Initially, the saturated acids were located on the chromatograms by forming the cupric d t s and then detect= the copper with potasaim ferrocyanide (3,11). Attempts to reduce the background coloration by thorough washing often resulted in the loss of the desired spots, presumably due to hydrolysis of the cupric salts. Subsequently, the mercuric salts (3)viere formed and the mercury was located nith sdiphenylcarbazide. The mercuric salts were &Me to prolonged washing, and the final spots were easier to detect than the spots from the cupric salts. Since resin acids are often mixed with the fattj- acids obtained from wood, the chromatographic behavior of lmown resin acids was investigated briefly. "he chromatograms were developed on oir-treated paper for 15 hours at 3 i 0 C. n-itn the acetic acid-formic acid-hydr+ gcn peroxide (6: 1: 1) developer, and the spots were located mith the mercury reagent. Thk reagent is not as sensitive for the resin acids as for the fatty acids. Small amounts of resins do not seem to interfere mith the chromatography of the fatty acids. When approximately 0.1 mg. of h o r n resin acid was applied to the paper, spots in the R , range of 0.55 to 0.60 were given by abietic, neoahietic, palustric, levopimaric. i-dextropimaric, dehydro-
abietic, and d i h y h b i e t i c acids. The two iatter acids gave a aecond spot at a b u t B, 0.25. These acids also gave two spots on chromatograms developed with acetic acid-water (4 to l), and it is probable that they contained a second constituent. Fatty acid mixtures were also pretreated n-ith peracid, and the reaction m i . were chromatographed with a plain acetic acid-water developer. This permitted the chromatography of both the saturated and unsaturated acids in the same chromatographic jar, but did not offer any other advantage over the peracid developers.
Memorial Institute and to Ray V. Lawrence, Naval Stores Station, Olustee, Fla., for samples of the known resin acids. High purity palmitic and stearic acids were obtained from the Hormel Foundation, and anrchidic, behenic, and lignoceric acids were obtained from Delta Chemical Works, Inc. LmRAnJllE CmD
This study waa initiated on a project sponsored by Union Bag-Camp Paper Carp., and the author is grateful
(1) Ashley, B. D.,Reatphal, E., A d . Biodrem. Bwphys. 56, 1 (1955). (2) Kaufmann, H. P., Fcttc, Seifen, Anstrichmitiel 58,492 (1956). (3) Kaufmann, H. P., Budwig, J., Fe#e u. Seifen 53,390,399 (1951). (4) Kaufmann, H. P., Mob, E., Fdtc. selfen, A n s t k h i t i d 609 165 (1958). (5) Kaufmann, H. P., S i k h , R. H., Ibid.,56, 154 (1954). (6)Mangold, H. K., Gellerman, J. L., Schlenk,. H.,. Federaiion Proc. 17, 26$ (1958). ( 7 ) Mangold, H. K., Lamp, B. G., Schlenk., H.., J. A m . C h .Soc. 77.6010 (1955). ( 8 ) Schlenl;, H., Gellerman, J. L., Tillob am, J. A., Mangold, H. K.,J . Am. Oil CAembfs' Soc 34,37i (195i').. (9) Saern, D.,Billen, G. S., Findleg., T. IT..Scanlan. J. T.. J.Am.Chem. Soc. 67. 1% (iwj. ' (ioj'TC mmies, G., Homiller, R. P., I&id., 64,3054 (1942). (11)K a p e r , H.,Abisch, L., Bernhard, K., Hela. Chirn. -4da 38, 1536 (1955;.
for permission to publish the procedure. He is indebted to G . A. Lutz of Battelle
RECEIVED for review March 1. 1959 -4ccepted June 11, 1959.
