~
RESULTS A N D DISCUSSION
Table 1.
-
Detn. 1 2 3 4 5 6
Mean Std. dev. Coeff. of variation, %
Reproducibility Experiments
(Glucose concentrations in micrograms) Absorbance X 100 50 100 150 200 250 300
350
400
450
29 42 87 111 139 161 191 220 243 133 164 197 219 248 31 45 81 107 134 192 224 247 161 81 107 28 41 139 196 223 248 161 29 44 85 113 112 134 164 195 220 248 87 31 41 137 193 220 248 167 83 110 29 45 39 43 83 110 136 163 194 221 247 f1.55 f 1 . W f2.76 f2.54 f2.68 f2.45 f2.37 f2.0 f 2 . 0 5.4
4.4
3.3
sponding regions of the nonsprayed paper are cut and dipped in tubes containing 10 ml. of methanol. The tubes are placed in a water bath at 65" C. for 20 minutes for a complete elution of the sugars from the filter paper. The methanol of each tube is transported in another tube 20 cm. in length and 2.5 cm. in diameter and 3 ml. of the following solution are added: 1-propanol: aniline: orthophosphoric acid = 75:0.5:
2.3
2.0
1.5
1.2
0.9
0.8
0.5 v./v. The tubes are placed in a boiling water bath for 20 minutes. To each tube are added 5 ml. of methanol and the yellow-brown color appearing during boiling is measured in a spectrophotometer at 400 mp. A blank is run on a piece of sugar-free paper dipped into methanol. Standard curves are obtained by plotting concentrations of known a m ple~ against density.
Table I shows the reproducibility of the determinations when 4OO-mp a m ples of known glucose concentration are measured after chromatographic development. The method can be employed for sugar concentrations from 20 up to 500 y with good accuracy by using a Hilger spectrophotometer. The standard deviation and coefficient of variation of other reducing sugars, such as galactose, lactose, and fructose, are approximately the same as those of glucose. The method is generally reproducible and the whole procedure is much facilitated by the simple way of eluting sugar from the filter paper. The accuracy of the method of sugar elution is shown from the coefficient of variation, ranging from 0.8 to 5.4%. LITERATURE CITED
(1) Bryson, J. L., Mitchell, T. J., Nalure 167,864 (1951).
RECEIVED for review December 5, 1958. Accepted June 11, 1959.
Paper Chromatography of the Saturated Fatty Acids M. A. BUCHANAN The institute of Paper Chemistry, Appleton, Wis.
b l n a new procedure for the paper chromatography of the saturated fatty acids, the developer reacts with the unsaturated acids to form products which readily separate from the saturated acids. Hydrogen peroxide and formic acid are added to the usual acetic acidwater developers used for reverse-phase chromatography of the fatty acids. The new procedure permits the separation of small amounts of saturated acids from large amounts of the unsaturated acids, and is suitable for tentative identification of the even-numbered straight-chain acids from lauric acid to lignoceric acid.
M
species of wood contain a p preciable amounts of fatty acids, resin acids, and sometimes other acids. r%verse-phase paper chromatography has proved to be a useful tool for the tentative identification and separation of the fatty acids. Stearic, oleic, linoleic, and linolenic acids are readily separated on paper treated with mineral oil ( I ) developed at. room temperature with acetic acid-water (6,7,8),but these conditions are not suitable for the detection of some of the saturated acids which are present in wood extracts. Kaufmanu (2) has reported the following ANY
critical pairs of acids which are not separated by the usual conditions: pafitic-oleic, myristic-linoleic, and h u ric-linolenic. In addition, the saturated acids are often minor components, and the detection of small quantities is difficult. The saturated acids can be separated from the unsaturated acids before chromatography, but this is an added step, and often chromatography is of value for testing the efficiency of the desired separation. Chromatography at -30" C. has been suggested (4, 8) for the separation of the critical pairs, and treatment with bromine has been used (7) to form addition products with the U n s a t u r a t e d acids on the chromatogram. They can then be separated from the saturated acids. Recently, Mangold, Gellerman, and Schlenk (6) reported a new procedure in which peracetic acid is added t o the acetic acid developer. The peracid reacts with the double bonds of the unsaturated acids to form oxygenated products with high R, values. They found the oxidation of the unsaturated acids to be quantitative. In these laboratories, 1 to 1 mixtures of fordaic acid and 30% hydrogen peroxide have been added to the acetic acid developer. Formic acid was used because it reaets
