·       Antioxidants Are Added To Oils And Fats To Prevent Oxidative Rancidity. Ethyl, Propyl, Octyl And Dodecyl Gallates, Butylated Hydroxyanisole (BHA), Butylated Hydroxy Toluene (BHT), Tertiary Butyl Hydroquinone (TBHQ) And Resin Guaic, Ascorbic Acid , Tocopheropl Are Permitted Under FSS, Rules And Regulation, 2011.

Qualitative Method:

·       Detection Of Propyl Gallate, BHA, BHT And Nordihydroquaiaretic Acid (NDGA) In Oils And Fats:

·       Reagents :

·       Barium Hydroxide (1%): Dissolve 1 Gm Of Ba (OH) 2,H2O In 100 Ml Distilled Acetonitrile (Methyl Cyanide) Solvent (CH3CN) Saturated With Petroleum Ether.

·       Ehrlich Reagent: Diazobenzene Sulfonic Acid (0.5%): Prepare 0.5% Solution Of Nano2 In Water And 0.5% Solution Of Sulfonilic Acid In Hcl (1+2). Prepare Nano2 Solution Fresh Every Three Weeks. Keep Solutions Refrigerated. Mix Nano2 And Sulfonilic Acid Solution (1+100)

·       Dianisidine Solution: Dissolve 250 Mg Of Dianisidine (3,3 Dimethoxy Benzidine) In 50 Ml Of Anhydrous Add 100 Mg Activated Charcoal, Shake For 5 Min. And Filter. Mix 40 Ml Filtrate With 60 Ml Of 1N Hcl. Prepare Daily And Protect From Light.

·       Activated Florisil Absorbent: 60-100 Mesh Activated At 260ºc Or 650ºc.

·       Test Florisil For BHT Retention As Follows: Add 0.2 Mg Of BHT In 25 Ml Petroleum Ether To Prepared Column, Elute With 150 Ml Petroleum Ether And Apply BHT Test After Evaporating The Solvent Just To Dryness. If BHT Is Not Eluted, Activate Remaining Florisil By Heating For 2 Hours At 650ºc, Cool And Add 6.5% Water By Weight And Homogenize By Shaking For 1 Hour In A Closed

·       Preparation Of Florisil Column For Cleanup Of BHT Extract: Insert Small Glass Wool Plug Into A Chromatographic Tube 20 (O.D.) × 250 Mm With Stopcock. Add 12 Gm Of Florisil With Gentle Tapping. Wash With Two 15 Ml Portions Petroleum Ether, Adding Second Portion When Liquid Level Is Just Above Top Of Florisil. Do Not Let The Column Dry.

Tests

·       Prophyl Gallate (PG): Weigh About 30 Gm Of Fat Or Oil, Dissolve In About 60 Ml Of Petroleum Ether And Transfer To 250 Ml Separator. Add 15 Ml Of Water And Shake Gently For 1 Min. Let Layers Separate And Drain Aqueous Phase Into 125 Ml Separator, Leaving Any Emulsion In Organic Repeat Extraction Of Petroleum Ether With Two Additional 15 Ml Portions Of Water And Reserve Organic Phase For Further Extraction With CH3CN. Add 15 Ml Petroleum Ether To Aqueous Extract And Shake For 1 Min. Discard Aqueous Phase And Evaporate The Solvent Just To Dryness In Small Beaker. Add 4 Ml Of 50% Alcohol To Residue, Swirl And Add 1 Ml NH4OH. It The Solution Colour Turns To Rose Colour, PG Is Present (Colour Is Unstable And Fades After Few Min.)

