Measurement and Use of Substrate and Partition Liquid Selectivities in Gas Chromatography EDGAR D. SMITH and JUNIOR 1. JOHNSON Universify o f Arkansas, Graduate lnsfifute of Technology, liifle Rock, Ark.
b The use of independently-determined substrate (solid support) and partition liquid selectivities has been studied in connection with the separation of two close-boiling isomeric ketones. Substrate selectivities were determined from measurements of the retention times of these ketones on columns containing only the bare support. Partition liquid selectivities were obtained similarly on columns containing relatively-large amounts of liquid on a practially inert support. Useful separations were quickly developed by combining substrates and partition liquids of similar selectivity characteristics, whereas all previous attempts to achieve this separation had failed. The systematic approach outlined herein may prove to b e of general use in achieving other difficult gas chromatographic separations.
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have reflected a growing awareness of the fact that the substrate (solid support) used in the preparation of a gas chromatographic packing can profoundly influence the separations obtained (9). The exact nature of the substrate interaction is not clear, but its existence is usually revealed by tailing peaks typical of nonlinear gas solid adsorption isotherms. Craig (9) and Martin (6) have shown, however, that the substrate may significantly effect gas chromatographic separations without producing asymmetric peaks. Both of these workers have suggested the possibility of a partition liquid-substrate interaction as the explanation of these results, but they have also recognized the possibility of direct gas solid adsorption effects with polar organic solutes. Regardless of the rewons for the influence of the substrate, it is an important factor which may prove very useful in achieving separations not possible on the basis of a pure partition process. The present work was undertaken to determine if substrate and partition liquid selectivities could be independently measured, then used to prepare superior, gas chromatographic packings. An excellent pair of test materials for this work was provided by 2- and 3pentanone. These isomeric ketones boil only 0.7" C. apart and are so similar ECENT ARTICLES
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structurally that their partition coefficients in most organic solvents are identical. For this reason, no satisfactory gas chromatographic separation of these compounds had previously been achieved despite several reported attempts to do so (4,8-10). EXPERIMENTAL
A Perkin-Elmer Model 154-L Vapor Fractometer having a thermistor-type detector was used with a Leeds & Northrup Speedomax H recorder. The recorder has a 1-mv. span, I-second response, and a chart speed of I/, inch per minute. The Vapor Fractometer was modified by connecting a small powerstat in the heating circuit to control independently the injection block temperature. Helium was used as the carrier gas. One-fourth inch copper tubing, 2 meters in length, was used for all screening columns with the exception of some of the bare substrate columns, where intense adsorption made the use of 1-meter lengths necessary. The various substrates mere obtained from a number of sources as listed below: Firebrick, regular (60/80 mesh, Wilkens Instrument and Research Inc., Cat. No. XA-165). Chromosorb-P, regular (35/80 mesh, Wilkens Instrument and Research Inc., Cat. No. XA-161). Chromosorb-W, regular (30/60 mesh, Johns-Manville Corp., Cat. No. A-472). Embacel (60/100 mesh, Perkin-Elmer Corp., Cat. No. 990-5151). Gas-Chrom Z (80/100 mesh, Applied Science Laboratories, Inc., Lot No. Z-1). Fluoropak (20/80 mesh, Wilkens Instrument and Research, Inc., Cat. No. XA-168-1). Partition liquids were obtained from various commercial sources, and used without further purification. The 2and 3-pentanone samples were reagent grade chemicals obtained from Distillation Products Industries, Inc., and were also used without further purification, A Hamilton 701-NCH microsyringe equipped with a Chaney adapter was used to inject the solutes. During the initial screening process, the operating conditions were held constant whenever possible. The column temperature was held at 50" C., the injection block a t 150" C., and the helium flow rate at 80 ml. per minute measured a t the outlet. Two-meter columns containing only bare substrate were prepared in conventional manner, and conditioned in a helium stream at 150" C. for 24 hours. Individual 1-pl. injections of 2- and 3pentanone were made and the retention
