Table IV. Excretion of Apomorphine in Urine of Sprague-Dawley Rats Administered 1-HC1 Amt of Np. of l r H C l ( m ) animals lnlected? Exp. No. injected animal
1 2= 3 4 5 6e
Amount of 1" (mg) excreted in w i n e 0-24 k
24-28 k
... ...
48-72 hr
2 2.1 0.37 ...b 2 3 .O 0.86 ...b d d 3 4.1 0.92 d 3 5.2 0.82 0.22 b 3 5.8 1.62 0.22 3 4.2 1.92 0.29 0.11 b b ve 3 5.1 2.13 a No more than trace levels of free 1 were detected in any experiment; values obtained from urine hydrolyzed to free 1 from its conjugates (probably glucuronides, see Ref. 18). Samples not examined. < In this experiment only, a 5.6% excretion of 1 as apocodeine was determined (qualitatively by TLC (Ref. 2 ) and GC (Ref. 4 ) ; quantitatively by GC ( 4 ) ) . No apomorphine detected. e During these experiments, animals drank 1%ammonium chloride solutions ad libitum in lieu of water.
...
...
... ... ...
...
drolyzed in 4N hydrochloric acid and subsequently adjusted to pH 8.6. Though recoveries are low (as indicated in Table 111), approximately 5 to 10% conversions of 1 to 2 or 3 could be detected in feces, assuming principal elimination via this route and dose levels of 16 to 20 mg/kg of 1 in rats. Metabolism Studies. The developed GC methods were employed in determining amounts of I (combined free and conjugate) excreted in the urine of Sprague-Dawley rats injected with 1-HCl. Results of these investigations appear in Table IV. Over all, the values obtained are about one-half those reported by Kaul et al. (15, 17) for a different inbred strain (Long Evens) of rat. Interestingly, significant increases in elimination of apomorphine in urine were observed in animals whose urinary pH was decreased by oral administration of ammonium chloride. This effect has also been observed in rabbits (15). The amount of I excreted over 72 hours by Sprague-Dawley rats seemed to be independent of dosage over the range studied (see Table IV). In the above experiments, the identity of 1 excreted in urine was confirmed by GC/MS of its T M S derivative. In one experiment, 2 was detected as a urinary metabolite of 1 (see Table IV). These results along with those of related experiments will be the subject of a future report. Pooled feces (collected for 72 hours following injection of 1-HC1) were examined for compounds 1, 2, and 3. Compound 1 was detected while its 0-methyl metabolites, 2 and 3, were not observed, within the limits of detections for these compounds.
During the development of the GC method for 1, compounds 4 (as its TMS derivative) and 6 were found to chromatograph with retention times different than those obtained for the TMS derivatives of 1,2, and 3 (see Figure 5 ) . Attempts were made to detect compounds 4 and 6 in hydrolyzed urine samples derived from rats injected with 1HC1. However, neither 4 nor 6 was observed in 72-hour samples.
CONCLUSION GC methods have been developed for determining apomorphine and some of its potential metabolites in urine. Further work is required to devise reliable assays for these compounds in feces. The methods described should permit further investigation of the metabolic fate of apomorphine in mammals. ACKNOWLEDGMENT We are grateful to Linda M. Lizak for excellent technical assistance during certain stages of this work. We also wish to express our thanks to Joseph G. Cannon of the University of Iowa for a gift of norapomorphine. LITERATURE CITED (1) R. V. Smith and S. P. Sood. J. Pharm. Sci., 60, 1654 (1971). (2) J. G. Cannon, R. V. Smith, A. Modiri, S. P. Sood, R. J. Borgman. M. A. Aleem, and J. P. Long, J. Med. Chem., 15, 273 (1972). (3) R. V. Smith and M. R. Cook, J. Pharm. Sci., 63, 161 (1974). (4) R. V. Smith and A. W. Stocklinski, J. Chromatogr., 77, 419 (1973). (5) R. V. Smith, M. R. Cook, and A. W. Stocklinski, J. Chromatogr., 87, 295 (1973). (6)P. N. Kaul, E. Brochmann-Hanssen, and E. L. Way, J. Am. Pharm. Assoc., Sci Ed.. 48, 638 (1959). (7) M. P. Cava, A. Venkateswarbu, M. Srinivasan, and D. L. Edie, Tetrahedron, 28, 4299 (1972). (8) R. V. Smith and S. P. Sood, Anal. Lett.,5, 273 (1972). (9) K. Rehse and G. Dreke, Fresenius'Z. Anal. Chem., 248, 179 (1969) (10) K. Rehse, Arch. Pharm. (Weinheim),302, 487 (1969). (11) K. Rehse. Arch. Pharm. (Weinheim),305, 625 (1972). (12) W. K. VanTyleand A. M. Burkman, J. Pharm. Sci., 60, 1736 (1971). (13) K. Parker. C. R. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (14) M. V. Koch, J. G. Cannon, and A. M. Burkman, J. Med. Chem., 11, 977 (1968). (15) P. N. Kaul, E. Brochmann-Hanssen. and E. L. Way, J. Pharm. Sci.. 50, 244 (1961). (16) H. H. A. Linde and M. S. Rageb, Helv. Chim. Acta, 51, 683 (1968). (17) P. N. Kaui, E. Brochmann-Hannssen, and E. L. Way, J. Pharm. Sci, 50, 248 (1961). (18) P. N. Kaul, E. Brockmann-Hanssen, and E. L. Way, J. Pharm. Sci., 5 0 , 840 (1961).
