ratio constant and changing the ethanolwater ratio while holding the p H constant. A third type of eluting mixture, which will be explored in more detail elsewhere, is changing to different organic solvents. Even though KD values vary, they tend to be large with respect to convenient separation of a several component mixture with a single type of eluting agent. Thus, in preliminary separation experiments, some overlapping and broad bands were observed except in cases where the components of the mixture had widely different KD values. An optimum elution behavior, however, can be ex-
i
l0
l
I
I
I’OPH
lil’,\d L 60
120
J 180
Aj 240
Volume of eluent, ml
Figure 2. A typical chromatograph illustrating separation and the advantage of gradient elution ( A ) pH gradient profile; ( B ) p-nitrophenol; (C) rn-nitrophenol. A 0.9- X 16.5-cm, 100 to 200 mesh, XAD-’2 column was used. The lower curve represents elution at pH 9.2 while the upper curve represents a pH gradient elution (see Table I I I for gradient conditions)
perienced with a gradient. Two possible types of gradients were employed; a p H change a t a fixed ethanol (10% by volume)-water ratio, and a changing ethanol-water ratio a t a buffered p H ( p H 1 2 ) . p H Gradient-Stepwise p H Elution. For most separations, a nearly linear p H gradient was employed. In general, a p H 11 phosphate buffer (0.1M) was delivered to a mixing chamber containing a phosphate-borate (both 0.lM) mixture usually a t p H 7 (10% ethanol was maintained). The presence of the borate provides buffer capacity in the region of p H 8.5 to 9.5 and, therefore, a nearly linear p H change is obtained. The advantage of a p H gradient for elution over elution with a fixed pH is illustrated in Figure 2 . The gradient elution chromatogram is also typical of the other separations reported in this paper. The order of separation of p - , o-, and m-nitrophenol by a p H gradient can be predicted from the KD data (Table 11) to be p-nitrophenol first, followed by ortho and then the meta derivative. This also happens to follow an elution order predicted from K , values. Experimentally, this separation was obtained. Similarly, a t pH 11.9, the separation of phenol, o-chlorophenol, and p-chlorophenol should elute in this order based on the minimal amount of determined KD values (Table 11). This order would not be predicted from their K, values. However, upon using stepwise elution ( p H 10.8, 11.3, and 12.3 buffers) in 10% ethanol, the order was o-chlorophenol, phenol, and finally p-chlorophenol. This order is substantiated by calculation of KD as a function of p H by Equation 3, since the calculated data show crossover of the KD values in the p H range of interest (see Figure 1). This experiment further demonstrates the versatility of Equation 3. Several other separations of the mononitro- and -chlorophenols are possible. A p H gradient was more suitable for complete separation of the nitrophenols while stepwise pH elution was more suitable for the monochlorophenols. A summary of the conditions and recovery for several separations are listed in Table 111. Ethanol-Water Gradient. The KD values (Table 11) for the di- and trichlorophenols are too large in 10% ethanol even a t p H of 12 for well-defined, sharp separations. However, the KD values can be easily changed by introducing more ethanol into the eluting system. This is also more easily accomplished experimentally in comparison to a pH gradient in the p H 12 region. Since the KD drops rapidly with increasing ethanol concentration, a successful gradient requires only a small change in the ethanol concentration. In general, the mixANALYTICAL CHEMISTRY, VOL. 45, NO. 8, J U L Y 1973
*
1351
Table 1 1 1 . Separation of Mixtures of Nitrophenols and Monochlorophenols p h gradienta Mixture
mg added
mg recoveredC
1. p-Nitrophenol o-Nitrophenol 2. p-Nitrophenol o-Nitrophenol 3. p-Nitrophenol rn-Nitrophenol 4. p-Nitrophenol rn-Nitrophenol 5. p-Nitrophenol rn-Nitrophenol
0.018
0.257 0.100 0.514
0.018 0.025 0.053 0.222 0.048 0.247 0.101 0.512
0.070
0.070
0.360
0.343
0.019 0.056 0.234
0.050
Stepwise pH elution* Eluent,
M
mg added
10.8 11.3 12.3 11.0 12.3 10.9 12.4
0.136 0.102 0.131 0.120 0.091 0.150 0.106
Mixture 6. o-Chlorophenol Phenol p-Chlorophenol 7. o-Chlorophenol p-Chlorophenol 8. o-Chlorophenol rn-Chlorophenol
pH pH pH pH pH pH pH
mg recoveredC 0.117 0.129 0.127 0.112
0.091 0.145 0.109
a A 0.9- X 16.5-cm column of 100 to 200 mesh resin was used. The gradient was formed with 70 m i of a 0 . l M p h 8.4 phosphate buffer in the mixing chamber and a 0 . l M p h 11 phosphate buffer in the reservoir: 10% ethanol was maintained. Flow rate was 1 . 5 ml/min. A 0.9- X 24.3cm column of 100 to 200 mesh resin was used. Flow rate was 1.5 ml/min and the buffers were phosphate buffers in 10% ethanol. In many instances, averages for several separations are given.
