Quantitative Analysis of Lipids by Thin-Layer
`Chromatography 1
`M. L. BLANK, J. A. SCHMIT and 0. S. PRIVETT, The Hormel Institute, University
`of Minnesota, Austin, Minnesota
`
`Abstract
`A procedure is described for the quantitative
`analysis of neutral and phospholipids by thin(cid:173)
`layer chromatography (TLC) employing densi(cid:173)
`tometry. The chromatoplates are prepared with
`the usual solvent systems. The spots are charred
`under standard conditions and analyzed with a
`Photovolt Corp. densitometer equipped with a
`special stage designed for holding 20 x 20 cm
`chromatoplates. Each spot on the chromatoplate
`gives a peak of density values which is used for
`quantitative analysis.
`Radioactive lipids are analyzed by autoradiog(cid:173)
`raphy by the densitometry of radiograms of
`chromatoplates developed from X-ray films.
`The precision of the method is demonstrated
`on model mixtures of mono, di- and triglycerides,
`neutral and phospholipids and C14 labeled lipids.
`Results of the analysis of several samples of
`rat liver lipids compared closely to those ob(cid:173)
`tained by silicic acid column chromatography.
`
`Introduction
`
`SINCE THE FIRST REPORTS of the fractionation of
`
`lipids by adsorption chromatography (1,2,3,4,5),
`this technique has undergone continuous refinement
`and development for analytical applications.
`lJipid
`class analysis by chromatographic methods has been
`demonstrated by Borgstrom (6), Fillerup and Mead
`( 7) and Barron and Hanahan ( 8). The systematic
`studies by Hirsch and Ahrens (9) brought particu(cid:173)
`lar attention to the factors involved in the quantita(cid:173)
`tive analysis of the lipid classes by silicic acid column
`chromatography and provided a general impetus to
`its routine application in biological studies.
`Column absorption techniques have also been ap(cid:173)
`plied extensively to the fractionation of the phospho(cid:173)
`lipids based largely on the studies by Lea et al.
`(11,12) and Hanahan et al. (13,14,15).
`Recognition that no single system was adequate
`for the complete fractionation of the lipid classes,
`Rouser et al.
`(16,17), developed multi-column
`schemes for the analysis of lipids. Techniques em(cid:173)
`ploying a combination of DEAE cellulose, silicic
`acid and silicic acid-silicate-water columns were de(cid:173)
`scribed for the analysis of several complex lipids.
`The general principle involved in the use of a com(cid:173)
`bination of columns is to reduce the complexity of
`each fraction with each succeeding step until pure
`components or fractions are obtained.
`Since TLC is rapid and efficiently fractionates,
`a wide variety of lipids, its application to the quan(cid:173)
`titative analysis of the lipid classes is well indicated.
`At present, quantitative analysis of lipids by TLC
`has been carried out mainly by recovering the sepa(cid:173)
`rated components of a mixture and analyzing them
`independently by established analytical methods ( 18,
`19,20,21,22,23,24). Snyder and Stephens (25) de-
`1 Supported in part by U. S. Public Health Service. Research Grant
`HE·05375.
`
`vel?ped_ a~ elegant_ method for the analysis of radio(cid:173)
`active hpids by this technique. The measurement of
`spot size has also been employed successfully for the
`quantitative analysis by TLC (26,27,28,29,30). We
`have empl?yed the densitometry of charred spots for
`the analysis of mono-, di- and triglyceride mixtures
`and component triglycerides ( 31,32). Described here
`are refinements in this technique and its application
`to neutral and phospholipids. The quantitative
`analysis of radioactive lipids by autoradiography by
`the same general technique is also described.
`
`Experimental
`Mr:terials. Highly purified reference lipids were
`obtamed from The Hormel Institute, Austin, Minn.
`Samples of lecithin and phosphatidyl ethanolamine
`were ~solated from egg lipid by the general procedure
`described by Rhodes and Lea (10,11). Characteristics
`of these preparations have been previously described
`( 33).
`Sphingomyelin was isolated from beef heart by the
`~ethod of Rapport and Lerner ( 34). This prepara(cid:173)
`t10n was homogeneous by TLC analysis and no im(cid:173)
`purities co1;lld be detected in it by IR analysis (35).
`Cere~roside was prepared from beef spinal cord
`employmg
`the general procedure described by
`Carter . ( 36_) and Radin ( 37). However, repeated
`crysta~hzati?n of the. preparation as described by
`these mvestigators failed to give a chromatographi(cid:173)
`cally homogeneous product by TLC and thus final
`purification was carried out by sili~ic acid chroma(cid:173)
`tography.