time of each component, as well as that of air, was measured. Relative selectivities of the various substrates (a values) were expressed as the ratio of the retention time of 2-pentanone to tJhat of 3-pentanone, both times being measured from the air peak. For the determination of partition liquid selectivities, Gas-Chrom Z was chosen as the solid support since it proved to be the most inert as well as the least selective of the available substrates. Several partition liquids were selected initially to survey a range of different solvent types, and others were added as the results indicated they were needed. Packings were prepared by the usual slurry technique to contain 2.73 grams of liquid phase on 11.00 grams of Gas-Chrom 2. This was the amount of packing necessary to fill a Zmeter column and gave roughly a 25% packing of partition liquid based on the weight of substrate used. (At the start of this work it was thought that Embacel would be the most inert substrate, and the 2.73 grams was the calculated amount to give a 30% packing on this support.) Various solvents were used in preparing these packings, depending on the solubility characteristics of the partition liquids, and these solvents were removed with the aid of a thin-film rotary vacuum evaporator. The columns were packed in conventional manner, then conditioned a t 70" C. for 24 hours in a helium stream. All packings were checked by direct weighing to be sure that they still contained the calculated quantity of partition liquid after these preparation processes. Samples of 2- and 3pentanone were injected, retention times mere measured, and (Y values were calculated in the same manner as for the bare substrates. Values of the separation factor (S), peak sharpness (Q), and resolution ( R )were also calculated using the relationships discussed by Jones and Kieselbach (6). RESULTS AND DISCUSSION
Table I summarizes the relative selectivity characteristics of the six substrates toward 2- and 3-pentanone. As noted in this tabulation, the selectivity values for Firebrick and Chromosorb-P were determined on 1-meter columns and a t 100" C., rather than with 2-meter columns and 50" C. as was "standard" for the other packings. This change was necessitated by the strong adsorption of the ketones on
these two very active substrates. Under standard conditions, no recorder deflection was observed for either of the ketones, the samples apparently being retained by the columns indefinitely. Even under the mod&ed conditions indicated, s?ecial techniques were required to obtain reproducible values for these substrates, The first sample injected into a freshly-conditioned column gave a very low peak with a relatively long retention time. Subsequent injections gradually increased the peak height and decreased the retention time until fairly constant values were obtained. Minor fluctuations in these values could be brought about by changing either the sample size or time allowed between injections. It was apparent that much of the sample was being retabled on the packing, and that the amount of this adsorbed material was causing the fluctuations noted in retention times and peak areas. To obtain meaningful retention time values under these conditions, it was necessary that the quantity of adsorbed ketone be maintained constant. This was accomplished by injecting repeated 1-pl. samples at exact 10-minute intervals. .4fter about the fifth injection, it was found that constant and reproducible retention time values were obtained, and these are the ones recorded in Table I. After obtaining retention data for one ketone, the columns were recclnditioned for 1 hour a t 150” C. before proceeding to similar measurements for the second compound. To check the validity of the selectivity values obtained by the modified procedures described above, similar measurements were carried out using Firebrick and Chromosorb-P a t other temperatures. The results of this work are given in Table 1:. Despite the wide variations noted n the retention times, the a values did not change significantly with chanses in temperature. This result is not in conflict with the theoretical consider:ttions of Sawyer and Barr (8), since calculations showed that the small changes in a values, which these workers predicted should occur with changes in temperature, were well within our limits of experimental error. Because of the pronounced tailing of the ketone peaks on these bare substrates, no attempt was made to calculate S, &, and R values and substrates were evrduated only on the basis of their a va1ut:s. I n evaluating partition liquid selectivities, the assumption was made that use of a large qusntity of liquid phase on a relatively inert, nonselective substrate should make any effect of the solid support negligible. Strong support for this idea is provided by the data in Table 111. In this series of tests, 2meter columns were prepared from
Table 1.
Relative Selectivities of Various Substrates
Retention time (minutes) Substrate 2-Pentanone 3-Pentanone Firebricka 3.75 2.35 1.11 0.92 Chromosorb-W 3.06 2.53 Chromosorb-Pa 2.14 2.45 Embacel 0.60 0.60 Gae-Chrom Z 0.79 0.83 Fluoropak a Data obtained on 1-meter columns at 100’ C. (see text).
Table
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Air
0.20 0.40 0.20 0.60
0.50 0.40
Effect of Temperature o n the Relative Selectivities of Firebrick and Chromosorb P
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Retention time (minutes)” Substrate Temp., O C. 2-Pentanone %Pentanone Firebrick 3.55 2.15 100 1.20 125 0.78 150 0.58 0.94 Chromosorb-P 100 2.86 2.33 125 1.20 0.98 150 0.65 0.53 a Correct’ed for air retention time of 0.20 minutes.
Table 111.
