This 29 page thesis reports on the efforts to apply columnar chromatography to the identification of a fatty oil and the separation of a fatty oil from a mineral oil

Newborn, G. Earl

English

Ursinus College

Ursinus College Digital Commons @ Ursinus College Chemistry Honors Papers Student Research 5-12-1953 Identification of Certain Fatty Oils by Chromatographic Methods G. Earl Newborn Follow this and additional works at: https://digitalcommons.ursinus.edu/chem_hon  Part of the Chemistry Commons Click here to let us know how access to this document benefits you. U r s i n u s C o l l e g e L i b r a r y , URSINUSIANA COLLECTION C l a s s 0 Boxdfci/Th.-'V lUENTIFICATION OP CEKTAHI FATTY OILS BY CHROMATOCTRAPHIC JffiTHODS This paper i s submitted to the faculty of Ursinus College in partial fulfillment of requirements for departmental honors in chemistry. Submitted: G, Earl Nowboni Chemistry Department Ursinus College May 12, 1953 Approved: TaW e of Contenta S e c t i o n "^age T I n t r o d u c t i o n 7. I I Purpose 7. I l l Procedure 7. I V F x p l a n a t i o n c f Data n h a r t s 4 V R e s u l t s and D i s c u s s i o n 33 R e f e r e n c e s ^9 I . Introduction Tv;o problems facing the a n a l y t i c a l chemist i n the fa t t y o i l industry are: This paper reports on the ef f o r t s to apcly columnar chromatography to the solution of these two problems. I I . Purpose 1 . To determine i f the use of a column of adsorbent alumina w i l l produce colored adsorption bands v i s i b l e i n natural or u l t r a v i o l e t l i g h t which are ch a r a c t e r i s t i c for each substance concerned. The appearance of sxich hands i s called a chromatogram. 2 . To determine hy chromatographic methods v/hether i t i s possible to separate f a t t y o i l s from mineral o i l s with subsequent recovery of both. Such a separation vfould make possible an examination of the f a t t y o i l components without changing t h e i r structures. (Such an examination i s not possible9rith the o f f i c i a l American O i l Chemists Society procedure ( l ) . I l l . Procedure The adsorbent, adsorptive alumina (80 - 2 0 0 mesh), was packed to a depth of 1 0 cm. i n a glass column 3 0 cm. X 1 . 3 cm. The packing was done i n the follox'Ving manner: After packing a h a l l of glass xfool into the column, one poured i n alumina u n t i l i t f i l l e d the column to a height of apnroximately 2 cm. above the sixrface of the glass wool. The column was then tapped v/ith the fingers u n t i l the adsorbent ceased to s e t t l e . This process of f i l l i n g with alumina to a 2 cm. height and s e t t l i n g t h i s adsorbent by tapping was repeated u n t i l the alumina height i n the column stood at 1 0 cm. Upon the upper sur-face of the adsorbent column thus produced 1 . 2 . I d e n t i f i c a t i o n of a f a t t y o i l . Separation of a f a t t y o i l from a mineral o i l . vras packed a b a l l of glass v/ool to keep the sur-face of the column intac t during the subsequent pouring of solvent upon i t . This column was then washed x^ith 1 5 ml. of petroleum ether (B. P. 3 0 - 6 0 ° C ) ; xfnereupon, the weighed sample, consisting of a single f a t t y or mineral o i l dissolved i n 2 5 ml. of petroleum ether vras poured into i t . The weight of the sample varied between 0 . 