`Samples of. rat liver lipid, for which analyses are
`reported herem, were obtained from animals receiv(cid:173)
`ing different fat supplements in nutritional experi(cid:173)
`ments. The results of these experiments will be
`reported elsewhere.
`Tripalmitin-1-C14, palmitic acid-l-C14 cholesterol-
`4-C14 and cholesterol palmitate-l-C14 .;ere obtained
`commercially and purified by TLC ( 28) . The ac(cid:173)
`tivity of all solutions was determined with a Packard
`Tricarb scintillation counter.
`
`General Procedure
`T~in-Layer Chromatography. The analyses were
`carr~ed out on 20 x 20 cm chromatoplates coated with
`a thm layer of silicic acid containing 10% calcium
`sulfate as a binder. The chromatoplates for these
`studies were dried and activated by heating them for
`2 hr at 110C in a convection type oven where they
`were allowed to cool to room temp. No special con(cid:173)
`ditions were. us~d for storage of the plates, but they
`~e.r~ use~ withm a day after their preparation. The
`sihcic acid used as an adsorbent for TLC in this
`study was prepared in our laboratory because com(cid:173)
`mercial pr?duct~, even tho~e compounded especially
`for TLC, mvanably contamed contaminant organic
`matter which made them unsatisfactory for prepara(cid:173)
`tive TLC. There are many aspects to the preparation
`of silicic acid. A consideration of the properties of
`
`371
`
`RIMFROST EXHIBIT 1176 Page 0001
`
`

`

`372.
`
`THE JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY
`
`VoL. 41
`
`300
`
`200
`
`1:1
`
`x
`
`100
`
`'/.
`
`0
`
`<(
`w
`0::
`<(
`
`~
`<(
`w
`CL
`
`10
`
`20
`ug CARBON
`
`30
`
`'l'l,C standard cu rve 8lrnwil1g rc\:1tio11ship of peak
`Yig. :i.
`m·ea vs. µ.g of carbou.
`0 Cholesterol pa.Imitate.
`0 Palmitic acid.
`X Tripalrnitill.
`
`Charring <>'. the spots was carri ed out hy heating
`the plates Ill an oven for 25 min at 180C after
`spraying them lightly with a saturated solution of
`K~Cr201 in 70 % by volume of aqtwous sulfuric
`acid (HD).
`Hadioactivc lipids are analyzc~d by autoradioO'raphy
`as follows. After the eh rornatoplat<~s are dev~lopeci,
`they are exposed to no-screen X-ray film in the
`holder shown in Figure 2. The holder is loaded and
`unloaded in the dark. The chromatoplate is placed
`on the stage CF'ig. 2) and the holder is closed. Then
`the chromatoplate is brought into contact with the
`X-ray film by removing the slide~ from the upper
`slot and placing it in the lowt•r slot.
`'l'he length of
`ti~n<~ the plate is left in contad with the X-ray film
`will depend on the activity in the spots. With Cl4
`labeled lipi~s, spots with 1000- 10,000 dpm can be
`measured with a 24-hr exposure period. Spots with
`100- 1000 dpm should be exposed up to ca. 10 days.
`~fter ~he. plates have been exposed for tlie r equired
`period of time, they arc developed photographically
`as described by Mangold, et al. (28,29) . The final
`radio gr~ms are trimmed to 20 x 20 cm and analyzed
`b:r: densitometry by the same general procedure ap(cid:173)
`plied to charred spots on chromatoplates as described
`below.
`Densitometry. A Photovolt Densitometer (Models
`5~C, 520A l_'hotomet~r, Photovolt Corp., New York)
`with a specially d esigned stage for holding 20 x 20
`cm ~hromatoplates was used for the densitometry.
`Detail~ ?f the constr~ction st~ge show in :B"'igure l.
`The shdmg metal strip ( C, Fig. l) contains a series
`of splits of different lengths (0.5 mm wide) to in(cid:173)
`sure high sensitivity regardless of the size of the
`spot. The metal strip is made of 3/ 16 in. material to
`col~imate the light. The small attachment (D. Fig. 1)
`wluch fits over the photocell also is for this purpose.
`It is important to eliminate all stray and reflected
`light; otherwise, standard curves do not pass through
`the origin. For this reason, also, all parts of the
`stage are painted black. The stage itself is made
`out of 1/4 in. Bakelite. The metal guides on the
`stage (E, Fig. 1) permit adjustment of the plate so
`that all spots pass directly over the slit. The photo-
`RIMFROST EXHIBIT 1176 Page 0002
`
`}'ig. 1. Modific(l stage for quantitative ']'LC.