CONCLUSION
Oil-treated paper developed a t 37' C. nith acetic acid-%% f o m k acid-30% hydrogen peroxide (6:l:l) provides a good separation of the even-numbered straight-chain saturated fatty acids from lauric acid to lignoceric acid when these are present in samples containing large amounts of unsaturated acids. ACKNOWLEDGMENT
0
Nephelometric Detection of Lipides in Chromat ogra phic Column Eff Iuents A. C. ARCUS Nutrition Research Department, Medical School, Dunedin, New Zealand ,A relatively nonspecific method of roughly estimatir+g lipides in chromatographic column effluents is based on the light-scattering power of the suspension formed when the lipide is precipitated from methanolic solution containing 0 to 1 mg. per ml. by the addition of water. The method is applicable only to substances soluble in pure methanol and insoluble in a mixture of 1 part of methanol with 2 parts of water, but suggestions are made for its extension to other substances. The smallest detectable concentratian is of the order of 2 to 20 7 per mi., depending on the substance. Application to chromatography is illustrated.
followed by absorption spectrophotometn, which has the advantage of specificity but the disadvantage of laving many lipides undetected. This paper describes an alternative method, based on the lightscattering power of the suspension formed when water is added to the alcoholic solution of the lipide. The method is relatively nonspecific but can be used for roughly estimating a wide range of lipides in concentrations up to about 1 mg. per mi. When used in conjunction with spectrophotometn, it gives a fairly complete picture of the way in which lipides are eluted from the c h r o m a b graphic column. MPERMNTAL
of Ythe chromatography of the nonsaponifiable matter of fiahliver oils a t this laboratory (3),the elution of lipidea from the column has been N THE S
~
Materials.
Methanol (analytical reagent grade) was obtained from Imperial Chemical Industries LM. Hexoestrol and methyltestosterone were obtained from Boots, and proges-
terone from British Drug Houses Ltd. The following were obtained from L. Light 8: Co. Ltd.: dehydroisoandrosterone, lumisterol, B-sit+ sterol, phytol, and stearic acid. Mj-rktir acid was obtained from Hopbrin & ffilliams Ltd., and cholesterol from F l u b . Vitamin d alcohol was made by saponification of vitamin A acetate (I). Vitamin A acetate, calciferol, and racemic cr-tocopherol acetate (British Pharmacopoeia grades) were obtained from Roche Products. The suspension is formed by controlled addition of Cater to the alcoholic solution in a special mkxer. After dilution with water, the light-scattering is measured in a nephelometer. Apparatus. MIXER (Figure 1). The motor, which run8 at lo00 r.p.m.? rotates the ballrace assembly and the test tube (6 X '/g inch) at 300 r.p.m. The removable test tube fib loosely in the polyethylene sleeve and is held in by the rim a t the upper end of the sleeve; t o allow the polyethylene t o expand as the tube is pushed pa&
Figure 1. mixer
Details of
,I r*
this rim the end of the sleeve is castellated. This sleeve fits tightly inside the bras sleeve, which itself is a snug fit in both the ballrace and the pulley, holding the two together. The taut rubber hand is a stop to position the test tube, nhich is inclined a t an angle of 30" to the horizontal. The tip of the syringe needle lies below the level of the liquid in the test tube. Constriction of the mouth of the test tube ensures that it does not pick up dirt from the h i d e of the polyethylene sleeve as it is pushed in or taken out. The height of the reservoir is set so that 2 ml. of rrater put in it run out a t the tip of the syringe needle in just 2 minutes. The reservoir, syringe needle, ballrace, and electric motor are clamped to a single stand, nhich can be lifted to remore the test tube from the water bath, which is kept a t 50' f 0.5"C. SEPHEU)VETER, COIlStruCted Of W o o d to bolt onto the photocell compartment of the Beckman Model DU spectrophotometer. A cubical 15-ml. cuvette of poly(methylrnethacr?.late) is illuminatcd by a 3" tungsten lamp through a condenser lens and the light scattered 3t right angles by the suspension in the cuvette is directed onto the multiplier photocell by a second lens. Masking and placement of the lenses ensures a minimum of stray light falling on the photocell when the cuvette is filled with clear liquid. Nephelometric standards are made of pieces of selected Bristol board (0.018 inch thick) of very iow transmittance fitting diagonally across the cuvette holder in such a way that only the transmitted light fa& on the photocell. blethod. One milliliter of the sample dissolved in methanol is run into the test tube, Khich is fitted t o the mixer, immersed in the water bath, and rotated for 1 minute to bring the wntents u p t c temperature. Then, 30 make the suspension, 2 ml. of water at room temperature are run into the m e m o i r . Two minutes later when t h e reservoir has emptied, rotation is stopped and the drop of water renaining in the syringe needle is blown aut. The test tube is removed from the apparatus, 10 ml. of mater a t room temperature are added to bring the total volume of liquid up to that of the cuvette, and the contents are mixed by inverting the tube several times, the end being closed by a thumb. The suspension is poured into the cuvette
*.
