C

FISHERIES AND MARINE SERVICE

Translation Series No. 3473

An apparatus for the microanalytical determination

of C-methyl and acet'yl groups

by E. Wiesenberger

Original title: Eine Apparatur fuer die mikroanalytischeBestimmung von C-Methyl- und Acetylgruppen

From: Mikrochim. Acta 1: 127-139, 1954

Translated by the Translation Bureau(EMF)Multilingual Services Division

Department of the Secretary of State of Canada

Department of the EnvironmentFisheries and Marine Service

Biological StationSt. John's, Nfld.

1975

A r RCHIVES

18 pages typescript

DEPARTMENT OF THE SECRETARY OF STATE

TRANSLATION BUREAU

MULTILINGUAL SERVICESCANADA

SECRÉTARIAT D'ÉTAT

BUREAU DES TRADUCTIONS

DIVISION DES SERVICES

DIVISION MULTILINGUES

TRANSLATED FROM - TRADUCTION DE

German

AUTHOR - AUTEUR

TITLE IN ENGLISH - TITRE ANGLAIS

INTO - EN

E. Wiesenberger

English

An apparatus for the microanalytical determination

of C-methyl and acetyl groups

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS)

TITRE EN LANGUE ETRANGÈRE (TRANSCRIRE EN CARACTÉRES ROMAINS)

Eine Apparatur fuer die mikroanalytische Bestimmunr

von C-RtethylA und Acetylgruppen

REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS.RÉFERENCE EN LANGUE ÉTRANGERE (NOM DU LIVRE OU PUBLICATION), Au COMPLET, TRPVSCRiRF. EN CARACTÉRES ROMAINS.

Mikrochimica Acta

REFERENCE IN ENGLISH - REFERENCE EN ANGLAIS

PUBLISHER- EDITEUR

not shown

PLACE OF PUBLICATIONLIEU DE PUBLICATION

not shown

REQUESTING DEPARTMENT EnvironmentMINISTÈRE-CLIENT

BRANCH OR DIVISIONDIRECTION OU DIVISION Fisheries Service

DATE OF PUBLICATIONDATE DE PUBLICATION

YEAR

ANNEE

1954

PERSON REQUESTINGAllan T. Reid - n _ 1-i _ Sh jtv ,DEMANDÉ PAR for Dr,

St, John's, Neivfoundli:tndYOUR NUMBERVOTRE DOSSIER NO

DATE OF REQUESTDATE DE LA DEMANDE April 8,_ 1975

676539

KMF

LINEDIM" D TRANSLATION

0;1 :;^l`/

TRaD:JCT3i7NIr)(0)•)u10i0n setcl^;r^aP

505.200-10-6 (REV, 2/68)

7630-2/•02G-0333

VOLUME

1

ISSUE NO.NUMERO

PAGE NUMBERS IN ORIGINALNUMEROS DES PAGES DANS

L'ORIGINAL

127 - 139NUMBER OF TYPED PAGES

NOMBRE DE PAGESDACTYLOGRAPHIÉES

18

TRANSLATION BUREAU NO.NOTRE DOSSIER NO

TRANSLATOR (INITIALS)TRADUCTEUR (INITIALES)_

MULTILINGUAL SERVICES

DIVISION •

SECRÉTARIAT D'ÉTAT

BUREAU DES TRADUCTIONS

DIVISION DES SERVICES

MULTILINGUES

DEPARTMENT OF THE SECRETARY OF STATE

• TRANSLATION BUREAU

CLIENT'S NO. DEPARTMENT DIVISION/BRANCH CITY

N° DU CLIENT MINISTE' RE DIVISION/DIRECTION VILLE

Environment Fisheries and Marine St. John's, Service Newfoundland

BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) N ° DU BUREAU LANGUE TRADUCTEUR (INITIALES)

676539 German KMF JUN 2 o Ï97 5

From the Department of Microchemistry of the Institute for Organic and Pharmaceutical Chemistry of the University of Graz‘ AUSTRIA) .

of C -InothIlftILL.n.0.-11.P2 * *

E. Wiesenberger**

With 2 illustrations .

(Submitted October 10, 1953)

alMIJIMX

An apparatus for the microanalytical determination of acetyl

groups is described with which it is possible to accomplish standard

saponification procedures, the determination of C-methyl groups, and

the application of the Freudenberg transesterification. Directions

for the various procedures are given.

