8
ANALYTICAL CHEMISTRY
reacted iodine, and titration with 0.1.4’ thiosulfate using a Gilmont ultramicroburet of 1-ml. rapacity. RESULTS
Two known mixtures of glucose and the three pertinent niono0-methyl-D-glucoses were analyzed. A comparison of the amount of each sugar weighed into the mixture and spotted on the chromatogram t o the amount of that sugar determined by elution and titration is presented in Table I. It is evident from this tablr that a quantitative separation of the mono-0-methylD-glucoses was obtained. An indication of the efficiency of the separations is given in the per cent error column. The colors of the spots of the three mono-0-methyh-glucoses were not identical: 2-O-methyl-~-glucose gave a lavender spot, whereas the 3-O-methyl-~-glucose spot was yellowish brown and the 6-0-methyl-n-glucose spot a brownish yellom-. ACKNOW L E I X MEYT
Grateful acknowledgment is made t o the American Viscose Corp. for sponsoring the research fellowship under which this
work was carried out. The authorh wish to thaiik R. 1,. Whistler, J. K. N. Jones, and R. E. Reeves for supplying the methylated glucoxcs used in this study. LITERATURE CITED
(1) Brown. F.,and Jones, J . K. K’.,J . Chem. SOC.1947, p. 1344. (2) Hirst, E.L.,Hough, L., and Jones, J. K. N., Ibid., 1949, p. 928. (3) Hough, L., Jones, J. K. N., and Wadman, FV. H., Zbid., 1950, p. 1702. (4) Jones, J. K.N., and Smith, F., ildvames in Carbohydrate Chem. 4, 243 (1949). (5) Lemieux, R. U.,and Bauer, H. F., Can. J . Chem. 31, 814 (1953) (6) Macdonald, J. T.,J . Am. Chem. Soc. 57, 771 (1935). (7) Mahoney, J. F., and Purves, C. B., Ibid., 64, 9,15 (1942). (8) Peat, S.,and Whetstone, C., J . Chem. SOC.1940, p. 276. (9) Rebenfeld, L.,and Pacsu, E., Teztile Research J . 24, 941 (1954). (10) Timell, T., “Studies on Cellulose Reactions,” Esselte Aktiebolag, Stockholm, Sweden, 1950. (11) Traube, W., Piwonka, R., and Funk, A . , Ber. deut. chevr. Ges. 69B, 1483 (1936). KECEIVEDfor review iZugust 5 , 1955.
.iccepted Segteiiiber 15, l’J5.5
Chromatographic Determination of Gum in Fuels H. S. KNIGHT, THURSTON SKEI, SIGURD GROENNINGS, and A. C. NIXON Shell Development Co., Emeryville, Calif.
A novel chromatographic method for the determination of gum in small fuel samples is presented. The gum content is related to the length of a brown zone observed when the sample is displaced over silica gel in a very small column with 1-methylnaphthalene as solvent and acetone as eluent. Although the method was developed for use in research on fuels of limited availability, its simplicity makes it more generally attractive. This chromatographic determination of gum appears to be applicable to soluble and insoluble gum in jet fuels, gas oils, and possibly gasolines. Additional data will be required to establish its merits for routine control purposes.
