V O L U M E 28, NO. 2, F E B R U A R Y 1 9 5 6
157
and qualities of the products, or the effect of some variable on these properties. T o compare results from two such operations it is necessary to know the least differences which can be considered significant. The least significant difference is a function of the precision with which the results are known, and this may be computed by application of the t test.
LSD
=
Table V.
Least Significant Difference of Typical Process Results
Yield or Quality C.-. wt. %
~ S D
Std. Dev. of Individual Detma
Least Significant Difference a t 95% Confidence Level for Total of 8 Testsb
zt0.6 10 9
0.8
where
S D = standard deviation of the difference = S & + z 1 S = standard deviation of the individual results -VI and N ? = number of individual results in each operation t = 1.96 for 95% confidence with infinite degrees of freedom
a Based on many determinations made in quality control program. b For example, in 8 total tests, 4 would be made on Catalyst A and 4 on Catalyst B . The “least significant difference” between Catalyst A and B , a t the 95% confidence level, is calculated as:
L.S.D.
= tS
d$, +
1 S,
where S = standard deviation of an individual deteryjnation, A’i and number of determinations in each operation and t = Student’s’’ 1.
.V2
=
(The meaning of t is explained in any standard book on statistics and its values a t any confidence level with various degrees of freedom are available in tables.) Some typical examples of the least statistically significant differences of process results are shown in Table V. It may be seen that differences in C g c yield, for instance, must exceed 1.2% before they may be considered statistically significant a t the 95% confidence level, based on the data given in the table. This type of information has been invaluable in appraising differences in results from pilot plant work.
better precision with the same number of runs. The value of this to a research program is often inestimable. An intangible benefit of a statistical quality control program is an awakening of the technical and nontechnical personnel alike to the need of quality in all steps of a process to achieve the desired results. This often results in an automatic reduction in variance in a short period of time.
CONCLUSION
ACKNOWLEDGMENT
Some 10% or less of the analytical time and the small pilot unit time is spent in the statistical quality control program. This expenditure has been amply justified by the results obtained. I n addition to the benefits indicated above, it has been possible, because of a knowledge of the variance distribution, to reduce the over-all variance of some of our units. This results in a reduction of runs necessary to achieve a given precision, or
The authors wish to thank C. E. Kling for coordination of the analytical quality control program and for computation of the many precision values, and E. bl. Charlet and L. M. Addison for their assistance in effectively applying the quality control system in the analytical laboratories. RECEIVED for review June 16, 1955. .4ccepted October 28. 1955. Division of Petroleum Chemistry, 126th Meeting, ACS, Nen York, September 1954.
Determination of High Boiling Paraffin Hydrocarbons in Polluted Water F. J. LUDZACK
and
C. E. WHITFIELD
Robert A. Taft Sanitary Engineering Center, Public Health Service, Cincinnati, O h i o
U. S. Department
Investigation of oil pollution in surface water has frequently been confused by analytical problems. The methods may have failed because of incomplete recovery of oily materials, lack of identification of the major components, or inadequate removal of interference. The proposed method includes an improved wet extraction procedure, infrared analysis for quantitative and classification purposes, and chromatographic separation to isolate mineral oils from animal or vegetable oils, soaps, and miscellaneous extractable components found in polluted water. This procedure makes it possible to determine the amount, type, and condition of oily residues in the presence of mixed components in polluted water.