In order to destroy the decomposition products, which are

formed to a considerable extent during acid and alkaline saponifica-

tion, the vapors drawn off during the acetic acid distillation are

passed through hot- chromo-sulfuric acid, removing the interfering side

products by oxidation.

S03.400-10..31

* Dedicated to Prof. Dr. Adolf Franke on the occasion of his 80th birthday.

**Address: Berlin-Siemensstadt, Im Heidewinkol 38. UNEDIT7D n TiqUAT!')N For infOtrOZ1101

TRADUCTION NON REVE

information st.:k.,.ment

Correct results were obtained using this principle with octa-

acetyl cellobiose when saponifying with either acid or alkali, but

only with alkaline saponification of pentaacetylglucose. A systematic

studi is needed to determine whether the procedure is generally applic-

able, particularly in the mono- and polysaccharide groups.

The shortcomings of equipment used in the microanalytical de-

termination of acetyl groups could largely be overcome by suitable

distillation apparatus 1 9 2 . The difficulties which nevertheless arise

in the determination of acetyl groups are due to the different be-

havior of acetyl derivatives toward saponification agents. The number

of compounds which yield only the pure saponification products is small

compared to those which, in addition, also form volatile acidic de-

composition products. These error-causing by-products formed during

saponification become more significant with increasing molecular weight

and increasing number of building blocks and side chains involved in

the structure of the acetyl compounds. It can therefore be stated almost

as a general rule that higher-molecular compounds cannot be analysed

according to the commonly used saponification methods. The acetyl de-

rivatives of the carbohydrates and all related compounds should again

be pointed out as typical and best-known examples for this group. The

interferences which arise with this class of substances cannot be eli-

minated by measures involving the apparatus, but can only be prevented

by the selection of a more suitable method of saponification. Thus

Freudenberg 3,4 found a method in the procedure according to Perkin

which proved an excellent one for the carbohydrate derivatives which

could hitherto not be analdzed without error. Furthermore, the great

dissolving power of p-toluenesulfonic acid in alcoholic solution used (128)

here for the first time, greatly expanded the range of application of . .

this method of determination. It subsequently became evident that to

date this method is the only one suitable for general application.

AS is well known, another possibility for eliminating the

additional by-products occurring in saponification is the utilization

of the oxidizing effect of a mixture of 5-n chromic acid and sulfuric

acid. Kuhn and LtOrsa5 e 6 used this oxidation mixture for the determi-

nation of C-methyl groups; in this procedure the formed oxidation-

resistant acetic acid which is free of interfering acidic decomposi-

tion products is distilled off. This chromosulfuric acid would thus

make an excellent saponification agent iî its applicability were not

restricted to only those saponifiable acetyl compounds which, except

for the acetyl group, contain no other terminal methyl groups bonded

to carbon.

A more promising way to utilize the favourable effect of the

chromosulfuric acid for the determination of saponifiable acetyl groups

despite the presence of C-methyl groups would be the combining of ordi-

nary saponification with the subsequent oxidation of the distilling

vapors. Some orienting experiments have already shown that the brief

contact period of vapor flowing through a heated chromosulfuric acid

bath suffices to oxidatively break down the decomposition products which

are carried over in the distillation.

Since it would be possible to analyze a large number of com-

pounds according to this principle without interference 1 I have

attempted to modify the apparatus mentioned earlier while retaining

4

the simple method of determination used tô date. Taking into considera-

tion the oxidizing purification of the vapors this method thus per-

mitted, beside the ordinary saponification methods, determination of

C-methyl groups as well as the esterification technique by combining

different parts of one and the same apparatus in various ways. This

provides the opportunity to utilize on one single apparatus that method

of determination which is most suited to the particular behavior of a

given acetyl product. In addition, a further simplification was

accomplished for the esterif ication process which will undoubtedly

facilitate more frequent application of this excellent method.

The modified apparatus,* is shown in Fig. 1. It is composed of

the following parts:

a) The saponification flask. (1)-, with the insert serving as a (129)

reflux condenser. I .

b) The connectizig.paxt with the washing vessel (3) with the

two condensation chambers serving to receive the chromosulfuric acid.

