a. Determination by Saponification with Heat. 

The determination of he ester content is of great importance in the evaluation of many essential oils. Since most esters which occur as normal constituents of essential oils are esters of monobasic acids, the process of saponification may be repreicnted by the following reaction :
where R and R' may be an aliphatic, aromatic, or alicyclic radical (R may also be a hydrogen atom).
Procedure: Into a 100 cc. alkali-resistant saponification flask weigh accurately about 1.5 g. of the oil. Add 5 cc. of neutral 95% alcohol and 3 drops of a 1% alcoholic solution of phenolphthalein, and neutralize the free acids with standardized 0.1 N aqueous sodium hydroxide solution.56 Then add 10 cc. of 0.5 N alcoholic sodium hydroxide solution, measured accurately from a pipette or a burette. Attach a glass, air-cooled condenser to the flask, 1 m. in length and about 1 cm. in diameter, and reflux the contents of the flask for 1 hr. on a steam bath. Remove and permit to cool at room temperature for 15 min. Titrate the excess alkali with standardized 0.5 N aqueous hydrochloric acid. A further addition of a few drops of phenolphthalein solution may be necessary at tins point.
56 This usually requires not more than 5 drops of the 0.1 N alkali.

In order to determine the amount of alkali consumed, carry out a blank determination, observing the same conditions but omitting the oil. The difference in the amounts of acid used in titrating the actual determination and the blank gives the amount of alkali used for the saponification of the esters. The blank should require an excess of about 100 per cent over the amount used in the determination. If insufficient excess is used, results will be obtained which are too low.
It is well to use saponification flasks made of "Jena Glass" or of the special alkaliresistant glass recently made available by Corning Glass Company. These flasks minimize the amount of alkali consumed by the action of the sodium hydroxide on the glass itself. More accurate results are thus obtained. This is of importance when the ester determination requires more than 1 hr. of refluxing, as for example, in the case of the isovalerates.
Saponification flask
DIAGRAM 4.7. Saponification flask.
The alcoholic 0.5 N sodium hydroxide solution is best prepared by adding 11.5 g. of metallic sodium of analytical grade to 1 liter of 95 per cent ethyl alcohol. (If larger amounts of solution are to be prepared, use 43.5 g. of sodium for each gallon of alcohol.) The sodium should be added slowly, a few small pieces at a time. After weighing out the sodium and cutting it into small pieces, it should be protected from atmospheric moisture until it is used by immersion in low boiling petroleum ether. After the required amount DIAGRAM 4.7. Saponification flask. of sodium has been added, the solution is set aside for several days to permit any carbonate to settle; it is filtered into the reagent reservoir and permitted to stand for a few days before it is used. A cleur, water white solution is thus obtained. The 0.5 N hydrochloric acid may be prepared by diluting 85 cc. of concentrated acid to 2 liters; it should then be carefully standardized.
Calculation of Results. The ester content may be calculated from the following formula :
Percentage of ester = am/20s
where: a = number of cc. of 0.5 N sodium hydroxide used in the saponification;
           m = molecular weight of the ester ;
           s= weight f the sample in grams.
This formula assumes that the ester is monobasic ; for esters of dibasic acids (e.g., dimethyl phthalate) and dihydroxy alcohols (e.g., glycol diacetate), the ester content is divided by 2; for tribasic acids (e.g., triethyl citrate) and trihydroxy alcohols (e.g., triacetin), by 3.57
The ester may also be expressed by the ester number, which is defined as the number of milligrams of potassium hydroxide required to saponify the esters present in 1 g. of oil. The use of the ester number is especially convenient when the ester present in the oil is unknown, since a knowledge of the molecular weight of the ester is not required.
Ester number =28.05a/s
Ester numbers are frequently used for oils which contain very small amounts of ester; e.g., oil of black pepper and oil of cubeb. A high ester number in such cases is usually indicative of adulteration.
The ester number may readily be converted to an ester content, expressed as a weight percentage, by the following formula if the acid radical of the ester is monobasic :
Percentage of ester = m(ester no.)/561.04
If the acid is dibasic, the result must be divided by 2; if tribasic, by 3. Also, if the alcohol radical contains two hydroxy groups, the result 58 must be divided by 2; if three hydroxy groups, by 3.
In 'Fable 4.10 are listed the molecular weights of those esters which are frequently encountered.
Modification of the General Procedure. Certain esters are not completely saponified in a period of 1 hr. by the procedure described above. Notable exceptions are the salicykites which should be rerluxed for 2 hr. ; terpinyl acetate, 2 hr. ; menthyl acetate, 2 hr. ; isovalerates, G hr. Certain esters of sesquiterpene alcohols require 2 hr. or more e.g., cedryl acetate, 4 hr. A solution of potassium hydroxide in a high boiling solvent (such as the monoethyl ether of ethylene glycol) has been recommended59 for the determination of difficultly saponifiable esters. Such a solution also permits of rapid saponification (ca. 15 min.) of other esters. Since such high temperatures may have an adverse effect upon some of the constituents of an essential oil, this method should be applied with caution.
57 This is based on the assumption that the ester is neutral in the case of di- and tribasi acids, and that all alcoholic groups have been estcrified in the case of esters of di- and trihydrox alcohols.
58 This is based on the assumption that the ester is neutral in the case of di- and tribasic acids, and that all alcoholic groups have been esterified in the case of esters of di- and trihydroxy alcohols.
59 Steet, Analyst 61 (1936), 687.

