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©TBS 2 020  
DRAFT TANZANIA STANDARD  
 
 
 
 
 
Methods for determination of organic preservatives in foodstuffs – 
Part 2: propionic acid and its salts  
 
 
 
 
 
 
 
 
 
TANZANIA BUREAU OF STANDARDS  
  TBS/AFDC 3(72) P3 
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©TBS 2 020 0. Foreword  
For protecting food from microbial deterioration, a number of methods as application of heat or cold, 
dehydration, fermentation, irradiation o r 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 o rganoleptic changes, be economical in use and be capable of being analyz ed. 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 wo uld not only ensure repeatable and 
reproducible results for their correc t interpretation, but would also facilitate inter -laboratory comparisons  
There are two classes of preservatives , class I and class II. Class I preservatives include common salt, 
sugar,  dextrose, glucose (syrup), wood smoke, spices, vinegar   honey, etc. cl ass II preservatives include 
inorganic substances such as sulphurous acid including salts thereof, nitrates of sodium or potassium and 
organic substances like benzoic acid including sa lts thereof, sorbic acids and including its sodium, 
potassium and calciu m salts and sodium and calcium propionate  
This standard, covering the determination of organic preservatives, is being issued in three parts. This 
part (Par t 2) covers the determinati on of propionic  acid and its salts in foodstuffs. The p art: 1 covers 
benzoic acid and its salts and part : 3 covers sorbic acid and its salts . In reporting the results of a test or 
analysis made in accordance with this standard , if the final value, observed  or calculated, is to be 
rounded off, it shall be done in accordance to  with TZS 4   
In the preparation of this standards assistance was drawn from  IS 12014 (Part 2):1986, Methods for 
determination of organic preservatives in foodstuffs – Part 2: propionic acid and its salts published by 
Bureau of Indian Standa rds  
1. Scope   
This standard  prescribes  the methods for  determination of propionic acid and its salts used as 
preservatives in foodstuffs  
2. Normative References  
The following standard , in whole or in  part, are normatively referenced in this document and are 
indispensable for its application. For dated references, only the edition cited applies. For undated 
references, the latest edition of the referenced document (including any amendme nts) applies  
TZS 59: Water for analytical laboratory use – Specification and test method  
TZS 4: Rounding off numerical values  
TZS 672: Automotive gasoline (premium motor spirit)  
3. Qualit y of reagents   
3.1 analytical grade chemicals and distilled water  conforming to TZS 59 shall be employed in tests    TBS/AFDC 3(72) P3 
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©TBS 2 020 4. General  
4.1 This standard  specifies three methods for determination of propionic acids and its salts , namely, 
paper and column chromatog raphic methods , titration and spectrophotometric methods. Paper 
chromat ographic methods shall be used for qualitative detection and  column chromatographic methods 
shall be used for quantitative estimation of propioni c acid  and its salts.  
4.2 Principle  
Prop ionic acid is so luble in water and organic solvents (alcohol) and being  non-volatile can remains as 
residues when the solution is constantly dried . In solution propionic acid can be determined by 
quantitative analysis using sodium hydroxide solution   
 5. Paper chromatogra phic method   
5.1 Reagents   
5.1.1 Mobile Solvent  
take t wo parts of acetone, one part of tertiary butyl alcohol, one part of n -butyl  alcohol and one part of 
liquid ammonia and mix them. This solvent should always be prepared fresh  
5.1.2 Chro mogenic  reagent  
add 200mg each of methyl red and bromothymol blue to a mixture of 100 mL formalin and 400 mL 