(4) is a tube with ground glass.joint and serves as a connecting piece

for the water-jet vacuum pump when washing (3)-

c) Cooler (5) and flask (6) which can be connected to collect

the distillate.

The correct performance of a determination requires the thorough

cleaning of all parts. This is best accomplished with chromosulfuric

acid. If the apparatus is in continual use, it is sufficient to clean

with chromosulfuric acid in intervals of approxo eight days. However,

it is always necessary when the distillate no longer drains freely.

* The apparatus can be obtained from the glassblowing Firm Paul Haack,Vienna IX., Garelligasse 4 and Kurt Bartelt, Graz, Alorellenfeldgasse 15.

I am grateful to both Companies for their support.

5

L

^t3 QJ^^A'VCmFO

Fig. 1

A thorough rinse with tap water is imperative. Particularly the ground

joints in the path of the draining distillate should be left under

running water for approximately 10 minutes because traces of acid

which might adhere can give rise to inexplicable errors. After rinsing

with very pure# distilled water_the apparatus is ready for the determi-

nation. The saponification flasks are always used dry.

Determination of acetyl groupé by saponification

The apparatus in the arrangement as shown in Fig. 1 with

attached condenser (5) is used for the determination of acetyl groups

by saponification and for chromic acid oxidation.

The entire apparatus is supported by the condenser (5) which

is held with a condenser clamp and all ground glass joints are secured

with springs. The substance is usually introduced horizontally into

.the dry saponification flask'with the aid of a long-stemmed weighing

(130)

tube and is only then emptied. It is desirable that the substance is

deposited onto the bottom of the flask. Hygroscopic substances are

weighed into thin—walled platinum weighing boats, are then dried and

placed into the flask with the boats. To achieve smooth boiling, two

or three platinum tetrahedra are added. It should be pointed out that

only sulfuric acid must be used to clean the platinum tetrahedra,

nover nitric or hydrochloric acid because traces of nitrate or chloride

may sometimes remain in the interior of the tetrahedra which would

distill as acids in the acetic acid distillation.

a) Acid saponification

It is not necessary to completely assemble the apparatus before

beginning the determination because only the insert (2) which serves

as reflux condenser is required at first. This is therefore cleaned

and clamped to a support. After the reflux condenser is in operation

the flask containing the sample is attached and the ground glass joint

is sealed with water. Through a funnel with ground glass joint 2 ml

of dilute sulfuric acid 1:2 (sp. gr . = 1.84) are added and the flask

is immersed in a preheated bath until both liquid levels are equal.

Saponification is carried out at a bath temperature of 160 to 170°C

and takes 30 minutes. This time is used to prepare the sample for the

next determination and to clean and assemble the other parts of the

distillation apparatus. In the meantime the washing vessel (3) is

charged with 2 ml chromosulfuric acid (4 parts 5—n chromic acid and one

part concentrated H2504, sp. gr . = 1.84) which is heated to 135 °C to

140 0t.After saponification is complete the flask is lifted from the

heating—bath, 2 ml water are added from the ground glass joint funnel

and the flask is then connected to the prepared apparatus.

After emptying the reflux condenser the distillation is started

by gentle heating of . the contents of the flask. Heating is accomplished

by means of a small Bunsen flame shielded by a chimney — the tip of the

flame -must not be allowed to touch the bottom of the flask. The dis-

tillation should be adjusted so that the distillate drops at the rate

of 1 ml per minute. This is collected in a freshly boiled quartz flask (131)

which has been rinsed with distilled water. To check the distilled

amount of liquid, the distillation flask is marked with a marking

pencil indicating 2.5 ml, the volume to which distillation ià carried.

Further, the expected final volume is marked on the quartz flask. The

distillation is now carried to the 2.5 ml mark. Then another 2 ml water

is added and the same amount of water is repeatedly distilled. The water

is added by lifting the ground glass stopper after removing the burner

and after waiting for the rising of the chromosulfuric acid due to

. cooling. When the acid has reached the spot marked with an arrow in

Fig. 1, the ground glass stopper is opened by carefully turning it and

care is taken that the water flows in slowly and uniformly. This takes

about 20 seconds and does not cause an appreciable interruption in the

distillation. After replacing the burner under the flask, distillation

recommences immediately and the entire process is repeated until the

distillate reaches the 26 ml mark on the quartz flask. If the distilla-

tion is carried out properly, it should be completed after 30 to 35

minutes. During distillation care should be taken that the temperature

of the heating bath of the chromic acid wash is maintained strictly

within the indicated limits. If the temperature is allowed to fall below,

the liquid volume rises rapidly through condensation of the vapors and

leads to undesirable disruptions. Exceeding the temperature causes a

concentration of chromic acid which may . lead to erroneous results.