T
HE American Petroleum Institute ( 1 ) includes as oily wastes the highly complex mixtures of petroleum and their sulfur, nitrogen, and oxygen derivatives. It classifies oily wastes according to the tendency to form films, emulsions, or sedimentary deposits. Rirschman and Pomeroy ( 7 ) define an oily
o f Health, Education, and W e l f a r e ,
waste as a relatively nonvolatile unsaponifiable liquid that contributes to the formation of oil films or deposits. Other definitions of oil are based primarily upon the method of analysis and include whatever is recovered by a particular procedure. Definitions of this type (2-dj 8,10-12) may lead to results that are lacking in specificity. The definition for oil used in this investigation includes the relatively nonvolatile components that exhibit the characteristic infrared curves of saturated aliphatic hydrocarbons. This definition excludes materials such as fatty acid soaps, animal or vegetable oils, and coloring materials, n-hich may be associated r i t h oils in a stream. Interferenre due to associated colloidal solids in the extract is minimized. The analyst could estimate the magnitude of significant interfei ence and the type of material that had to be removed to obtain a specific result. Investigation of oil in surface water frequently is more complex than determining oil in an isolated industrial waste. Stream concentrations of oil are generally lower; interference is increased by domestic and other industrial wastes. Living and dead microorganisms in the stream not only absorb oil but also tend to stabilize emulsions between sample and solvent during most
ANALYTICAL CHEMISTRY
158
extraction procedures. Several of the established oil determinations that were satisfactory on fresh industrial wastes failed 11-hen applied to samples containing mixed wastes and biological detritus. Extraction difficulties were controlled by a modified liquidliquid extraction of the sample with carbon tetrachloride, followed by infrared determination of oil in the same solvent. Interference revealed by the infrared curve was removed by chromatographic separation and a corrected curve was obtained on the specific class of hydrocarbons. 4hALYTICA L PROCEDURE
Apparatus (Figure 1). Liquid-liquid extractor, l-liter>Sargcnt No. 31414. Boiling flasks, 500-ml., with ball joint socket 25/35 mm. Allihn-type condenser, 600-mm., water-cooled, 29/42 joint. Glass air inlet line (8-mm. diameter) of sufficient length to extend from the top of the condenser to about 15 cm. from the bottom of the extractor column. Separatory funnel, 2-liter capacity. Soxhlet flask with condenser and distillate trap. A small bulb should be blown in the flask near the bottom to serve as a collecting well to facilitate extract removal with a transfer pipet. Infrared spectrophotometer, sodium chloride optics used. Liquid cell, path length approximately 0.35 mm. Chromatographic column, 10 X 300 mm. Fritted-glass filter apparatus for vacuum operation. Air compressor of a type which will minimize oil contamination. Activated carbon air filter, 4 inches X 4 feet. Reagents. Analytical grade carbon tetrachloride. This material should have no significant infrared absorption from 4000 to 1'700 cm.-l Hydrochloric acid, concentrated. Anhydrous sodium sulfate, powder. Chromatographic alumina, Fisher S o . A 541/2, 80 to 200 mesh, was suitable. Reference standard. This material should be representative of the oil to be determined. Eastman octadecane was used in this work. Procedure. The assembled apparatus should be diy; use a current of warm air if necessary After drying, clean the entire apparatus by using it as air condenser for solvent distillation. To clean apparatus connect the 500-ml. flask to the side arm and add approximately 300 ml. of solvent to the extractor column. Add Berl saddles to the column to a point slightly above the junction of the solvent return arm. Tamp glass ~vool(extracted if necessary) in place lightly to form a return solvent filter of 1 to 2 em. in thickness above the Berl saddles. Add the sample (1 liter or aliquot). The solvent-sample interface should be located 2 to 3 cm. above the glass wool. Do not permit the glass wool or the solvent vapor arm to be ivet ivith water. Add concentrated hydrochloric acid to reduce the sample p H to 3.5 or below. Check the amount of acid required on a separate aliquot. A lower pH may be desirable for breaking a persistent emulsion. Connect the condenser to the column and lower the air inlet to within 2 to 6 cm. of the solvent-sample interface. When solvent is refluxing into the