·       Nordihydroguaiaretic Acid (NDGA): Extract Petroleum Either Solution From By Shaking For 2 Min With 20 Ml CH3 Let Layers Separate And Drain CH3CN Into 1 Litre Separator. Repeat Extraction With 2 Additional 30 Ml Portions Of CH3CN And Discard Petroleum Ether Phase. Dilute Combined Acetonitrile Extracts With 400 Ml Water, Add 2- 3 Gm Nacl And Shake For 2 Min With 20 Ml Petroleum Ether. Let Layers Separate, Drain The Diluted CH3CN Layer Into Second 1000 Ml Separatory Funnel. Extract Dilute CH3CN Layer With Two Additional 20 Ml Portions Of Petroleum Ether And Reserve Dilute CH3CN Solution For Further Extraction. Combine Petroleum Ether Extracts In 100 Ml Beaker And Set Aside For BHA And BHT Tests. Add 50 Ml Ether + Petroleum Ether (1+1) To Diluted CH3CN From (B) And Shake For 2 Min. Let Layers Separate, Discard CH3CN And Evaporate The Solvent Just To Dryness In A Small Beaker. Add 4 Ml Of 50% Alcohol, Swirl And Then Add 1 Ml Of 1% Barium Hydroxide Solution And Mix. If NDGA Is Present The Solution Turns To Blue And Fades Rapidly.

·       Butylated Hydroxyanisole (BHA): Take 1/3 Of Combined Petroleum Ether Solution Reserved For BHA And BHT Tests And Evaporate Just To Dryness, Using Gentle Heat, Under Air Current. Add 2.5 Ml Of Alcohol To Dissolve Residue And Dilute With 2.5 Ml Water. Swirl, Add 1 Ml Of Ehrlich Reagent Followed By 1 Ml Of A 1N Naoh And Swirl Again. If Solution Turns Red Purple, BHA Is Present.

·       Butylated Hydroxytoluene (BHT): Pass Remaining 2/3 Combined Petroleum Ether Through Florisil Column And Elute With 150 Ml Petroleum Ether. Collect Eluate In 200 Ml Beaker And Evaporate Just To Dryness. Add 2.5 Ml Alcohol Swirl And Dilute To 5 Ml With Water And Mix. Add 2 Ml Of Dianisidine Solution And Mix. Add 0.8 Ml Of 0.3% Sodium Nitrite (Nano2) Solution, Mix And Let It Stand For 5 Min, Then Transfer To A Small Separator. Add 0.5 Ml Chloroform (Chcl3) Chcl3 Shake Vigorously For 30 And Let Layers Separate. If Chcl3 Layer Turns Pink To Red. BHT Is Present. Confirm BHT By Comparing Spectrophotometric Curve Of Coloured Chcl3 Extract Obtained From Reference Standard BHT By Dissolving Approximately 15 Mg In 5 Ml Aqueous Alcohol (1+1) And 2 Ml Dianisidine.

Thin Layer Chromatographic Detection Of Antioxidants

Principle

·       The Sample Oil Is Dissolved In Petroleum Ether And Extracted With Acetonitrile. Acetonitrile Extract Is Evaporated In Vacuum In A Rotary Evaporator At A Temperature Not Exceeding 40ºc. The Residue Is Dissolved In Alcohol, Applied To TLC Plates And After Development, Spots Are Visualized By Spraying With Gibb’s Reagent.

Apparatus

·       Separatory Funnel (250 Ml); Flask;

·       Rotary Evaporator;

·       TLC Equipment; Silica Gel G.

Reagents

·       Acetonitrile

·       Petroleum Ether

·       Developing Solvent: Petroleum Ether : Benzene : Acetic Acid

·       Spray Reagent: 2, 6-Dichloroquinone Chlorimide (Gibb’s Reagent); 1% In Alcohol

·       Standard Solution (0.1%): Dissolve Propyl Gallate (PG), Octyl Gallate (OG), Dodecyl Gallate (DG), Butylated Hydroxy Anisole (BHA) And Butylated Hydroxy Toluene (BHT) In Methanol.