RECEIVEDfor review December 23, 1974. Accepted March 12, 1975. This work was supported in ?art by grants NS04349 and NS-12259, National Institute of Neurological Diseases and Stroke.
Chromatographic Determination of Phenols in Water Colin D. Chriswell, Richard C. Chang, and James S. Fritz Ames Laboratory-USAEC and Department of Chemistry, lowa State University, Ames, /A 500 10
Phenols in natural waters and treated drinking water are determined by sorption on macroporous anion-exchange resin, elution with acetone, and measurement by gas chromatography. Techniques are given for preventing phenol losses caused by chlorination, oxidation, and other reactions during their determination. Common inorganic ions and many organic substances cause no interference; neutral or-
ganics that are retained by the resin can be removed by a methanol wash. The method gives accurate results for phenol, alkyl-, and chloro-substituted phenols in the ppb to ppm concentration range.
As part of an extensive effort to develop improved, practical methods f o r determining trace concentrations of orANALYTICALCHEMISTRY, VOL. 47,
NO. 8,
JULY 1975
-
1325
ganic contaminants in drinking water and surface waters (1-61, we have become interested in developing more effective methods for determining acidic substances such as phenols, carboxylic acids, and sulfonic acids. Phenols are of particular interest because many substituted phenols have been found to be harmful to fish and other life forms found in natural waters ( 7 ) , and phenol and many substituted phenols contribute an unpleasant medicinal odor and taste to drinking water even a t low concentrations. A number of sensitive colorimetric methods have been developed for determining phenols in water based on the intensely colored compounds formed by their reactions with 4-amino antipyrine and related compounds (8, 9). Although the colorimetric methods are sensitive, they cannot differentiate between substituted phenols and are used primarily for determining total phenol concentrations. Also, the color-forming reagents will not react completely with most para-substituted phenols, and thus the results obtained for total phenol concentrations may be significantly in error. Some of these problems are alleviated by a procedure which involves absorption of phenols on carbon, extraction of the carbon with chloroform to remove the phenols, concentration of the phenols in the chloroform by evaporation, and determination of phenols in the chloroform extract by gas chromatography (10). The most significant limitations of this method are that it is difficult to avoid losses of phenols caused by incomplete absorption on the carbon, incomplete extraction with the chloroform, or evaporative losses during the concentration of the chloroform. Another limitation is that this procedure is not specific for phenols, and other organic compounds present in water can interfere with the gas chromatographic determination of phenols unless elaborate cleanup procedures are performed on the chloroform extract ( 1 1 ) . The absorption of phenols from acidic solution on porous polymer resins such as Amberlite XAD-2 has been shown to form the basis for a simple and accurate method for determining organics, including phenols in water (5, 12). XAD-2 resin effectively sorbs phenols and other organic compounds from water, although low recoveries are obtained for phenol itself. A method was sought that would concentrate all phenols effectively and would permit separation of phenols from other organics that might be present in water. Preliminary experiments showed that phenols are effectively retained from water when samples of water containing low concentrations of phenols are made basic and passed through a column containing an anion-exchange resin (A-26). Exploratory experiments in our laboratory indicated that low concentrations of neutral organic compounds such as naphthalene were only 0 to 3% retained by the anion exchange column. Thus, a reasonable selectivity for phenols over other organics was indicated. The basis of the analytical method reported in this paper is as follows: Phenols are taken up as phenolate ions by passing an alkaline water sample through a column of A-26 anion-exchange resin in the hydroxyl form. Any neutral organic compounds retained by the resin are removed by washing with alkaline methanol. Phenolate ions continue to be held by the resin during this washing step and are then converted to the molecular form by washing the column with aqueous hydrochloric acid. The phenols are subsequently eluted from the column with acetone-water. The hydrochloric acid and acetone-water effluents are each extracted with methylene chloride. The organic phases are concentrated by evaporation, and the phenols are separated by gas chromatography. 1326
