TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020   
 
 
 
 DRAFT TANZANIA STANDARD  
 
 
 
Methods for determination of organic preservatives in foodstuffs – 
Part 1: benzoic acid  and its salts  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TANZANIA BUREAU OF STANDARDS  
 
  TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  0. Foreword  
For protecting food from microbial deterioration, a n umber of methods as application of heat or cold, 
dehydration, fermentatio n, irradiation or addition of certain chemicals are employed. Besides extending 
the periods of use of food a chemical preservative should be safe for human consumption, should not 
impart undesirable organoleptic changes, be economical in use and be capable  of being analyzed. While 
the use of preservative to be safe under conditions of use is governed by law, it is considered necessary 
to prescribe methods for their analysis. The use of  these methods would not only ensure repeatable and 
reproducible results for their correct interpretation, but would also facilitate inter -laboratory comparisons  
There are two classes of preservatives, class I and class II . Class I preservatives include c ommon salt, 
sugar, dextrose, glucose (syrup), wood smoke, spices, vinegar    honey, etc. class II preservatives include 
inorganic substances such as sulphurous acid including salts  thereof, nitrates of sodium or potassium and 
organic substances like benzoic  acid including salts thereof, sorbic acids and including its sodium, 
potassium and calcium salts and sodium and calcium propionate.  
This standard, covering the determination of organic preservatives, is being issued in three parts. This 
part (Part 1) cove rs the determination of benzoic acid and its salts in foodstuffs. Part 2 covers propionic 
acid and its salts and part 3 covers sorbic acid and its salts .  
Benzoic acid and its salts are commonly used as preservatives in non -alcoholic wines, squashes, 
crush es, fruit syrups, cordials, fruit juices, barley water, sweetened mineral  water, jam, marmalade, 
canned cherry, fruit jelly, pickles and chutney made from fruits or vegetables, tomatoes and other sauces, 
syrups and sharbats.  
In the preparation of this standard assistance was drawn from IS 12014 (Part 1):1986, Methods for 
determination of organic preservatives in foodstuffs – Part 1: benzoic acid and its salts published by 
Bureau of Indian Standards  (BIS)   
In reporting the results of a test or analysis made in accordance with this standard, if the final value, 
obser ved or calculated, is to be rounded off, it shall be done in accordance with  TZS 4  
1.  Scope   
This Tanzania standard prescribe  the methods for determination of benzoic acid and its s alts used as 
preservatives in foodstuffs  
2.  Normative References  
The fol lowing referenced standards referred to in the text in such a way that some or all of their content 
constitutes requirements of this standards.  For dated references, only the edition  cited applies . For 
undated references, the latest edition of the referen ced document (including any amendments) applies   
TZS 59: Water for analytical laboratory use – Specification and te st method  
TZS 4: rounding off numerical values  
TZS 672: automotive gasoli ne (premium motor spirit)   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  3. Quality of reagents    
3.1 analytical  grade chemicals and distilled water  conforming to TZS 59 shall be employed in tests.  
4. General  
4.1 this standard s pecifies thre e methods for determination of benzoic acids and its salts n amely, thin 
layer chromatography, titration and spectrophotometric methods. Thin layer chromatographic methods 
shall be used for qualitative detection and titration or spectrophotomet ric methods s hall be used for 
quantitative estimation of benzoic acid  
4.2 Principle  
benzoic acid acids are isolated from food by extraction with diethyl ether and successive partitioning into 
aqueous NaOH  and CH 2Cl2. Acids are converted to trimethylsilyl ( TMS) ester an d determined by  gas 
chromatography ( GC). Phenyl acetic acid is used as internal standards for benzoic acid  
5. Thin layer chromatographic (TLC) methods   
5.1 Reagents   
5.1.1 Magnesium sulphate (MgSO 4.7H 2O) 
5.1.2 Phosphoric Acid  
5.1.3 Sodium Hy droxide -1N 
5.1.4 concentrated  hydrochloric acid – analytical grade   
5.1.5 Chloroform  
5.1.6 diethyl e ther  
5.1.7 Solvent – A mixture of n -hexane and acetic acid in the ratio of 96:4  
5.1.8 Bromocresol Green  
5.1.9 Ethanol – 50 percent and 95 percent  
5.1.10  Standard Ben zoic Acid  
5.2 Apparatus  
5.2.1 Kjeldahl Flask  
5.2.2 separat ing funnel  
5.2.3 Ultraviolet lamp  
5.2.4 Ultraviolet Spectrophotometer   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  5.2.5 TLC Plate - made up of a layer containing kieselguhr GF 254 kieselguhr G (1:1) to a thickness of 
0.25 mm. A ctivate at 10 0 ◦C for 1hour  
5.3 Preparation of the Sample  
5.3.1 Liquids – accurately weigh 50 to 60g sample directly in a kjeldahl flask with a small portion of water 
and proceed;  
5.3.2 Solids  - accurately weigh 40g into a high-speed  blender. Add 100 mL distilled water , blend until 
homogeneous and transfer to a kjeldahl with a small portion of water.  
5.3.3 Semisolid and Solid Liquid Mixture - Blend the whole sample in high speed blender to get a 
homogeneous mixture. Weigh 50 to 60g of the blended sample in a kjeldahl fl ask with a small portion of 
distilled water and proceed.  
5.4 Procedure  
5.4.1 Extraction - To the prepared sample (see 5.3), add 200g of magnesium sulphate and 25 mL of 
phosphoric acid. Wash the neck of the flask till the total volume is 350 to  375mL. steam  distil the 
preparation into separating funnel containing 50 mL of sodium hydroxide collecting 725 to 750 mL of 
distillate in 90 minutes. Rinse the condenser with 20 mL of water. Acidify the distillate to litmus with 20 mL 
of hydrochloric acid.  M ake the extra ction solution by mixing two volume of chloroform and one vo lume of 
ether. Then extract the distillate with 100 mL and four 50 mL portions of the extraction solution, each 