1.65 1.37 1.22 1.20 1.00 0.91
Q
1.65 1.60 1.62 1.23 1.23 1.23
Effect of Substrate on Retention Times and Relative Selectivities Using Adiponitrile as the Partition Liquid
Retention time (minutes)” Substrate LY %Pentanone 3-Pentanone Firebrick 38.5 37.6 1.02 Chromosorb-W 31.8 31.2 1.02 Chromosorb-P 30.3 1.02 29.9 33.4 33.1 Embacel 1.01 Gas-Chrom Z 33.0 32.3 1.02 31.3 Fluoropak 30.9 1.01 Corrected for the retention time of air, which was 0.50 minutes for all columna. All columns were 2 meters in length and contained 2.73 grams of adiponitrile. Operating conditione were 50’ C. and SO-m1.-per-minute helium flow.
each of the various substrates and 2.73 grams of adiponitrile. Despite the wide variations in adsorptive strengths, film thicknesses, and selectivity characteristics represented by these substrates, the resulting a values cal: culated for the adiponitrile agreed very well. In fact, the variations noted in individual retention times were also calculated to be within the limits of experimental error except for the values on Firebrick. Good agreement of this nature should not be expected with less polar partition liquids which are less able to “cover up” active adsorption sites (8, 7). This was also briefly tested and nonpolar partition phases, such as squalane, gave significantly longer retention times when coated on Firebrick than when coated on Gas-Chrom Z. However, even with this completely nonpolar partition phase, the retention times and selectivity values obtained from columns using Embacel or Chromosorb W as the substrates agreed closely with those found for Gas-Chrom Z. Since Gas-Chrom Z is an appreciably
weaker and less selective ketone adsorbant than either Embacel or Chromosorb W, it was felt that valid partition liquid selectivity values were being obtained by the procedures discussed. Table IV summarizes the data obtained on a representative group of partition liquids. These liquids were selected to show a range of a values. Numerous attempts were made to correlate the observed selectivity values with observed structural features of these compounds, but few satisfactory generalizations could be made. One difficulty in evaluating the data shown lies in the fact that we do not know what the a value should be for a truly nonselective partition liquid. Since 2-pentanone is slightly lower boiling than 3-pentanoneJ an a value of 1.00 actually represents a slight selectivity for the 2-pentanone. Possibly, the lowest a values noted in this work, 0.91 for Fluoropak and 0.88 for squalane, are the theoretical values for true neutrality. Because of these complications, however, it was decided VOL 35, NO. 9, AUGUST 1963
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Figure 1. Separation of 2- and 3-pentanone on 4 meters of 5% ethylene glycol on Firebrick
Figure 2. Separation of 2- and 3-pentanone on 2 meters of 25y0 squalane on Gas-Chrom Z
Operating conditions: 27' C., 80-ml.-per-minute helium flow
Operating conditions: 27" C., 1 00-ml.-per-minute helium flow
best to trcat these data empirically, and to consider an (Y value of 1.00 as indicating a neutral substrate or partition liquid. On this basis, all of the polyhydroxy compounds (except Carbowax 1540 and Carbowax 20M) were selective for 2-pentanone, and there seemed to be a definite correlation between hydroxyl content and 2-pentanone selectivity. The partition liquids with no important functional groups, such as the silicone fluids and the hydrocarbons, were at the opposite end of the scale and showed the least selectivity for 2-pentanone. Finally, there was numerous compounds, showing intermediate selectivity values, for which no correlation of selectivity and structure seemed possible. Having established the selectivity characteristics of the various substrates and partition phases, it was now possible to test the main thesis of this work.
Table IV.
