QUANTITATIVE METHODS​

Titrimetric Method:

  • Principle: Benzoic acid is separated from a known quantity of the sample by saturating with NaCl and then acidifying with dilute HCl and extracting with chloroform. The chloroform layer is made mineral acid free and the solvent is removed by evaporation. The residue is dissolved in neutral alcohol and the amount of benzoic acid is determined by titration against standard alkali.
  • Reagents:
    • Chloroform-distilled
    • HCL (1+3)
    • Sodium hydroxide (10%)
    • Standard NaOH solution (0.05N)

Preparation of Sample:

  • Beverages and liquid products: Mix the sample thoroughly and transfer 100 gm of the sample into a 250 volumetric flask, using saturated NaCl solution. Make alkaline to litmus paper with 10% NaOH solution and make upto volume with saturated NaCl solution. Shake thoroughly and let it stand for 2 hours. Filter the sample and use the filtrate for determination.

  • Sauces and Ketchups: Add 15 gm salt to 150 gm of weighed sample and transfer into volumetric flask. Rinse with saturated NaCl solution, Add 15 gm pulverized NaCl and then add 10 ml of 10% NaOH solution and make upto volume with NaCl solution. Let it stand for 2 hrs. with occasional shaking. Filter and use the filtrate for determination.

  • Jams, Jellies, Preservatives and Marmalades: Mix 150 gm of sample with 300 ml saturated NaCl solution. Add 15 gm pulverised NaCl. Add 10 ml of 10% NaOH solution. Transfer to 500 ml volumetric flask and dilute to volume with saturated NaCl solution. Let it stand for 2 hrs with frequent shaking, filter and use the filtrate for determination.

    • Determination: Pipette 100 ml to 200 ml of the filtrate into a 250 ml separatory funnel. Neutralize to litmus paper using HCI (1+3) and add 5 ml excess. Extract carefully with 40, 30, 30 and 20 ml portions of chloroform. Avoid formation of emulsion by shaking gently with rotatory motion. If emulsion forms, break it by stirring CHCl3 solution with a glass rod after each extraction, but do not drain any of the emulsion with chloroform layer. Transfer the combined chloroform extract in to a separatory funnel and wash it free from mineral acid by shaking gently and rinsing with water. Drain off the water phase. Dry the chloroform layer over anhydrous sodium sulphate and distil off the solvent. Remove the last traces of the solvent under a current of air at room temperature. Dry the residue overnight or until no residue of acetic acid is detected if the product is a ketchup. Dissolve residue in 30-50 ml of alcohol neutralised to phenolphthalein and titrate with 0.05 N NaOH

Spectrophotometric method:

  • Principle: Benzoic acid is extracted from prepared sample using diethyl ether and the absorbance of the ether layer is measured at 272 nm, 267.5 nm and 276.5 nm in the UV region. From the corrected absorbance and the calibration graph obtained using standard benzoic’ acid solution, the amount of benzoic acid is determined.

  • Reagents
    • Diethyl ether distilled
    • HCl (1+3)
    • Saturated sodium chloride solution
    • Ammonium hydroxide (0.1%)
    • Standard benzoic
  • Procedure : Prepare solution of benzoic acid in ether containing 20, 40, 60, 80,100 and 120 mg/l. Determine absorbance of these solutions in a spectrophotometer at points B, C and D. For each concentration average absorbance at Band D subtract from absorbance at C. Preparation of standard curve: Prepare solution of benzoic acid in ether containing 50 mgs/l. Determine absorbance of this solution in tightly stoppered cell in Beckman DU or recording spectrophotometer between 265 and 280 nm at 1 nm intervals. Plot absorbance against wavelength and record wavelength of minimum at approximately 5 nm as point B. Other minimum at approximately 276.5 nm as point D and highest maximum at approximately 272 nm as point C.  Preparation of sample: Mix sample thoroughly. Transfer 10 gm or 10 ml to separator and dilute to 200 ml with saturated NaCl solution. Make solution definitely acidic to litmus with HCI and mix well. Determination: Extract prepared solutions with 70, 50, 40, and 30 ml portions of diethyl ether, shaking well to ensure complete extraction (break emulsions by standing, stirring or centrifuging). Drain and discard aqueous phase. Wash combined ether extracts with 40 and 30 ml portions HCl (1+1000) and discard HCl washings (if extraction requires no purification,. proceed to next para). Extract ether solution with 50, 40, 30, and 20 ml portions of 0.1% ammonium hydroxide and discard ether. Neutralize combined ammonium hydroxide extracts with HCI and add 1 ml excess. Extract the acidified solution with 70, 50, 40 and 30 ml ether.

HPLC Method:

  • Principle: Benzoic acid is extracted and separated by liquid chromatography (LC) on C18 column, detected by ultra violet absorbance at 230 nm, and quantitated by standard calibration plot.

  • Apparatus :
    • Liquid chromatograph equipped with pump, injector, and integrator or data system, and UV Operating conditions: flow rate, 1.0 mL/min isocratic; column temperature, ambient; detector, 230 nm, 0.05 absorbance unit full scale (AUFS); and injection volume, 20 µL.
    • LC – C18, 4.6 X250mm length, 5µm.
    • Ultrasonic
  • Reagents :
    • Solvents.—Acetonitrile and water (LC grade).
    • Sodium Benzoate Standard
    • Potassium phosphate monobasic buffer. — Prepare 0.05M potassium dihydrogen orthophosphate; adjust the pH 3.0 with ortho phosphoric acid
    • LC mobile —Acetonitrile–phosphate buffer (40 + 60). Combine 400 mL acetonitrile with 600 mL 0.05M potassium dihydrogen orthophosphate. De-gas in ultrasonic bath for 2 min and filter through 0.45µ polyvinylidene fluoride filter.
  • Procedure

    • Standard preparation: Weigh accurately 25.0 mg of sodium benzoate std & transfer it into 100 ml volumetric flask. Dissolve it in water by sonication & make upto the volume. This corresponds to 250 ppm of sodium benzoate. Dilute 1, 2, 4, 6, 8 and 10 ml of this standard solution to 50 ml with buffer, this corresponds to 5, 10, 20, 30, 40 and 50 ppm of sodium benzoate respectively. Filter these standards and inject. Plot a graph with concentration (ppm) against area and calculate the slope.

    • Preparation of Test Solution: Weigh accurately 25.0 mg of sample & transfer it into 100 ml volumetric flask. Dissolve it in water by sonication & make upto the volume. Dilute 5 ml of this solution to 50 ml with buffer.(In case of thick samples weigh the sample in beaker dissolve using sonicator and then transfer to 100ml volumetric flask).Filter the extract through 0.45µ syringe filter. Use this filtered solution for the HPLC analysis. Inject the sample 20µl in HPLC.

    • Chromatographic Condition:

      • Detector  : UV – Visible

         

      • Wavelength  :   230nm

         

      • Flow rate : 1 ml/min

         

      • Mobile Phase  : Acetonitrile: Buffer (40:60)

         

      • Injection volume : 20 uL

         

      • Diluent  : Water

      • Column temperature  :  Ambient

      • Run time : 20 min

         

      • Retention time : 5 – 6 min

    • Calculation: Inject separately 20 mL of standard solution, record the chromatograms. Develop calibration plot for standards and plot the regression equation for standard benzoate solution. Inject sample solution record the chromatograms and measure the peak responses and calculate the quantity of sodium benzoate.