In the case of salicylutcs, benzoates, and phthalates, an addition ot 5 cc of water should be made before the ester is heated on the steam bath to provent the separation of the sodium salts of the acids during the saponification.
If the oil contains large amounts of free acids, these should be determined separately by the procedure described under "Determination of Acids."
The saponification number, representing the sum of the acid number and the ester number, is then determined for the oil using the general procedure described above, except that the free acids are not neutralized before the addition of the 0.5 N alkali.
In the case of oils containing large amounts of esters (e.g., oil of wintergreen), or esters of low-molecular weight (e.g., methyl formate), or esters of dibasic or tribasic acids, it becomes necessary to vary the size of the sample and the amount of alkali employed. If 10 cc. of alkali is insufficient, 20 cc. may be used. For synthetic esters, it is often necessary to decrease the size of the sample ; usually 1 g. (of the pure synthetic) is used and 20 cc. of alkali. In the case of esters of low molecular weight or esters of polybasic acids, a 0.5 g. sample and 20 cc. of alkali may be required.
Relatively small samples are also required in the case of certain darkly colored oils. It may also be necessary to dilute the saponified oil with alcohol in order to ascertain the end point of the titration, and to use a spot plate. The use of thymolphthalein (in place of phenolphthalein as an indicator) has been suggested60 for determinations involving red or brown solutions, such as result during the saponification of oleoresins. Thymolphthalein changes from a deep blue to colorless in the range pH 9.3 to pH 10.5.
The determination of the ester content by saponification will not yield satisfactory results if the oil contains appreciable amounts of aldehydes, unless the aldehydes are removed and the residual oil saponified.
It has been reported that certain phenols also may interfere with the ester determination.61 In addition to esters, lactones may be determined quantitatively by saponification.

b. Determination by Saponification in the Cold. 

As an analytical procedure, saponification in the cold is not generally applicable. In most cases, long periods of time are necessary to complete the process; furthermore, side reactions frequently occur which give rise to inconsistent and deceptive results.
Saponification in the cold has a definite value for the determination of those esters which are very easily saponified; this is particularly true for certain formates. Thus, cold saponification is used in the analysis of geranium oils to determine the amount of "actual formate," since the standard procedure for the determination of esters with a reflux period of 1 hr. saponifies not only the geranyl formate but also other esters including geranyl tiglate.
For the determination of geranyl formate in geranium oils, the following procedure has given satisfactory results.
Procedure: Into a 100 cc. saponification flask, weigh accurately about 1.5 g. of the oil. Add 5 cc. of neutral alcohol and 3 drops of a 1% alcoholic solution of phenolphthalein, and neutralize the free acids quickly with standardized 0.1 N aqueous sodium hydroxide solution. Add 10 cc. of 0.5 N alcoholic sodium hydroxide solution, measured accurately from a burette or pipette, and titrate the excess alkali immediately with standardized 0.5 N aqueous hydrochloric acid. Calculate the ester content as geranyl formate in the usual manner.
In the case of pure synthetic formates, it is advisable to add 5 cc. of water to the flask in order to dissolve the sodium formate which otherwise may precipitate out of solution.

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