`A. Chromatoplatc;
`B. llcight adjustment for photocell;
`Ci. Rtrip en111ailling various Hlit sizes ;
`n. Rlit cap for photocell;
`K A<l;inst:ible stage to hold ehrom:itoplaks;
`F . . Motor fo ,. eonti1111 ous stage move111 e11 t.
`
`these adsorbents for the separation of lipids has been
`reviewed hy '\Vrcn (88).
`'l'he following procl'dnrc was used for the prepara(cid:173)
`I11 our liters
`tion of silicic acid used in this study.
`40% so<li11111 silicate (Sargent Co.) is diluted with
`]~ liters distilled water, and the si licic acid is prc(cid:173)
`C'.ipitatcd by the addition of 2 liters conce n UCL The
`Pxcess allid is removed by washing the precipitate
`with <listilled water. When the washings arc neutral
`to met.b y l orange, the silicic acid (ca. 1400 g)
`is
`washl'd :i timPs with ('.a. 10 liters acdouc, then 3
`ti 111cs with ca. 10 liters of a l: l mixtltre of acetone
`a 11d diethy I ether and, finally, twice more with about
`;, liters diethyl ether.
`'.l'he silicic acid is then dried
`i11 vacuo, grnund with gla;;s marhl cs ( l cm OD) in
`a small laboratory ball mill, and sieved through a
`i\o. 200 mesh screen. The y i<'lcJ of final product was
`I lOO g. 1kagcHt grad e calcium sulfate ( 10% by wt )
`was tJw11 rnixt~d with it to serve as a binder.
`'I'he neutral lipids were separated with various
`ratios of 1lidhyl ether, petroleum ether and acetic
`1wid, an<l tht~ phospholipids with ehloroform, meth(cid:173)
`anol and water mixtures as described with the r esults.
`
`Fig. 2. Film holder for exposing chromatoplates to X-ray
`film.
`
`A. Slot for X-ray film.
`B. Chromatoplate on platform.
`C. Slot by which slide D raises chromatoplate against
`the X-ray film.
`
`

`

`MAY, 1964
`
`BLANK E'l' AL.: QUANTITATIVE ANALYSIS OF LIPIDS BY Tl1C
`
`373
`
`TABLE I
`Standard Curve Analysis
`
`Compound
`
`Cholesterol palmitate ................................................ ............ .
`Tripalmitin ........................•.........•............................... ••.........
`Tristearin .................................... ............ .......... .•...................
`Triolein ....................•...........•..........•.........•.........•...................
`Palmitic acid .......... .. ............ ................................................ .
`Stearic acid ......................... .............................. ................... ..
`Linoleic acid ............................................................ ............. .
`Cholesterol.. ........................................................................... .
`Hydrogenated egg lecithin ..................................... .............. .
`Purified egg lecithin ........................... ......... ........................ .
`Purified soybean lecithin ................ ..................................... .
`Dipalmitin ................................ ......... .•............................... ....
`Monopalmitin ......................................................................... .
`Sphingomyelin .................................... .. ......... ........................ .
`Phosphatidyl ethanolamine .................................................. .
`
`Slope
`
`.504
`.504
`.504
`.508
`.498
`.498
`.501
`.492
`.492
`.494
`.495
`.508
`.501
`.643
`.658
`
`cell support (B, Fig. 1) permits fine adjustment of
`the photocell above the chromatoplate.
`The chromatoplate may be placed either adsorbent
`side up or down on the stage. When a dark room is
`available, it is preferable to make the measurements
`with the adsorbent side up so that the adsorbent
`layer is not disturbed. After the chromatoplate is
`properly positioned on the stage, a slit is selected
`which is ca. 1/4 again as long as the diameter of
`the spot. The intensity of the light, with no filter on
`the instrument, is adjusted to 100% transmission
`when the slit is between the spots, and 0% transmis(cid:173)
`sion when the shutter in front of the photocell is
`closed. Analysis of the spots may be carried out semi(cid:173)
`automatically by plotting the readings on the pho(cid:173)
`tometer against millimeters of travel or automatically
`by means of a strip chart recorder ( Photovolt Vari(cid:173)
`cord Model 43 recorder). Each spot gives a peak
`of density values; the area under the peak is a func(cid:173)
`tion of the size and density of the spot and is used
`for quantitative analysis.
`
`Results and Discussion
`Analysis of the charred spots of a large number
`of lipids showed that the peak area vs. the amount
`of sample gave a linear relationship which passed
`through the origin, as demonstrated in Figure 3.