readily with hydrogen peroxide to form the peracid (9, IO). The exact composition of the peracid developers has not been determined, but i t seems likely that they consist of acetic acid, performic acid, formic acid, and water, but some peracetic acid and/or unreacted hydrogen peroxide may be present. Fatty acid mixtures also have been pretreated with peracids, and the resulting mixture has been chromatographed with the usual acetic acid developers. EXPERIMENTAL PROCEDURES
Chromatography with Peracid Developers. Strips of Whatman No. l
filter paper (7.25 X 24 inch) were drawn through a solution consisting of 7 grams of water-washed USP heavy mineral oil in 100 ml. of USP ether ( 1 ) . After hanging in the hood lor a few minutes, the treated papers were ready for spotting. Approximately 0.005 ml. of a solution of the fatty acids in chlorofmn (or any other suitable solvent) was spotted 3 inches from the end of the oil-treated paper. The concentration of the solutions used for spotting was varied according to the proportion of saturated acids in the sample. For the chromatograms reported in Tables I and 11, the solutions of known acids contained 10 grams per liter of the individual satu-
rated acids, and sufficient oleic and linoleic acids to make the total concentration 500 grams per liter. For the acids obtained from wood extracts, the solutions were usually prepared by using 2 ml. of chloroform per gram of acids. For samples with a very low concentration of saturated acids, the liquid acids were spotted without dilution. After the solvent had evaporated from the spot, the paper was heated in an oven a t 80" to 90" C. for 5 minutes to fuse the acid sample in the paper. When this was not done, the higher saturated acids sometimes crystallized on the paper and then formed streaks on the chromatogram. Developer solvents were prepared each day by mixing equal volumes of 88% formic acid and 30% hydrogen peroxide, and adding this solution to glacial acetic acid. When desired, water \vas then added to this solution. The proportion of total water in the developer was varied by using the following: acetic acid-formic acid-hydrogen peroxide (9: I : 1, v./v.) and (6:1: l), and acetic acid-formic acid-hydrogen peroxide-water (9 : 1:1:1) and (6:1:l:1). Because formic acid and 30% hydrogen peroxide are very corrosive to the skin, rubber gloves were worn when preparing and handling t.he developer. Usually, the chromatograms were developed by the descending technique in 12 X 24 inch glass jars which were kept in a constant temperature room for the higher temperatures. In some instances, chromatograms were run on 4.75 X 18 inch strips of treated paper in a 6.25 X 18 inch glass jar which was heated in a large oven. After removal from the jars, the chromatograms were allowed to air dry in a hood. In the early work, the acids were located on the chromatograms by forming the cupric salts and treating with potassium ferrocyanide as described by Wagner and coworkers (11). Later, t.he mercuric salts \I ere formed by immersing the air-dried chromatograms for 15 minutes in a O.lyomercuric acetate solution containing 0.5 mi. of acetic acid per liter. Excess mercuric acetate was removed by washing the chromatogram in running tap water for 45 minutes. After air drying, the chromatogram was sprayed with a 02% solution of sdiphenylcarbazide in 95y0ethyl alcohol. The purple-colored spots were stable for several days. Pretreatment with Peracid. A 1gram sample of the fatty acids was dissolved in 14 ml. of acetic acid, and 2 nil. of a freshly prepared 1 to 1 mixture of 88% formic acid and 30% hydrogen peroxide were added. After standing overnight a t room temperature the solution was diluted with 15 ml. of water and extracted with 2 ml. of chloroform. The chloroform solution for spotting on oil-treated paper was used either directly or it, was first diluted if the proportion of saturated acids was high. For samples containing the higher saturated acids, the chromatograms were developed a t 3 7 ° C . with acetic acid-water (85 to 15).
-Table I.
Oil-Treated Paper Developed 15 to 16 Holrx 6: 1 : 1 Develoner' 9 : 1 : 1 : 1 Developer6 a{. at 37' C. _ _ Room Temp. (20-30" C.) ___ A v . Rjc Range in f l j Av. K,P Range in I?:. . . 0.01 ti. 0 1-0, 0i .. ... 0.04 0.02-0.0; 0.09 Ci ,08-0.12 0'67 0.05-0.12 0 l W J 2-1 0.17 0.12-0.24 27-cl.40 .. ... 0.5i 0 454.51: .. ... 0.62 0 594.68 ~
Acid Lignoceric Behenic Arachidic Stearic Palmitic Myristic Lauric Unsaturated acid reaction products 0.58 0 . 5 W .62 0.73 * Acetic acid-88% formic acid-30% hydrogen peroxide-water, v./v. i! Acetic acid-88% formic acid-30% hydrogen peroxide, v.!~. Results are averages for 10 or more chromatograms. Table
li.