column a t a suitable rate, adjust the air flow into the column to break the surface film of solvent and reduce the size of solvent globules. Time the extraction from the point of air adjustment. If the oil emulsion is broken in less than 1 hour, 4 hours' total time should be adequate for extraction. At the end of extraction, rinse the air inlet line and condenser with fresh solvent. Pour the contents of the column into a 2-liter separatory funnel through the glass wool filter. Combine sample container rinse and residual solvent from the column with the extract. Add approximately 10 grams of anhydrous sodium sulfate to the combined extract and mix. Filter the dried extract through fritted glass, using suction. Transfer the sodium sulfate to the filter and break up any lumps, before rinsing. Transfer the filtrate to a 125-ml. Soxhlet flask by increments and distill to approximately 10 ml. a t a rapid rate. Use a steam bath for final concentration to approximately 2 ml. Sweep the vapors off with a gentle stream of nitrogen (preferred) or dry air. Adjust the final volume of the extract on the basis of infrared absorption results or prior sample information. The sample should never approach the solvent disappearance point a t any stage of the operations and should be protected from moisture condensation. Calibrate the infrared instrument for zero adjustment with glass in the sample position; adjust for 100% transmittance in the critical areas with solvent in the sample cell. Establish the
absorbance of a known concentration of a reference standard several times during a series of sample runs. Use a low gain setting and slow speed a t the critical absorption bands. The iesults of a sample determination a t this stage includes everything absorbing at 2925 cm.-l in terms of the reference equivalent. Hydrocarbons and oxygenated extractables (Hc Ox) may be shown. Qualitative data and a rough quantitative estimate of contamination can be obtained from the position and intensity of extra absorption bands. After this infrared determination, concentrate the sample to 2 to 3 ml. Prepare a column of activated alumina (10 X 150 nun.) and prewet with two bed volumes of carbon tetrachloride (20 ml.). Allow the prewet solvent to drain to a dull top surface and add the sample. Rinse the sample container with solvent and add more solvent to collect 30 ml. of eluate. Concentrate the eluate to a suitable volume as indicated by the previous deteimination. Perform a second infrared determination on the chromatographed sample. This result includes only the mixed hydrocarbons (Hc) in terms of the reference equivalent. Appearance of interference in this fraction indicates improper technique in chromatographing, column overloading, or an unsuitable adsorbent.
+
SAMPLE PREP4RATION
,411 fresh samples of hydrocarbons were dispersed in water nith a Waring Blendor. The resulting emulsion was stable for an indefinite period in a sterile system. Appreciable dilution did not affect it. Other samples were homogenized in an Eppenbach Type QV6-1 vertical mill if an aliquot was required for anal? sis. Best results viere obtained when the sample was taken in a separate bottle and the entire amount used for analysis. EXTRACTION
The sample was extracted by continuous downflow application of freshly distilled solvent. Countercurrent air flow caused a decrease in solvent globule size and greater interface area, increased solvent retention in the column, frequent reformation of interface area by collision of the solvent and air, and better distribution of solvent, suspended solids, and sample. mithout countercurrent air 70% extraction of an octadecane emulsion was obtained in 23 hours; with air, more than 90% extraction was obtained in 2 hours. The agitation described did not produce excessive emulsification of sample and solvent. Only clean extract returned to the boiling flask. The solvent filter broke the slight emulsion of sample and solvent and retained suspended solids in the sample column. Success or failure of the extraction was indicated early in the procedure by observation of sample clarification. If it did not calarify within a reasonable period, additional chemical treatment, different treatment on a new aliquot, or a smaller aliquot could he started without an excessive time loss.
A T E D CA980k 41R FILTER
5AIhER WOOL F i ' l T E D CLmSS FILTSR
R U4B, BRE RP UT M U aPl N G
t SOLVENT REBOILER SAMPLLiN SOLVEN TERFb GLASS F l L TWECi
Figure 1.
Extraction assembly
V O L U M E 2 8 , NO. 2, F E B R U A R Y 1 9 5 6
159
I
w
:0 3 0 L
0 20
0 0
0
2925
Figure 2.