`The amount of sample is expressed as carbon so that
`all compounds may be equated on the same basis. The
`proportionality (slope of the curve, Fig. 3) of the
`relationship of peak area and amount of sample
`agreed closely for a large number of lipids as shown
`in Table I. This relationship depends on the extent
`of the conversion to carbon. Thus, the charring con(cid:173)
`ditions are important. The extent of the conversion
`to carbon is influenced by structure and mol wt. The
`conversion to carbon appears to be nearly quanti(cid:173)
`tative for most of the common lipids under the
`conditions employed. Phosphatidyl ethanolamine,
`sphingomyelin and methyl esters of long-chain fatty
`acids gave anomalous high values which cannot be
`explained at present, however.
`It was shown previously ( 39) that when the char(cid:173)
`ring was carried out at high temp (2500) and with
`a weak oxidizing agent ( 50% H 2S0 4 ) the amount of
`carbon formed resulted from two reactions, oxidation
`and evaporation. Accordingly, since unsaturated
`compounds are oxidized much faster than their satu(cid:173)
`rated analogues, they gave much higher yields of
`carbon. Surprisingly enough, an appreciable amount
`of evaporation of tripalmitin also occurred under
`these conditions.
`Variations due to differences in yield of carbon
`can be normalized by comparison with known amounts
`of suitable standards applied to the same plate.
`Apart from small differences due to carbon density,
`
`350
`
`300
`
`..
`•
`
`<(
`w
`a:
`<(
`
`~
`<(
`w
`CL
`
`250
`
`200
`
`Rf
`Fig. 4. Relationship between Rt value and peak area with
`tripalmitin.
`
`there is little variation in the extent of the conversion
`of compounds of the same class to carbon, as shown
`in Table I. Thus, a compound need not be identical
`with that being analyzed to serve as a standard.
`Regardless of the technique employed, poorly de(cid:173)
`veloped chromatoplates should not be used for quan(cid:173)
`titative analysis. Samples for quantitative analysis
`also should be applied to chromatoplates in low boil(cid:173)
`ing petroleum ether or other highly volatile solvents
`in order to avoid a chromatographic effect within
`the spot itself.
`Influence of R 1 Value. Generally, spots become
`larger the higher they are allowed to migrate on the
`chromatoplate. The increase in size is compensated
`by a decrease in density, giving the same densi(cid:173)
`tometrically determined peak area only when the
`spot is migrated sufficiently to form its spot charac(cid:173)
`teristics and not so close to the front to become dis(cid:173)
`torted. This relationship is illustrated with tripal(cid:173)
`mitin in Figure 4. These results show that the peak
`area is essentially constant from an Rf of about 0.3-
`0.8.
`The effect of Rt can also be normalized by the use
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`A
`
`B
`
`c
`
`• • • •
`
`Fig. 5. Chromatoplate of separated major lipid classes
`of natural and reference mixtures on silicic acid with 15%
`diethyl ether in petroleum ether + 1.0% acetic acid.
`A. Sterol esters;
`B. Triglycerides;
`C. Free fatty acids;
`D. Cholesterol;
`E. Phospholipids (even numbers are standard mixtures;
`odd numbers, samples of natural lipid).
`
`RIMFROST EXHIBIT 1176 Page 0003
`
`

`

`374
`
`A
`
`B
`
`2
`
`THE JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY
`D
`
`c
`
`TABJ,E II
`TJ,c Analyses of Standard Mixtures of Mono·, Di·, and Tripalmitin
`
`VoL. 41
`
`2
`
`3
`
`4
`
`3
`
`4
`
`5
`
`2
`3-5
`
`1-4
`
`5
`
`Tri-
`Di·
`Mono-
`
`Tri-
`Di·
`Mono-
`
`- - - - - - ·
`'I1ri-
`Di-
`Mono-
`--------·--
`
`#1
`
`68.0
`20.0
`12.0
`
`#1
`
`14.0
`45.6
`40.4
`
`#l
`
`6il.O
`t:l.O
`24.0
`
`Sample #I
`
`Abs.
`Avg
`Known
`#3
`#2
`Error
`- - - - - - - - - - - - - - -
`63.5
`64.0
`65.2
`-1.5
`66.7
`24.5
`24.4
`22.9
`23.1
`-0.2
`12.0
`11.6
`10.2
`11.8
`+1.6
`
`Sample #II
`
`- - - - - - ___ , , - - - - - -
`
`Avg
`
`Known
`
`12.4
`47.2
`40.4
`
`11.3
`47.0
`41.7
`
`Abs.
`Error
`
`+Ll
`+0.2
`-1.3
`
`#2
`
`11.l
`47.6
`41.3
`
`#3
`
`12.2
`48.3
`39.G
`
`Ramp le #TH
`
`#2
`
`(i5.ti
`12.G
`21.H
`
`#:I
`
`(i(),(J
`I 2.1
`21.f'i
`
`Avg
`
`fi4.n
`12.fi
`22.fi
`
`Abs.