F
UEL deterioration during storage in the presence of air re-
sults in the formation of various oxidized products, which leave a gummy brown residue on evaporation. Often precipitation of insoluble material also occurs. Prevention or minimization of gum formation is a continuing problem which is particularly severe with cracked fuels. The problem is becoming more acute as our dependence on cracked fuels increases and the engines in which the fuels are consumed become more complex and more critical. Gum is normally determined by evaporation of a 50-ml. sample of fuel a t elevated temperatures in a jet of air or steam. The residue is weighed and the gum content is reported in milligrams per deciliter (1). This method is time-consuming, requires special equipment and relatively large samples, and sometimes gives inconsistent results. Also, the results are affected by the end point of the fuel ( 4 ) . The volume requirement is not significant normally, but becomes important in studies on experimental or salvaged fuels of limited availability. I n connection with a study of stability of experimental fuels, adsorption on silica gel was employed as a means of isolating the gum fraction. During the elution step it was noted t h a t a considerable brown zone developed. On the basis’of this observation an investigation was made to see if the length of the dark
zone could be related to the gum content. As fuel volumes frequently were limited, these studies were mads in equipment similar t o that used for small scale chromatography as applied to hydrocarbon group analysis ( 2 ) . This paper introduces the resulting chromatographic or “chromatogum” method which hab been found suitable for a wide variety of jet fuels, as well as foi gas oils. Incomplete data suggest that it may also be applied to gasoline. To meet the needs of the above stability study it was necessai v to show a correlation between the chromatographic and the steam-jet gum results for jet fuels and gas oils having a wide range of gum contentg. For routine use on unaged samples more emphasis on the low gum region is desirable. I n the long view, it may be preferable to use the chromatographic method as a new approach, independent of the evitporation methods, for determining oxidized material in fuels. SUhIhlAHY OF METHOI)
-4small glass adsorption column nith a long mpi1lar)- extension is packed with fine activated siliva gel and 0.5 t o 1 ml. of sample is introduced. This is followed hy a small quantity of I-methylnaphthalene and then aretone eluent. The length of a brown zone containing gum is measured :tiid enipiricallv correlated ~ i t the h steam-jet gum content. DEVELOYMIIYT OF METIIOI)
A typical jet fuel sample 1%as divided and one portion was aged with oxygen a t elevated temperature to increase its gum content. These samples were subjected to chromatography to see whether zones were obtained that could be related to the gum contents. When a sample was added to a long, narrow column of Davison’s Grade 923 (100 to 200 mesh) silica gel and displaced by isopropyl alcohol, a brown zone was formed adjacent t o the alcohol front. This zone was unexpectedly long for the small amount of gum present, and its length was approximately proportional t o the gum content. Thus the feasibility of the chromatographic approach was indicated. -4dditional samples of jet fuels, as well as gasolines and gas oils, covering the range of gum concentrations of
V O L U M E 28, NO. 1, J A N U A R Y 1 9 5 6 interest, were used ill t,hc stud!. of the variables affecting the malysis. Eluents. Eluents investig:itetl include isopropylamint,. dimethylformamide, isopropyl :i,l(*ohol,methanol, pyridinr, formic acid, and acetone. S o n e of t,hese displaced all of t,he brown Inaterial from all of the samplrs. However, acetone, isopropylamine, anti inethanol Ivere aniong the best and were :tbout equally good. .4c*rtont, was selected :is the eluent for futuw nmrk for reasons of wnvc~nirnre.
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Adsorption column
Column Design. A special coIuinn, designed t o provide sh:irp b r o m zones with very small snniplcs, is shown in Figure 1. I t consists of it relatively broad “c*h:ugrr” section at the tOlJ, a narrower “separator” section in thr middle, and a true-bore capillary “analyzer” section a t t,he bottom in which the Lon(’ measurements are made. Sample Size. The sniall gum column requires only 0.5 to 1.0 ml. of jet. fuels or gas oils. For greater accuracy in the low gum region, : m i p:trticularly for gasolines (which normally exhibit very low chromatographic gum contents), it may be necessaqto employ up to 5 ml. of sample. This requires a preliminary, rapid gum separation in a second column. The data in thip report, however, were all obt,ained by the one-column technique. Flow Rate. The brown zone becomes longer and more diffuse :is the pressure and flow rat? are incwascd. I t was found that, pressures of up to 10 pounds per squ:ti-c inch can be employed when the brown zone is in the upper part of the column, but t,hat when it reaches the lower part, n-here readings are taken, the pressure should he decretwrd to 2 or 3 pounds per square inch. Inhibitors. Ionol (2,6-di terf-butyl-4-methylphenol) and Tenamine-1 (~l’-n-butyl-p-aniinophcnoI) in abnormally high conccntration (0.03% or 10 pounds per 5000 gallons) had little effect on the broxvn zone obtained with an aged thermally cracked jet fuel. \Vith the same amount of LOP-F) (:V>.V-di-sec-butyl-p-phenylenedi:tmine) there was a 10 to 15y0 increase in gum zone length. It is therefore concluded that the inhibitors in pract,ical concentrations ( 1 pound per 5000 gallons) would not affect the results sign; ficsantly.