It is also necessary to ensure that the amount of water to

be distilled always corresponds to . the added amount and that the 2.5 ml

mark on the flask is observed within small, permissible fluctuations.

The funnel with ground glass joint must never be completely emptied

during the addition of water and it is therefore advisable to affix

two marks between which a volume of 2 ml is contained.

b) Alkaline saponification

The apparatus and sample are prepared in the same manner as

described above.

2 ml of a 1—n methylalcoholic sodium hydroxide solution which

is used for saponification is placed directly into the flask before it

is attached to the reflux condenser. After boiling the solution for

20 minutes (temperature of heating bath 130 — 140 °C) the substance is

saponified. The heating bath is then removed, 2 ml water is added from

the funnel with ground glass joint, the reflux condenser is emptied

and the flask is heated to boiling with a small, open flame. The (132)

methylalcohol vapor excapes into the atmosphere without condensation.

The alcohol is boiled off twice more after prior addition of water,

evaporating down to the 2.5 ml mark in both cases. The flask is then

connected to the prepared distillation apparatus and 2 ml of the dilute

sulfuric acid 1:2 is slowly allowed to enter through the funnel with

ground glass joint. The last drop of sulfuric acid seals the ground

glass stopper after which the funnel is immediately filled with water.

The subsequent acetic acid distillation is carried out as described

above. The methylalcoholic sodium hydroxide solution has great dissolving

9

power and is therefore used preferentially as an alkaline saponifica-

tion agent. In rare cases it is necessary to'use more concentrated

caustic alkaline solutions. Where this is the case, the determination

is carried out according to the same instructions. The aniount of

sulfuric acid to be added must, however, be adjusted to the concentra-

tion of the caustic alkaline solution used.

Determination of the C-methyl groups according to

the chroinic acid oxidation method2 s6 ,7.

The preparations for this determination are the same as described

for the acidic saponification. To cxidize the substance, 2 ml chromic

acid (four parts 5-n chromic acid and 1 part conc. H2SO4, sp.gr. :-.

1.84) is used which is introduced into the flask through the ground

glass joint funnel. The oxidation is carried out at the boiling tempera-

ture of the chromic acid mixture, the heating bath being heated to

165 - 170°C. The heating time is determined by the nature of the sub-

stance, 1 to ll/z hours generally being sufficient. The change-over to

acetâc acid destillation is carried out in the same manner as with the

acidic saponification.

For this determination, filling the washing vessel with chromo-

sulfuric acid can be omitted. Nevertheless, I do recommend it, because

the intermediate vessel must be heated anyway to prevent condensation

of the vapors. Furthermore, it does not affect the simplicity of the

distillation process.

If it is necessary to use 4 ml chromosulfuric acid for the oxi-

dation of the substance where larger samples are involved, 30 ml are

distilled off for the titration rather than 26. The acetic acid distilla-

tion is carried out in the usual manner.

-10-.

The determination of saponifiable acetyl groups, in which the

vapor stream was scrubbed with chromic acid was first tested on simple

acetyl compounds which produce no interference products in the saponi-

fication. It was observed that modification of the apparatus has no

detrimental effect on the quality of the results. It was therefore (133)

possible with this arrangement to attempt determinations with sub-

stances which form acidic, volatile decomposition products during

normal, alkaline or acidic saponification.

When analyzing pentaacetylglucose and octaacetyl cellobiose

it was observed that in both substances the decomposition products

formed during acid saponification are oxidatively destroyed by the

chromic acid scrub. Table 1 shows several examples of the obtained

results.

ai-ppleSubstance lireirht

Tiig

11t•11(lu.etyl(;3vrtÀ.uxw .

t )ktacotylo©llobiotio . . .