4 5 0 and 0 . 5 5 0 gms. The solvent issuing from the column a f t e r the addition of t h i s l i q u i d x-zas collected i n a weighed 5 0 ml. beaker. ¥h.en flovr thru the column had ceased, the column ws.s further v/ashed xfith t\.ro successive 3 5 ml. portions of petroleum ether followed by three successive 3 5 ™i• volumes of dieth y l ether. F i n a l l y 3 5 ml. of acetone x-/as poured into the column. The procedure for the collection of the effluent caused by these s i x 3.dditions of solvent x-ras the same as that used i n collecting the flox'T caused by the sample solvent as described above. After evaporation of the solvent from the beaker, the l a t t e r vras x«7eighed to the nearest milligranx, and the per cent of the sample eluted into i t v/as calculated. I t was hoped that an examination of the elution c h a r a c t e r i s t i c s of individual o i l s X'7ould enable one to predict xfhether a p a r t i c u l a r f a t t y o i l could be separated from a pa r t i c u l a r mineral o i l by t h i s procedure. A great difference i n elution c h a r a c t e r i s t i c s xfould indicate a ready separation; a s i m i l a r i t y of such cha,racteristics x-rould indicate the impossihilit]'- of separation by t h i s chromatographic method. The o i l s examined by th i s method xxere: 1 . A v/hite mineral o i l - U. S. P., 340 S. U. v i s c o s i t y at 1 0 0 ° F. 2 . A paraffinio mineral o i l -"Topaz B" of At l a n t i c Refining Comnany, 1 0 0 S. U. v i s c o s i t y at 1 0 0 ° F. 3 . A naphthenic mineral o i l -" 5 6 1 O i l " of Gulf O i l Company, 100 S. U. v i s c o s i t y at 1 0 0 ° F. 4. A glycerol t r i o l e a t e - commercial grade. o 5 - Neatsfoot o i l - 2 0 cold t e s t . 6 . Herring o i l . 7. Salmon o i l - B r i t i s h Golumbitan Crude. 8. Menhaden o i l . 9. Coconut o i l . 1 0 . Soybean o i l . 1 1 . Linseed o i l . 1 2 . A blovm (air-oxidized) soybean o i l . 1 3 . Castor o i l - technical grade. 14. Sperm o i l - 3 8 cold t e s t . 1 5 . A methyl oleate - technical grade. In the case of blovm soybean o i l and castor o i l , both of vrhich are p r a c t i c a l l y insoluble i n petroleum ether, the procedure v/as altered somexfhat. With these tvro o i l s the sample v/as dissolved i n 3 5 ml. of diethyl ether and treated i n the column vrith successive portions of 3 5 ml. of diethyl ether, and 3 5 ml. of acetone. Follox^rins the termination of solvent flov; from the column, the position and color of the chromatogram bands vrere noted. The chrom.atogram vras observed i n both v i s i b l e and u l t r a v i o l e t light.•'^ The exhibition by an o i l of a unio_ue chromatogram v/ould indicate that the o i l could he i d e n t i f i e d hy means of t h i s chromatogram. ^The u l t r a v i o l e t lip;ht source v/as a Hanovia Mercury Vapor Arc Lamp producing a vmve length of 3 6 6 0 A°. IV. Explanation of Data Charts A chart for each o i l examined i s given showing the observed elution data and u l t r a v i o l e t chromatogram c h a r a c t e r i s t i c s . The v e r t i c a l columns i n a chart designated 1 to 7 re f e r to the chromatograms produced hj the p a r t i c u l a r solvents added i n the seauence shov/n: Column #1 - Solvent I n which sample i s dissolved. Golunm # 2 - F i r s t petroleum ether ad-dition . Column #3 - Second petroleum ether addition. Column #4 - F i r s t diethyl ether addition. Colximn # 5 - Second diethyl ether addition. Column #6 - Third diethyl ether addition. Column #7 - Addition of acetone. An upward extending gray bar i n a column of the chart indicates that elution occured during that addition The per cent of sample weight eluted i s given by the height of the bar and can be read from the left-hand mar-gin of the chart. The colored hands i n the upper part of a column of the chart show the color and position of the u l t r a -v i o l e t chromatogram bands present following the t r e a t -ment represented by the column. In order to f i x the position of these bands the alumina column was graduated from 0 mm. at the bottom to 100 mms. at the top. This alumina column graduation i s represented, i n the l e f t -hand, margin of the charts, so a band position can be read from t h i s margin. An uncolored handmarked "¥" represents a white chromatogram hand. The striped bar i n the v e r t i c a l column designated " A " gives the per cent of sample unrecovered (as read from the left-hand margin) i f i t i s red. I f i t i s blue, i t gives the per cent of sample recovered i n excess of the sample weight. This excess recovery ( F i g . #1 and #2) may be caused by i n s u f f i c i e n t drying of the eluted samples. 1 h O fii 0 j l ai < < I o ! I I I I ! I I I 1 4 4 1 I m I I ! I 11 F . G 3 ' Nl AP HTHE N ^ \ H l i E a A L O l U 1-1 ' ' I X ? 4 JT t 7 A —(--1 o p m y UJ 2 0 0 1 z TT li. o X_4-FIG. 4- 8 -t—i-I a i 4 E 7 A S o L Y E M T ADDITIONS 0̂  o r.a 7 ALMOK> O I L I P pi lii > 2 o lij ^ I D -J p. < y o p z < q : D D IT l o M S ICD Ho ?5 Xo PO A<\ 46 ^< 3o as' 1^ I o W w W V/ V V/ V w w y V/ V w w w w w w w V V o I ! ! 102: Fi 6 8i3 E N H A D E N 0 IL I a 3 4 S' L 7 A t n — —1 TiuT -TTTI o 1 JJ EL -M 1 0 Ui a P cQ rnti O loo 90 So 7a Si a. 4 o j 3.̂  30 3-0 10 M I ! I ! I M M J . L J L t x n J ,L j 1 1 . 1 i ! M l I I I I I l _ 13 COCLONOT OIL "TTtrtT r r m I a 3 4 5 t 7 A 0/ o 1 (X -2-0 a y p O F i g lOu SOTBEAM OIL 1 a 3 4 V 6 7 A p Ui lii 3 rr z Q Ji O J) ^5 46 7 o 51̂-1 4S^ 40 3 0 a o lo f irtz # I a i 4- < L 7 A rtrr kt4T .! 111 hjirr: o n T ' e i m 15 L O IL. T T - r t t r n M l M i l M iiro H \ y 4< 1 1, ^1- 4 JJ / 1 -/ N 1 Gr 4_ 4H \ 1 > 1 o v i s t / S N i n ki w > / 1 L 'I \ ^ S i _ — * V f< Ml \ r \ » A ... < k/ vi i [ P I 2 . M l l«l sc 1 c • " iT L > C ' a r c r 0-i •i 1 1 VI V >• Q-6 l - 1 1 I u - Ui W UJ r > • - 1 r T T Ui P 1 X X • •a "5 "5 "3 H i 2 ^ < 1 / / / / .\ / / / it' "A ;̂ A. A b • / • L - / / / f / 1 — 1. / u / / / / 1- A A / / j / / •/ f\ • 2 3 ^ i L 7 A -t r h ZtM T SOD I n o u s . 4 - - W\ # 4 -.... 1 V --M > mm r r t 1 1 b » r i t <?/ > R L J I L • c 1 >. * ,i /' i J u r ( i 4-T l i Li J ( 1 s 31 J — n 0 1 i \ 1 u «© T I "t 9 1 \ . I L » i l l n r • r m A — 4 c ' • CQ •4 • 1 1 1 I 1. is r w \ ] ( s J .'3 A 1 . t f 1 1 1 1 (. \ n i J n I-i fa H s L ) I V E N T / STi I T 1 O k l 9 h H4 -hHh hB-H4- h-m-fT-MM . n*. 1 • HI P J . . . — 8 L h \ j w 1 ( % m. 3r .3, J 1 X \ J: t -J D — M r 3< kV • • fi : 1; » i: I fi ili J ill n # _ l ( S li % B _/ )- n II '— \ c L 1 m L r n / n J • £.L —, • 1 • H • Xi 1 • n 0 — i 9 1 — il in i ) • f H < MU 5 ry 9 ii « y > S S JU 3c HI ^ HI < J Bi 9 y <^ M H H / I . ii c n 9 1 p " B • 1 c r ' (8 / / • / • 1 '— -> 1 / >• R 1 / # I IB / B B y H f H 1 5 7 It A s NTT D D I T I O - 4 : 4 1 1' 1 too ^ M m 1 1 y \ i - ! ^ t PS > 1 11 r 1 <; 1 1! 