extraction should be shaken for 1 minute vigorously. Collect the combined  extract in a  beaker and 
evaporate carefully on a steam -bath and take in a 25mL volumetric flask  
5.4.2 Spot on the plate 100µ L of the sample solution with the standard benzoic acid. Place the plate in a 
chamber containing the solvent and develop up to 10cm . air-dry the  plate for 10 minutes. See the benzoic 
acid spots under the ultraviolet light. For the better visibility, spray the plate with 0.5 percent bromocresol 
green ethanol. Spots are permanent and can be observed for several days.  
5.4.3 Approximate q uantitative e stimation by visual comparison and quantitative estimation b y 
densitometric comparison of the intensity of the colour of the spot of the sample with those of the 
standards may be done. Quantitative estimation may also be done by scraping off t he spot of th e sample 
in unsprayed condition identified with the help of the ultraviolet light. Trasfer  the scrapped sample into 
centrifuge tube also prepare a blank . To each add 7 mL of 95 percent ethanol. Shake and centrifuge. Take 
supernatant in 5cm micr ocell. Read i n spectrophotometer from 310 to 350nm against the blank. Cal culate 
as under:  
Percent benzoic acid x 1.18 = percentage sodium benzoic  
6. Titration Method   
6.1 reagents  
6.1.1 Saturated sodium chloride solution  
6.1.2 Pulverized sodium chlorid e 
6.1.3 Calci um hydroxide solution 1:3 solution of milk of lime   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  6.1.4 Di lute hydrochloric acid - 1:3 
6.1.5 Chloroform  
6.1.6 Ethyl Alcohol - Made Neutral with an alkali using phenolphthalein as an indicator  
6.1.7 Phenolphthalein indicator solution - prepa red by dissol ving 0.1g in 100 mL of rectified spirit 
conforming to TZS 672  
6.1.8 Standards Sodium Hydroxide – 0.05N  
6.2 Preparation of the Sample  
6.2.1 General Food Stuffs - Weigh accurately about 150g of the material  put into digesting tube , (grind if 
nece ssary ) Add about 300 mL of saturated sodium chloride solution. Digest the material and then add 15g 
of pulverized sodium chloride. Make the solution alkaline to litmus by adding required amount of calcium 
hydroxide solution. Transfer the solution to a 500 mL volumetric f lask and make up to volume with 
saturated solution of sodium  chloride. Allow it to stand for at least two hours while shaking frequently; 
centrifuge if necessary, and filter.  
6.2.2 Catsup - weigh accurately about 150g of the sample and add to it 15g pulver ized sodium chloride 
and transfer the mixture to a 500 mL volumetric flask rinsing with 150 mL saturated sodium chloride 
solution. Make slightly alkaline to litmus paper with 10 percent sodium hydroxide solution and dilute to 
volume with saturat ed sodium chl oride solution. Allow it to stand for 2 hours shaking freque ntly centrifuge, 
if necessary and filter.  
6.2.3 Jellies, Jams Preserves and Marmalades - weigh accurately 150g sample and digest in 300 mL of 
saturated sodium chloride solution. Add 15 g of pulveriz ed sodium chloride and make it alkaline to litmus 
paper with  calcium hydroxide solution. Transfer the solution to a 500 mL volumetric flasks and make up to 
volume with sodium chloride solution. Allow it to stand for 2 hours shaking frequently c entrifuge, if  
necessary and filter.  
 6.2.4 Cider containing alcohol and s imilar products - weigh accurately 250g sample make it alkaline to 
litmus paper with 10 percent sodium hydroxide solution and evaporate to steam bath to 100 mL. Transfer 
the solution t o a 250 mL volumetric flasks and add 30g of pulverized sodium chloride an d shake until 
dissolved. Dilute to volume with saturated sodium chloride solution. Allow it to stand for 2 hours shaking 
frequently centrifuge, if necessary and filter.  
 6.2.5 Salted o r Dried Fish - Accurately weigh 50g of sample and grind it. Transfer the sample into a 
500mL volumetric flask with a little amount of distilled water. Make the solution slightly alkaline to litmus 
paper with 10 percent sodium hydroxide solution and dilute to volume wit h distilled water. Allow it to stand 
for 2 hours shaking fre quently centrifuge, if necessary and filter. Pipette 300 mL of  the filtrate into  another 
500mL volumetric flask  and add 90g of pulverized sodium chloride. Shake until sodium chloride d issolves 
and dilute to volume with saturated sodium chloride solution. Mi x thoroughly and filter off the precipitated 
protein and other extraneous matter.  
6.3 Procedure  
6.3.1 Pipette accurately 100 to 200 mL of the filtrate (see clause 6.2) into a separati ng funnel. Ne utralize it 
to litmus with hydrochloric acid and add 5 mL in excess. Extract with 70, 50, 40, and 30 mL of chloroform  TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  shaking with a rotary motion to avoid an emulsion. If an emulsion forms, break up with a glass rod, by 
drawing off into a secon d funnel  and giving one or two sharp  up-and-down shakes or by centrifugin g. A 
neutral hygroscopic reagent like calcium chloride may be used for breaking the emulsion. Avoid drawing 
off the aqueous layer  
6.3.2 Transfer the extract to a porcelain dish rinse  the containe r   several times with a small quantity of 
chloroform , and  evaporate at a room temperature in a current of dry air. Alternatively, transfer it into an 
Erlenmeyer flask and distil slowly to one -quarter of its volume. Transfer it to the dish, rinsing the fla sk with 
a small amount of chloroform and evaporate to dryn ess at room temperature in a current of a dry air. Dry 
the residue overnight in a desiccator containing concentrated sulphuric acid . 
  Note – if necessary,  the chloroform extract may b e washed with distilled water to make it free from  
              hydroch loric acid as the presence of the acid may vitiate the results . 
6.3.3 Dissolve the residue in 30 to 50 mL of ethyl alcohol, and add 10 mL of water and one or two drops of 
phenolphthalei n indicator so lution. Titrate this solution with a standard sodium hydrox ide solution and 
note the volume of the sodium hydroxide  
6.4 Calculation . 
   