`Kno\vn
`l~rror
`- - - -·· ---~
`-2.8
`G7.7
`11.6
`f l.O
`20.7
`+1.8
`
`111:1.ior lipid classes
`
`l•'ig. 6. Chrornatoplak of the f:eparatPd
`ill 4 Holvcut HyHtemH 011 the s:m1e plate.
`A. 5%, ether in pdroleurn ether
`B. 50% ntlwr + O.fi<Y,, HAC in
`petroleum ether
`C 15'/r' nthn I 1% llAC in
`pl"1roll'11111 dh1"·
`ll. 711::111:4 <~llC\,,:~froH:ll,O
`
`1. Choi<•sterol olcatc
`rrrio]ein
`•>
`:). Oleic acid
`4. < 'holcHkrol
`iJ. Lecithi11
`i ..... 'HZh
`
`of suitabk rd1•n•11cc standards. MorPOVPr, siuce most
`natural mixt11rcs of lipids <~ontain !'omponnds with
`widr>Jy difforl'llt polarities, the use of r1•forence stand(cid:173)
`ards is recomnwuded whenever thPy are available,
`h1•1·a11s1• they pPrmit the analysis to be carried out
`011 a sing-IP plate as wl'll as Pli111inati11g- variations
`duc to charring-. 'l'he general f>rO<'<'<h.1r1• in the analy(cid:173)
`sis of a sarn pie with standards
`is illtu;trated
`in
`l•'ig11r1• G.
`l ls11ally, 4 sarnpl<'s of both rder1~11ee stand(cid:173)
`anl and samp]p arc spotted on a singl<~ plate. Sine<~
`
`T.LC PATH METHOD
`B
`C
`
`A
`
`D
`
`Fig. 7. Chromatoplate illustrating path method for the
`analysis of lipids with tripalmitin developed to different R,
`values.
`A. 5% et20 in petroleum ether.
`B. 10% et,O in petroleum ether.
`C. 15% et20 in petroleum ether.
`D. 20% et20 in petroleum ether.
`
`the standard curve for each <·ompo11111l pass1•s through
`the origin, 4 samples of the same 1~011c11 can be ana(cid:173)
`lyzed and the average of tlw 4 valtws <'.omparcd
`diredly with that of the sampl<'. F'or example, if
`the avcraw' area for a 10 0 g-
`load of the stan<lard
`triglyc<'rid<~ was 400 aud the avcrag-<' of thn 1111k11own
`sample was 200, it could be 1•oncl\l(IPd that tlw 1m(cid:173)
`known sampl1~ contained G fLg" trigly1·Pridc.
`'l'hc per(cid:173)
`eentag-c eom position
`is
`th1·n calen lakd fron1
`the
`amount of sample spotted on the plate.
`I 11 addition
`to the use of standards, or if standards arc not
`available for all components of a mixt11r1', each com(cid:173)
`pound may be migrated to an Hr vahw h1·i w<'<'n o.:3-
`0.8 for d ired corn parison of areas.
`I 11 order to
`eliminate variations from one chrornatoplatc to an(cid:173)
`other, such an analysis is usually carriPd 011t on the
`same plate by dividing it ( scori ug the adsorbent
`layer) into sections, one section for Nteh diffon~nt
`sol vent system.
`'l'his technique is demonstrated 011 a 111odel mixture
`of the lipid classes in B'igure fi. After the sample
`in the first section is developed, the adsorbent layer
`is scraped from the bottom of the chromatoplate
`above the height of the solvent in
`the chromato(cid:173)
`graphic jar. Then another sample is spotted in the
`second section of the chromatoplate. After it has
`developed, the procedure is repeated until all of the
`compounds of the mixture have been developed to an
`Rr in the range of from 0.3-0.8. For the greatest pos(cid:173)
`sible accuracy, this technique may be applied along
`with reference standards.
`In
`the analysis of radioactive lipids, reference
`standards of known activity must be applied to the
`same plate as the sample in order to normalize the
`conditions of exposure to the X-ray film and the
`photographic development of the film.
`Path Method of TLC. This method is illustrated
`in Figure 7. The sample is developed between lines
`scored in the adsorbent. The lines fix one dimension
`of the spots; the other dimension, as well as the
`maximum density of the spots, is determined by
`passing the spots over a slit shorter than the distance
`between the lines. The product of the area of the
`spot (calculated from the dimensional measurements)
`and the max density form a linear relationship with
`the amount of material in the spot. The disadvantage
`of this method is that the efficiency of separations
`is decreased because relatively large samples must
`be applied to the plate. We have applied the prin(cid:173)
`ciple of the path method in the analysis of triglyc-
`RIMFROST EXHIBIT 1176 Page 0004
`
`

`

`BLANK ET AL.: QUANTITATIVE ANALYSIS OF LIPIDS BY TiiC
`TABLE III
`TABLE IV
`Silicic Acid Column Chromatography vs. TLC Analyses of
`TLC Analyses of Standard Mixtures of Lipid Classes
`Rat Liver Lil)ids
`(animals on different fat supplements)
`Sample #1
`10% Corn Oil
`Fat-Free
`#2 I #3
`Column
`TLC
`TLC
`Column
`- - - - -- - - -
`S.E.