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
EXPERIMENTAL Reagents a n d Apparatus. The A-26 resin was obtained from the Rohm and Haas Chemical Company. The resin, as received, was first screened to remove any resin beads smaller than 60 mesh, which restrict flow through absorption columns, and then subjected t o a thorough cleaning procedure to remove organic impurities left by the manufacturing process. A procedure we have found effective consists of placing the resin in a sintered glass filter attached to a suction flask, and with the vacuum adjusted so that solvents will flow slowly through the resin, wash in sequence with 2M sodium hydroxide, purified distilled water, 4M hydrochloric acid, purified distilled water, and acetone. This sequence of washings is repeated until no color is apparent in the final acetone wash (generally one to three times). The resin is then extracted with acetone for 24 hours in a Soxhlet extractor. Following this cleaning, the resin requires only routine generations to maintain its effectiveness for at least a hundred phenol determinations. A ?$-in. X 6-in. glass chromatographic column with a 1000-ml reservoir is prepared by first placing a glass wool plug in the bottom to retain the resin and then pouring a water slurry of the cleaned A-26 resin into the column until the resin bed is within about one-half inch of the top of the column. The column is placed in the hydroxy form by passing approximately 20 ml of 0.1M sodium hydroxide solution through the resin. Excess sodium hydroxide is washed from the resin bed with 50 ml of purified distilled water. Distilled water is further purified to remove trace organic contaminants by passing it through a column packed with XAD-2 resin and activated charcoal as previously described by Junk et al. ( 5 ) . Acetone is distilled to remove any high boiling impurities before use. All other chemicals are of reagent quality, if available, or the highest purity available. Solutions of phenols in acetone-methylene chloride are concentrated by evaporation in specially designed flasks with an attached Snyder column. The design of these flasks and their significant advantage have been previously reported ( 5 ) . A Hewlett-Packard Model 5 7 l l A gas chromatograph equipped with dual flame ionization detectors and a six-foot stainless steel column, %-in. 0.d. (5% OV-17 on chromosorb W AW DSCS SO/lOO mesh), and a Hewlett-Packard Model 5750B equipped with dual flame ionization detectors and a 18-in. X fi,-in. 0.d. stainless steel column packed with Tenax-GC 60/80 mesh, were used for the determination of phenols. A DuPont Model 21-490-1 combination gas chromatographmass spectrometer was used to identify certain chloro-substituted phenols. P r o c e d u r e f o r Determining Phenols. 1) Take a 500-ml sample of water. If phenol standards are added to water containing chlorine, add 15 to 25 mg of hydroxylamine hydrochloride and allow to stir for a t least 5 min before proceeding. Add 15 to 25 mg of sodium hydrosulfite to the sample and adjust the pH to between 12.0 and 12.5 with 2M sodium hydroxide. 2) If a precipitate forms, coagulate it by allowing the sample to sit for about 15 min. Decant the supernatant liquid through a medium porosity, 150-ml sintered glass filter attached to a suction flask. Wash the precipitate into the filter with a minimum amount of water, then wash thoroughly with approximately 50 ml of distilled water. 3) Pour the filtered water and washings into the reuervoir of the absorption column and allow to flow through the resin column a t a rate of 10 to 15 mlimin. When the liquid level reaches the top of the resin bed, wash the column with 25 ml of basic methanol ( 2 ml of 2M sodium hydroxide in 23 ml of methanol) and 25 ml of distilled water. 4) Place a 125-ml separatory funnel under the column and elute the column with 25 ml of 4M hydrochloric acid, then with 25 ml of distilled water, Extract the solution in the separatory funnel with 25 ml of methylene chloride. Allow the phases to separate and become clear, then drain the lower methylene chloride layer into a second 125-ml separatory funnel and discard the aqueous layer. 5) Elute the column with 30 ml of 5:1 acetone-water and 50 ml of distilled water in sequence into the separatory funnel containing the methylene chloride. Shake, allow the phases to separate and clear, then drain the lower organic layer into an evaporating flask. 6) Add a small boiling chip to the flask, attach a Snyder column, and evaporate the solvent over a steam bath until the volume is reduced to approximately 0.5 ml. Remove the evaporating flask from the steam and immediately spray the outside with acetone to condense the vapors inside. Adjust the volume to exactly 1.0 ml with acetone.