4,7tt75,162^i,075

4,4605,1624,2323,590

^kVerbraueh nilO,Ut•u \uUfi

(3,12a,6 :36,49

5,276,115,015,30

°n CH^ . co

Ca^.cm found

55,16 55,0355,2;i55,O-4

50,75 :itl,lt(iaill,Sl,i5U,S1115U,ît4

Table 1

Sulfuric acid saponification

* Consumption

In the case of saponification in alkaline solution, the two

substances behave differently. As in acidic saponification, the de-

composition products of the octaacetyl cellobiose are destroyed and

thus true results are obtained here also. The acetyl values of the

pentaacetyl glucose, by contrast, are too high by 1 to yo. Reaction

products are formed through the action of alkaline substances which

produCe acetic acid in the subsequent oxidation with chromic acid.

Table 2 contains examples of saponification in methylalcoholic 1—n

sodium hydroxide solution.

Sample r 0/,c14.0) Substance weight vprbwiehmi

— mg • 0 . 01-nnfflu calcul. , found

I •

l'entamtylghlèm' • • • • 3,888 4,82 55,16 1 56,26

3,266 4,27 56,28

4,450 5,89 56,97

4,390 5,81

1,U4welyke1l01)jilse _, 4,059 4,78 50,75 50,89

4,579 5,38 iitt. -oi

5,304 6,25 50,72

4,372 5,165 50,85

Table 2 •

Alkaline saponification

*Uonsumption

This fact must be taken into consideration when applying this (134)

method to carbohydrate derivatives. The solubility of the acetyl com-

pounds of the monosaccharides in sulfuric acid is sufficiently great

so that saponification can always be carried out in acid solution. The

acetyl derivatives of the oligo— and polysaccharides, however, are

hardly or not at all soluble in sulfuric acid, making saponification

in methylalcoholic sodium hydroxide solution the preferred method. A

systematic study of numerous acetyl derivatives of these types of

compounds should still be carried out*. For acetyl derivatives of

*Circumstances beyond our control have to date prevented continuation of these investigations.

12 -

glycoside: only sulfuric acid saponification is applicable because

these behave like the acetyl compounds of pure monosaccharides. Their

low solubility in sulfuric acid however, prevents the use of this

method so that this class of substances must still be analyzed accord-

ing to the esterification technique.

It was therefore logical when modifying the apparatus for

acetyl determinations to also consider its applicability to this

excellent method. The use of ground glass joints which fit into each

other permits different ways of joining the various component parts,

so that the apparatus can always be assembled according to the chosen

method of determination. For the transesterification the intermediate

part (3) can be omitted. The apparatus is assembled from parts (1),

(2) , (5), (6) and (?a).

Determination of the acetyl groups according to

the esterification method.

The apparatus is cleaned in the manner described above. After

washing with distilled water the apparatus is then rinsed with the

ethyl alcohol used for the determination and is assembled still damp

with alcohol. The saponification flask is used dry and the sample is

weighed in in the manner described earlier.

To start the. determination, 2 ml solution of a 50 mg p-tolu-

enesulfonic acid in 1 nil ethyl alcohol is allowed to enter through

the funnel with ground glass joint and the solution is heated to boiling

point for 10 minutes under a reflux condenser. The flask dips into the

heating bath which is heated'to 95 to 100°C so that the liquids are

at the same level. After the first heating period the cooling water

_13..

is blown from the reflux condenser without interrupting the heating

process and the alcohol-ester mixture is distilled for the first time.

The distillation is carried out at uniform temperature and takes

approximately 10 minutes. The reflux condenser is then activated

again andl ml ethyl alcohol is added to the flask from the ground (135)

