a. Determination by Acetylation. 

The alcoholic constituents of an essential oil are determined by acetylation; i.e., the oil is acetylized with acetic anhydride and the ester content of the resulting oil is determined; from this value the percentage of alcohol in the original oil may be calculated.
The basic chemical processes involved in this determination may be summarized by the following equations :
The basic chemical processes involved in this determination may be summarized by the following equations

where Rl, R2 and R3 may be a hydrogen atom, an aliphatic, aromatic or alicyclic radical.
For this determination, a special acetylation flask of approximately 100cc. capacity is employed. This flask is equipped with an air-cooled condenser attached to the flask by means of a ground glass joint (see Diagram 4.8). A condenser 1 m. in length is to be preferred in order to prevent the loss of volatile constituents.
Procedure: Introduce into a 100 cc. acetylation flask 10 cc. of the oil (measured from a graduated cylinder), 10 cc. of acetic anhydride (similarly measured) and 2.0 g. of anhydrous sodium acetate. Attach the air condenser, and boil the contents of the flask gently for exactly 1 hr. on a sand bath suitably heated by an open Bunsen flame or an electric hot-plate. Permit the flask to cool for 15 min. and introduce 50 cc. of distilled water through the top of the condenser. Heat the flask on a steam bath for 15 min. with frequent shaking to destroy the excess of acetic anhydride. Transfer the contents of the flask to a separatory funnel and rinse the flask with two 10 cc. portions of distilled water; add these rinsings to the separatory funnel. Shake thoroughly to assure good contact of the aqueous layer with the oil. When the liquids have separated completely, reject the aqueous layer and wash the remaining oil repeatedly with 100 cc. portions of saturated salt solution, until the washings are neutral to litmus ; this usually requires three washings. Dry the resulting oil with anhydrous sodium sulfate and filter. (If the oil has been washed properly, not more than 0.2 cc. of 0.1 N aqueous sodium hydroxide solution should be required per gram of acetylized oil in order to neutralize the remaining trace of acetic acid.)
Saponify the acetylized-oil, using the procedure described under "The Determination of Esters," p. 265.
In order to secure accurate and reproducible results it is important to use exactly 2.0 g. of sodium acetate and to reflux the mixture for exactly 1 hr. A notable exception occurs in the case of citronella oils, which require a reflux period of 2 hr.
Calculation of Results. If the original oil contains a negligible quantity of saponifiable constituents, the free alcohol may be calculated by the following formula :
Percentage of alcohol in the original oil =am/20(s-0.021a)
where: a = number of cc. of 0.5 N sodium hydroxide solution required for the saponification of the acetylized oil ;
            s = weight of acetylized oil in grams used in the saponification ;
            m molecular weight of the alcohol.
For oils which have not been thoroughly investigated and whose alcoholic constituents are not well known, it is frequently more convenient to report the result as an ester number after acetylation.
Ester number after acetylation =  28.05a/s
The ester number after acetylation is numerically equal to the number of milligrams of potassium hydroxide required to saponify the esters present in 1 g. of the acetylized oil.
If the original oil contains an appreciable amount of esters (as indicated by the ester number), the percentage of free alcohol may be estimated by the following formula:
Percentage of free alcohol in the original oil = dm/(561.04 - 0.42d)
where d = (ester number after acetylation ester number) .
Although this expression is not mathematically precise, nevertheless it is sufficiently accurate for all practical work  and has been used traditionally by essential oil chemists.
For the evaluation of essential oils, it is often desirable to know the percentage of total alcohol; i.e., the percentage of free alcohol plus the percentage of alcohol combined as ester present in the original unacetylized oil.
Percentage of total alcohol in the original oil

where e = ester content in per cent. This formula assumes that all of the esterified alcohol present in the original oil is combined as the acetate.
All formulas in this chapter that calculate the result of an acetylation as a percentage actually refer to all constituents which are capable of acetylation under the experimental conditions, calculated as a specific alcohol. Thus for example, the "total alcohol" in citronella oils includes not only the geraniol, free and as ester, but also all other acetylizable constituents and their esters, such as, borneol, citronellol, sesquiterpene alcohols, and thealdehyde citronellal, all calculated as geraniol. These formulas further assume that the alcohol is a monohydroxy compound. Table 4.11 gives the molecular weights of alcohols frequently encountered in the analysis of essential oils.


* All molecular weights have been calculated from the values of the Internationa Atomic Weights adopted by the Committee on Atomic Weights in 1938.
Limitations and Modifications of the General Procedure. As mentioned above, acetic anhydride employed under the experimental conditions described in the "Procedure" will react with certain compounds found in essential oils other than alcohols. Phenols will be quantitatively converted into the acetates. Certain aldehydes and ketones are partially acetylated and partially destroyed, or are converted to other compounds which are capable of acetylation.
Furthermore, some tertiary alcohols are not quantitatively converted to the acetate by this process of acetylation; the most important alcohols in this class are terpineol and linalool.

b. Determination of Primary Alcohols. 