7) Inject 2 p1 of the acetone solution into the gas chromatograph, with the temperature programmed from 115 to 230 O C at a rate of 16O/min and hold at 230 " C for 4 min (OV-17 column). Identify the phenols by comparing their retention times with standards. Determine their concentration by comparing either their peak heights or areas with a previously prepared calibration curve. Note. If the acetone solvent peak interferes with some of the individual phenol peaks, the acetone may be removed by adding approximately 7 ml of pentane to the evaporated (1.0 ml) acetone extract. By reevaporating and diluting to exactly 1.0 ml with pentane, the acetone is volatilized by azeotropic distillation. Injection of a pentane solution permits operation of the gas chromatograph at significantly lower attenuation settings without interference from the tailing edge of the solvent peak.
RESULTS AND DISCUSSION Development of Analytical Method. In developing the analytical method the effect of various experimental parameters was studied by adding 1.0 ml of an acetone solution containing known concentrations of various phenols to 500-ml water samples. The recovery of phenols was determined by comparing the peak heights of the recovered phenols with the peak heights of phenols in the standard acetone solution. As expected, the recovery of phenols was found to be a function of the p H of the sample. In general, phenols are completely retained on the resin if the p H of the sample is at least two units higher than the pK, of the phenol, and all phenols studied are retained a t p H between 12.0 and 12.5. Quantitative recovery of phenols can be attained from sample volumes up to 1000 ml, but not from larger sample volumes. Some very hard water samples contain sufficient bicarbonate to form a copious precipitate of calcium or magnesium bicarbonate when the sample is made basic with sodium hydroxide, which restricts flow through the column. Attempts to avoid precipitation by first adding a complexing agent such as EDTA or tartrate were not effective. The carbonate precipitate can be effectively removed by filtration. This is best done by allowing the precipitate to coagulate for 15 to 20 min, then filtering it through a sintered glass filter. Paper filters require tedious and lengthy cleaning procedures to avoid introduction of impurities from the paper. Appreciable fractions of phenols are lost through oxidation in basic solutions and during filtration and column sorption if preventative measures are not taken. Standing a t p H 12.5 results in complete loss of phenol within 4 hours and p-cresol within 24 hours. Loss of phenols during slow filtrations through filters clogged with calcium carbonate was variable but generally was appreciable, ranging up t o 40% of the amount added. No phenols were found adsorbed on the precipitate and thus the losses were attributed to oxidation. When sodium hydrosulfite was added, phenol losses were negligible in basic solutions and were reduced during slow filtrations. Allowing the precipitates to coagulate prior to filtration and decanting the clear water quickly through the filter reduced losses during filtration to undetectable levels. Elution of phenols from the anion exchange column is accomplished by first converting them to the molecular form with hydrochloric acid. However, phenols in the molecular form are still partially retained on the column by sorption so an organic solvent must also be used for elution. Aqueous hydrochloric acid mixed with either acetone or acetonitrile is more effective than other common organic solvents. However, evaporative concentration of acetone or acetonitrile solutions containing hydrochloric acid often produced dark colored solutions and subsequent gas chromatographic analysis showed low results for phenols. Possi-
I
W
I
I
I
2
I
4
I
I
I
6 TIME, MINUTES
I
e
I
I
I
10
Figure 1. Separation of a standard mixture of phenols on OV-17 column
Peak order: ( A ) solvent impurity; ( E ) phenol: (C) o-cresol; (D) p-cresol; ( E ) 3,5dimethylphenol; ( 4 p-chlorophenol; (G)4-chloro-3-methylphenol; (H) 2,4,6-trichlorophenoI; and (0 pentachlorophenol. Separation obtained on a '/&. X 6-ft S.S. column packed with 5 % OV-17 on Chromosorb W-AWDMCS, 60180 mesh. Helium flow rate was 27 ml/min. Temperature was programmed from 115 to 230