`6.2
`7.2
`7.5
`7.0
`T.G.
`48.0
`49.4
`20.7
`18.8
`F.F.A.
`none
`none
`2.5
`1.5
`Sterols
`2.4
`3.8
`5.8
`7.0
`P.L.•
`43.4
`39.6
`62.:l
`66.9
`• By difference on both column and TLC.
`S.E. = Sterol esters.
`T.G. =Triglycerides.
`F.F.A. =Free fatty acids.
`P .L. = Polar lipids (consisting mostly of phospholipids).
`
`MAY, 1964
`
`C.E.
`T.G.
`F.F.A.
`CHOL.
`H.L.
`
`C.E.
`T.G.
`F.F.A.
`CHOL.
`H.L.
`
`#1
`
`19.5
`19.2
`23.0
`20.0
`18.3
`
`#1
`
`9.0
`27.7
`31.6
`25.6
`6.1
`
`- - - - - - -
`19.0
`18.6
`20.2
`18.8
`23.3
`23.4
`20.3
`19.8
`19.6
`18.0
`Sample #II
`
`#3
`#2
`- - - - - -
`8.5
`7.0
`26.9
`26.8
`31.0
`30.6
`I
`26.8
`28.3
`6.8
`7.3
`Samvle #Ill
`
`Avg
`Known
`- - - - - -
`19.0
`18.8
`19.4
`18.8
`23.2
`23.4
`20.3
`20.0
`18.6
`20.1
`
`Abs.
`Error - - -
`
`+0.2
`+o.6
`+0.2
`-0.3
`-1.5
`
`Avg
`Known
`- - - - - -
`7.4
`8.2
`26.1
`27.1
`31.1
`30.4
`28.2
`26.9
`6.7
`7.9
`
`Abs.
`Error - - -
`
`+o.8
`+i.o
`+o.7
`-1.3
`-1.2
`
`375
`
`10%
`Column
`- - - - -
`2.8
`69.5
`1.0
`3.0
`23.7
`
`Lard
`TLC
`- - -
`3.7
`67.6
`4.0
`6.5
`18.2
`
`tometry of a spot on the origin did not give reliable
`results. The results of the analysis by the simple
`system described above show in Table V.
`The analysis of this fat was also carried out by a
`corresponding simple silicic acid column chroma(cid:173)
`tographic method. The results also show in Table V.
`The column analysis was carried out by the stepwise
`elution of a 2-g sample from a column 20 cm long
`and 4.7 cm in diam. The elution was started with
`petroleum ether (bp 35-60C) and continued until
`all of the sterol ester fraction was eluted as deter(cid:173)
`mined by TLC analysis. The elution was then con(cid:173)
`tinued with 10% ethyl ether in petroleum ether until
`all the triglycerides were eluted. The sterol fraction
`which consisted mainly of cholesterol was eluted next
`with 50% ethyl ether in petroleum ether. When the
`free fatty acid content of the lipids is less than 5%,
`the amount of it which contaminates the other frac(cid:173)
`tions is extremely small, and the bulk of it is eluted
`in the last fraction with ethyl ether containing 1 %
`acetic acid.
`The polar lipids were eluted with methanol; but,
`since frequently the complete recovery of these com(cid:173)
`pounds was not practical, they ":ere estimated by
`difference. Comparison of results m Table V shows
`that the results of the TiiC method agree well with
`those of column method and indicate that TIJC may
`be used for the quantitative analysis of the neutral
`lipids.
`The neutral lipids of some fats may contain ap(cid:173)
`preciable amounts of lipids other than those listed
`above· for example some fish oils contain appreciable
`amou~ts of glycer~l ethers. These may be analyzed
`by the same general technique using the appropriate
`standards. It may, of course, be desirable to use
`more than one solvent system to fractionate all the
`components. In this case, one component which gives
`a measurable spot in both solvent systems is used to
`normalize all the values. Separations in different
`solvent systems may be carried out on the same plate
`by employing the technique i~lustrated in Figure 6.
`Polar lipids are analyzed m the same manner as
`the neutral lipids with the appropriate solvent sys(cid:173)
`tem on a small sample isolated by preparative TLC.