·       Developing Solvent

·       A – Petroleum Ether -Benzene – Glacial Acetic Acid

·       B – Petroleum Ether -Benzene -Ethyl Acetate – Gl. Acetic Acid 

·       C – Chloroform- Methanol – Gl. Acetic Acid

Procedure

·       Dissolve 10 Gm Of Oil Or Melted Fat In 100 Ml Of Petroleum Ether And Transfer Into A 250 Ml Separatory Funnel. Add 25 Ml Acetonitrile Saturated With Petroleum Ether To The Separator And Shake Gently. Run Off The Acetonitrile Into A Second Separator And Repeat The Extraction Three Times. Transfer Acetonitrile Extracts To Rotary Evaporation Flask And Evaporate At Less Than 40ºc Temperature Just To Dryness. Dissolve The Residue In 2 Ml Methanol, Filter If Not Entirely Soluble Prepare 20 X 20 Cm Silica Gel G Plates With A 0.25 Mm Layer Using 30 Gm In A Slurry With 60 Ml 1% Citric Acid Solution. Dry The Plates In Air, Activate At 30°C For 1 Hour And Store In A Desiccator. Saturate The Developing Chamber With A Freshly Prepared Solvent Mixture. Line The Tank With Filter Paper, Allow To Stabilize For 1-2 Hrs In The Dark. Apply 10 To 20 µg Extract Solution Along With Standards (4 µl) 2 Cm Apart On A Start Line 2 Cm Above The Bottom Edge.

·       Develop The Plate To A Distance Of 15 Cm And Allow It To Air Dry. Spray With Gibb’s Reagent And Dry At 103 ± 2ºc For 15 Min. Compare The Colour And Rf Values With Standards. Cool The Plate And Place In A Tank Containing Ammonia And Note The Characteristic Colour Change As Indicated Below:

Antioxidant

R_f Value With Developing Solvents

Gibb’s Reagent

Gibb’s Reagent Followed By

A

B

C

Ammonia

PG

0.12

0.25

0.55

Brown

Gray–Green

OG

0.22

0.40

0.64

Brown

Gray–Green

DG

0.27

0.45

0.66

Brown

Gray–Green

BHA

0.62

0.87

0.92

Brown-Red

Gray

BHT

0.82

0.99

0.95

Brown– Violet

Gray

Quantitative Method:

Spectrophotometric Determination Of Propyl Gallate:

·       Principle: Oil Or Melted Fat Is Dissolved In Petroleum Ether And Extracted With Ammonium Acetate Solution And. Water. The Combined Extract Is Treated With Ferrous Tartarate And The Absorbance Of The Coloured Solution Is Read At 540 Nm. The Amount Of PG Present In The Sample Is Calculated From The Calibration Graph.

Reagent

·       Petroleum Ether Reagent: Mix One Volume Of 40-600 C 167C Pet. Ether With 3 Volumes Of 80-100ºc Petroleum Ether. Shake For 5 Min With 1/10th Of Its Volume Of H2SO4. Discard Acid Layers, Wash Several Times With Water, Then Again With Water Until Washings Are Subsequently Neutral. Discard All Washings And Distill Petroleum Ether In All Glass Apparatus.

·       Ammonium Acetate Solutions: 1.25%, 1.67% And 10% Aqueous Solutions. Solution Containing 1.67% NH4OAC In 5 % Alcohol May Also Be Required.

·       Ferrous Tartarate Reagent: Dissolve 0.1 Gm Of Feso4.7H2O And 0.5 Gm Of Rochelle Salt (Nakc4h4o6.4H2O) In Water And Dilute To 100 Ml. Reagent Must Be Used Within Three Hours Of Preparation.

·       Propyl Gallate Standard Solution: 50 µg/Ml. Dissolve 50 Mg In Water And Dilute To 1000 Ml With Water.

·       Preparation Of Standard Curve: Place 7 Aliquots Of Standard Solution Ranging From 50 To 1000 µg In 50 Ml Erlenmeyer Flasks. Add Exactly 2.5 Ml Of 10% NH4OAC To Each Flask, Dilute Exactly To 24 Ml With Water And Pipette 1 Ml Of Ferrous Tartarate Solution Into Each Flask. Let The Solution Stand For 3 Min. Measure The Absorbance At 540 Nm Against Solution Containing 20 Ml Of 1.25% Ammonium Acetate Solution, 4 Ml Water And 1 Ml Ferrous Tartarate Solution. Plot The Calibration Curve.

