Unit 5: Antioxidant Activity - Subjective Questions

Antioxidants are substances that inhibit or delay the oxidation of other molecules by neutralizing free radicals or reactive oxygen species.

• Free radicals contain an unpaired electron and can damage lipids, proteins, and nucleic acids.
• Antioxidants donate an electron or hydrogen atom to stabilize these reactive molecules.
• Important dietary antioxidants include vitamin C, vitamin E, carotenoids, flavonoids, and phenolic compounds.
• In food, antioxidants prevent rancidity, color changes, nutrient loss, and deterioration of flavor.
• In biological systems, they help protect cells from oxidative stress and may reduce the risk of chronic diseases.

The DPPH assay is based on the reduction of the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by antioxidant molecules.

• DPPH has a deep violet color and shows strong absorbance near .
• An antioxidant donates a hydrogen atom or electron to DPPH.
• The radical is converted into the non-radical form, diphenylpicrylhydrazine.
• This reaction causes the violet color to fade.
• The decrease in absorbance is proportional to the radical-scavenging activity of the sample.

The percentage inhibition is calculated as:

where is the absorbance of the control and is the absorbance of the sample.

A general spectrophotometric procedure includes the following steps:

1. Sample preparation: Weigh a representative quantity of the food sample and extract its antioxidant compounds using a suitable solvent such as methanol, ethanol, or aqueous alcohol.
2. Filtration or centrifugation: Remove insoluble particles from the extract.
3. Preparation of reagents: Prepare the radical or oxidant reagent and a suitable blank and control.
4. Reaction: Mix a measured volume of the sample extract with the reagent and incubate for the specified time.
5. Measurement: Measure absorbance at the wavelength appropriate for the assay, such as for DPPH.
6. Calculation: Compare sample absorbance with the control absorbance and calculate antioxidant activity.
7. Reporting: Express the result as percentage inhibition, equivalent concentration of a standard, or antioxidant capacity per gram of sample.

All measurements should be performed using replicates to improve reliability.

Blank: A blank contains the solvent and reagents but no food extract. It is used to correct for the absorbance contributed by the reagents or solvent.

Control: A control contains the antioxidant reagent and solvent but no test sample. It represents the maximum absorbance of the reacting radical or oxidant before antioxidant action.

Their importance includes:

• Correcting background absorbance.
• Establishing the initial reaction intensity.
• Allowing accurate calculation of percentage inhibition.
• Detecting contamination or reagent deterioration.
• Improving the reliability and comparability of results.

Without an appropriate blank and control, the measured antioxidant activity may be artificially high or low.

Let represent the absorbance of the control and represent the absorbance of the sample.

The decrease in absorbance caused by the antioxidant is:

The fraction of radical scavenged is obtained by dividing this decrease by the control absorbance:

Multiplying by gives the percentage radical-scavenging activity:

If a blank correction is required, corrected absorbances are used:

where is the blank absorbance.

Several experimental and sample-related factors influence the measured antioxidant activity:

• Solvent polarity: Different solvents extract different classes of antioxidants.
• Extraction time and temperature: Excessive heating or prolonged extraction may destroy sensitive compounds.
• Sample particle size: Finer particles generally improve contact between the sample and solvent.
• pH: The ionization state and reactivity of antioxidants can change with pH.
• Reaction time: Incomplete reaction gives low values, whereas excessive reaction time may produce misleading results.
• Reagent concentration: Incorrect reagent concentration affects absorbance and sensitivity.
• Light and oxygen exposure: These may degrade antioxidants or reagents.
• Food composition: Pigments, sugars, proteins, and turbidity can interfere with absorbance measurements.
• Temperature: Reaction rates and antioxidant stability are temperature-dependent.

Therefore, extraction and assay conditions must be standardized.

A standard curve relates the response of the assay to known concentrations of a reference antioxidant, commonly ascorbic acid or Trolox.

1. Prepare a series of known standard concentrations.
2. React each standard with the antioxidant reagent under identical conditions.
3. Measure the absorbance or percentage inhibition for each concentration.
4. Plot the response against standard concentration.
5. Obtain the regression equation, commonly written as:

where is the measured response, is the slope, is the standard concentration, and is the intercept.

6. Substitute the sample response into the equation to determine its equivalent antioxidant concentration.
7. Correct for dilution and sample mass.

Results may be reported as milligrams of Trolox equivalents per gram of sample, written as .