`The recovery of the polar lipids is usually ca!rie.d
`out by scraping this fraction from .a plate while it
`is still wet with solvent. The material scraped from
`the plate is slurried with water and placed in a small
`
`TABLE v
`TLC Analysis of a Standard Mixture of Polar Lipids
`
`#1
`
`#2
`
`21.9
`
`23.9
`42.4
`11.8
`
`Abs.
`Avg
`Known
`#3
`Error
`- - - - - - - - - - -
`20.6
`21.9
`20.8
`+i.1
`
`24.2
`42.0
`13.2
`
`24.3
`41.9
`11.9
`
`23.3
`44.8
`11.l
`
`+i.o
`-2.9
`+o.8
`
`Cerebrosides ............. 23.3
`Phosphatidyl
`ethanolamine ........ 24.9
`Lecithin .................... 41.2
`Sphingomyelin .......... 10.6
`RIMFROST EXHIBIT 1176 Page 0005
`
`#3
`- - -
`9.6
`45.0
`10.8
`10.4
`24.2
`
`Avg
`Known
`- - - - - -
`10.2
`9.1
`44.8
`45.9
`10.7
`11.2
`10.8
`10.0
`23.3
`24.3
`
`Abs.
`Error
`- - -
`+1.1
`-1.1
`+o.5
`+o.8
`-1.0
`
`#2
`#1
`- - - - - -
`C.E.
`10.0
`11.1
`T.G.
`46.0
`43.3
`F.F.A.
`11.1
`11.8
`CHOL.
`10.2
`11. 7
`H.L.
`22.7
`23.1
`C.E. =Cholesterol Palmitate
`T.G. = Tripalmitin
`F.F.A. = Palmitic Acid
`CHOL. = Cholesterol
`H.L. = Hydrogenated Lecithin
`
`eride structure (32). Since the components in the
`analysis of these compounds have very similar struc(cid:173)
`tures, they give spots of the same shape and char(cid:173)
`acteristics. Thus, it is not necessary to develop the
`spots in channels to .control ~he~r s1:ape. Althoug.h
`this method has special application m the analysis
`of triglyceride structure, it has no particular ad(cid:173)
`vantage over the normal procedure using a slit just
`longer than the diam of the spot especially when
`reference standards are available.
`Analysis of Standard Mixtures. The precision of
`the method for the analysis of the lipid classes which
`is frequently found as a major component of lipi<_ls
`shows in Tables II, III, IV. The absolute error m
`these analyses is about ± 1.5%. The solvent systems
`used for these separations may be varied somewhat
`from that employed in this study for ideal separa(cid:173)
`tions, depending on the activity of the chromato-
`plates.
`.
`..
`Analysis of Natural Fats. Rat liver lipid was se(cid:173)
`lected to illustrate the application of the method to
`a natural fat because it contains appreciable amounts
`of both neutral and phospholipids, and because this
`lipid is frequently the subject of biological studies.
`The lipid was extracted from the livers of animals,
`killed by exsanguination, with chloroform-methanol
`( 2: 1, v /v) in a Servall Omni-Mixer. The solutions
`from 3 extractions were combined and evaporated
`to near dryness. Then the aqueous fatty resi?ue was
`dissolved in ca. 100 ml diethyl ether and dried over
`anhydrous sodium sulfate. By repeating this process,
`the water as well as the chloroform and methanol,
`was remo~ed. The solutions were maintained under
`an atmosphere of nitrogen throughout all operations
`to avoid oxidation.
`The composition of the neutral lipids was d~ter­
`mined first on an aliquot containing a known weight
`of the sample dissolved in low boiling petroleum
`ether ( 35-60C) by the method illustrated in Figure
`5. Cholesterol oleate, triolein, oleic acid, cholesterol
`and hydrogenated lecithin were used as standards
`for the analysis. The "polar lipids" were estimate?
`by difference because, as
`indicated above, densi-
`
`

`

`376
`
`TnE J ouRN AL OF THE AMERICAN OIL CHEMISTS' SocrnTY
`
`VoL. 41
`
`TABLE VI
`Analysis of Rat Liver Polar Lipids
`(10% corn oil supplemented diets)
`
`Phosphatidyl ethanolamine ......................... .
`l..recithin ........................................................ .
`S1>hingomyelin .............................................. .
`Others ........................................................... .
`Loss .............................................................. .
`
`Percent Composition
`
`Silicic
`Acid
`Column
`
`26.0
`52.4
`14.0
`2.7
`4.9
`
`TLC
`
`31.0
`50.6
`16.0
`2.4
`
`sintered glass funnel through which it is filtered
`by means of suction. When most of the water has
`been filtered, the adsorbent is washed with methanol
`several times and finally with chloroform. The sam(cid:173)
`pl(~ may be recovered as previously described or by
`(~vaporation of tlw solvent under reduced pressure.
