ANALYSIS OF CHEMICAL PRESERVATIVES

ANALYSIS OF CHEMICAL PRESERVATIVES

SULPHURDIOXIDE (SO2), SULPHITES AND, BISULPHITES

As aqueous solution SO2 exit as sulphurous acid (HSO3), sulphite (SO3) and bisulphate ions (HSO3)- . The microbial growth inhibiting effects of SO2 are most intense when it is in the unionized form as H2SO3. They are more inhibiting to bacteria than yeast and molds. Major role of SO2 and its salts as antimicrobial agent is in beverages and fruit preservation

SO2 react with vitamin B1 and makes it unavailable. Therefore it is recommended for food which serve as major sources of this vitamin in the diet

SO2 decolourises foods which derive their colour from anthocyanin

ESTIMATION OF SO2

The presence of SO₂ is ascertained before commencing its estimation :

A piece of filter paper dipped in lead acetate is dried.

A few piece of sulphur free zinc are added to a conical flask containing 10 to 15 gm of sample in solution and 20-25 ml of dil. HCL.

The lead acetate paper is folded in a cone shaped and placed into the mouth of the conical flask

The H2S generated in the presence of sulphites turn the exposed portion of the lead acetate paper black.

The available methods estimate the free or total SO₂ , Free SO₂ is estimated by direct titration with iodine whereas total SO₂ is estimated by liberating the combined SO₂ by any of the two methods :

(1) Treatment with excess alkali at room temperature , subsequent acidification to block recombination and iodinmetric titration

(2) Distillation from acid solution and titration

FREE SO2

50 ml of sample ( juices, squash, etc diluted with water if needed)

Acidified with 5 ml of dil. H2SO4

500 mg Na₂CO₃ is added to expel the air if present

Titrate rapidly with 0.02N standard iodine solution using starch as indicator .

The blue coloured end point should persist for a few minutes.

Let this titre value be a

A similar aliquot of the substance is acidified with similar 5 ml of dil. H2SO4

10 ml of 38+-2% formaldehyde is added

Kept for 10 min

Titrated against 0.02N iodine solution to faint but permanent blue colour

Let this titre value be b

Volume of iodine used by free SO2 present in the sample = a-b ml

TOTAL SO2

A. To two similar aliquot of sample 5ml of 5 N NaOH is added

Content gently stirred to avoid air introduction into the solution and kept for 20min

To one of the sample 7 ml of 5N HCL is added with stirring to avoid local concentration .

1 ml of 1% starch solution as indicator is added

Titrated immediately with 0.02N iodine to a definite dark blue colour

Let titre value be c

Reducing substance beside sulphite need to be determined, therefore the second sample is acidified

10 ml formaldehyde added to bind the sulphite and kept aside for 10 min , mix using a mechanical stirrer

Starch indicator is added and contents titrated rapidly against 0.02 N iodine solution till a dark blue colour persist for more than 15 sec

Let this as d

Volume of iodine used by the total SO₂ present in the sample= c-d ml

Calculation

1 ml of 0.02 N iodine = 0.64 mg of SO₂

SO2 (ppm) = titre * 0.64*1000

Wt. of sample

Combined SO₂ = total SO₂ – free SO₂

B. H₂O₂ method

Reagent :

1. 0.1% Bromophenol blue solution in water

2. 0.1% phenolphthalein solution in 50% ethanol. 100 mg is dissolved in 50 ml of ethanol and then diluted to 100 ml with water

3. 3% of H₂O₂ solution

APPARATUS :

Procedure :

20 ml and 5 ml of 3% H₂O₂ are added to flask D and trap E respectively.

Cooling the condenser C by circulating cold water

50 gm of Homogenized sample is transferred through gas inlet in to flask B using 300 ml of water, replacing the gas inlet A immediately .

20 ml of conc. HCL is slowly introduced through gas inlet and the position is tightened

CO₂ washed through Na₂CO₃ solution or nitrogen gas at 18 bubbles /minutes is passed through the gas inlet and flask B is heated to boil the contents first vigorously and then slowly for 30 min

H₂O₂ solution from the trap is washed in to flask D, the trap also rinsed with water

Content are titrated against 0.05 N NaOH using 3 drops of bromophenol blue indicator .

End point is pale sky blue . A blank on 20 ml H₂O₂ is done , correcting the net titre value .

CALCULATION

1 ml of 0.1 N NaOH = 3.2 of SO₂

SO₂ = Titre*normality of NaOH *32*1000

Wt. of sample

C. Accurately weigh 25 gm of the food sample is pulverized and slurried in water

20 ml of conc. HCL + 60 ml of boiled water is added through quick fit stop cock filter funnel.