  
Where  
N = normality of the standards sodium hydroxide solution  
V = Volume of the standards  sodium hydrox ide solution  
V1 = Volume of filtrate taken ( 6.2); and  
M = m ass in g, of the material.  
7. Spectrophotometric Method  
7.1 Reagents  
7.1.1 Crystallized Tartaric Acid  
7.1.2 Sodium Hydroxide – 1N  
7.1.2 sodium hydrogen carbonate  (NaHCO 3) 
7.1.3 Po tassium Dichro mate – aqueous solution of 33 to 34g  
7.1.4 Dilute Sulphuric  Acid – Obtained by diluting t wo volumes  of concentrated sulphuric acid with one 
volume of water  
7.1.5 Diethyl Ether – freshly redistilled   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  7.1.6 Standard Benzoic Acid Solution – 100mg/L prepared in diethyl ether  
7.2 apparatus and equipment  
7.2.1 Volume tric flask – 50mL capacity  
7.2.2 Beakers – 50 and 100 mL capacity  
7.2.3 Pipette – 20mL 
7.2.4 Graduated pipette  
7.2.5 Separating funnel  
7.2.6 Homogenizer  
7.2.7 Ultraviolet spectrophot ometer – provi ded with a 0.5mm monochromator with silica cells of thickne ss 
20mm, fitted with ground lids  
7.3 Procedure   
7.3.1 Preparation of the sample  
7.3.1.1 Liquid products – homogenize the sample well  
7.3.1.2 Thick products homogenize the sample for  analysis afte r carefully mixing it  
7.3.1.3 Solid products – cut a porti on of the sample for analysis into small pieces and remove the stones. 
Take about 40g of the product and crush them in a homogenizer or a motor  
7.3.2 Test Portion  
7.3.2.1 Liquid pro ducts – take 2 0mL of the prepared sample free from suspended matters with  the help of 
the pipette, mix them with about 50 mL of distilled water and transfer them into a 500 mL separating funnel. 
In the case of the pulpy liquid product, place 20 mL of the prod uct in a morta r and dilute them with 20 mL 
of distilled water. After decan tation, filter the liquid. The residue is dissolved twice with 20 mL of water. 
The filtrates are directly collected in the separating funnel.  
7.3.2.2 Thick or solid products – weigh to  the nearest 0 .01g, about 10g of the prepared sample. Entrain in 
a 250 mL borosilicate glass flask with ground -glass stopper with 30 to 40 mL of distilled water; add about 
50mg of sodium hydrogen carbonate (see note), shake and place on a water bath at 70 to  80◦C for 15 t o 
30 minutes. Pour the contents of the flask on a fluted filter paper and rinse twice with 15 to 20 mL of 
distilled water. The filtrate and the washing are collected in a 500 mL separating funnel. Allow to cool.  
  NOTE - The purpose of adding sodiu m hydroge n carbonate  (NaHCO 3) is to salify the benzoic acid,  
   the traces of which could be lost by volatilization  
7.3.2.3 Extraction of the benzoic acid - introduce 1g of tartaric acid into the separating funnel containing 
the aqueous dilution of the produ ct to be analyzed  (see 7.3.2.1 and 7.3.2.2) , add 60 mL of diethyl ether 
and shake carefully.   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  Allow to settle and then collect the other layer in another separating funnel. Wash the aqueous phase in 
the first funnel a second time with 60 mL diethyl ether. Al low t o settle and add once again the other layer 
to the first one. Carry out in the same manner a third extraction with 30 mL of diethyl ether.  
Add successively 10 mL, then 5 mL of sodium hydroxide solution and two sets of 10 mL of distilled water to 
the combi ned e ther extract. Shake and after allowing to settle down, collect the aqueous phase in a dish. 
Place the dish on a water -bath until about half of the alkaline solution is evaporated so as to eliminate the 
dissolved diethyl ether.  
7.3.2.4 Purification of benzo ic acid - after cooling, transfer the contents of the dish to a 250 mL flask with a 
ground -glass stopper containing a mixture of 20 mL of dilute sulphuric acid and 20 mL of potassium 
dichromate solution. Stopper the flask, shake and allow to remain in co ntact  for at least an hour.  
  NOTE1 – the extension of time of this oxidation does not create any difficulty because the benzoic  
    acid withstands this oxidation mixture  
  NOTE2 – when the original product also contains sorbic acid it necessary to exten d by 24 hours the 
    oxidation  in order to ensure the complete elimination of this anti -fungal agent.  
7.3.2.5 Prepa ration of the ether solution – extract the benzoic acid by treating twice the chromic solution 
with 20 to 25 mL of diethyl ether solutions. W ash t he ether  solution twice with a few millilit re of 
distilled water. After carefully decanting on a dry filter paper, collect the filtrate in a 50 mL volumetric flask. 
Wash the filter with a few millilit re of diethyl ether so as to bring the filtrate to 50mL 
7.3.2.6 Determination – place the diethyl ether solution in a silica cell with a ground lid of thickness 20mm 
and measure the absorbance of this solution with respect to the absorbance of the pure diethyl in a 
similar silica cell.  
The differential mea surem ent of the emergence at 272nm is calculated as follows:  
                                                           