1 • n 4 T \ \ • < t p r P-/ < a' \ MB t 1 ' (1 * :̂  ~ 0 r u t > < >. tr ? (/ .i > 1 r ry Li. / I • L a« A X 1 — • m < • J J \ 4 ] J 1. A £ b * A d 1 1 c B-1 a Bi / / / 1 / 1 / / H / 1 / > / / / • ( / 1 1 i4z5*\-0 fa 1 n 0 L V fc-M T DIl i T l ON4S 44-J M4+ - H 4 4 - M 4 4 h4-h f-f-1 L / V X 1 S 3 ...J \ \ 1 6 'k L J • 1 j i 1 1 — 1 1 1 1 i 1 W' 0 / t A T T S T O O T \ f 1 r j —j • j -1 t IT.,. •••i ' I t D I (SI<o > 1 j j m I 1 1 I I F M CFk 43 1 t l t - / \ 6 1 » \ 1 • 1 X k g P E • • c 1 a I . |U vr . . . - I A / z H * i r t > j k C/] Q ' fi k C •* • — j f ' 1 ly y /, 1 • 1 1 / 1 / • / / X L / / ./ ( 3 I I * M a . ./L X 1 p S Ifcr C 5 D I 1 * O I N V E N T i ) iTl DNS r T V. Results and discussion A. Elution c h a r a c t e r i s t i c s of individual o i l s . 1. Mineral o i l s a. Wnite mineral o i l ( F i g . l ) Wnite mineral o i l i s completely eluted hy the three volumns of petro-leum ether. b. Naphthenic mineral o i l ( F i g . 3) Naphthenic mineral o i l i s com-pletely eluted by the three volumns of petroleum ether plus the f i r s t d i e thyl ether development. c. P a r a f f i n i c mineral o i l ( F i g . 2) P a r a f f i n i c mineral o i l behaves s i m i l a r l y to naphthenic mineral o i l . 2. Fatty o i l s a. Glycerol esters ( F i g . 4-13) A l l glycerol ester f a t t y o i l s were p a r t i a l l y eluted hy diethyl ether and acetone but not at a,ll by petroleum ether. The per cent of sample recovered a f t e r diethyl ether and acetone treatment varied betvxeen 57% i n the case of menhaden o i l and 98% for castor o i l . b. Alcohol esters Sperm o i l ( F i g . 14) 7% of the sperm o i l was eluted by the second petroleum ether addition and 83% by the f i r s t and second diethjrl ether washings. 10% \fas unrecovered. Methyl oleate ( F i g . 15) 36% of the methyl oleate was eluted by the f i r s t and second petroleum ether additions aznd 49% by the f i r s t d i ethyl ether addition. 15% was unrecovered. B. Separation of f a t t y o i l s from mineral o i l s . 1. Separation of glycerol ester f a t t y o i l s from white mineral o i l . Since t h i s l a t t e r o i l i s eluted 100% by the petroleum ether developments ( F i g . 1) vAiile glycerol ester f a t t y o i l s are not eluted by t h i s solvent ( F i g . 4-13)j a glycerol ester f a t t y o i l may thus be readiljr separated from vrhite mineral o i l by use of t h i s chromatographic method. The following procedure further supports t h i s conclusion. ViThen a mixture of 51% by vreight of neatsfoot o i l and 49% by weight of white mineral o i l i s chromato-graphed, i t i s apparent that the elutions produced by the f i r s t and second washings represent mineral o i l while the elutions produced hy the fourth thru seventh represent neatsfoot o i l components.