The results of Tables I and I V show that the best chance of separating the ketones-utilizing substrate interaction-was with Firebrick and ethylene glycol. Glycol and diglycerol were ruled out on practical grounds because, although they showed good selectivity, they gave broad tailing peaks as well as short retention times. The tailing observed appeared to be of the type described by Martin (6) for adsorption a t the gas-liquid interface, and it is interesting to note that these were the only two cases where this phenomenon was observed. Since the Firebrick was to be the chief factor in bringing about the separation, it was important that the final packing contain only enough liquid phase to reduce tailing to an acceptable degree. -4few trials showed that 5% by weight of ethylene glycol fulfilled this requirement, and that the predicted separation was obtained. Figure 1
Relative Selectivities of Representative Partition Liquids"
Retention time ( minutes)b 2-
3-
Partition liquid Pentanone Pentanone 01 S X lo2 Q R X lo2 1.69 12.5 1.40 1.11 7.40 1.55 G1 cero1 48.8 5.58 1.10 8.56 6.93 6.32 Etxylene glycol 1.04 6.1 1.95 5.83 2.10 1.08 Diglycerol 42.6 6.99 6.09 10.50 1.07 9.85 Diethylene glycol 27.2 6.53 4.17 1.04 10.45 10.00 Triethylene glycol 1~.02 7.35 13.8 .~ 1.88 23.10 23.55 Octanoic acid 28.8 10.15 2.83 18.65 1.02 19.20 fl,8'-oxydipropionitisile i7.i 5.08 2.11 1.02 33.00 32.30 Adiponitrile 15.4 1.85 8.33 13.15 13.00 0.99 Carbowax 1540 17.2 8.88 1.94 12.45 0.98 12.20 Carbowax 20M 53.4 6.25 9.18 22.35 21.00 0.94 Dinonvl Dhthalate Poly-.&-p'henyl ether, 5.32 39.3 0 . 9 3 7.41 16.20 6-ring 15.00 67.3 7.28 9.25 0.93 8.70 DC 200 fluid 8.10 70.2 8.24 8.50 0.92 13.90 DC 710 fluid 12.80 8.23 94.5 11.48 10.90 0.89 Hexadecane 9.72 7.74 90.8 0.88 11.73 7.60 8.55 Squalane All data were obtained on 2-meter columns containing 2.73 g r a m of the indicated liquid on Gas-Chroni Z. Operating conditions were 50' C. and SO-ml.-per-rninute helium flow.
Corrected for air retention time, which varied from 0.50 to 0.60 minute.
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shows the practically base-line scparation finally achieved on a 4-meter column of this packing. As a final test of the ideas proposed, an attempt was made to separate the ketone isomers in inverse order to that shown in Figure 1. Again, inspection of Tables I and IV indicated that this should be possible by combining squalane (or hexadecane) with Fluoropak. Preliminary tests showed that this would be a difficult combination with which to work, probably because of the poor wetting characteristics of Fluoropak (7, 9). Very broad peaks with short retention times were obtained, showing that the squalane was not being made available for the necessary partition interactions. Since Gas-Chrom Z was the only other possible substrate, all others being selective for %pentanone, it was next chosen for study. It was realized that any separation obtained would have to be brought about by the selectivity characteristics of the partition phase, so that it seemed likely that a relatively high percentage of the latter would be required. This proved to be the case, and a 10% column of squalane on Gas-Chrom Z gave significantly poorer separation of the ketones than the 25% column used in the original screening studies. Figure 2 shows the separation obtained on the original 25% column by reducing the operating temperature and optimizing the flow rate. KO attempt was made to improve the resolution, but presumably this could be done by the use of a longer column and possibly by increasing the quantity of partition liquid. In conclusion, it is felt that this study has adequately demonstrated the usefulness of independent measurements of substrate and partition liquid selectivities. For critical separations, we feel it is imperative that the substrate and partition phase act in unison to effect the desired separation. It is recognized that other substrate variables-e.g., sur-
face areas (1)-are als2 important and work is continuing to evaluate the effect of these variables on gas chromatographic separations. LITERATURE CITED
R. H., Purnell, J. H., J. Chem. SOC. 1960, b44. . (3) Craig, B. M., “Gas Chromatography,” pp. 37-56, N. Brenner, J. E. Callen, and M. D. Weiss, e&., Academic Preas, New York, 1962. (4) Fry, A., Eberhardt, M., Ookuni, I., J . Org. Chem. 25, 1252 (1960). (5) Jones. W. L.. Kieselbach., R... ANAL. CHEM.30,1590(1958). (6) Martin, R. L., Ibid., 33,347 (1961). (7) Ottenstein, D. M., Seventh Detroit Anachem. Conference, Detroit, Michigan, October, 1959. .
(1) Baker, W. J., Lee, E. W., Wall, R. F., "Gas Chromatography,” pp. 21-31, H. Noebels, R. F. Wall,,and N. Brenner,
e&., Academic Press, .New York, 1961. (2) Bohemen, H., Langw, S. H., Perrett,
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(8) Sawyer, D. T., Barr, J. K., ANAL. CHEM.34, 1052 (1962). (9) Ibid., p. 1518. (10) Young, J. R., Chem. Znd. (London) 1958,594. RECEIVEDfor review March 25, 1963. Acce ted May 27, 1963. Presented at
the Eouthwest Regional Meeting, ACS, Dallas, Texaa, December, 1962. The authors ex reas their appreciation to The Crossett Crossett, Ark. (now the Crossett Division of the Georgia-Pacific Corp.), for providing the Research Grant under which this study wria conducted.