glass joint funnel. The content of the flask is again heated to boiling

point under reflux for 10 minutes. This is followed by the final dis-

tillation of the alcohol-ester mixture for which the temperature of

the heating bath is increased to 105 - 110°C. A total of 10'm1 al-

cohol are distilled off and care is taken that approximately 1 ml

liquid remains in the flask during the distillation. Each distilled

cubic centimeter is immediately replenished from the funnel with ground

glass joint. The distillate is collected in a-100-ml Erlenmeyer flask

(6) of Jena apparatus glass which is attached to the ground glass joint

of the condenser (5). To saponify the ester, 1 ml of a 0.5-n sodium

hydroxide solution is used. After the distillation is completed the

ground glass joints connecting (2) and (5) are opened, the condenser

(5) is rinsed with 1 to 2 ml alcohol and is then sealed with the soda-

lime tube (7). The distillate is then heated to a low boil for 15

minutes under reflux and the saponification of the ester is thus com-

pleted. For cooling the flask is placed into a beaker containing water

and in the meantime the cooling and ventilating tube is rinsed with a

total of 10 ml distilled water. The flask is then removed from the con-

denser and one starts to boil off the alcohol. For this purpose the

solution is heated to boiling over an open flame with a continuous

swirling motion, i•rhâl.e the flask is held tilted with rubber-covered

crucible tongs. No losses of substance will occur through spattering if

— 14 —

the solution is kept in a steady swirling motion. In three to five

minutes the alcohol is evaporated and the soiution is reduced to

approximately 3 to 5 ml.

It goes without saying that a standard 0.02—n sodium hydroxide

solution can also be used for the saponification of the ester which

is titrated with 0.02—n hydrochloric acid following removal of the

alcohol.

However, I personally prefer a concentrated sodium hydroxide

solution because this considerably reduces the saponification time of

the ester. Furthermore, the alkaline solution which contains sodium

acetate can be converted into acetic acid solution by exchange with

a cation exchange resin8 and can he titrated with 0.01—n sodium hydro-

xyde solution according to the usual technique.

At this point some indications should be offered concerning

the method of the ion exchange reaction. Wofatit K or Wofatit KS with

a grain size of 0.2 to 0.3 mm was used as ion exchange resin. The finer

particles are removed by wet—screening. The fraction intended for ana-

lysis is activated with e hydrochloric acid by pouring the acid over .the resin in a beaker which is allowed to stand for approx. 30 minutes.

The mixture is subsequently poured through a filter tube the size of

which depends on the amount of exchanger to be prepared in advance.

Approx. 50 ml hydrochloric acid are then passed through the resin

followed by a wash with very pure distilled water until it is completely (136)

free of acid. The resin is stored under water and is ready for use.

This product is used in the amount required for the exchange reaction.

The reaction is carried out in small tubes which can easily be fabricated

•

— 15 —

from a Jena apparatus glass tube (outside diameter =-1 8 mm). Fig. 2

shows two shapes of reaction tubes. Before charging the tube, a few

absorbent—cotton Libers are loosely placed into the spot where the

outlet tube is attached. After filling with water the ion exchanger

is added up to the point where the tube widens into a funnel.

Ahh. 2.

-Fig. 2

For the quantitative completion of the reaction care should be

taken that the layer of ion exchanger never runs dry during passage of

.the solution to be converted. In such a case air bubbles would become

enclosed between the resin grains when the solution is poured in which

might result in a reduced rate of filtration and would affect the quanti-

tative conversion. With shape b) this unfavourable condition cannot

occur because the solution can never completely drain from the tube.

The capillary tube which is bent upwards prevents complete draining and

keeps the ion exchanger under water at all times.

To finish the determination of the acetyl groups the alcohol-

free, alkaline sodium acetate solution is then filtered through the

- 16 -

exchanger tube. By using a thin glass rod it is easy to quantitatively

transfer the solution from the Erlenmeyer flask which has a pouring

spout. The flask is washed with approx. 2 ml dist. water which is

passed through the exchanger as well. Washing is continued until an

amount of 20 ml filtrat.e is obtained. A 100-ml quartz Erlenmeyer flask

is used to collect the acetic acid solution. The solution is now ready

for titration. No matter which method is used for the determination of

the acetyl, groups, one always obtains an acetic acid distillate of 20

to 30 ml. To determine the acetic acid content the carbon dioxide must

be removed by boiling before the end point is reached, for which I con-

sider a 20-second boiling time as sufficient. Numerous determinations (137)

have confirmed that no losses of acetic acid occur with these dilu-

tions even if the boiling time is doubled. Comparative experiments

using a cold finger acting as reflux condenser as recommended by Kainz9

showed no differences in the results. However, the use of such a reflux

condenser no doubt increases the reliability of the procedure and, in

addition, permits several repetitions of the titration.