gently to avoid bubbles being emied d o n and sticking to the n-alls, and after 1 minute its light-scattering power is compared in the nephelometer with that of a suitable standard. The test tube is rinsed with 2 ml. of 95% ethyl alcohol and allowed to drnin for 1 minute. The appcimtus is then ready to use again. The p M i c cuvette h3s the advantage of shedding pnctically all the liquid if the contents are poured out slowly. By employing t n o k t tubes, analyes can be d e a t the mte of 17 per hour. Distilled water md alcohol are p x d through a So. 3 s i n t e r e d - g k filter before use and care is taken to keep dust from getting into the suspension. RESULTS AND DISCUSSION
The lightiscsttering power of a suspension is espressed as the ratio s,'&, v-here S and SI are the nephelometric m d i n g s for the suspension and the KO.1 standard, respective1)-, with the readings made one after the other. The actual resdings are made on the transmission scale of the spectrophotometer; with the multiplier photocdl a t its lowest sensitivity setting and the sensitivity h o b of the spectrophotometer fully a n t i c l o c h (highest optical sensitivity), standard No. 1 has a reading of about 12. (In a weU-lit room, this standard appears to be quite opaque.) For distilled, filtered water, 8/81 = 0.30, while for a sample of methanol carried through the mixing procedure, S/SI is approximately 0.4. A suspension for which S/S1 = 20 is just noticeably cloudy to the eye. For readings of S/S1 greater than 10, the sensitivity of the nephelometer is decreased about 10 times by inserting a cardboard diaphragm containing an aperture 0.25 inch in diameter between the cuvette and the photocell; this method, which reduces the light trans mitted to the photocell, is preferred to the alternative method of lowering the sensitivity of the instrument with the sensitivity knob, because fluctuations of the photocurrent become excessive when the photccell is strongly illuminated. A range of substances for which the method is seellgitive i s shown in Figure 2. The 6gure shows graphs of S/S, against
concentration for the subshnces, which include high molecular weight alcohols and sterols, I o n g d a i n fatty acids, and esters. As the readings were made on serial dilutions, it has been conrenient to use logarithmic scales on both axes. The curves hare not been superimposed but placed one above the other and the vertiml scale is indicated on each by short horizontal lines labeled a t log S,'SI = 0 and 1. The lowest messureable concentration, talien arbitrarily as the concentration for n hich S = SI (log S/S1 = O), lies between 2 y per nil. (cholesterol) and 20 7 per ml. (myristic acid). .4n idea of the reproducibility of the method can be obtained from the duplicate readings shown in Figure 2, each of a pair being joined by a hesvy vertical line. Most substances produce turbidities that are stable for half nn hour or more, but thcse of \itamin A and myristic acid increase more or less npidly for seven1 minutes after the suspension is fornied, while an u n h o n n constituent of cod-lirer oil produces 3 turbiditity that decreases; for these substances it is neem to make the readings a t a definite time after nixing. The visible form of a strong turbidity depends to some exAent on the substance; for e m p l e , \-itamin X produces a &-blue suspension in which the individual particles are invisible, while &&sterol produces a granular suspension containing large, visible particles or clumps of particles. The S 'SI value^ for these two subst3nres, howeyer, are quite comparable oyer the nhole range of concentrations studied (Figure 21. This individuality is sometimes useful in the identification of substances eluted from columns. The method, as it stands, is not s u i b able for detecting steroid hormones and some related compounds. Table I shows that these are not detected in concentrations weaker than about 500 y per ml., presumably because they are too soluble in the 33% methanol used for precipitation. No doubt the method could be modified for detecting these compounds, as described later. In 11942, Davis and Parke (9)measured the 30lubibty of polyc\-clic ammatic hydrocarbons in water by a -
Table L Application of Method to Steroid Hormones and Related CM-
&s
sub&Anors HeXOe8tl.01
Concn.,
y/?tll.
s/s,
125
0 49 053 0 66 0 75 7.4
250
m Methyl testosterone 1320 Dehydroisoandrosterone LOO0
Progesterone
150
m
VOL 31, NO. 10, OCTOBER I959
7.7 2 0 0 1
6
77 66 11
1619
Figure 2.