* Altho mineral o i l has been almost 100% recovered by elution, only 84% of the neatsfoot o i l has been eluted from the column. That fra c t i o n of the 16% of neatsfoot o i l unrecovered which represents gl3''cerol esters could possibly be recovered by treating the column with a more e f f e c t i v e solvent. However, i t has been shown ( 2 ) , ( 3 ) , (4) that the more highly adsorbed glycerol esters can be recovered from an alumina column by extruding the column and vmshing the collected adsorbent with die t h y l ether v/hereupon the adsorbed glycerides are dissolved i n the solvent from which they are e a s i l y recovered. Since i t has been determined (5) that f a t t y acids react with active alumina, i t i s doubt-f u l that s l i g h t part of the unrecovered sample made up of free (non-esterifled) f a t t y acids could be recovered hy the above method. *Fi g . 1 6 shov;s the elution c h a r a c t e r i s t i c s of the mix-ture. Assuming the separation of the two o i l s to be complete, Figs. 1 7 and 1 8 show the elution character-i s t i c s of mineral o i l and neatsfoot o i l , respectively. The s i m i l a r i t y of these two figures with Figs. 1 and 5, respectively , i s evident. Separation of alcohol ester f a t t y o i l s (sperm o i l and methyl oleate) from white mineral o i l . a. Sperm o i l An examination of the elution character-i s t i c s of sperm o i l ( F i g . 14) shov/s 7% of the sample eluted by the second addition of petroleum ether, 77% by the f i r s t diethyl ether addition and 6% hy the second. VThile mineral o i l , on the other hand, I s 9 9 % eluted hy the two petroleum ether treatments ( F i g . 1 ) . A ready separation of these two o i l s by the above method i s i n -dicated. The recovery of that portion of the sperm o i l remaining i n the column would proceed as described under VBl, Separation of glycerol ester f a t t y o i l s from white mineral o i l . b. Methyl oleate An examination of the elution character-i s t i c s of t h i s o i l ( F i g . 1 5 ) reveals that 17% of the sample I s eluted by the f i r s t a,nd 19% by the second petroleum ether addition, and 4 9 % hy the f i r s t d i e t h y l ether treatment. Since white mineral o i l i s also eluted by two petroleum ether treatments ( F i g . 1 ) , a complete sepa.ration of these o i l s using the solvent-adsorbent method as outlined i s doubtful. Separation of f a t t y o i l s from naphthenic and p a r a f f i n i c mineral o i l s . Unlike white mineral o i l , these two o i l s a t t a i n 100% elution only upon addition of the f i r s t portion of diethyl ether ( F i g s . 2 and 3 ) . Since a l l fa.tty o i l s give elutions under the solvent action of t h i s f i r s t diethyl ether treatment ( F i g s . 4 - 1 3 ) , i t i s doubtful that a complete separation of the tvro o i l types could be made by use of a 1 0 cm. column of alumina. I d e n t i f i c a t i o n of a f a t t y o i l by the appear-ance of i t s chromatogrs-m. 1. I n u l t r a v i o l e t l i g h t Each sample examined gave a highly d i s -t i n c t i v e chromatogram i n u l t r a v i o l e t l i g h t . I t i s , therefore, possible to iden t i f y a f a t t y o i l hy mea-ns of i t s u l t r a v i o l e t chromatogram. In addition i t i s to be observed that a l l f i s h o i l s tested gave a brown band ( F i g . 6-8) while no other o i l s showed t h i s c h a r a c t e r i s t i c . 