The titration of the acetic acid is carried out in the follow-

ing manner: the obtained acetic acid distillate is treated with 6 drops

of a 1% phenophthalein solution in alcohol and is titrated first with

0.01-n sodium hydroxide solution until it turns clearly pink. The solu-

tion is then acidified with 0.5 to 0.8 ml 0.01--n hydrochloric acid and

using the cold finger boiled for 20 seconds, while shaking. A washing

bottle held ready with slowly boiling water serves to rinse the cold

finger; 1 to 2 ml are suffi.cient. The titration is then quickly carried

to the end point. The titration is repeated, adhering to the same con-

ditions. To calculate thé acetyl content, a blank value is always

-17 • IMO

subtracted from the consumption of 0.01—n sodium hydrochloride solu-

tion which must be determined separately for.each method of determina-

tion. This blank value, which usually amounts to approx. 0.1 ml 0.01—n

sodium hydroxide solution only has to be tested now and then because it

remains constant as long as the same solvents are used. It does there-

, fore not affect the determination and is determined without substance

according to the method used.

Reagents

Even if the best reagents are used, very low blank values are

not always readily obtained. Howeve:2, before undertaking any purifica-

tion of the freshly prepared solutions it is expedient to perform a

blank value determination for each technique. Only if the blank values

are too high — the values generally lie around 0.1 to 0.15 ml 0.01—n

sodium hydroxide solution — attempts should be made to purify the

individual solutions.

For sulfuric acid and chromosulfuric acid solutions the best

effect is achieved by prolonged boiling under reflux and subsequent

distillation of the easily volatile, acid constituents. The distilled

water is replaced with the same amount of freshly distilled water in

order to maintain the same concentration of the solutions.

The same procedure is followed when preparing the alcoholic

solution of the p—toluenesulfonic acid and after boiling under reflux

the solution is repeatedly distilled off with freshly distilled alcohol. (138)

Finally, the portion remaining in the distillation flask is topped up

with alcohol to make up the original volume. The ethyl alcohol used

.for this purpose is boiled under reflux over sodium carbonate before

use and is subsequently distilled.

- 18 -

The toluenesulfonic acid solution is prepared in small amounts

only which are used'up after a few days. Longer storage is not practical

because the blank values of older solutions increase.

The methyl alcohol used for the preparation of the methyl-

alcoholic sodium hydroxide solution is deacidified by boiling with the

addition of potassium hydroxide and then distilled.

To prepare pure, distilled waters filtration over a combination

of a cation and anion exchanger is recommended if the available dis-

tilled water does not meet the exacting demands outlined above.

It is my pleasant duty to thank the Director of the Tn.,titute,

Prof. Dr. A. Zinke for his support and interest in this work. Prof. Dr.

T. Reichstein, University of Basle, has aided me greatly by lending to

me an acetyl apparatus and sending various reagents; for this I would

like to express my sincere thanks. The I'arbenfabriken Bayerg Leverkusen,

made available to me samples of Wofatit for which I am grateful. Iam

further indebted to Dr. Robert Kretz for the performance of numerous

analyses.

L3.terature

V. N'irxru6rrf)rr, Miki-n<-lu-m. 30, 241 ( 111•12).E. Il•irerrthrrflrr, .llikrom)ir•rn. 11$, fil (1947).K. F'rrtulrnhrrri tnul M. (Irunfrr, Ann. ('hr•nt. 4713, 2:10 (1112:9).

K.Errurlrri h,rg tiuti E. tl'rLrr, Z. unHow. (.'ho ► n. 71H, •lH(t (14125).

It. htufur unit F. 1.'Or.vrt. G. utt};ew. ('hem. 44. H47 (111;i 1 1.

H. liulur und H. Ilrrrh, litv. tltsr•It. chtatt. C r•v. (ili• 1274 ( 193:3).1'.l'rryl, Iht tlttantitttlkt or}Lnnir+tlte A1il:rrrtnul^'vt, ntuhcurbtittt t•ou

f{. Aufl. N. 24H. Wirw til)"ingorNr•rlnu. 1049.K. !{'ir•srribrrgrv•• \liluunt•l•tn. tlll, 176 (11142).

, (%. Krrir+.. \liknw)irrn. 1P). K9 (19511).

7. Quantitative organic microanalysis, revised by H. Roth, 6th Hdition,

p. 248. Vienna: Springer Verlag 1949.