A novel microtechnique for the measurement of acoustic properties of lipids

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laert-.E,AP~FEL.and.DonaldC, MALINS

l ~ r t h . ~ . ~ ~ a ::~ter, Nationals.MarineFt~herie~~gerrtce,National Oceanicand ~mo~ ..~c Admi~ffat~on,.~and.Cy~eac!~.tr.J, Department,.Secttle University.,Seattle, . ~ , 98122.a~ .f~:.£A,J:~ m e n t o f Englneertng:_andAppl~d Sctenceo Ma~on Inborato~, YMeUnl~erllt.y,•New-llaven, Conn. 06520, USA .

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Received September 21st,1976,

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accepted November 25th. i 976

Application of anew microtechnique is described for the derivation of acoustic properties (uRrasonicvelocity, adiabatic Comprfutibility and acoustic impedance) of lipids. The technique u ~ v e t y . . . s m a l l v..0tumes (4 X i0 -3 - 4 × 10 -6, ml) of lipids and thus overcomes the requiremerit for !arge volumes inhel~,nt in the use of conventional velocimete~s. The use of the microt .echnique ~ W s jeoustic.pmpe,tries to be included among the variety of other physical proper-tiCs~¢ratinely measured in the characterization of liplds, including those isolated in small amounts from complex.biological mixtures.

I. Introduction ~ter.est "Inthe biochemistry of echo locating porpoises aad whales has focused

.Ol!:i[elati~~ting bet~n]ipidstructure and sound properties in acoustic tissues of!t~/:~ad[!,.3],Moreover, sotrod velo~tyof fats and oils i~ considered to be an i m ~ a n t p h Y ~ ~o~ty: of potential valuein characterization of these compounds ::[4,$~ Yet an undemanding of relations between molecular structure and acoustic ,lXO.perties ~ ~ severelyhampered by an inability to .measure ultrasonic veloci. t~Zt!t.~ ~ t amunts of hi,ghlypure !ipi~ that are attainable from biological ~ U ~ , . l~esentlycmp~yed velocimeters(i.e., the sing,around type) require any•

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.:~e.from/::$ i.~ .65::~-of tiquid.:for acc~ate measurement.ofultrasonic velocities ~::|!,4,$|,:~en. tly, a~:~w,m i c r 0 t ~ u e for determiningultrasonic velocitiesof . ~ ~.ljquidi!sam~(4X 10- 6 - 4 X 10-3 rot)has been developed [6]. This tethe r . . ! ~ ~ - a . ¢ ~ ~ n : ~ O f the.~nditiom required f:,r the acoustic levitation : : ~ ~ ~ / i O ~ ! ~ ~ , ~ . !i!~d .a t a : . ~ . ~ition in a.kno~ , immiscible'host' li~ : . i!:~~ d ~ e / ~ ~ n / 0 f a / : ~ e g ~ c e ~opletl of ~own: propertiesat the-~me po-i ;~~.~i ~ i i ~ U ~ ; t ~ . q ~ . ~ ~ !~ ,nsJ'~bk promise for overcoming/the i ~ ¢ ~ t ~ i : ~ t i i n , t~~i-iWof con~fi~l:~t~imeters, . . . . . . :. : i!i:ii~! ~ i t . i ~ r ~ i ~ ~ . i , ~ _ a~liea~n 0fthe,le~tationtecJmiqueto the:

logical samples, including compoUn:~:f0und in. cetacoana:cous.~c tin~i(e,g~; iso~: : i . valeroyl~ycerides a n d ~ estm).Close.c0mp,m .s0m iwerefound~weeni:~tr a. •:::.~

synthetic andnaturalmiXtures, :: : :~.:,: . : :/i :~ ~::~::i-! : .::i: ~: i: :i- . :i .i!:~:~~i-:ii :i:::ii , ..... :!:::ii:::~; >:-~/-:: ::ii -::~ /?:. >i"!::::i•:: .~::•: :: i"••-•:-;:".i.:!'.~Li,i,:i.,::::/~:~:i::•>:!:!::i:i:i •i`~•~:::'~/~::!:!i `:!•!' H. Experimental:: .`.> :':: • • : @='=='r=~'~=~:''=""@.'',' @"--':a'~:: ::"~' ~'~0: ~"=f~=" 0~+''='¢:: ~@>~" ='~" ''''::