                                         
 Where  
         B = absorbance at 272 nm,  
         A = absorbance at 267.5 nm,  
         C = absorbance at 276.5 nm  
7.3.2.7 plotting of the calibration curve – into a series of six 50 mL volumetric flasks, introduce 5, 7.5, 10, 
12.5, 15 and 20 mL of the standard solution of benzoic acid. Make up the volume to 50 mL with diethyl 
ether. The solut ions obtained contain  10, 15, 20, 25, 30 and 40µg of benzoic acid per litre. Carry out on 
the determinations on these  solutions the differential measurements by proceeding as indicated in  
7.3.2.6.  
Plot the curve giving the differential measurements as a fu nction of the number of micrograms of benzoic 
acid per litre indicated above.  
7.4 Expression of the Results   TBS/AFDC 3(71) P3 
ICS 67.250  
  
  
©TBS 2020  7.4.1 Liquid Products – ‘‘Benzoic acid ’’  
  
   Where;  
         m2   = mass in milligram of benzoic acid read on the calibration curve.  
The results can also be expressed in milligrams per kilogram  
7.4.2 Thick or Solid Products – “Benzoic acid’’, 
  
 Where  
 m1 = mass in grams of the product contained in the test portion, and  
m2   = mass expressed in milligram of benzoic acid read on the calibration curve