OC
at 16'/min and held at 230 OC for 4 minutes
bly condensation reactions in the organic solvent are catalyzed by hydrochloric acid. These difficulties were avoided by eluting the resin with aqueous hydrochloric acid and then with water, followed by extraction of any phenols with methylene chloride. The remaining phenols are eluted from the resin with acetone (or better with 5 : l acetone-water), followed by extraction of the eluted phenols into methylene chloride. This procedure effectively elutes phenols from the column and removes the bulk of the water from the acetone-methylene chloride solution of the phenols. Trace amounts of water remaining in this solut'ion are removed by virtue of a ternary, low boiling azeotrope formed between methylene chloride, acetone, and water and thus no drying step is required. Although acetone tails to a lesser extent than acetonitrile on the OV-17 gas chromatographic column, it does tail to the extent that relatively high attenuation factors must be used t o prevent interference with early eluting phenols. This presents no problem when the concentrations of phenols are above a few hundred parts per billion (in the original water) but does prevent determination of phenols a t lower concentrations even though they are present a t concentrations well within the limits where accurate determinations can be made if the full sensitivity of the instrument were usable. Complete evaporation of acetone solutions requires high temperatures with potential loss of phenols. An effective way of removing the acetone is to add approximately 7 ml of pentane to the acetone. Acetone and pentane form an azeotrope containing 80% pentane and 20% acetone which boils a t 32 "C, compared with the boiling points of acetone and pentane of 56 "C and 39 "C, respectively. Thus, all acetone is removed as the azeotrope, leaving a solution of phenols in pentane. Pentane tails significantly less than does acetone on the OV-17 column, is eluted well before the first phenol peak, and contains no interfering impurities. With the substitution of pentane for acetone, the full sensitivity of the instrument can be utilized to detect phenol concentrations a t the parts per billion level. ANALYTICALCHEMISTRY, VOL. 47,
NO. 8,
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Table I. Recovery of Phenols Added to Iowa S t a t e University T a p Water Compound
Concn, PPb
Recovery, 16"
Concn, ppb
Recovery, %*
Phenol 500 93 25 95 0-Cresol 94 300 15 90 p-Cresol 96 800 40 80 p - Chlorophenol 45 95 100 900 4-Chloro-3 -methyl800 100 40 95 phenol 2,4,6-Trichloro1100 102 55 95 phenol Pentachlorophenol 1700 89 85 80 3,5-Dimethylphenol 700 95 35 90 2 -Naphthol 95 500 ... ... 0 Using calibration curve; average of 9 analyses except for 2naphthol. busing only a single standard, a single analysis is reported to the closest 5%. 0
2
4
6 8 TIME, MINUTES
IO
1 2 1 4
Flgure 2. Separation of a standard mixture of phenols on a Tenax
GC column
Table 11. Determination of Phenols in Refinery a n d Petrochemical P l a n t Effluents Concenhation of Phenols Found in Various Streams by Chromatographic Method, ppm
Peak order: ( A ) solvent impurity: (6)phenol: ( C ) ocresol; (D)3.5dimethyC 2-naphphenol: ( E ) 4-chloro-3-methylphenol: ( F ) 2,4,6-trichlorophenol: thol: and (M pentachlorophenol. Separations were obtained on a %-in. X 18-in. S.S. Tenax GC column. Temperature held at 190 OC for one minute. then programmed at 10 OCImin to 270 OC and held at 270 OC for 4 minutes
(a
Type of Phenol