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Hexadecylisovalerate and t,3-.dii~valez0yi,2:laexadecanoyl~ycero]i Were.syntlte,:: sized in our laboratory [ 9 ] : a n d : p o r ~ . ~ t r ' m c . . y ! # y c e r o l s ~ n d w a x : e s . t e r s : w e ~ e i s o + . lated from the melon o f a p o ' p o i s ¢ , - T W s i O p s t~,n~tus,as dmcfi~d:previously. [10, 11]. Other Epid standards were p u r c ~ d tromNucheckPrep,,"~yflan)-~nn. and Applied Science ~ b o r a . t ~ ~,i.State=~g0, ~ , ! ~ - : l i ~ . . cmlfl0ye d: in i ~ ~A levitation instrument d~gned; by A p f e l ~[..6.]wa~.us0d:" mobtam~ng" ..... .a..known-- ' function Ofadiabati.c compressibilitya.ndd.emity.-of!lipidsam~i ~Thedeflgn Of:<:.:. the instrument is descri~, d elsewhere i[6]"~ d e t a l ! . : ~ . p t O c e d . m e " i n v o l v ~ a c e t i c levitation of a droplet'0f a liquid(4 x i.0 -~ ml,.4.ix ~l:0y6:.I.~:)..~.a glasscolumn~ " containing an immiscible .host liquid, such. as water.Ac, ousttclevitation of ~oplets of liquids in the host liquid column depends o n balancing o f acoustic,force and buoy-

ancy. A droplet of reference liquid (benzene) oflmown adiabatic compressibili, ty f#s*) and density (9*) was levitated at a Certain positionin thehost liquid.w.hose. adiabatic compressibility ~ ) ~ d d e ~ t y , ~ ) . ~ r e ~ k n . o ~ . ~ e voltage input (V*.) : corded. A droFtet..ofa,!ipid:'.~s,theal..~~!iat:.:~~lyi~~~~i.:~here-:i ~ the reference droplet.was:suspen~.tn!the.Wat~ c o i U _ ~ ! i . ~ e . : ~ 6 i ~ e : : ~ ) ! i ~ m " ~ l > . to levitate the.lipid droplet w a s . . , n o t e d ; i A i m i ~ u n t o : ~ ~ ~ . . / . ~ O ~ i w e ~ : . . : ; , . - : ,. ii. carried out for each ~mpte,- ~e:a~o.mtic~tation,:~Ob-~!~e.~i:~WO.~, . cMculated fromthe followingequafi0n:, :-.- ' ~,;..... :,:..,:.... ~>-:::..,:..... .,:" . . ' , , , - : - , "--. .

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Because.values of-#, p*,/~and ~ are known and V* and V' were determined expefimerRalty,eq, (1)maybe used to yield a known function o'f p'/p and g'/gs de. scn'bed.as,th¢ .Co.mpremdensity function [6]. When the density (05 of the unknown : ::s a ~e iisl ~ ~ i ~ p ~ t e i y , t h ¢ . a d i a b a t i c compressibilit y (~:i)may be derived --.;-£rom;t~..¢~pressadensity funct~Jon,,Once..-p:and-.~- :.are:known, the ultrasomc veto. :... at.y, C--;0f:the..un~own~S-.obtalned: trom the equa_tlon • " " ;

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In the present work;densitiesof most of the lipid samples were obtained using • ::either a p y ~ o m e t e r [I] or.a densitome.ter.0 f a typesimilar to tlmt produced by the Metrier :~rporation~ The den-sities were determined at 23 +-.0.5°C and had a preCision of ± 0 , 0 0 3 g - c m - 3

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• :The determination Of sound properties required relatively large volumes of sample until thedevelopment of the levitation technique of Apfel [6]. This requirement severelyhampered the acquisition of data on biological samples and synthetic mixtures whi,?h were difficult to obtain in gram amounts in high purity. These limita. tions.eontributed to the fact that only a few reports exist on the sound properties oflipids. Gouw and Vlugter [7 ], employing a commercial interferometer requiring 5ml of liquid, determined the ultrasonic velocities of a variety of pure triacylglyccrols and .natural oils. Hustad et al. [8] also obtained altrasonic velocities of several fatty acids and triacylglycerols on 35 ml liquid samples using a veiocimeter of the Sing-around..type.Moreover, Varanasi et aL [ 1] reported ultrasonic velocities of por:PO"~.t.da~lg!y~rols-and wax esters t/ch in isovaleric acid using a sing-around velo.ci~ter.r-.~ring 65 ml of liquid. Recently Blomberg and Jensen [3] and Flewellen ::and Mo.r~ [2] reported ultrasonic velocities of head oils from pilot whale and ,~perm whale, r c s ~ i v d y A n e a c h of these reports,, relatively large volumes (> 4 mI). of

