Harold T. McKonel St. Joseph College West Hartford. CT 0 6 1 17
I
High Performance Liquid Chromatography of Essential Oils
Stenm distillation is an effective means of selectively separating volatile organic rnmpounds from a complex matrix. Consequently, most introductory organic courses include a t least one experiment employing this technique. Spices are nonular substrates for steam distillation exoeriments because of modest cost, low toxicity of t h e distillate and high student interest resultine from the use of "real-world" substances (1-5). We have expanded this popular experiment by including high performance liquid chromatography (LC) for distillate separation and analysis. Since LC is fast becoming a favored analytical method for foods, biological fluids, drugs, soils and polymers, we felt a need t o incorporate i t into our undergraduate organic laboratory curriculum.
. ~ ~ . ~ ~ ~~
~
~
lnstrumentatlon T h e instrument used was a Waters ALCIGPC-204 liquid chromatograph equipped with a Model 6000A Solvent Delivery System (Waters Associates, Milford, MA 01757), a n d a Houston Omni-Scribe Recorder. Model 5211-1. T h e w e packed analytical column for this "reverse-phase" separation consisted of 10p m silica particles bonded with octad&ylsilane ( ~ B o n d a p a k I C t aWaters Associates). For a comprehensive discussioh of reverse phase chromatography see reference ( 6 ) . T h e mobile phase was a 8 5 1 5 mixture of methano1:water which was filtered and degassed before use. The flow rate was 1.0 mllmin and the absorbance detector was fixed a t 254 n m with a sensitivity of 1.0 absorbance units full scale. The essential oil was isolated by steam distilling 10-15 g of spice using standard procedures (7-9). Analytical samples were prepared bv dissolving 25 d of the extracted oil in 10 ml of methanol. To extend column lifeuand to minimize oressure huild-uo. .. all solutions were filtered hefore LC analysi~.~The diluted samples (5-10+1) were analyzed by LC and the results compared to chromatagrams of standard essential oil extract^.^ Results and Discussion The results of the steam distillation and LC separation are summarized in the table. Six common spices, moderately rich in readily available essential oils were chosen for analvsis. A discussion of the comnositinn of a varietvof ~, snices . mav he found in references (10-12). The liquid chnvndiugrams of the d~tilledeisential ois were relatively simple ru interpret tor rwg, rensms: ( 1 I theessenrinl oil ofrnrh sluce chosen was rich in one substance; and (2) the detector only recorded those components that had some absorbance at 254 nm ~
~~~
.
~
~
1 presentaddress: Waters Associates, Milford, MA 01757. 2 Information on sample filtering may he obtained from Waters
Associates, Milford MA 01757 (Brochure #D43). 3 Standards may he purchased from chemical supply houses or from supermarkets, drug and specialty stores.
698 / Journal of Chemical Education
Results of Essentlal011 Analysis Spice Allspice Clove
Anise Fennel Dill
Caraway
Number of K Value and Identity % Essential Oil Found Literature Components by LC of Major Component' 3.0% 5.1 2.4 4.1 2.5 3.9
3 - 4.5% 6 -17 1.5- 4 3 - 4 2.5- 4 3 - 7
2 2 2 2 1
1
0.55 Eugenol 0.55 Eugenol 1.60 Anethole 1.60 Anethole 0.90 (+) Carvone 0.90 (+) Carvone
k'(CBpacify factor)b a measure 01 me retention of a component on a mlmn. n is deflnsd as me dlstanoe between me mlmn void volume (vd am the band center of me m m p a n e r & ( V l l d Wby Yo.Differences lnmlumne(ticlmcym a y r s ~ u ~ i n r l i g h tdin& ly K valuer. Eugmol la the mn@r u v nhsorhing esent~nloil of horh allspice and clovr.although thrrr isa mnrkrd d~ifwenreinodor hptween rhr rwu oh,. Anerholr omnr-I-I/>-mt.rhox\.phenyIrproprnel LS rhr prpdominant compound of both onisennd fennel; the latter hring much richpr in i h od~ than rhe f m m r . r + ) ( h w n e 1s rhaonly detertahle uv absorbing constituent of both dill and caraway oii. The chromatograms of essential ail standards were in general agreement with those of the steam distillate exceot that the anise standard (from a hotrle of "pure aniee ertract"~had m e peak as