Percentage inhibition indicates the proportion of radicals neutralized by a particular concentration of sample.

is the concentration of sample required to inhibit of the initial radical activity.

• Percentage inhibition is simple and useful for screening at a fixed concentration.
• requires testing several concentrations and constructing a dose-response curve.
• A lower indicates stronger antioxidant activity because less sample is needed to achieve inhibition.
• Percentage inhibition values cannot always be compared if different sample concentrations are used.
• is more useful for comparing samples tested under identical conditions.

Both measures depend on the assay system, reaction time, solvent, and experimental conditions.

Important precautions include:

• Use clean, dry, and scratch-free cuvettes.
• Select a wavelength appropriate for the assay.
• Prepare fresh reagents when required, especially light-sensitive radical solutions.
• Protect DPPH and other sensitive reagents from strong light.
• Use accurately calibrated pipettes and balances.
• Maintain identical reaction times and temperatures for standards and samples.
• Mix samples uniformly before measurement.
• Remove turbidity by filtration or centrifugation.
• Use appropriate blanks and controls.
• Perform measurements in duplicate or triplicate.
• Avoid bubbles in the cuvette because they scatter light.
• Record absorbance only after the instrument has been properly zeroed.

These precautions reduce analytical error and improve reproducibility.

A single assay cannot represent all antioxidant mechanisms because antioxidants act through different pathways.

• Some assays measure hydrogen atom transfer, whereas others measure electron transfer.
• Antioxidants may react differently with different radicals.
• Solvent and pH conditions may favor certain compounds.
• Colored food extracts can interfere with absorbance readings.
• The assay may not reflect antioxidant behavior in the human body.
• Antioxidants that react slowly may appear weak if the reaction time is short.
• Some compounds may interfere with the reagent without actually functioning as biologically important antioxidants.

For more reliable evaluation, different methods can be combined, such as DPPH, ABTS, ferric reducing antioxidant power, and phosphomolybdenum assays.

Thin-layer chromatography (TLC) is a separation technique used to separate and identify components of a mixture.

Its principle is based on the differential distribution of compounds between:

• A stationary phase, usually a thin layer of silica gel or alumina coated on a plate.
• A mobile phase, which is a solvent or mixture of solvents moving through the stationary phase by capillary action.

Components that interact strongly with the stationary phase move slowly, while components more soluble in the mobile phase move faster. Separation depends on polarity, adsorption, solubility, and the chemical nature of the analytes.

TLC is rapid, inexpensive, requires a small sample volume, and can be used to examine food pigments, phenolics, and other antioxidant compounds.

The general TLC procedure is as follows:

1. Select a silica gel TLC plate and handle it only by the edges.
2. Draw a light baseline approximately to from the lower edge using a pencil.
3. Apply small spots of the food extract and reference standards on the baseline using a capillary tube.
4. Allow the spots to dry.
5. Place the plate in a developing chamber containing a suitable solvent system. The solvent level must remain below the baseline.
6. Close the chamber to allow solvent vapor saturation.
7. Allow the solvent to rise by capillary action.
8. Remove the plate when the solvent front is near the top and immediately mark the solvent front.
9. Dry the plate.
10. Visualize separated spots using visible light, ultraviolet light, iodine vapor, or a suitable spraying reagent.
11. Measure the distance traveled by each spot and calculate its value.

The pattern and values are compared with standards for tentative identification.

The retardation factor, or , expresses the relative movement of a compound on a TLC plate.

It is calculated as:

Both distances are measured from the baseline. Since the solute spot cannot travel farther than the solvent front, the value normally lies between and .

For example, if a compound travels and the solvent front travels :

Uses of include:

• Comparing an unknown compound with a standard.
• Assessing the number of components in a mixture.
• Monitoring purification or chemical reactions.
• Comparing samples under identical TLC conditions.

The value depends on the stationary phase, solvent system, temperature, humidity, and sample loading.

In normal-phase TLC, silica gel is polar and acts as the stationary phase.

• A polar compound forms stronger interactions with silica through hydrogen bonding, dipole interactions, or adsorption. It therefore moves slowly and has a lower value.
• A less polar compound interacts less strongly with silica and is carried farther by the mobile phase. It generally has a higher value.
• Increasing the polarity of the mobile phase usually allows compounds to move farther because the solvent competes more effectively with the analytes for adsorption sites on silica.
• If the solvent is too nonpolar, spots may remain near the baseline.
• If the solvent is too polar, most spots may move with the solvent front, producing poor separation.