absolute alcohol. Adjust to pH 6.2 with 0.1N sodium hydroxide  
5.1.3 Sodium Hydroxide - 1N 
5.1.4 Phosphotungstic acid - 20 percent solution in distil led water  
5.1.6 Crystalline Magnesium Sulphate - MgSO 4.7H 2O 
5.1.6 Sulphuric Acid - 1N. 
5.1.7 cresol red indicator  
5.1.8 propionic acid Standard solution  
pipette 1 mL of propionic acid into a 100 mL volumetric flask and dilute to volume with distilled wate r. 
Pipette 1 mL of this stock into  a 25mL beaker and neutralize the acid  with 0.1N  sodium hydroxide using 
cresol red indicator avoiding excess alkali. Evaporate to 0. 5mL in a water -bath  
5.1.9. Congo Red Indicator Paper  
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©TBS 2 020 5.2 Apparatus  
5.2.1 Chromatographic  tank  
5.2.2 Pi pette - with 0.1 mL graduations  
5.2.3 Chromatographic pap er - Whatman No.  1, 20  x 20cm, sheet  
5.2.4 Steam Distillation Apparatus  
5.2.6 Beakers - 25mL capacity  
5.3 Procedure  
5.3.1 Sample Preparation  
5.3.1.1 all types of bread not containi ng fruit   
take one or half loaf of bread and cut it in slices of 2 -3mm thickness. Spread the  slices on the paper and 
let them dry in a   room  temperature  until sufficiently crisp and brittle to grind well. Grind entire sample to 
pass through 860-micron  sieve, mix well an d keep in an air -tight container before proceeding for analysis  
5.3.1.2 Bread containing raisins and fruits  
proceed as in 5.3.1.1 except comminute by passing twice through food chopper instead of grinding and 
dry the air-dried  sample in an  uncovered dish  for 16 hours at 70 ºC under pressure of less 0.07999bars  
 5.3.2 Distillation  
weigh accurately 10g of air-dried  bread and transfer it to a 1 60mL distillation flask.  Add 40 mL of distilled 
water and 10 percent of 1N suphuric acid, mix thoroughly and add 10 mL of 20 percent phosphotungstic 
acid solution . Mix the contents well and add 40g of magnesium sulphate. Swirl the contents well and 
make the solution acidic to Congo  red indicator paper with 60 percent sulphuric acid. Connect the 
condenser and steam genera tor and distil 200 mL in 36-40 minutes.  
Immediately neutralize the distillate using cresol red and 0.1N sodium hydroxide. Evaporate the solution 
to 0.6mL or evaporate to just dryness and then take up in 0. 6mL distilled water.  
5.3.3 Paper Chromatography  
5.3.3.1 Take  a Whatma n No.  1 (see 5.2.3) unwashed chromatographic paper and rule the starting line 
2.6cm from the bottom edge with a hard pencil. Spot two 1µ L spots with  1µl pipette on the paper 2. 6cm 
apart from each other, leaving at least 2. 6cm margin, the first spot being o f propionic acid standard 
solution a nd the second of unknown sample. Let the paper dry and clip it to a glass rod and suspend it in 
the chromatographic tank with 60mL of mobile solvent in a trough. Do not saturate the tank with mobile 
solvent before insert ing paper. Seal the glass cover with cellophanes or other suitable tape and let it 
develop until solvents reaches 2. 6cm from  top of paper. Remove the paper from the tank and let it air dry .  TBS/AFDC 3(72) P3 
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©TBS 2 020 5.3.3.2 Spray chromogenic reagent on front side of the paper. Spra ying should be uniform and rather 
heavy but not to the extent that chromogenic reagents runs or drips. Faint yellow spots indicate presence 
of propionic acid  
5.3.3.3  To intensify the acid spots, place the paper in the atmosphere of ammon ia fumes momentari ly (by 
placing 60mL ammonium hydroxide in a 2litre beaker and exposing to fumes by placing each end in 
beaker momentarily), entire paper immediately turns green.  
5.3.3.4 Remove  paper from ammonia fumes, propionic acid gradually appears as  red spots  and pre sence 
of propionic acid in the sample may be determined by comparing its Rf value with that of standard 
propionic acid. Since colour of the acid is not stable, mark the spot with the pencil as soon as they are 
completely developed.  