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Spectrophotometric Determination of Nitrate with 2,6-XyIenoI Reagent A. M. HARTLEY and I?. 1. ASAll Deporfment o f Chemistry, University o f Illincis, Urbana, 111.
b Nitrate in a solution of sulfuric acid-water-acetic acid reacts with 2,6-xylenol (2,6-dimethylphenol) to produce the corresponding 4-nitro2,6-xylenol. The readion mixture so produced has an absorption maximum at 320-4 m,u with a molar absorptivity of 7900 which has bee1 identified with the 4-nitro-2,6-xylenol by extraction and comparison with the authentic compound. Under the optimum conditions of: complete removal of chloride and nitrate, solvent composition of 6 : 3 : 1 v./v. sulfuric acid-wateracetic acid (or 4 : 4 : 1 : 1 sulfuric acidphosphoric acid-water-acetic acid) and room temperature, the absorbance at 320-4 mp is a linear function of nitrate added in the range of 2 i o 30 p.p.m. with an over-all relative standard deviaiion of 0.1 to 1.2%. A procedure has been developed from these conditions which requires 7 to 10 minutes per sample determination. The time, precision, and accuracy compare favorably with existing methods.
T
HE determination of nitrate or oxides of nitrogen in trace concentrations is of interest in connection with problems of air and water pollution, sanitation, and allied areas relating to public health (15). Eoltz has summarized the existing methods for the determination of nitrate and/or nitrite in a monograph (4). ldacdonald had discussed the spectrophotometric and electrometric methods in two review articles (17, 18). For the most part these procedures suffer from the disadvantages attendant cn nonstoichiometric reactions, nonlinear working
Present address, Department of Chemistry, University of Nevada, Reno, S ev.
curves, limitations of usable concentration range, and overlong time per determination. The most common spectrophotometric methods involve either nitration of a suitable reagent to form the corresponding nitro compound or oxidation by nitrate to yield, usually, quinones. Kitration of 2,4-xylenol has received considerable attention (1, 5, 19, 26, 29). In this method the 6-nitro-2,4-xylenol is formed, extracted or steam-distilled, and subsequently measured as the yellow nitro-phenolate in aqueous alkali. Phenoldisulfonic acid, presumably the 2,4-disulfonic acid derivative produced by gently warming a solution of phenol in concentrated sulfuric acid, has been utilized in similar fashion (7, 9, 23-26). Brucine, when treated with a sulfuric acid solution containing nitrate, produces a yellow color which may be made a measure of nitrate (8, 10, 16,20, d2,27, 28). The reaction is exceedingly empirical; color development is dependent on reaction time, nitrate level, and source of the alkaloid. Each of these methods suffers from a common list of difficulties. Kone will distinguish between nitrate and nitrite. Halides are a serious interference although less so for the brucine method. Time per determination is a minimum of 30 minutes in each case. The absorbance-nitrate working curves are, for the most part, nonlinear. In 1949, Holler and Huch published a method based on the nitration of a xylenol (3,4-dimethylphenol) in sulfuric solution followed by steam distillation of the nitro-xylenol and subsequent visible colorimetric estimation of the yellow nitro-phenolate in aqueous alkali (14). This method depends upon the predominant formation of the ortho-nitroxylenols since these are readily
codistilled with water. For this reason Holler and Huch limited their investigation to the five isomeric xylenols which have at least one open ortho position; the remaining isomer, 2,6dimethylphenol, is obviously unsuitable since both ortho positions are blocked. Recently we have developed a series of methods for the determination of nitrate, nitrite, or both based on the nitration of this isomer ( 2 ) . A brief report containing only a recommended procedure for the determination of nitrate in water has been published elsewhere (12). -4polarographic modification of this method has been reported (13). I n the latter two articles the optimum conditions were offered without comment. Since the effects of temperature, acidity, and the like, can be considerable, a more detailed exposition is offered herein. EXPERIMENTAL
Apparatus. All spectral data were recorded using a Cary Model 14 recording spectrophotometer and matched quartz cells. Unless otherwise indicated, the latter were of 1-cm. path length. Reagents. All reagents were of highest quality obtainable. Sulfuric acid, acetic acid, and phosphoric acid were the concentrated c.P., glacial, and 85% reagent grades, respectively. Eastman White Label (No. 1772) 2,6xylenol was used without further purification for the most part; in the later stages of this investigation oxidative deterioration of the xylenol required recrystallization from aqueous ethyl alcohol until the melting point improved t o the literature value. The method of von Auwers and Markovits (3) was used to prepare 4nitro-2,6-xylenol. The compound was obtained as pale-yellow plates melting VOL. 35, NO. 9, AUGUST 1963
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