An appropriate solvent mixture provides distinct, compact, and well-separated spots.

Adsorption chromatography separates compounds according to their degree of adsorption onto a solid stationary phase.

• The stationary phase is usually silica gel or alumina.
• Strongly adsorbed compounds move slowly.
• Separation depends on polarity and surface interactions.

Partition chromatography separates compounds according to their distribution between two phases.

• One phase is stationary and the other is mobile.
• Compounds distribute between the phases according to their relative solubilities.
• Separation depends on partition coefficients.

Most commonly used silica gel TLC is primarily an adsorption process, although partition effects may also contribute when the stationary layer contains bound moisture or a modified liquid phase.

TLC spots may be visualized by physical or chemical methods:

• Visible light: Naturally colored compounds such as carotenoids and chlorophylls may be observed directly.
• Ultraviolet light: Fluorescent indicators in the plate allow UV-absorbing compounds to appear as dark spots.
• Iodine vapor: Many organic compounds form temporary colored complexes with iodine.
• Spraying reagents: Specific reagents react with functional groups to produce colored spots.
• Antioxidant-specific reagents: DPPH spray can be used to identify radical-scavenging compounds. Antioxidant spots may appear as yellow or pale zones against a purple background.
• Ferric chloride reagent: Phenolic compounds may produce colored complexes.

The plate should be handled carefully, and visualization should be performed promptly when spots are unstable or colors fade.

TLC can provide a qualitative profile of antioxidant constituents in a food extract.

1. Extract the food sample using an appropriate solvent.
2. Apply the extract and antioxidant standards to a silica gel plate.
3. Develop the plate using a suitable solvent system.
4. Visualize the separated compounds under UV light or with a reagent such as DPPH.
5. Locate spots that decolorize the DPPH background or produce characteristic color reactions.
6. Calculate the values of the active spots.
7. Compare the spot positions and colors with known standards.

A single extract may produce several spots, indicating the presence of multiple antioxidant compounds. TLC is mainly qualitative; confirmation of identity generally requires additional techniques such as HPLC or mass spectrometry.

Poor TLC results may result from several causes:

• Overloading: Applying too much sample produces broad or streaked spots.
• Large application spots: Large spots reduce resolution.
• Wet baseline: Immersion of the sample spot in the solvent causes sample loss.
• Improper chamber saturation: Uneven solvent vapor conditions affect migration.
• Unsuitable solvent system: Poor solvent strength causes overlapping or stationary spots.
• Damaged or contaminated plates: Irregular surfaces interfere with solvent movement.
• Uneven solvent level: This produces nonuniform development.
• Tilting the plate: The solvent front becomes irregular.
• Failure to mark the solvent front: Solvent evaporation leads to inaccurate measurements.
• Incomplete drying: Residual solvent causes spreading during development.

Using small concentrated spots, clean equipment, and a properly prepared chamber improves separation.

Spectrophotometric analysis and TLC provide different types of information.

Spectrophotometric analysis:

• Measures the overall antioxidant response of an extract.
• Usually gives quantitative or semi-quantitative results.
• Requires a spectrophotometer and suitable reagents.
• Does not necessarily distinguish individual antioxidant compounds.
• Results may be expressed as percentage inhibition or standard equivalents.

TLC analysis:

• Separates individual components before visualization.
• Gives a qualitative fingerprint of the sample.
• Requires a TLC plate, solvent chamber, and visualization method.
• Can indicate which separated spots possess antioxidant activity.
• Provides values but generally not accurate total concentrations.

The methods are complementary: spectrophotometry estimates total activity, whereas TLC reveals the number and relative movement of active constituents.

Laboratory safety is essential during both experiments.

• Wear a laboratory coat, gloves, and protective eyewear.
• Handle organic solvents in a fume hood because many are volatile, flammable, or toxic.
• Keep solvents away from flames and ignition sources.
• Avoid direct contact with DPPH, iodine, staining reagents, and corrosive chemicals.
• Never pipette by mouth.
• Label all extracts, reagents, and waste containers clearly.
• Dispose of organic solvent waste in designated containers rather than in the sink.
• Dispose of contaminated TLC plates, capillaries, and gloves as chemical laboratory waste.
• Clean spills according to institutional procedures.
• Wash hands after completing the experiment.
• Consult safety data sheets before using unfamiliar reagents.

Proper disposal prevents environmental contamination and protects laboratory personnel.