2. In v i s i b l e l i g h t As seen from the following table, the chromatograms observed i n v i s i b l e l i g h t are le s s num.erous and less d i s t i n c t i v e i n color than the u l t r a v i o l e t chromatograms. I t i s therefore impossible to identify a fa t t y o i l by means of i t s chromatograra as observed i n v i s i b l e l i g h t . I t i s to be observed that a l l f i s h o i l s give a jellOM band i n v i s i b l e l i g h t as do certain other o i l s , butthis band when produced by a f i s h o i l i s not to be observed under vi l t r a v i o l e t l i g h t although when produced by another o i l i s present i n u l t r a v i o l e t l i g h t as v/ell as v i s i b l e l i g h t . 27 APPEARANCE OF CHROMATOCRAI IS IN VISIBLE LIGHT O i l Color Position Developments Color of of Band of Band * 1 During VPnich Band In Band Present *2 U l t r a v i o l e t Light Herring Olive 9 0 - 1 0 0 1 , 2 , 3 , 4 , 5 , 6 Olive Herring Olive 9 0 - 9 9 7 Olive Herring Yellovj 9 9 - 1 0 0 7 Not Present Menhaden Brovrn 73-82 1 , 2 , 3 Brovm Menhaden Yellov/ 9 9 - 1 0 0 7 Not Present Menhaden mite 84 - 9 0 1 , 2 , 3 iiThite Salmon Tan 9 1 - 1 0 0 1,2 , 3,4 , 5 , 6 Tan Salmon Tan 9 1 - 9 9 7 Tan Salmon Yellow 9 9 - 1 0 0 7 Not Present Linseed Yellow 5 2 - 5 4 1 Blue Linseed Yellow 3 5 - 3 9 2 Blue Linseed Yellow 3 1 - 3 4 3 Blue Sperm Yellow 9 8 - 1 0 0 1 Yellow Sperm Yellow 9 7 - 1 0 0 2 Yellow Sperm Yellow 9 6 - 1 0 0 3 Yellow Naphthenic Brown 9 9 - 1 0 0 1 Yellow P a r a f f i n i c Yellow 9 9 - 1 0 0 1 Gray *1 The scheme for positioning bands i s given i n IV, Explanation of Data Charts. *2 The scheme of designating developments i n t h i s tahle i s sim i l a r to that used to designate them i n the charts. For an explanation of t h i s scheme see IV, Explanation of Data Charts. Future work. Since separation of chromatographic adsorption hands depends upon differences i n the rate of t h e i r descent thru the column, t h i s separation increases as the length of the column increases. . In certa-in cases the u l t r a v i o l e t chrom-atogram hanids of an o i l were poorljr separ-cated and thus hard to define. Increase of the column length over the 10 cm. column length used i n t h i s experiment should correct t h i s d i f f i c u l t y . Increase of the column length would also give more informative data concerning f a t t y o i l — m i n e r a l o i l separations. Future work extending the preliminary work presented i n t h i s paper should therefore be done on a column greater i n lena;th than 10 cm. REFERENCES 2 9 2. Segregation of the Component Glycerides of Linseed O i l hy Chromatography. F. T. Walker and M. R. M i l l s J . Soc. Chem. Ind. 6 1 , #8, 125-8 (1942) 3 - Segregation of the Component Glycerides of Linseed O i l hy Chromatography I I . F. T. Walker and M. R. M i l l s J . Soc. Chem. Ind. 62, #7, 106-9 (1943) 4. Segregation of the Component Glycerides of Linseed O i l by Chromatography I I I . F. T. Walker J . O i l Colour Chem. Assoc. 28, 119-34 ( 1 9 4 5 ) 5 . Separation of Higher Fat Acids by Selective Adsorption. Jaime Gracian Y. Tons and Agustin Vioque Pizarro Anales f r s y quin (Madrid) 42, 1 0 9 - 2 2 ( 1 9 4 7 ) 

Identification of Certain Fatty Oils by Chromatographic Methods

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Identification of Certain Fatty Oils by Chromatographic Methods

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