The liberated SO₂ is collected in a beaker already containing water+ a few drops of starch solution + 0.1 ml of 0.02 N iodine solution in KI solution.

Simultaneously titration carried on against 0.02N iodine solution till colour reappears

1ml of 0.02N iodine solution = 640 ppm of SO₂

ESTERS OF p-OH BENZOIC ACID, SYN. PARABENS

The esters of p-OH benzoic acid were produced mainly to replace benzoic acid and salicylic acid which were mainly effective in highly acidic products.

CH3, C3H7, C4H9, C7H15, esters of p- OH benzoic acid despite having better antimicrobial activity than the parent acid , less acceptance in food preservation due to low solubility and not so good organoleptic properties

Minimum inhibitory concentration against bacteria and fungi are 12-400 ppm

DETECTION OF PARABENS AND p-OH BENZOIC ACID

A. TLC Method:

The sample is acidified and extracted with (C2H5)2O and the extract is concentrated and subjected to TLC

Reagent:

1. Silica gel G

2. Developing solvent: toluene: methanol: acetic acid (45: 8: 4)

3. 2% solution of acid and parabens

4. Denige’s Reagent: It is prepared by mixing 5gm HgO and 40 ml of water , cooling in ice-salt mixture , adding freezing cold 20 ml of H2SO4 very slowly and stirring the contend

5. H2SO4

6. NaNO2 2%

7. Na2SO4- anhydrous

8. Solvent ether

Procedure:

5ml H2so4 is added to 10 gm of sample and ground withNa2SO4 in a mortar until the sample is dry

Further grinding is done with small successive quantities of solvent ether and ether is decanted

The ether extract is filtered and the filtrate is evaporated at a low temperature .

The residue is dissolved in 1ml of methanol

20ml is spotted and chromatogram is developed with developing solvent

The plate is view under UV (254 nm)

Parabens show black spots and the area is marked with a pin and sprayed with Denige’s reagent.

Parabens gives a white spot

After heating at 100⁰C for 5 min it is sprayed with NaNO2 , red spots appear indicate presence of parabens

B. Qualitative Test for 4-OH Benzoic acid:

The test is done on neutral ammonium salt. The food if not acidic is acidified and then 4-OH benzoic acid is extracted with ether and desolventised. The residue is dissolved in few drops of NH4OH solution in a test tube

Millon’s Reagent is prepared by dissolving 3ml mercury in 27 ml cold fuming HNO3 and diluted with an equal volume of H2O. A few drops of this reagent are added to above solution. Presence 4-OH benzoic acid is revealed by rose-red colour

Estimation of parabens:

Parabens present in the sample are hydrolysed with alkali and the released p-OH benzoic acid is extracted with (C2H5)2O after acidification of the sample. After re-extraction with NaOH from ether, colour is developed with Denige’s reagent and the absorption is read at 518 nm.

REAGENTS

1. Denige’s reagent: 5g of HgO in 20ml of conc.H2SO4 and diluted to 100ml with water.

2. K4Fe(CN)6 15% in water

3. ZnSO4 30% in water

4. NaOH 5% in water

5. NaNO2 2% freshly prepared in water

6. Dil H2SO4 ;100ml conc H2SO4 in 300ml water

PROCEDURE

To 2g of the sample 60ml water at 50

PROCEDURE

To 2g of the sample 60ml water at 50°C is added.The Ph of the contents is adjusted to 7.5 with and is heated at 50°C for 30 minutes with occasional stirrings.2ml of is added and mixed carefully.2ml of is mixed ,the volume made upto 100ml and then filtered.50ml of this filtrate and 1 ml are mixed and then extracted with 3×50ml portions of (C2H5)2O.The combined ethereal extracts are washed with water,a drop of phenolphthalein is added and then shaken with 3ml of 0.25M NaOH solution.After washing with 3 ml water,the alkaline extracts are combined,traces of ether,if any,are removed on a hot water bath and the volume is made upto 10ml.5ml of this solution and 5ml of are mixed and heated in a boiling water bath for 5 minutes,cooled,then 5 drops of are added and contents left aside for 45 minutes.A pink colour develops.The absorbance of this solution is measured at 518nm.

50,100,200,400 and 600mg of the ester under determination is dissolved separately in 3ml of 0.25M NaOH.The volume in each is made upto 5ml,the previously described procedure is followed.5ml of is added as usual before recording absorbance of the individual tube for preparation of the calibration graph.