*
Volume of effluent, m l
Figure 3. Chromatogram of a four component mixture illustrating separation by an ethanol gradient ( A ) Phenol; ( E ) 2,6-dichlorophenoi: ( C ) 2,5-dichlorophenol: ( D )3,5dichlorophenol. A 1.5- X 24.2-cm, 100 to 200 mesh, XAD-2 column was used. See sample 8 in Table I V for gradient conditions. At E the ethanol concentration in the reservoir was increased to 30%
>-
----A-
& 70
n
2
4
6 8 Flow rate, ml/min
Figure 4. Tailing of each peak in function of flow rate
a
IO
i I
I2
two-component mixture as a
( A ) 0-Chlorophenoi, ( E ) 2,5-dichiorophenol
ing chamber contained 15% ethanol and the reservoir 22% ethanol, the p H of 12 was maintained with a 0.01M phosphate buffer. The gradient profile was convex and the ex1352
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
tent of curvature in the profile depends on the volume in the mixing chamber (33). Several different mixtures were successfully separated to illustrate the eluting conditions. Data for these are shown in Table IV. A typical four-component chromatogram is shown in Figure 3. Five-component mixtures were also separated. Since bands tend to broaden for the last of the eluted components, increased eluting power is recommended for the more retained components (see Figure 3). Several separations a t concentrations other than 1: 1 solute ratio were made. For example, phenol and 2,4-dichlorophenol a t about 60:l and 1:60 molar ratios were separated (see samples 11 and 12 in Table IV). Since the peaks are broader (retention volumes are the same) at the higher concentration, whether complete separation is possible or not will depend on the magnitude of the difference in the retention volumes and the efficiency of the column. In general, in these two separations, the upper limit for loading a t the column dimensions stated in Table IV was shown by experiment to be about 10 mg. Column Parameters. A convenient way to establish the optimum column parameters, such as flow rate, column length and diameter, and particle size, is by determining the height equivalent to a theoretical plate (HETP) and/ or number of theoretical plates as a function of the parameter. For separation 6 in Table 111, where stepwise elution was used, the first compound had a retention volume of 51.5 ml. From this, the number of plates and HETP were calculated to be about 43 plates and 5.7 mm, respectively. For compound C in Figure 3, where a constant eluting solution was used, the values were calculated to be about 47 plates and 3.5 mm. These are typical values for the columns when they are used with constant eluting systems. One of the requirements for the equations that describe the number of theoretical plates and the HETP is that the K D (or eluting power of the eluting agent) remain constant. In gradient elution, this is not the case and, therefore, the usual equations cannot be used to calculate these parameters. Since gradient elution, in general, appeared to provide better eluting conditions, the column parameters, such as particle size, column diameter, and column length were investigated by separating known mixtures by varying each of these parameters while controlling the others and reproducing the gradient. These investigations are summarized in the following. Separations were possible on both a 60 to 80 and 100 to 200 mesh resin. The latter resin provides better resolution and sharper bands. The larger column diameter permits a higher loading. Increasing the column length leads to an increase in resolution without excessive band broadening. Also, a reduction in tailing was observed for the longer columns. This latter trend is not chromatographically predictable and is probably the result of differences in packing of the columns. The effect of flow rate on elution behavior was investigated by separating a mixture of o-chlorophenol and 2.5dichlorophenol on a 1.5- x 24.0-cm column of 100 to 200 mesh resin using an ethanol gradient as a function of flow rate. (Number of plates and HETP values were not calculated since a gradient was used.) Complete base-line separation was obtained a t all flow rates. In addition, the retention volume for each component was independent of flow rate. The maximum flow rate investigated was 11.2 ml/min and it appears that faster flow rates could be used. Flow rates as high as 35 ml/min have been successfully used for amine separations on porous cation exchange resins (33).Pressure build-up for the separation of (331 T C Gilmerand D J Pietrzyk,Ana/ Chem 43. 1585 (1971)