Used... ."-. l a ' : t ~ . ~ . ~ n t paler we investigated the application of the levitation technique . !:. to t~ .".~~:of a variety of lipid classes., Prior to this time..,the levitation techni-- .qUe.wasl~ l y employed for the det~mination.of the acoustic properties of a lira"i i ~ : a ~ r , of volatile organic compounds, Such as n-pentane and benzene [6,12 ]. • .Tab~ i giv~ ultrasonic ~locity, adiabatic compressibility, and acoustic trope. :..i~:i~a..:~0~=i..a:~!..yOf.fipi.~.~r¢ possi'bl¢,, ultrasonic velocities and adiabatic ::.":: :"":::::"~~~:i0btaincd by-the!~tatiOn technique are compared with previously :. "..:~~~i.~S~-obtaincdby-con~tional..ma.erotechniques. Compared.values were . ~~i:::d~r ~dwithin [he:li~tS Of reln.oducibility of either analytical proce• :,:-~:::-:.~...:~i~ m ~ : ~ ~ : ~,~ :-~-."-.~'ted"toshow that acoustic properties of micro-droplets ': '::~~""i~"~:"~:~aie~:~Sured b y t h e levitation technique. Such small quant i•

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Table 1 C o m p a r i s o n o f acoustic properties of synthetic and natural ~-pids measured by levRat~on t e c h ~ q u e and other techniques,

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•~ties, of l i ~ are readily obtainab!,. ftomcomplo~: .biological mixtures by procedures, such~ thin, layer Chx0mat~aphy~ [13, I4]. Moreover, the.data show that the levita•. , . t i o n : : : t ~ ~:can:5:.~:applie.d : s ~ ~ f u l ! y :to:different types of l i p i d s . . . •.":::iiiii:~id,at~i ~:~t,a b l e i ! : ; ~ . : t l t a t : s ~ ~ t ~fferen~.insound. properties exist in :":~ ~*::":':~~::0' ~ :~.[ptd,:,:.,.....!.,_, ':::"' : s i ~ ~ e ~..,:,~.. i.~.~u~.~,o~.: re~0Us~~ .::t: ~ ~n:,t ,.:....,~...... : ,..p.,-...,~..... :,,[ l~. ]~":Notable . . . . . . . d~ifferencesin • . . ultrai. , ~ { R l ~ i : m e o b ~ o m bet~,)Rde~lociad~noat¢ and hexadecylisovalerate / a " ~ ~',eat~i:i~!d:::in i~tacea,rt ~.~~:,S~Mtantiat diff©renc~ in sound propertiesal:-!Sole m t i ~ : ~ i : ! i ~ ~ v a l e r o Y ! i . a y m i ~ a n d trioctadecenoyl ;0ycerol. Moreover, :i: i t h e i n t ~ o n b f . . a : . ~ n d d 0 m e ~ . ~ n d ] n t o the hydrocarbon chain of octadeccne ::|0we~ t ~ . URt~nicvelocRy:ftom!i,436 mlsec.to 13Sl mlse.c. Interestingly, how, -.everi an ~dmo~lof ~ e i.~te double.-bondproduces a slightincrease in the velocity - o f o ~ c a t t i e n e . t O l 3 6 8 m t s e ¢ . ~is.findigg implies that minor differences in lipid !.:str~cture ~ t h i n a Class canbringabout substantial alterations in acoustical proper-tiesi These results, are in agreement with.studies carried out by several workers show~ingthati fin addition, to c ~ g e s i n molecular weight, subtle structural variations such ,esnum~rsOf double;bon~,.branc~g, or cis-trans isomerisation can cause signifi~ntchangesin:sound velocity and adiabatic compressibility values [ 1,7,8]. • ::~TheicqUi~nt forthe levitation procedure does not ,require. special skill to manu, ~ faetme and application tothe analysis of lipids is both'simple and rapid. Thus it be:"Corns ~ b l e to include s o l d properties among a host of other phyliical proper"ties Whi~im.measured routinely in the characterization of lipids. Moi:eover, it is • measure with great accuracy the compressadensity factor oi,~lipid samples e..fromroom temperature up to 150°C. This broadens the scope of the le,vitation technique to include acoustic properties of samples that are liquid within a wide tempera.ture range. Ourf'mdings suggest that the levitation technique should fi~!tdwide appli.~'-.tO.the:analytis of small amounts of individual lipids and li#d miixtures, •

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We thar~ J.R. Maloney and R.E. Young for technic~:lassistance. U.~Varanasi and D,C ~ thank the Iliolosjcadand Medical Sciences Division and R.E. Apfel thanks the l t h ~ ~ o n Of the U.S. Office of Naval Setearcl~,for financial support.

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