9 Adsorbelits and Sample Composition. The relatively long hrown zones obtained on Grade 923 si1ic.a gel with samples containing a t most, only a fraction of :L p e r cent of gum, may be esplained on two bases: The gum molecules are too large to enter the pores of the adsorhcnt (32 iz. average pore diameter); and the gum forms an adsorption azeotrope or “asorhotrope” with some of the sample aromatics. This asorbotrope, or mixture which is not ,separated f)y passage over t,he adsorbent ( S ) , is more strongly adsorhcd than the remaining sample aromatics and, hence, ;tppe:trs hrtween them and the acetone front. The brown zone is long, lircause the concentration of gum in the asorbotrope is low. These hypotheses have been studied and both have some merit. Several adsorbents with larger pores than the Grade 923 silica were employed for gum separation, including Grade 70 silica gel (100 A , ) , alumina (40 .4.)> and silicic acid (50 to 100 A.). In all cases the gum was much mort: strongly adsorbed and in a shorter zone than on Grade 923; i n fact the gum often could not be displaced by any available eluent. Adsorbents of smaller pore size \\-ere not studied. Since Grade 923 gel appeared to he witable, it was used for all subsequent work. Fuel composition also affects the length of the zone. For example, when benzene was added to certain jet fuels, or gas oils, the zone became much shorter. Gasoline gum zones are also very short, because perhaps of the presence of benzene or toluene. These observations suggested that an asorbotrope of gum with nromatics (which, like the gum, are adjacent to the eluent front) is formed. Apparently the length of the brown zone depends on the concentration of gum in the asorbotrope, which in turn depends on the nature of the aromatics present. Hence a search !vas made for a single-component solvent that would displace the multicomponent, unpredictable aromatics. Asorbotroping Solvents. In order to be suitable for chromatographic gum determination the solvent must meet, two adsorbsbility requirements. Firat, it must, displace the sample aromatics, but not the gum. Thus both solvent and gum appear ns an ftsorbotrope just below the eluent front. Second, the so!vent must form a reasonably long brown zone for a given gum content . I t was found that anisole, ethylene dichloride, isopropyl chloride and 1-methylnaphthalene are in the right adsorbability range. Of these, 1-methylnaphthalene forms the longest b r o m zones and hence provides the greatest precision. Enough l-methylnaphthalene (0.1 ml.) is employed to form an asorbotrope x i t h all of the gum, the excess I-methylnaphthalene being dipplaced by the asorbotrope. The 1-methylnaphthalene reduced the very long gum zones obtained with certain gas oils and increased t,he zones obtained with gasolines, but had little effect on jet fuels. Thus all fuels were put on a common chromatographic basis. Since the composition of jet fuel is not rigidly specified, it is cor,sidered preferable to employ the solvent for t,his, as well as for other types of fuel. Dilution of Benzene-Free Fuels. When gas oils were analyzed ivithout dilution, the flow through the column was so slow that 5 or 6 hours were required for the analysis. I n addition the gum \vas developed downward during the sample addition and in ~ s treme cases it had entered the analJ-zer before the 1-methJ-1naphthalene was added. Dilution of the sample x i t h about 5051, of its volume of benzene corrected bot,h of these difficulties. Thc brnzene is displaced by the I-methylnaphthalene, which is added after the sample. Dilution of gas oils and other benzene-free fuels-for example, Diesel fuel or gasoline saturates-is reronimended. Use of Dye for Light-Colored Fuels. Fuels containing vcry little gum are so light in color that the brown zone is barely prrcseptible. I n such cases a red dye may be added to intensify thc c$olor. This should not be adopted as a routine measure because, if the brown zone is very long, the dye may not, color the entire zone, but only t,he upper part of it. The dyed portion could then
ANALYTICAL CHEMISTRY
10 be mistaken for the entire gum zone. The dye is also used when determining the “blank” brown zone due to the l-methylnaphthalene solvent added. Purification of I-Methylnaphthalene. The Eaetman “practical” grade 1-methylnaphthalene is very dark, but can be adequately decolorized chromatographically as described in the section on analytical procedure. All data in this paper were obtained with 1-methylnaphthalene, yielding a blank brown zone about 15 mm. long in the presence of the red dye.