3725
690
450 1900 I370 WAVE NUMBERS I N Cm
1300
I40
Relative infrared absorbance of samples at various band positions
15.0 mg. of sample per 5 ml. of carbon tetrachloride
Infrared absorptivity of the motor oil was appreciably less than that of octadecane. Absorptivity varied with different oil samples. Results expressed as an octadecane equivalent, were, therefore, minimum results. Other physical and chemical characteristics of the reference and oil were similar except for the additives, the nonparaffinic hydrocarbons, and the relatively wide volatility range of the oil. Infrared absorption a t 2925 em.-' \+asthe most suitable analytical band for the desired concentration range of oil. As this absorption also is characteristic of the carbon-hydrogen bands of alicyclic, olefinic, and aromatic hydrocarbons or their sulfur, nitrogen, or oxygen derivatives, man? substances could interfere a ith the determination of paraffin series hydrocarbons. Significant interference was indicated and classified by the nature of the infrared curve. Hrdrocaibon absorption conformed with Beei's lav. Base line technique similar to that of Heigl ( 5 ) and Simard (IS) was used, eucept that the base line was obtained from the solvent curve. Sample calculation reduced to: mg. reference/unit volume Rlg. sample/unit volume = AsampleX Areierence FT here A = sample or reference absorbance (6).
[
-1
INTERFEREYCE RElMOV4L
53
0
0
2.5
1
I
5.0
75
I
I
I
10.0
12.5
'5.0
Mp O C T A D E C A N E / 5 m l
Figure 3.
I
I
17.0
240
I 22.5
25.0
CCI,
Calibration curve of octadecane absorption z?s. concentration
Blank determinations, consisting of extraction of oil-free water should be made to check effectiveness of air and apparatus cleaning. A b w and consistent blank requires careful operation. Use of the carbon tetrachloride for both extraction and infrared measurement minimized errors associated with solvent removal, such as overheating, creeping, sample volatilization or ouidation, and identification of the solvent disappearance point. Loss of sample was insignificant as long as the temperature did not ex(seed the solvent boiling point or the sample vapor pressure was below 5 mm. of mercury a t 100" C. INFRARED DETERMINATION
A Baird recording spectrophotometer with sodium chloride optics was used as a single-beam instrument. A solvent curve was recorded on each chart with sample rurves superimposed and color coded. Duplicate readings were obtained. The curve included the spectral region from 4000 to 1000 cm.-l in which carbon tetrachloride absorption introduced significant interference only a t 1600 to 1500 and 1260 to 1190 em.-' Drift or other instrument variables were detectable by the curves of the solvent, reference sample, or a polystyrene film. The path length of the cell was suitable for the desired sensitivity range for oil, but did not obscure significant sample information by excessive solvent absorption. Octadecane, 99+ mole % purity, was selected as a reference material, so that all results could be expressed in terms of a definite material. The molecular --eight of the reference was relatively close to that of the average for medium weight oil.
Chromatographic technique was used to remove interference from the extract. Interference in the persistence study on motor oil (9) consisted mainly of the oxidation products of hydrocarbons. Biochemical oxidation of motor oil could result in the formation of peroxides, alcohols, aldehydes, acids, esters, ethers, and other oxygenated materials as intermediates in the path toward carbon dioxide and water. Alumina was the most satisfactory adsorbent for these intermediates. When a sample containing oxygenated compounds passed through the alumina column, hydrocarbons appeared first in the carbon tetrachloride eluate. The results of the procedure can be illustrated by the behavior of a carbon tetrachloride solution of octadecane, methyl stearate, and oleic acid. The sample was concentrated to a volume of 3 ml., applied to a 10 X 150 mm. column of alumina, and developed with carbon tetrachloride. The first two bed volumes of eluate contained all of the octadecane. Methyl stearate appeared in the fifth bed volume of eluate. Oleic acid was not completely recovered by protracted development of the column. Shorter columns and overloading resulted in separation failure. Infrared was invaluable for the identification of the various fractions as well as in the control of technique. RESULTS
Figure 2 is a bar graph of the absorbance us. band position in cm.-l of fresh samples and oxidation system extracts. Each sample was based on 15 mg. of material, or the octadecane equivalent thereof, in 5 ml. of carbon tetrachloride. The analysis band for oil was located a t 2925 cm. - I ; secondary bands appeared a t 1450 and 1370 cm.-l. The difference in absorptivity of oil and octadecane was apparent from samples 1 and 2. Absorbance a t 1725 to 1690, 1400, 1300, and 1140 cm.-' (sample 3) was produced by sample components other than h2-drocarbons. This type of absorption was common whenever motor oil was subjected to biochemical Oxidation. Sample 5, oleic acid, shows similarity to sample 3. It also shows that an estimate of hydrocarbons could not be obtained without purification. Absorption a t 1725 to 1690 cm.-' is a characteristic of carbonyl compounds. This absorption served only to indicate interference, not to correct for it. Sample 4, Figure 2, shows the effect of alumina chromatographj- on a sample similar to 3. Only the hydrocarbon absorption was found. Assuming equal concentration and absorptivity, the infrared curve was identical to that of fresh motor oil, Figure 3 is a calibration curve of octadecane absorbance us.