Effect of concentrationof pure substances on S/S,
b
Vertical scales are marked on individual curves
CHOLESTEROL
II
II
VITAMIN A
VITAMIN A ,
A FRACTION
i1
NO
Figure 3. Application of method to chromatography of cod-liver oil
method based on the lighbscattering power of the suspensions formed when 1 ml. of acetone containing the sample was run slowly from a pipet below the surface of 5 large volume of water in a vigorously shaken flask. When the present author applied this method to the estimation of cholesterol, the suspensions obtained were not reproducible and had a low light-scattering power. Experiments showed that the best result was obtained when the water was added to the sample solution a t a rate not greater than 1 nil. per minute. The present method of mixing was adopted after both shaking and stirring with a propellor were found t o give variable results. The position of the tip of the syringe needle does not matter as long as it is below the surface of the liquid; if it touches the side of the tube, a slightly higher blank reading occurs, possibly because of material worn off the needle and the test tube. The present nephelometer was built after the Beckman fluorescence attachment for the DU spectrophotometer was found to show a large variable scattered light when the cuvette was filled with solvent alone. (In fluorescence work this is removed with optical filters.) The time a t which the nepielometer is read is important in cases where the reading is changing. Temperature of mixing is quite important-for example, a cholesterol sample containing 10 y per n.1. mixed at 33", 43")50", and 60" C. p r (1s S 'SIvalues of 2.25, 3.28,3.81, :ii>tl 4.11,rcqcctively. The dust content of the final mixture must 1)eLvpt low. An excellent method of oqing this for substances that form fine suspensions-e.g.. cholesterol, vitamin A . :tnd vitamin D-is to pass the mixture through a No. 3 sintered-glass 1620
0
ANALYTICAL
CHEMISTRY
CONCENTRATION, MG. PER ML.
filter just before reading. However, other substances-e.g., 6-sitosterolform suspensions that are almost entirely removed by the filter, and for this reason the solvents rather than the final suspensions are filtered. It may be possible to separate pairs of substances--e.g., @-sitosterol and vitamin A--by means of this effect. Figure 3 shows an example of the use of this method in chromatography. The nonsaponifiable matter of cod-liver oil was eluted from a chromatographic column by a continuously increasing concentration of methanol in water. Of the 17 or more peaks in the elution curve, only the labeled ones have been identified. The method described can be used for detecting only those substances that are soluble in pure methanol and practically insoluble in methanol diluted with 2 parts of water. These restrictions could no doubt be removed--e.g., in the detection of steroid hormonesby using other water-miscible organic solvents or by adding a larger proportion of water for the precipitation, or both. Doubtless, a fluorescent substance whose wave length of excitation
is longer than about 350 nip would also show up in the method even if not precipitated; vitamin A, for example, would give an augmented S/S1because of fluorescence. This is no disadvantage, as the method was designed to show up as many substances as possible. The technique described in this paper has proved useful for follon ing the elution of certain lipides from chromatograms, and modifications would no doubt adapt it for use with compounds other than the class of lipides for which it has been specifically designed. Although the results are only seniiquantitative and relatively nonspecific, a wide range of substances can be roughly estimated in concentrations of the order 10 y per ml., rapidly and with simple apparatus. LITERATURE CITED
(1) British Pharmacopoeia, London, p. 846. - - . -19.53. - - -. (2) Davis, W. W., Parke, T. V., Jr.. J . Am. Chem. SOC.64,101-7 (1942). (3) Dunckley, G . G., Macfarlane, Y . J.. J . Sn'. Food Agf. 6,559-64 (1955).
RECEIVED for review December 1, 1958. Accepted June 8,1959.