6. Col umn chromatographi c method  
6.1 Reagents  
6.1.1 Sodium Hydroxide  - 0.1 and 1N  
6.1.2 Phosphotungstic  acid solution  – 20 percent in distilled water  
6.1.3 Crystalline Magnesium Sulphate - MgSO 4.7H 2O 
6.1.4 Sulphuric Acid – 1N.  
6.1.5 Formic Acid - 0.01N  
6.1.6 Alphamine Red R I ndicator  
6.1.7 Ammonium Hydroxide - 1N 
6.1.8 Silicic Acid - 100 Mesh  
6.1.9 Chloroform  
6.1.10  butyl alcohol  
6.1.11  Sulphuric Acid - 50 percent  
6.1.12 Absolute ethanol  
6.1.13 anhydrous sodium sulphate.  
6.1.1 4 Butanol in Chloroform - 1 percent.  
Remove alcohol in chloroform by washing three time with water. Add 10 mL of n-butyl alcohol to 1 litre of  
washed chloroform in separating funnel, shake vigorously, add 25 mL of distilled water and shake again.  TBS/AFDC 3(72) P3 
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©TBS 2 020 Let it stand until the lower layer cle ars. Drain and dis card the upper aqueous layer. Store it in contact with 
granular sodium sulphate.  
6.1.1 5 Cresol Red Indicator  
dissolve 50 mL of O – Cresol – Sulphonphthalein in 20 mL of absolute alcohol, add 1.3 mL of 0.1N sodium 
hydroxide and dilute to 50 mL with distilled w ater. Use 2 drops for each 25 mL of aqueous solution . 
6.1.1 6 Barium Hydroxide Standard Solution  - 0.01N  
6.1.1 7 Sodium Acetate -sodium chloride solution  
Dissolve  12g of sodium chloride and  25g of sodium acetate in distilled water and dilut e to 500 mL  
6.1.1 8 standard propionic acid solution  
6.2 Apparatus  
6.2.1 Chromatographic Tube – approximately 15 x 250mm.  
6.2.2 Rubber Bulb  
6.2.3 Micro -funnel - 2mL capacity  
6.2.4 Steam Distillation Apparatus  
6.3 Procedure  
6.3.1 Sample Preparation  
6.3.1.1 All type of b read not containing fruit  
take one or half loaf of bread and cut it in slices of 2 -3mm thickness. Spread the slices on the paper and 
let them dry in a   room temperature until sufficiently crisp and brittle to grind well. Grind entire s ample to 
pass throu gh 860 -micron sieve, mix well and keep in an air -tight container before proceeding for analysis  
6.3.1.2 Bread containing raisins and fruits  
proceed as in 5.3.1.1 except comminute by passing twice through food chopper instead of grindin g and 
dry the air-dried  sample in an uncovered dish for 16 hours at 70 ºC under pressure of less than 0.07999  
bars  
6.3.2 Distillation  
 weigh accurately 10g of air-dried  sample and transfer it to 150 mL distilling flask. Add 40 mL distilled water 
and 10 mL of 1N sulphuric acid, m ix thoroughly and add 10 mL of 20 percent of phosphotungstic acid 
solution. Mix the content well and add 40g magnesium sulphate. Swirl the contents well and make the 
solution acidic to the Congo red paper with 50 percent sulphuric acid.  Connect the condense r and steam  TBS/AFDC 3(72) P3 
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©TBS 2 020 generator and distill 200 mL in 35 -40 minutes. Transfer the distillate to 400 -600mL beaker, add 10 mL 
0.01N formic acid, make alkaline to phenolphthalein with 1N sodium hydroxide and evaporate to 5 mL. 
transfer it into 225 mL glass stoppered test t ube rinsing the beaker with three portions of distilled water. If 
insolub le matter adheres to the beaker, rinse with 1N sulfuric acid. Make this solution alkaline to 
phenolphthalein and evaporate to dryness just by insertin g the test t ube in a steam bath.  
6.3.3 Chromatographic Separation  
6.3.3.1 Preparation of partition column  
take 5g silicic acid in glazed porcelain evaporating dish and add 1 mL of alpha mine  red R indicator and 
just enough 1N ammonium hydroxide to alkaline colour of  the indicator (1 drop is enough) . Add maximum 
amount of distilled water that the silicic acid will hold without becoming sticky or agglomerating  in the butyl 
alcohol -chloroform solution (this amount shall be determined for each  of silicic acid and usually varies 
from 50 to 75  percent of the weight of the silicic  acid). Homogenize the mixture thoroughly in a pestle. 