Phthalic anhydride reacts wit primary alcohols forming an acid phthalic ester.
Phthalic anhydride reacts wit primary alcohols forming an acid phthalic ester

Under the experimental conditions described below, this reaction takes place readily at a temperature of about 100 in the case of primary alcohols; for secondary alcohols, the time required for reflux is greatly increased; for tertiary alcohols, no appreciable reaction occurs.
It is important that the phthalic anhydride does not contain free phthalic acid. This may be ascertained conveniently by shaking 1 g. of the anhydride with 10 cc. of benzene and warming to 40 ; a clear solution indicates the absence of appreciable amounts of phthalic acid.
Procedure:62 Into a 100 cc. acetylation flask introduce about 2 g. of powdered phthalic anhydride, accurately weighed, and about 2 g. of the oil, accurately weighed. Add 2 cc. of benzene, measured from a graduated cylinder. Heat the flask on a steam bath with frequent shaking for 2 hr. Then permit the flask to cool for 30 min. Add 60 cc. of 0.5 N aqueous potassium hydroxide solution, accurately measured from a pipette or burette. Stopper the acetylation flask with a ground glass stopper and shake thoroughly for 10 min. Titrate the excess of alkali with standardized 0.5 N hydrochloric acid, using 3 drops of a 1 per cent phenolphthalein solution as indicator.
Run a blank determination omitting the oil, and from this calculate the amount of alkali which would be required for the weight of phthalic anhydride used in the actual determination.
Calculate the percentage of primary alcohol by the following formula :
Percentage of primary alcohol = m(b-a)/20.w
where : m = the molecular weight of the primary alcohol ;
             b = the calculated number of cc. of 0.5 N potassium hydroxide required for the amount of phthalic anhydride used in the determination ;
             a = the number of cc. of 0.5 N potassium hydroxide consumed in the determination;
             w = weight of oil in grams.
62 Ber. SchimmeL & Co., October (1912), 39.

c. Determination of Tertiary Terpene Alcohols.

Most tertiary alcohols suffer partial or complete breakdown and dehydration when treated with acetic anhydride. In the event that an oil contains a large percentage of such easily dehydrated alcohols, special techniques are required.
I. The Method of Glichitch. The Glichitch method of formylation for the estimation of easily dehydrated alcohols has been successfully employed for the determination of linalool and terpineol.
Procedure: Introduce 15 cc. of aceto-formic acid reagent in a 125 cc. glass-stoppered Krlenmeyer flask. Cool in an ice bath and add slowly 10 cc. of the oil to be tested. Allow the mixture to stand for not less than 72 hr. at room temperature. The ice in the bath should not be renewed. At the end of this interval pour the contents of the flask into a separatory funnel. Shake well with 50 cc. of ice cold water and allow to stand for 2 hr. Separate the oil and wash successively with 50 cc. of cold water, 50 cc. of a 5% sodium bicarbonate solution, and then with two 50 cc. portions of water. Separate the oil and dry with anhydrous sodium sulfate. Filter and saponify by refluxing with 0.5 N alcoholic sodium hydroxide. Calculate the alcohols in the usual way on the assumption that they are present as formates.
Preparation of the Aceto-Formic Reagent. To 2 volumes of acetic anhydride, previously cooled to at least 0, add slowly 1 volume of 100 per cent formic acid.64 Mix thoroughly and then heat to 50 for 15 minutes and immediately cool in an ice bath.
II. The Method of Boulez.65 The Boulez method of acetylation makes use of a diluent in order to lessen the dehydrating effect of acetic anhydride. The period of acetylation, however, must be prolonged. This gives satisfactory results for linalool and terpineol if the prescribed conditions are rigidly followed.
The original method suggested oil of turpentine as a diluent in the ratio of 1 part of the oil under examination to 5 parts of oil of turpentine. chemists of Schimmel & Company67 modified the procedure by substituting xylene as a diluent in the ratio of 1:4. The period of acetylation is very important ; for terpineol 5 hr. are required, longer or shorter periods give low values ; for linalool, 7 hr.
63 The Bull, soc. chim. [4] 33 (1923), 1284.
64 It is very important to use a highly purified formic acid of substantially 100 per cent
strength. The usual A.R. grade of formic acid (specific gravity = 1.20; HCOOH = approximatel 87 per cent) is useless for the preparation of this reagent.
65 Butt. soc. chim. [4] 1 (1907), 117.
66 Boulez later suggested an even greater dilution namely, 1 g. oil to 25 cc. of xylene. Butt. soc. chim. [4] 35 (1924), 419.