The brown zone length may be empirically converted to milligrams of gum by reference to graphs such as shown in Figure 2. From the equations for the lines the gum contents may be calculated in milligrams per deciliter as follows: For JP-4 jet fuels
0.61 Gum, mg./dl. = - (L - B ) Ti For gas oils 0.54 Gum, mg./dl. = - (L - B )
v
ANALYTICAL PROCEDURE
The 1-methylnaphthalene is decolorized by passing it over about half its volume of silica gel with acetone as eluent. The purified material should have a blank not exceeding 15 mm. in the presence of the indicator dye, ethyl red. The indicator is conveniently employed in the form of dyed gel, made by slurrying 0.10 gram of dye and 100 grams of gel with methanol, and then evaporating the solvent a t room temperature in a stream of air with occasional stirring until the dyed gel is free-flowing. The end of the column is plugged with cotton and packed with silica gel to a point 30 to 40 mm. below the top of the separator section. If the sample is light colored, or when the blank is to be determined, 10 mm. of dyed gel is added. Soluble and insoluble gums require somewhat different techniques. For soluble gum in jet fuels or gas oils 0.50 ml. of sample is introduced, except when the gum content is below about 30 mg. per dl. in which case 1.00 ml. is added. Holvever, any convenient sample size in this range can be used. If the benzene plus toluene content is not known, or is less than lo%, the sample should be preceded by about half its volume of benzene. The sample is added immediately to promote mixing with the benzene before the latter is taken up by the gel. For the determination of soluble qum in l o w g u m fuels, including many gasolines, a 5-ml. sample may be required. Then, to save time, a preliminary rapid gum separation is made on a column of 5 mm. inside diameter by 150- or 200-mm. length. The sample (diluted with benzene if necessary) is followed by isopropyl chloride, which displaces the hydrocarbons, and the gum is eluted with acetone and collected in a small flask. The eluent is removed a t room temperature in a stream of air, and the gum is dissolved in chloroform and transferred to the gum column. S o benzene diluent is necessary here. A pressure of 3 pounds per square inch is applied until the liquid front reaches the middle of the analyzer section, when the pressure may be increased to 5 or 10 pounds per square inch until the sample is taken up. Then 0.10 i 0.02 ml. of purified 1methylnaphthalene is added, the walls are rinsed with a few drops of acetone, and the charger is filled with acetone. Pressure is again applied at 3 pounds per square inch ( 2 pounds per square inch for gasoline) until the brown zone has progressed well into the analyzer. The length of the brown zone is measured against a white background. Jt is the distance between the highest point of strong brown or yellow color and the lowest point where a color change is still apparent. Usually the column is white below the brown zone, but it may be uniformly yellow if the sample contains high boiling colored aromatics. The reading is repeated at 20- to 30mm. intervals until the zone length is stable. Readings should not be taken when the acetone front is mithin 80 to 100 mm. of the bottom of the column, as the zone length tends to increase somewhat in this region. Occasionally the brown zone becomes severely skewed in the separator and does not reach equilibrium in the analyzer. This can be corrected by employing a loJTer pressure after the acetone is added and until the gum zone enters the analyzer, and then reverting to the specified pressure. Insoluble gum, by definition, is the precipitate retained on a “fine” (Pyrex brand) sintered-glass filter after washing with nhexane. The gum thus isolated, is dissolved in a polar solvent and sampled for the chromatographic determination. If chloroform is employed as a solvent, the procedure is the same as for soluble gum. When the commonly used, strongly polar solvents such as acetone or ethyl acetate are employed, these must be removed, and the gum redissolved in chloroform. N o benzene diluent is necessary. The column may be cleaned by rinsing out the gel with a jet of water from a hypodermic tubing, or, it may be left under pressure until dry, inverted, and the gel removed by tapping gently.
where Ti = volume of sample, ml., L = length of brown zone, mm., and B = 1-methylnaphthalene blank, in mm. Similar expressions may be derived for other fuels as data become available. DISCUSSION O F RESULTS ON J E T FUELS AND GAS OILS
The jet fuels tested represent straight-run, thermally cracked, catalytically cracked, and blended samples from various sources. This selection is considered to be a good cross section of emergency-type JP-4 jet fuels. Most of the gas oils consist of a catalytically cracked stock having a 90% point of 585” F. (310’ C.) and 40° F. distillation fractions of this material. It is believed that other gas oils in this boiling range would behave similarly.
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Figure
2.