160
A N A L Y T I C A L CHE M 1STRY
concentration. Within the region bounded by an absorbance of 0.10 to 0.55, linearity was good. Instrument response was poor and the results were erratic outside of this range. Extract concentration was regulated to use the optimum instrumental range, from 5.0 to 20 mg. of octadecane equivalent in 5 ml. of extract or 0.5 t o 4.0 mg. in 1 ml. of extract. Precision and recovery data on manipulation including extraction, concentration, and infrared on fresh octadecane emulsions are shown in Table I. Four replicates a t three concectrations have a standard deviation of 1 mg. or less except when a dilution factor was involved as in series 3. Preliminary recovery data on extraction of motor oil or octadecane (16 samples, in concentrations from 35 to 140 mg. per liter) varied from 89.5 to 99%. There was no tendency for higher concentrations to show decreasing recovery, as shown in Table I.
Table I.
Precision and Recovery Data on Fresh Octadecane Emulsions
Series Added octadecane, mg./liter Sample S o . x -1 x -2 x-3 x-4
-X , mean, mg./liter
S, standard deviation, nig. Coefficient of variation = 100 (S/T),% Recovery, %
1 4.0 3 3 3 5
2 25 7 Found, Mg./Liter 25 8 24 3 24 3 23 5
9 2 6 0
3 64 5 59 4 60 9
57 9 56 1
3.9 0.8
24.5 0.9
58.6 2.4
21 97.5
4 95.3
4 92.0
Table 11. Precision of Hydrocarbon Analysis on Biochemical Oxidation Systems with Rlotor Oil Feed (Hc Ox reported)
+
Replicate S o .
Found, hIg./Liter so. 2 11.5 10.5
KO.l 4
6.4
11.1 11.1
No. 3 36.6 36.4 34.8 35.8
1.1
11.1 0.4
35.9 0.8
3 6
2.2
7.3 8.2 5.6
S mc.
Cbefficient of variation, % 16.0 Heavy growth of algae present.