Procedure

·       Dissolve 40 Gm Of Fat Or Oil In The Petroleum Ether Reagent And Dilute To 250 Ml With Reagent (Gentle Warming May Be Necessary To Obtain Complete Solution). Pipette 100 Ml Of Fat Solution Into 250 Ml Separator. Extract With 20 Ml Of Aqueous 1.67 % Ammonium Acetate Solution By Gentle Shaking For 2.5 Min. Allow The Layers To Separate And Drain Aqueous Layer Into 100 Ml Volumetric Flask (Some Shortenings Show Strong Tendency To Emulsify During Aqueous Extraction). To Prevent Emulsification, Add 2 Ml N-Octanol To Fat Solution Before Beginning Extraction And Use 1.67 % Ammonium Acetate Solution In 5% Alcohol For Extraction In Place Of Aqueous Solution. This Procedure Is Used Only When Usual Method Fails. Repeat Extraction Twice With 20 Ml Portion Of Ammonium Acetate Solution And Collect In Volumetric Flask. 

·       Finally, Extract Fat Solution With 15 Ml Water For 30 Sec. And Combine Aqueous Layer. Add Exactly 2.5 Ml Of 10% NH4OAC Solution To Combined Extract And Dilute To Volume With Water. Filter Through Filter Paper (No.4) To Remove Any Turbidity And Develop Colour The Same Day The Extract Is Prepared.

·       Pipette Aliquot Of Extract (About 20 Ml) Into 50 Ml Er1enmeyer Flask. Dilute To 20 Ml With 1.25% Ammonium Acetate Solution. Add Exactly 4 Ml Water And Pipette 1 Ml Ferrous Tartarate Solution. Mix Well And Measure Absorbance At 540 Nm Against A Solution Containing 1.25% NH4OAC Solution, 4 Ml Water And 1 Ml Ferrous Tartarate Solution. Calculate The Amount Of Propyl Gallate From The Calibration Curve.

Spectrophotometric Determination Of BHA:

Principle

·       BHA Is Extracted From Oil Or Melted Fat Sample With 95 % Methanol. The Extract Gives Colour With Gibb’s Reagent Which Has Maximum Absorption At 610 Nm.

Reagents

·       95% (V/V) Methanol

·       Sodium Tetraborate Decahydrate: 0.5%

·       2, 6-Dichloroquinone Chlorimide (Gibb’s Reagent): 01% In 95% Methanol.

·       N-Butanol

·       BHA-Standard: Prepare 25 Mg/L In 95% Methanol.

Procedure

·       Vigorously Shake 10 Gm Of Warm Liquid Sample Or Melted Fat With 25 Ml Of 95% Methanol For One Minute In A Centrifuge Tube. Place In A Water Bath At 40-50ºc And Allow To Separate For About 15 Min. Pour The Upper Layer Into A 50 Ml Calibrated Flask, Repeat The Extraction With 20 Ml Of 95% Methanol, Transfer The Upper Layer To The Flask And Dilute To Mark. Add One Gram Of Calcium Carbonate, Shake And Filter Through A Paper (Whatman No.1 Or Equivalent) Rejecting The First Few Ml Of Filtrate. The Amount Of Calcium Carbonate Is Not Critical But Must Be Enough To Ensure A Clear Filtrate. To Exactly 2 Ml Of This Extract Add 2 Ml Of 95% Methanol, 8 Ml Of Borax Solution And 2 Ml Of Gibb’s Reagent. After 15 Min. Dilute Exactly To 20 Ml With N- Butanol. Prepare The Blank And Standard Using The 25 Mg/L BHA Solution.

Spectrophotometric Determination Of BHT:

Principle

·       The Sample Is Steam-Distilled And BHT In The Steam Distillate Is Determined By The Colour Reaction With Q-Anisidine And Sodium Nitrite.