0 . (594.76
Comparison of Different Developers, Temperatures, Times, and Papers
Paper treatment Developera Time, hours Temp., O C.
Oil Oil Oil Oil Oil Oil Oil 6:l:l:l 6:l:l 6:l:l 6:l:l 9:l:l 9 : l : l 9 : l : l 44 40 24 39 15 15 48 37 37 40 37 40 45 37
6:l:l 19 37
Distance6 R, .Urn.
Mm. Mm. Acids Lignoceric .. 25 46 0.01 0.03 105 113 83 Behenic .. 0.04 0.07 167 170 49 81 Arachidic .. 77 152 0.09 0.14 266 228 161 Stearic 53' 143 246 0 18 0.23 385 292 216 Palmitic 100 .. 378 0.28 0.35 346 264 hlyristic 202 .. 0.46 0.52 .. 390 298 Lauric 279 . . .. 0.60 0.63 .. .. Unsaturated acid reaction products 305 .. . . 0.71 0.67 . . .. , . 4 Acetic acid-%% formic acid-30% hydrogen peroxide-water (water waa added only t o developer in first column). Each value is the average of two chromatograms. In most instances the solvent ran off the paper, and the distance of component travel is expressed in millimeters. Mm.
Mm.
Silicone
Mm.
RESULTS AND DISCUSSION
The 9: 1 :1 :1 developer is suitable for the separation of palmitic and stearic acids on oil-treated paper when developed for 15 hours a t room temperature. Under these conditions the reaction with the unsaturated acids is slow, and the reaction products form a streak which moves ahead of palmitic acid, but which obscures myristic acid. In addition] temperatures below 30" C. are not very satisfactory for the Cna t o Cw saturated acids. A greater range of acids can be separated by decreasing the proportion of water in the developer and increasing the temperature of development. The even-numbered saturated acids from C,? to Clr are readily separated on oiltreated paper developed a t 37' C. with the 6: 1:1 developer. Development for 15 hours separates all of the acids, but a somewhat better separation of behenic and lignoceric acids is obtained when the time is extended to 48 hours. Average results for the two developers are given in Table I, and results obtained by other modifications are presented in Table XI. The results in Table I are averages from 10 or more chromatograms, while those in Table 11 are the averages of only two chromatograms. Under all of the con-
Rj
ditions there was often appreciable variation in the R, values, and it seemed necessary to have a known mixture on each chromatogram. The results in Tables I and I1 were obtained on acid mixtures which consisted chiefly of unsaturated acids with approximately 2% of each individual saturated acid, but as little as 0.2% palmitic acid has been detected in a mixture consisting chiefly of oleic ana linoleic acids. The large amounts of unsaturated acids do not interfere, since the reaction products with the developer tend to move with the solvent, front. With the longer development times. the developer and the more mobile constituents ran off the paper. In these cases the travel distance was expressed in millimeters. Somewhat better results were obtained for lauric and myristic acids, with the developers containing the larger proportion oi water, while the best separations of behenic and lignoceric acids were obtained when the amount of wItter was decreased by reducing the proportimi of formic acid and hydrogen peroxidc. There did not seem to be any advantag? in increasing the temperature beyond 3 7 O , nor was there any apparent advantage to the use of silicone-treated paper ('7). VOL. 31, NO. 10, OCTOBER 1959
1617
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. 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
This study waa initiated on a project sponsored by Union Bag-Camp Paper Carp., and the author is grateful for permission to publish the procedure. He is indebted to G . A. Lutz of Battelle
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
(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. SOG. 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;.
RECEIVED for review March 1. 1959 -4ccepted June 11, 1959.
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
N THE STUDY of the chromatography
of the nonsaponifiable matter of fiahliver oils a t this laboratory (3),the elution of lipidea from the column has been
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 IO00 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; to allow the polyethylene to expand as the tube is pushed pa&