Table I V . Separation of Mixtures of Mono-, Di-, and Trichlorophenols on 100 to 200 Mesh XAD-2 Resin Mixture 1. 2,5-Dichlor~phenol~ 3,5-Dichlorophenol
2. 2 , 5 - D i c h l o r ~ p h e n o l ~ 3,5-Dichlorophenol 3. 2 , 6 - D i c h l o r ~ p h e n o l ~ 3,5-Dichlorophenol
4. o-Chlorophenold 2,6- D ic hlorop heno I 2,5-Dichlorophenol 5. o-Chlorophenold 2,6-Dichlorophenol 2,5-Dichlorophenol 6. 2 , 6 - D i c h l ~ r o p h e n o l ~ 2,5-Dichlorophenol 7. o-Chlorophenold 2,5-Dichlorophenol 8. PhenolC 2,6-Dichlorophenol 2,5-Dichlorophenol 3,5-Dichlorophenol 9. PhenolC 2,4-Dichlorophenol 1 0 . PhenolC 2,4-Dichlorophenol 11. Phenold 2,4-Dichlorophenol 12. Phenold 2,4-Dichlorophenol
Eluenta 130 m l of 10-’M p H 12.2 in 1 5 % ethanol, 10-’M p H 12.2 in 22% ethanol S a m e as 1 120 mt of 1 0 - 2 M pH 12.2 in 1 5 % ethanol, 10-’M p H 12.2 in 22% ethanol 125 m l o f 10-’M pH 12 in 15% ethanol, lO-’M p H 12 in 22% ethanol Same as 4
S a m e as 4 120 m l of 10-’M p H 1 2 in 15% ethanol, 10-’M pH 12 in 22% ethanol Same as 7 After 2,5-dichlorophenol was eluted, 10-’M p H 12 in 3 0 % ethanol was placed in reservoir 70 ml of lO-’M p H 1 2 in 15% ethanol, 10-’M p H 12 in 22% ethanol Same as 9 Same
as 9
Same as 9
mg added
mg recoveredb
0.369 0.395
0.365 0.332
0.554 0.592 0.337 0.286
0.538
0.602 0.576 0.771 0.482 0.461 0.61 7 0.319 0.577 0.389 0.598
0.593 0.599 0.788 0.485 0.483 0.622 0.327 0.576 0.386 0.608
0.372 0.41 2 0.576 0.748
0.369 0.423 0.580 0.754
0.166
0.205
0.163 0.206
2.244 0.228 7.274 0.199 0.087 8.68
2.221 0.254 7.308 0.21 1 0.086 8.51
0.550 0.334 0.268
a The first solution was placed in the mixing chamber. The second was for the solvent in the reservoir. Flow rates were 1 to 2.5 ml/min except sample 7 where flow rates as high as 11 ml/min were used. In many
instances averages for several separations are given. A 0.9- X 24-cm column of resin was used. A 1.5- X 24-cm column of resin was used.
the phenols was not measured; however, in other studies column pressure increased linearly up to 35 ml/min (34). The fact that the fast flow rate does not result in poor chromatographic behavior was demonstrated by calculating a tailing factor for each component of the mixture as a function of flow rate. The tailing factor (35) compares the base of the second half of the chromatographic peak to the first half and, therefore, a value of 100 implies a completely symmetrical peak. Figure 4 shows a plot of tailing factor us. flow rate. The degrees of tailing of both components are similar. The important observation, however, is that as the flow rate increases, tailing does not increase as might be expected but rather becomes less. These minimum amounts of data do not permit a quantitative conclusion regarding why the tailing decreases. For example, this could be the result of a change in packing of the col-
umn beads since the pressure drop across the column increases with increased flow rate. Or, the sorption process may involve a kinetic step which is slow enough so that the sorption a t fast flow rates is reduced. It can be concluded, however, that favorable chromatograms are possible a t a fast flow rate. The data in Tables I11 and IV reflect optimum column dimensions and particle size based on these experiments that are suitable for the separations. Faster flow rates than those listed can also be conveniently used. Preliminary experiments with a 0.25- x 27.0-cm, 200 to 400 mesh column also suggest that these data can be extended to high pressure liquid chromatography. A pressure of 400 to 500 psi, a flow rate of 0.2 ml/min, and an ethanol gradient were used in these experiments.
(34) T. C. Gilmer. Ph.D. Thesis, The University of Iowa. May 1971 (35) H . M. McNair and E. J. Bonelli. “Basic Gas Chromatography,” Varian Aerograph, Walnut Creek, Calif., 1965.
Received for review October 3, 1972. Accepted January 8, 1973. Taken from the Ph.D. Thesis submitted by M.D.G. to the University of Iowa, July 1972. Support for M.D.G. was provided by the National Aeronautics and Space Administration in the form of a Traineeship.
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