Apparatus

·       Distillation And Volumetric Flasks:

·       Separatory Funnels 60 Ml Capacity;

·       Steam Distillation Apparatus;

·       Oil Bath At 160ºc;

·       Steam

Reagents

·       Chloroform

·       Magnesium Chloride Solution: Dissolve 100 Gm Of Magnesium Chloride Hexahydrate In 50 Ml Water.

·       O-Dianisidine Solution: Dissolve 0.25 Gm In 50 Ml Methanol, Add 100 Gm Of Activated Charcoal, Shake For 5 Min And Filter. Mix 40 Ml Of This Clear Solution With 60 Ml Of 1 N Prepare Daily And Protect From Light.

·       Sodium Nitrite: Prepare 3% Solution In Water.

·       Standard Solution Of BHT: Dissolve 50 Gm In Methanol And Dilute To 100 Ml With Prepare Working Standards Containing 1-5 µg/Ml By Diluting With 50% (V/V) Methanol.

Procedure

·       Add 15 Ml Of Magnesium Chloride Solution And 5 Gm Of Fat To The Distillation Flask. Preheat The Bath For The Distillation Flask To 160±10⁰C. Adjust The Steam Generator To Distill About 4 Ml Water Per Minute. Connect Condenser And Steam- Generator To The Distillation Flask And Immediately Immerse The Later In The Oil Bath. Steam Distillation Must Be Vigorous. Collect The First 100 Ml Of The Distillate In A 200 Ml Volumetric Flask Containing 50 Ml Methanol. Disconnect The Distillation Flask From The Steam Generator And Remove It From The Oil Bath. Wash The Condenser With 5 Ml Portions Of Methanol Adding Washing To Volumetric Flask. Cool To Room Temperature And Adjust The Volume To 200 Ml With Methanol And Mix.

·       Clean And Dry Three 60 Ml Separating Funnels Of Low Actinic Glass Or Painted Black. To The First One Add 25 Ml Of 50% Methanol, To The Second One Add 25 Ml Of 1-3 µg/Ml Standard And To The Third One Add 25 Ml Of Distillate. To Each Funnel Add 5 Ml Of O-Dianisidine Solution, Stopper The Funnel And Carefully Mix The Contents. Then To Each Funnel Add 2 Ml Of 0.3% Sodium Nitrite Solution, Stopper The Funnels And Thoroughly Mix The Contents. Let Them Stand For 10 Min. Then Add 10 Ml Of Chloroform To Each Funnel. Extract The Coloured Complex By Vigorously Shaking Funnels For 30 Sec. Let The Layers Separate.

Antioxidants By High Performance Liquid Chromatography

·       (Applicable To Propyl Gallate (PG), Trihydroxybutyrophenone ( THBP) Ter– Butyl Hydroquinone (TBHQ), Nor Dihydroguaritic Acid (NDGA), Butylated Hydroxyl Anisole (BHA), Butylated Hydroxyl Toluene(BHT) At 20- 200 Ug / Gm In Oils And Fats 10-100 Ug /Gm In Butteroil And To Octyl And Dodecyl Gallate At 10 – 100 Ug / Gm In Butter Oil)

Principle

·       Antioxidants Are Extracted Into Acetonitrile Extract Is Concentrated And Diluted With 2 Propanol. Antioxidants Are Separated By Liquid Chromatograph And Measured By Uv Detection At 280 Nm.

Apparatus

·       Liquid Chromatograph (LC) – Equipped With 10 Mv Recorder Or Integrator To Electronically Measure Peak Heights, 10 Ul Sample Loop Injection Valve And Detector To Measure Absorbance At 280 Nm. Typical Operating Conditions – Detector Sensitivity 0.05AUFS, Temp Ambient , Flow Rate 2ml / Min

·       LC Column – Packed With C 18 Bonded Spherical( Preferred) Silica Or Equivalent

·       Glass Ware – Rinse All Glass Ware With Chloroform, Acetone And Methanol Successively And Blow Dry With Nitrogen.