Chromatographic determination soluble gum
of
Soluble Gum. The soluble gum contents of these fuels were determined by a steam-jet evaporation procedure (1, at 500” F.). The chromatographic gum values were then determined as described. The JP-4 jet fuel and gas oil results are presented in Figure 2, in which the actual weight of gum in the sample is plotted against the brown zone length. Thus the plots are independent of sample size. Two lines have been drawn, one for each type of fuel, because it is statistically probably that the lines should be separate. However, more data would be required to prove this rigorously. The scatter of the points may
.. 11
V O L U M E 2 8 , NO. 1, J A N U A R Y 1 9 5 6 be attributed t o the limited repeatability of both steam-jet and chromatographic methods. The standard deviation of the repeatability was about i %in both cases, and the standard deviation betrveen the steam jet and chromatographic methods was about 14%. The jet fuels of Figure 2 were aged in the desert. -1few additional samples that had been subjected to accelerated aging were studied, and the data indicated t h a t the steam jet-chromatographic correlation for jet fuels is valid up t o an aging temperature of 100’ C. The gas oils were aged a t 110’ F. (43’ C.) in a constant temperature room. Additional results from samples aged a t 70” C. indicated that these could be treated by the same correlation. HoTvever, gas oils aged a t 100” C. gave a much steeper curve, and hence the chromatographic method is suitable for only a rough determination on such samples. Insoluble Gum. Available data are not conclusive, but it appears that Figure 2 can be used for both soluble and insoluble gum in jet fuels (JP-4) and gas oils. Honever, earlier work under different analytical conditions indicated that results from fractions of narrow boiling gas oil of 220 t o 2-10 molecular weight and with high insoluble gum content mav diverge considerably from the line drawn. DISCUSSION OF PRELIhlINARY RESULTS ON G 4 S O L I y E
Preliminary tests have indicated that there may be a correlation between the chromatographic brown zone and the steamjet gum (500” F.) content for gasolines. This observation is interesting and encouraginp. However, gasoline gums are normally determined by air-jet evaporation a t 330” F. h study of
the relation between the air-jet and chromatographic deterniinations of gum is in progress. EVALUATION OF CHROMATOGRAPHIC METHOD
The chromatographic method for determination of gum gives values that appear t o he acceptably close to the steam-jet results for soluble and insoluble gum in the jet fuels and gas oils tested. The reliability is uncertain in the case of gas oils aged above 70’ C. and of insoluble gum from severely aged gas oils of high molecular weight. Only about 1 ml. of sample is required, and one operator with facilities for 10 columns can make 15 to 30 determinations in 8 hours. Preliminary data indicate that the method may he applicahle to gasoline. ACKlVOTC L EDGM En-T
This work was carried out under sponsorship of the Materials Laboratory, Kright Air Development Center, U.S.Air Force. LITERATURE CITED
(1) 4 m . SOC.Testing Materials, Philadelphia, Pa., “A4STMStandards
on Petroleum Products and Lubricants,” D 381-54T,1954. (2) Criddle, D. W., and Le Tourneau, R. L., ANAL.CHEM.23, 1620 (1951). (3) Knight, H. S., and Groennings, S., Ibid., 26, 1.549 (1954). (4) Nixon, A. C., Cole, C. A., and Walters, E. L., “Factors in Jet Fuel Stability. Occurrence and Measurement of Gums in Jet
Fuel,” unpublished. RECEIVEDfor review May 9, 1955. Accepted September 26, 1955. Division of Petroleum Chemistry, 128th Meeting, ACS, Minneapolis, hIinn., September 1955.
Determination of Specific Activity of Carbon4 4-labeled Sugars on Paper Chromatograms Using an Automatic Scanning Device H E N R Y R. ROBERTS and FREDERICK J. C A R L E T O N National Dairy Research Laboratories, Inc., Oakdale, L. I., N.
A simple method was needed for determining the quantitative distribution of carbon-14-labeled sugars and sugar derivatives in solutions resulting from biological studies. The paper chromatographic separation of these carbon-14 substances and the localization of active sites on the chromatogram were possible by scanning the developed chromatographic strip with an automatic scanning device. This device is applied to the quantitative determination of the specific activity of carbon-14-labeled sugars by two methods. One method employs a summation equation involving the areas under the scanning curves and the total specific activity of the solution being assayed. The second uses known carbon-14-labeled sugar solutions which serve as standards. The heights of the scanning curves of the standards were measured and a straight-line standard curve was prepared. The heights of the scanning curves of the unknowns were measured also, and their specific activities calculated from the standard curve. Determination of the specific activity of carbon-14labeled lactose, galactose, and glucose by either method results in an error no greater than 37’0.
T
Y.
H E distribution of radioactivity among a number of labeled components in a solution or mixture can be of considerable importance t o an investigator. The application of paper chromatography for the separation of radioactive components and their subsequent localization by autoradiographic techniques is well known. The autoradiographic technique has, in the past, suffered from being time-consuming and/or inefficient, especially when dealing with R-eak beta emitters of low specific activity. The scanning of a paper strip by a systematic manual exploration with a Geiger counter is also, t o a certain extent, inefficient and tedious. The difficulties encountered with autoradiography and hand scanning have been offset by the construction of devices for automatically scanning paper radiochromatograms ( 2 , 3, 5, 7 , 9, 10, 12-14). Each consists of a mechanism for moving a paper strip past a collimating slit in front of a Geiger counter or ionization chamber and an apparatus for recording the counting rate as a function of the position of the paper. TKOmethods of using the automatic scanning device for the quantitative determination of the specific activity of carbon-14labeled sugars after separation on paper chromatograms are reported herein. The first method is based on the determination of the specific activity of the unknown solution (two or