In this work, emphasis was placed on developing an effective method for isolating and concentrating phenols quantitatively from water. Conditions for gas chromatographic separation of typical phenols were developed, but no attempt was made to develop a universal separation method for phenolic mixtures. The phenols studied are resolved satisfactorily on an OV-17 column (Figure 1).Somewhat less tailing of solvent peaks can be achieved on a Tenax-GC column (Figure 2). Analytical Results. Known amounts of several phenols were added to Iowa State University tap water (-450 ppm hardness) and analyses were performed by the procedure described above. Results are summarized in Table I. A sample containing the first eight phenols was analyzed nine times with the average results as given in the first recovery column. Here the average phenol recovery was 97.7% with a standard deviation of 9.7%. The result for 2-naphthol is an average of three separate determinations in a sample containing 2-naphthol plus several other phenols. In this case, the gas chromatographic separation was done on a TenaxGC column. The results in column five of the table show that the method is applicable for analysis of phenols a t concentration levels as low as 15 to 85 ppb. These results are for a single experiment and are given to the nearest 5%. Other samples were analyzed in which various phenols were added to distilled water or well water. The results were similar to those reported in Table I. Phenols were determined in water samples from an oil refinery-petrochemical plant complex by the chromatographic procedure. The samples undoubtedly contained other organic compounds in addition to phenols. Before analysis, the specific phenols present were not known, but the chromatographic peaks were identified by comparison of their retention times with those of phenolic standards. The quantitative results show reasonably good agreement with those obtained by the refinery group using a colorimetric method (Table 11). The colorimetric method would be expected to give lower results because para-substituted phenols do not react completely with the reagent. 1328
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
Once-thru
Petm-
Biologically
refinery
chemical
treated refinery
cooling
plant process
wastewarera
water b
wastewater"
Phenol 0.01 0.02 0.36 Cresols 0.22 0.22 0.04 Dimethyl phenols 2.59 0.02 0.40 Trimethyl phenols 0.71 0.03 0.17 Other phenolics 0.31 0.07 0.21 Total phenols 3.83 0.36 1.18 Total phenols by 2.3 0.39 1.03 colorimetric method Major components: 2,5-dimethyl phenol (0.87 ppm), 3,j-dimethyl phenol (0.82 ppm). Major components: m - and p-cresol (0.20 pprn). Major components: phenol (0.36 ppm), 3,4-dimethyl phenol (0.32p p m ) , Since copper sulfate is commonly used as a preservative in samples of water taken for determination of organics, special studies were performed to determine its effect on the procedure. At concentrations up to 1000 ppm, copper sulfate has no deleterious effect on the determination of phenols. Inorganic anions in water, such as bicarbonate, can compete effectively with phenolate ions for exchange sites unless sufficient excess resin capacity is present. The recommended H-in. X 6-in. columns have enough capacity to allow for very high concentrations of other anions. A number of neutral organic compounds are retained by the A-26 resin. However none of these compounds affected the recovery of phenols (Table 111). They are eluted from the resin by basic methanol prior to elution of phenols and thus do not interfere with the gas chromatographic determination of phenols. Carboxylic acids also cause no interference with the recovery of phenols using the recommended procedure, but can interfere with the gas chromatographic determination of phenols on OV-17 columns by overlapping the phenols and cresol peaks. When a Tenax-GC column is used, acids elute well before phenols. Chlorination of Phenols. Chlorine in drinking water has been reported to chlorinate phenols (13). During the present study, it was observed that low recoveries of phenol
Table 111. Interference Studya Added compound
Hexyl alcohol Benzyl alcohol 2 -Phenoxvethanol Methyl cellusolve Naphthalene Acenaphthene Hexanoic acid Octanoic acid Butanoic acid
1 1
r
Result
procedure are used, pentachlorophenol losses will be negligible as will losses due to chlorination and oxidation of other phenols.
ACKNOWLEDGMENT No interference
1 -2 to 3% recovery without MeOH wash. No interference after MeOH wash
We thank M. D. Grieser and M. D. Arguello for their preliminary work on the sorption of organics by A-26 resin, and M. Avery for mass spectrographic identification of chlorination products of phenols. The authors are also grateful to H. J. Svec for valuable discussions and suggestions, and to J. J. Richard and G. A. Junk for their assistance.
