Qualitative Test:

Principle

·       Volatile Acids Such As Acetic, Propionic, Butyric And Valeric Acids Are Steam Distilled And The Distillate Is Concentrated After Neutralization. Separation Of These Acids Is Achieved By Paper Chromatography And Visualized By Spraying With Methyl Red And Bromothymol Blue.

Reagents

·       Developing Solvent- Acetone: Tert-Butanol: N-Butanol: Ammonia (2+1+1+1) Prepare Fresh

·       Spray Reagents: Add 200 Mgs Of Each Of Methyl Red And Bromothymol Blue To A Mixture Of 100 Ml Of Formalin And 400 Ml Of Alcohol. Adjust Ph To 5.2 With 0.1n Naoh.

·       Standard Acid Solution: Pipette 1 Ml Each Of Acetic, Propionic, Butyric And Valeric Acids Into 100 Ml Volumetric Flasks Separately And Dilute To Volume With Water. Pipette 1 Ml Of Each Stock Solution Into 25 Ml Beakers And 1 Ml Each Into Another Beaker (Mixture), Neutralise With 0.1 N Naoh Using Cresol Red Indicator And Evaporate To Dryness Without Charring. Dissolve In 0.5 Ml Water. Use These Solutions For Chromatography.

Procedure

·       Steam-Distill 20 Gm Of Well Mixed Sample And Collect 200 Ml Distillate. Immediately Neutralise The Distillate With 0.1n Naoh Using Cresol Red Indicator And Evaporate Just To Dryness And Dissolve In 0.5 Ml Water. Spot 1-2 µl Along With Standards On Whatman No.1 Paper And Allow Them To Air Dry. Clip Paper To Glass Rod And Suspend In A Tank With 50 Ml Mobile Phase In A Trough (Since Mobile Phase Is Heavy, Use 3 Clips To Hold The Paper To Glass Rod To Prevent Sagging).

·       Develop The Chromatogram Approximately To 2.5 Cm From Top Of The Paper, Remove And Let Air Dry. Spray With The Spray Reagent (Spray Should Be Uniform). Faint Yellow Spots Indicate Presence Of Acids, Heavier Blue Spots Are Due To Sodium Ion. To Intensify Spots, Expose Paper To Ammonia Fumes. Entire Paper Immediately Turns To Green And Acids Gradually Appear As Red Spots. Since Colour Of Acids Is Not Stable, Mark Spot With Pencil As Soon As They Are Completely Developed. Identify Them In Sample From The Rf Values Of Standard Acids.

An Alternate Gas Chromatographic Method For Analysis Of Acetic Acid And Propionic Acid Can Also Be Followed:

Principle

·       The Volatile Organic Acids Such As Acetic Acid, Propionic Acid, Butyric Acid And Valeric Acid Are Steam Distilled And The Distillate Is Used For Gc Analysis. The Results Can Be Confirmed After The Analysis Of Their Respective Standards.

Chemicals

·       Standard Acetic Acid, Propionic Acid, Butyric Acid, Valeric Acid – Ar Grade Deionized Water

Procedure

·       Steam-Distill 20 Gm Of Well Mixed Sample And Collect 200 Ml Distillate. Distillate Can Either Be Directly Used For On-Column Injections Or Can Be Dissolved In Any Appropriate Solvents.

Gc – Method

·       Column: Ffap Column 30 M Length X 53 Mm Id X 0.5 µ Film Thickness

·       Carrier Gas: Nitrogen/Argon

·       Detector: Fid/Ms

·       Injection Temperature: 250˚c

·       Oven Temperature: Initial 80˚c – Final 200˚c

·       Detector Temperature: 250-300˚c

·       Injection Volume: 1-2 µl

Reference

·       Beihoffer And C. Ferguson, J. Chromatogr. Sci., 1994, 32, 102-106.

·       Aoac Official Method 950.35 [Ref. Jaoac 33, 677(1950); 34, 284(1951); 36, 769(1953)] Acetic And Propionic Acids In Bread/Cakes -Chromatographic Method May Also Be Referred As Alternative Method.