`'fo illustrate the applieation of the method to a
`natural rnixtnn~ of the polar lipids, a sample isolated
`from the liv1•rs of 1he corn oil suppl<>mented group
`of rats was analyzed ('l'able VI).
`!<'or eomparison,
`a sample also was analyz(~d by silicic aeid colnrn11
`ehromatography by essP11tially
`the same elution
`s<'l1rnH· d<'i·Wri lwd by Rho<IPs a11d
`fJea
`( 10, 11). Tn
`this sdH•nw, the dntion was started with <'hloroforrn
`whi<'11
`t«'lllO\'Pd
`t nt<'<'s ot'
`\111k11ow11 mat Prial.
`'l'h<•
`plwsphat idyl dhanolarn ill!' t'ral'tion was elnted nPxt
`with a !l: 1 ( "/") ratio of d1 loroform-nwt hanol. vVhell
`all b11t a 1nwe of this t'rndion had <~mergccl, the
`Pllltio11 was (•011ti111H•(l with a 7:2 (v/v) ratio of
`<'hlorofonn-rn!'!ha11ol. A small intenm•diat<: fradion
`was ob1aincd, after whid1
`the main bulk of the
`k<'ithi11 was Pln1<>d with a 7 ::l ratio of thesP solvents.
`Toward the <•nd of the Pllltion of the lecithin, some
`sphingolipids started to (~llH~rgc from the column.
`'l'hc•sp fractions were collcd('d separately, then the
`sph i 11gol ipicls W<'re elutc•d with methanol.
`'l'he entire
`f!-ac·tio11atio11 was followed hy 'l'fJC analysiR. Analy(cid:173)
`sis of the mixed fractions was detPrrnined by the
`qlla111i1a1iYP 'l'fJC teehniq1H•, m; well as by IH spectral
`analysis, as cles<'ribed by S!lli1h and Freeman ( 40).
`Th(• rP1mlts in Table VI W<~re caleulated from the
`WPights of
`the pure fractions corrected for
`the
`amo1111ts in 1ht mixed fraetions. The values obtained
`by tlw two m<'thods agn~ed well, and indicated that
`the nwthCHl is applicable to polar lipids.
`?\o att(•111pt was made to make a d<~tailed analysis
`of the corn poncnts of the liver polar lipids because
`resolution of many acidic from nonacidic lipids can(cid:173)
`not be effoct<>d 011 silicic acicl ( 16). However, since
`Ruc·h s<~para1 ions are readily performed on DEAE
`columns (17) the problem of the analysis of complex
`lipids is lwi11g attacked by a combination of this
`technique and quantitative TT,C in a collaborative
`f'ffort by the authors with George Rouser and his
`co-workers.
`Analysis of Radioactive Lipids. Results in Figure
`8 demonstrate that the activities in the spots form
`a linear relationship with peak area of the spots on
`the X-ray film determined by densitometry as de(cid:173)
`scribed above. Thus, it is .Possible to determine the
`distribution of radioactivity among the components
`of the mixture by the same general technique em(cid:173)
`ployed with charred spots.
`By combining the charring technique with auto(cid:173)
`racliography, specific activity can be determined.
`When care is taken to insure against saturation of
`the X-ray film, the radioactivity of unknown com(cid:173)
`pounds can also be determined. Radiometric methods
`of analysis used in conjunction with TLC have the
`
`ISO
`
`<{
`w
`0::
`<{
`
`100
`
`so
`
`0
`
`0
`
`400
`
`800
`DPM
`l•'ig. 8. Standard curve of cholesterol palmitatc-1-C" Hhow(cid:173)
`illg relatio11shir between penk area :rnd r:1dio:1ctivity.
`
`1200
`
`1600
`
`advantage of speed and gn~ater s<>11sitivity whell
`S('intillation counters are availabl<). However,
`in
`many i11stmwes, particularly with poorly rpsolv<>d or
`f'Olltplex rnixt11res, it is JJPl'.!'ssary to l(wa1<> tfw posi-
`1 ions of radioac:tive lipids by <UL1oradiography. 1'!111s
`1hP above proccdnn~ SPI'V<'s as a good adjunct to
`radiometric m<>thods, allfl, i11 <·0111bi11atio11 with <·arbo11
`analysis as d(~serib<•d above, p<'r111its tlw <kkrrni11atio11
`of SJH'<'.ifi(, adivity.
`
`Ji; . • Ta:vko,
`
`.T. Biol.
`
`l1J. ~Jayko, .1. Am. Chr,m