Add 25 mL of 1 percent butyl alcohol in chloroform and mix to form a slurry that pours readily. Pour this 
slurry into a chromatographic tube cont aining a small cotto n plug in the neck of the constricted end. To 
avoid air pocket, tilt the tube slightly while pouring. If air bubbles form while pouring, eliminate by stirring 
suspension in tube with long glass rod. Clamp the tube vertically in ring sta nd. In the tube inse rt a one -
hole rubber stopper fitted with a glass tube bent to 90˚ angle and hold in place by a Bunsen clamp against 
the pressure to be exerted. Connect the bent glass tube to a pressure source. Adjust the pressure to 5 -10 
psi (0.3447 -0.6895 bars)  so that excess solvent is forced through the column dropwise.  
During remo val of excess solvent, gel packs down. As the column packs down,  particles of gel adhere to 
the wall of the tube, but eventually the gel leaves the wall of the tube relativ ely clean. This is the point of 
optimum density of the column, and the column is rea dy to use. Apply pressure until solvent reaches the 
surface of the column. If solvent passes below surface causing drying or ‘cracking of column’ or if air 
pocket s are pres ent extrude packing from the tube, re -slurry with the solvent and repack the column.  
6.3.3.2 Preparation of standard propionic acid solution  
Prepare stock solution of propionic acid by diluting 5 mL of propionic acid to 250 mL with distilled water. 
Pipette 1mL of stock solution into a 125 mL Erlenmeyer flask and titrate with 0.01N sodium hy droxide, 
using cresol red as indicator, to pink colour which persists for 45 seconds. mg acid/ mL standard solution = 
titre value (mL)  x normality  of sodium hydroxide used  x Factor (F)  
                  F = 7.40  for propionic acid  
6.3.3.3 Preparation of kno wn samples – pipette out 50 mL of standard solution into a 50 mL beaker and 
just neutralize with 0.01N sodium hydroxide solution, using phenolphthalein and add 10 drops in ex cess. 
Evaporat e it to dryness on a steam bath.  
6.3.3.4 Column separation - to the dr y residue add 2 mL of 10 percent butyl alcohol in chloroform solution 
and while stirring with glass rod add  50 percent sulphuric acid dropwise until the sodium sal t are 
conv erted to free acids ( acid to congo red paper)  and add 1g of anhydrous sodium sulphat e. Place a 
50mL graduated cylinder under  the column as receiver. Decant the supernatant onto column, pouring it 
slowly down the side   of the tube without disturbing level surface of the column. Apply pressure until  TBS/AFDC 3(72) P3 
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©TBS 2 020 solvent reaches surface of the gel. Wash  the beaker with 1 mL solvent and pour into column. Apply 
pressure until the solvent just disappears into sodium sulphate layer. Fill the tube with the solvent and 
apply the  pressure. Once the band reaches the point 2 -5mm above the narrowest portion of cons triction 
of tube, record the volume and remove the receiver.  
Transfer the elute to a 125 mL Erlenmeyer flask, rinsing the cylinder with three 5 mL portion of distilled 
water.  Add one drop of cresol red indicator and titrate with 0.1N barium hydroxide solutio n. As its end 
point approaches, stopper flask and shake vigorously to completely extract acid from solvent phase. 
Correct titration for blank as follows. Collect 25 mL of bu tyl alcohol - chloroform mixture from column before 
the acid is transferred, add 15 mL boiled and cooled distilled water and titrate as above with 0.01N barium 
hydroxide solution.  
6.4 Calculation   
Calculate the results for propionic acid as mg/100g sample  
 Propionic acid, mg/100g = 7.4 mL x 0.01 N barium hydroxide solution  
                Calcium propionate = propionic acid contents x 1.256  
6.5 Identification of propionic acid . 
Acid separated in butyl alcohol -chloroform solution may be further identified by p aper chromatography as 
described in 5.3.3