Interference with phenol and cresols on OV-17
='Samples contained approximately 1 ppm each of several phenols and 20 ppm of each added compound. and alkyl phenols added to tap water coincided with enhanced recoveries of chlorinated phenols. Addition of low concentrations of chloramine T to very dilute aqueous solutions of phenols caused similar results. Letting 3,5-dimethylphenol solutions prepared in chlorinated tap water stand for a few minutes resulted in -40% loss. New GC peaks from extracts from this solution were positively identified by mass spectrometry as being the di- and trichlorodimethylphenols. Chlorination reactions during the determination of phenols can be prevented by addition of hydroxylamine hydrochloride. On real samples, however, it must be recognized that chlorination reactions may have occurred before the sample was taken. The recovery of pentachlorophenol was affected by the amount of reductants used to prevent oxidation and chlorination. The more reductant added, the lower its recovery. If the amounts of reductants specified in the recommended
LITERATURE CITED (1) A. K. Burnham, G. V. Calder. J. S. Fritz, G. A. Junk, H. J. Svec, and R. Willis, Anal. Chem., 44, 139 (1972). (2) A. K. Burnham. G. V. Calder, J. S.Fritz, G. A. Junk, H. J. Svec, and R. Vick, J. Am. Water Works ASSOC.,65, 722 (1973). (3) J. J. Richardand J. S.Fritz, Talanta, 21, 91 (1974). (4) J. L. Witiak, G. A. Junk, G. V. Calder, J. S. Fritz, and H. J. Svec, J. Org. Chem., 38, 3066 (1973). (5) G. A. Junk, J. J. Richard, M. D. Grieser, D. Witiak, J. L. Witiak, M. D. Arguello, R. Vick. H. J. Svec, J. S. Fritz, and G. V. Calder, J. Chromatogr., 99, 745 (1974). (6) J. J. Richard, G. A. Junk, M. Avery. N. Nehring, J. S. Fritz, and H. J. Svec, J. Environ. Qual., (submitted for publication.) (7) W. D. Beer, Wis. Z. KarlMrxUniv., 8, 67, (1956/59). (8) R. L. Whitlock, S. Siggia. and J. E. Smola, Anal. Chem., 44, 532 (1972). (9) "Standard Methods for Examination of Water and Waste Water", 13th ed., American Public Health Association, New York, NY, 1971 (10) A. W. Briedenbach, J. J. Lichtenberg, C. F. Henke, D. J Smith, J. W. Eichelberger. and H. Steirli, U.S. Dept. of Interior Pub. WP-22 (Nov. 66). (11) J. S. Eichelberger, R. C. Dressman, and J. E. Longbottom, Environ. Sci. Technoi,, 4, 576 (1970). (12) J. A. Vinson, G. A. Burke, B. L. Flager, D. R. Casper. W. A. Nylander, and R. J. Middlemiss. Environ. Lett., 5, 199 (1973). (13) R. B. Dean, News Environ. Res. Cincinnati, July 5 , 1974.
RECEIVEDfor review January 13, 1975. Accepted March 13, 1975. Appreciation is expressed to the National Science Foundation (Grant No. GP-32526) for financial support.
Modern Liquid Chromatography on Spherosil Jean Vermont, Michel Deleuil,' A. J. de Vries,' and C. L. Guillemin Centre de Recherches Rhbne-Progil, 93308 Aubervilliers, France
The separation ability of various types of Spherosil, as a packing materlal for high performance Liquid-Solid and Liquid-Liquid Chromatography, has been studied as a function of various parameters: column geometry, particle size, specific surface area, and coating by a stationary phase. The effect of column geometry with respect to particle size has been demonstrated by introducing a dimensionless number, the Knox-Parcher ratio: dpL/dc2,which should be as small as possible in order to obtain maximum column efficiency. Fast separations have been obtained by Liquid-Solid Chromatography under low pressure drops (5 bars), with columns of short length (5 cm), packed with small particle size (5-10 M ) of Spherosil of high specific surface area (600800 m2/g). In Liquid-Liquid Chromatography, an appropriate heating treatment of Spherosll, coated with large amounts of &p'-oxydipropionltrile, allows rapid separations without base-line drift and without presaturatlon of the liquid carrier.
Present address, Centre de Recherches RhBne-Progil de la Croix de Berny.
The first chromatographic applications of Spherosil have been started with liquid phase exclusion chromatography as early as 1966 by De Vries and Le Page (1-3), followed by numerous other studies in the field of liquid (4-11) and gas chromatography (12-16). For the purpose of the present study in Liquid-Solid (LSC) and Liquid-Liquid Chromatography (LLC), we have used a number of commercial and experimental batches of Spherosil with particle sizes below 40 p , fractionated by sieving in order to obtain particles of different size ranges, between 10 and 40 p. Moreover 5- to 10-p particles directly generated a t that size were used without any subsequent sieving. The physical properties of the materials used are given in Table I.
EXPERIMENTAL Apparatus. Systematic experiments were performed on a homemade liquid chromatograph using a membrane pump Orlita, type M3S 4/4 and a LDC detector: R.I. model 1103, or U.V. model 1205 (254 nm), depending on the nature of the mixtures to be separated. Before reaching the pump, the solvent was continuously degassed, then passed through a filter. Downstream from the pump, two ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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