Table I1 s h o m the same type of data on motor oil oxidation system extracts. Results were similar to those in Table I. Table I11 includes the results of oxidation extracts before and after chromatographic separation. Precision \vas similar but the results show the removal of more than one third of the octadecane equivalent. Ti% would have appeared as a positive error in t,he results for oil, as defined here. Results of alumina chromatography on fresh oil or octadecane, extracts of oil oxidation systems and of sewage are shown in Table 1ir. Seventeen samples of oil or octadecane were processed without significant loss of oil. Hydrocarbon in the extracts of the motor oil oxidation systems varied from 66 to 28%. Sewage extracts showed a relatively small percentage of hydrocarbon. The relatively large error possible in the determination of oil in a mixed system without suitable correction is obvious. This analytical procedure was designed to obtain specific information on oii in the presence of relatively large concentrations of nonhomogeneous inteiference. I t n-ould be useful in the interpretation of results of the more rapid routine procedures used on a consistent type of sample. I t was the authors' experience that anomalous results frequently c o d d be resolved by the greater specificit,y of this method. Alkane series hydrocarbons were separated from oxygenakd products by alumina. Alumina was less effective for the separation of alkanes from aromatic interference. This and other separations would be possible by suitable selection of column technique, adsorbent, and developing solvent. CONCLUSIONS
Continuous liquid-liquid extraction Kith air agitation W ~ effective for recovery of oil from nonhomogeneous samples. Infrared results provided a quantitative measure of oil concentration and information on the specificity of the result. Chromatographic separation v a s effective for the conversion of mixed systems into homogeneous fractions. ACIPJOFLEDGMENT
The authors gratefully acknowledge the helpful suggestions by numerous individuals during the progress of this work. A. A. Rosen, Organic Analytical Unit, this laboratory, was particularly helpful on chromatographic and infrared technique. LITERATURE CITED
Table 111. Precision of Results on Oil Oxidation System Extracts before and after Alumina Chromatography Sample S o . 1 x-2 x-3 x-4
x-
-
Found, Mg./Liter Before (Hc f Ox) After (Hc) 24.7 15.8 23.7 16.0 23.7 15.2 22.4 14.8
Am. Petroleum I n s t . , " M a n u a l o n Disposal of Refinery Wastes, See. I, "Waste W a t e r Containing Oil," 4th ed., 1949. Am. Petroleum I n s t . , " M e t h o d s for Sampling a n d Analysis of Refinery Wastes," 1st ed., 1953.
A m . Public H e a l t h Association, "Standard M e t h o d s for t h e Examination of W a t e r and Sewage." 9th ed., 1946. Assoc. Offic. Agr. Chemists, "Official J I e t h o d s of Analysis," 1950. Heipl, J. J., Bell, 11. F., and W h i t e , J. U., d x - a ~CHEM. . 19, 293
(1947). H u g h e s , E. K . , Ibid., 24, 1349 (1952). (7) Kirschman, H. D., and Ponieroy, R . , Ibid., 21, 742 11949) (8) Levine, JT'. S..N a p e s , G. S., a n d R o d d y , hI. J., Ibid., 25, 1840 (1953). (9) L u d z a c k , F. J . , a n d K i n k e a d , D i a n a , "Persistence Table IV. Octadecane Equivalent on Samples before and after of the hlotor Oil Class of Hydrocarbons in PolAlumina Chromatography luted W a t e r under Aerobic Conditions," 127th Found, Mg./Liter Meeting. ACS, Cincinnati, Ohio, 1955. Before After Found, 70 Sample (10) N e l p o l d e r , F. hI., Warfield, C. W., a n d HeadingKO. (HC ox) (Ere) HC/(HC iOX) t o n , C. E., ANAL.CHEM.25, 1453 (1953). 97-102 (fresh preparations, (11) h l u s a n t e , .4. F. S., Ibid., 23, 1374 (1951). ... ... Y1in.-Max. Octadecane l 7 samples) Reported (12) Ruchhoft, C . C., hfiddleton, E'. M.,Braus, H a r r y , a n d Rosen, A. A., I n d . Eng. Chem. 46, 284 hIotor oil oxidation system extract 19.2 12.8 66.7 13.1 6.6 50.4 (1954). 18.4 7.3 39.7 (13) S i m a r d , R. G., Hasegawa, Ishiro, B a n d a r u k , 18.9 6.6 34.9 William, and Headington, C . E., ANAL.CHEM. 12.8 3.6 28.2 23, 1384 (1951). Fresh sewage extract X , mg./liter 23.6 S mg. 0.9 Cbefficient of variation, 4 Loss as octadecane equivalent, 70
h'otoLpil
I
15.5 0.6 4 34.5
_
_
-
\
+
RECEIYED for review June 20, 1955. Accepted November 9 , 1955. Division of T a t e r , Sewage, and Sanitation Chemistry, Symposium on Problems in Stream Pollution, 127th Meeting. ACS. Cincinnati, Ohio, %larch-April 1956.
E