Reagents

·       Solvents – Acetonitrile, 2 Propanol, Hexane ( HPLC Grade)

·       Mobile Phase – (1) 5 % Acetic Acid In Water (LC Grade),

·       Acetonitrile – Methanol (1+ 1, V /V ) LC Grade

·       Run Linear Gradient From 30 % (1) To 100 % (2) Over 10 Minutes With Hold Until Last Antioxidant (DG) Is Eluted. For Test Solutions Only Increase Flow Rate To 4 Ml/ Min At 100 % (2) Over 6 Minutes Or Until Non Polar Liquids Are Eluted. For Test Solutions And Standards Return To 30 % (2) In (10 Over1 Min At 2 Ml / Min And Let Baseline And Pressure Stabilize. Reduce Flow Rate And Proportionally Increase Rinsing And Equilibrium Times If Excessive Back Pressure Results. Run Blank Solvent Gradient (No Injection) To Ensure That No Peaks Interfering With Any Antioxidant Are Present. To Remove Or Reduce Peaks Arising From Elution Solvent (1) Replace In Let Filter With Pre Rinsed Solid – Phase C 18 Extraction Cartridge And Use In Line Filter. If Small Interfering Peaks Are Not Eliminated, Substract Peak Height Of Gradient Interference From That Of Relevant Standard Or Test Solution

Standard Solutions

·       Prepare In 2 Propanol – Acetonitrile (1 + 1 , V/V). (Caution – TBHQ Is Readily Oxidized Especially In Light. Refrigerate All Antioxidant Solutions And Store Out Of Direct Light). Monitor TBHQ Response Relative To PG Or THBP And Prepare Fresh Standards If Response Decreases More Than 5 %

·       Stock Standard Solution – 1 Mg / Ml Accurately Weigh 50 Mg To Nearest 0.1 Mg Each Antioxidant And Transfer To Single 50 Ml Volumetricflask. Dissolve Dilute To Volume And

·       Working Standard Solution – 0.01 Mg / Ml . Pipette 1 Ml Of Stock Standard Solution Into 100 Ml Volumetric Flask, Dilute To Volume And Mix.

·       Extraction Solvents – (1) Saturated Hexane – Saturate About 300 Ml In Separatory Funnel By Adding Acetonitrile Until Two Layers Remain After Shaking 2 Mins. Discard Acetonitrile Lower

·       Saturated Acetonitrile = Saturate About 300 Ml Acetonitrile In Separatory Funnel By Adding Hexane Until Two Layers Remain After Shaking 2 Mins

·       Determination

Extraction

·       Accurately Weigh To The Nearest 0.01 Gm, 50 Ml Beaker Containing About 5-6 Gm Liquid Or Butter Oil Or 3 Gm Lard Or Shortening ( Liquefied In Water Bath At 60⁰C And Swirled Or Shaken To Ensure Homogeneity). Decant As Much Test Portion As Possible Into 125 Ml Separatory Funnel Containing 20 Ml Aturated Hexane. Reweigh Beaker To Determine Test Portion Weight, Mix Test Portion With Hexane And Extract With Three 50 Ml Portions Of Saturated Acetonitrile. If Emulsion Forms Hold Separatory Funnel Under Hot Tap Water For 5-10 Seconds. Collect Extracts In 250 Ml Separatory Funnel And Let Combined Extracts Slowly Drain Into 250 Ml Round Bottom Flask To Aid Removal Of Hexane – Oil Droplets.

Chromatography

·       Using Sample Loop Injection Valve, Inject 10 µl Sample Extract And Eluate With Solvent Gradient Programme With Mobile Phase 1 And 2. Before And After Every 3-4 Test Injections Or More Frequently If Differences Between Standard Peak Heights Are Found To Be More Than 5 %, Inject 10 µl Of Antioxidant Working Standard Solution( 10 µl / Ml ) And Elute With Solvent Gradient Programme For Standards. For Analyte Peaks Off Scale Or More Than 3 X Standard, Quantitatively Dilute Test Extract With 2 Propanol – Acetonitrile (1+1) And Re Inject. Identify Peaks By Comparison With Retention Times Of Standard.