This modified procedure gives reproducible data. Great care must be exercised during this determination since any error introduced will be multiplied by 5 in the final result.
III. Dehydration Methods. Dehydration methods are based upon the  catalytic decomposition of tertiary alcohols and the splitting off of water.
The amount of water obtained is determined from which the percentage of tertiary alcohol may be calculated.
Such a method has been described by Ikeda and Takeda,68 using zinc chloride. A very satisfactory dehydration catalyst is iodine. Additions of approximately 0.5 per cent of catalyst to the oil will prove sufficient. Such dehydration methods offer the advantage that only tertiary alcohols are determined, primary and secondary alcohol being unaffected. This is an advantage not found in the other methods described here. Hydroxy ketones and hydroxy aldehydes will interfere in this procedure, since both split off water under the experimental conditions. A convenient method for the determination makes use of the distillation trap of Sterling and Bidwell.
Procedure: Dry the oil thoroughly by permitting it to stand overnight in contact with anhydrous sodium sulfate. Into a 1 liter, round bottom flask, introduce a sufficiently large sample, accurately weighed, to yield about 5 cc. of water upon dehydration of the tertiary alcohol. Add 0.5% of solid iodine as catalyst and 500 cc. of xylene. Connect the flask to a standard Sterling and Bidwell water-trap; attach a water-cooled, straight tube condenser. Heat the flask by means of an oil hath. Proceed as in the "Determination of Water Content," p. 323. Measure the amount of collected water and calculate the percentage of tertiary alcohol.
This method docs not yield highly accurate results, but is a convenient method for the determination of the tertiary alcohol content.
IV. Acctyl Chloride-Dimethyl Aniline Method. This method, originally described by Fiore, other tertiary terpene alcohols indicate that this may prove to be a valuable method for many tertiary alcohols.
67 gives exceptionally concordant and satisfactory result in the case of linalool and linalool-containing oils. It has been carefull evaluated by the members of the Essential Oil Association of the U. S. A and adopted by that body. Preliminary experiments with terpineol and 7 Ber. Schimmel & Co., April (1907), 128.
68 J. Chem. Soc. Japan 57 (1936), 442. Chem. Abstracts 30 (1936), 5907.
69 News Capsule (Essential Oil Association of U.S.A.), Vol. 1, No. 15 (1943).

The method is described below in the final form in which it was accepted by the Essential Oil Association for the determination of linalool.
Procedure: Ten cc. of linalool or essential oil containing linalool, previously dried with sodium sulfate, is introduced into a 125 cc. glass-stoppered Erienmeyer flask cooled with ice and water. To the cooled oil is added 20 cc. dimethyl aniline (monomethyl free) and the contents thoroughly mixed, then 8 cc. acetyl chloride (reagent grade) and 5 cc. of acetic anhydride are added, the anhydride serving as a solvent to prevent crystallization of the reaction mass. The mixture is cooled for a few minutes and permitted to stand at room temperature for onehalf hour after which time the flask is immersed in a water bath maintained at 40oC. 1 for three hours. At the end of this time the acetylated oil is washed three times with 75 cc. of ice water, then with successive washes of 25 cc. of 5% sulfuric acid until the separated acid layer fails to liberate any dimethyl aniline with an excess of caustic. After removal of the dimethyl aniline, the acetylated oil is washed with 10 cc. of 10% sodium carbonate solution and then finally washed neutral with water.
The oil is separated, dried over anhydrous sodium sulfate and the ester number determined in the usual manner. The linalool content can thus be obtained directly from saponification tables or by substitution in the following formula :
Percentage of linalool = cc.N/2KOHx15x.14/20(sample-cc. N/2 KOHx0.021)
As this test is further to be used for other oils containing linalool, besides linalool itself, a correction factor is necessary with oils containing significant amount of esters. For such oils, the following standard formula is recommended :
Percentage of total linalool = A.77.07/(B-(A.0.21)x(1-Ex0.0021)
where: A = cc. half normal alkali required for saponification ;
           B = weight of sample ;
           E = per cent of esters calculated as linalyl acetate in the original oil.

d. Determination of Citronellol by Formylation. 

Most terpene alcohols are dehydrated by strong formic acid, giving rise to nonsaponifiable terpenes. A notable exception is citronellol which is converted almost quantitatively to the corresponding formate. This results in a convenient and satisfactory method for the determination of citronellol in the presence of geraniol and linalool.
Formylation has become a standard procedure for the determination of citronellol in rose oils. The procedure to be followed is identical to that described under "Determination by Acetylation," p. 272, with the exception that the 10 cc. of acetic anhydride is replaced with 20 cc. of 100 per cent formic acid, and the anhydrous sodium acetate is omitted.
Place in the flask short pieces of glass tubing to permit heat transfer throughout the mixture. This is particularly important if the oil contains a high percentage of geraniol, since the dehydration which results may dilute the formic acid sufficiently to cause the formation of two layers in the flask. Should this occur, there will be some danger of the lower layer becoming overheated and violently throwing out the contents of the flask through the air condenser. A small clay chip should also be placed in the flask to help prevent such overheating.
The percentage of alcohol (citronellol) in the original oil may be calculated from the amount of alkali consumed in the subsequent saponification.
Percentage of alcohol in the original oil = am/20(s-0.014a)
where : a = number of cc. of 0.5 N sodium hydroxide solution required for the saponification of the formylated oil ;
            m = molecular weight of the alcohol ;
            s = weight of formylated oil in grams.

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