<<–2/”>a href=”https://exam.pscnotes.com/5653-2/”>h2>FRAP: A Powerful Tool for Studying Antioxidant Capacity
What is FRAP?
FRAP, which stands for Ferric Reducing Antioxidant Power, is a widely used spectrophotometric method for measuring the antioxidant capacity of biological samples, including food, beverages, and biological fluids. It assesses the ability of antioxidants to reduce ferric ions (Fe3+) to ferrous ions (Fe2+) in the presence of a specific reagent.
The FRAP Assay Principle
The FRAP assay relies on the following principle:
- Ferric-tripyridyltriazine (Fe3+-TPTZ) complex: A colored complex is formed when ferric ions (Fe3+) react with 2,4,6-tripyridyl-s-triazine (TPTZ) in acidic conditions. This complex has a strong absorbance at 593 nm.
- Antioxidant reaction: When antioxidants are present in the sample, they reduce the Fe3+-TPTZ complex to the ferrous form (Fe2+-TPTZ). This ferrous complex is colorless, resulting in a decrease in absorbance at 593 nm.
- Measurement: The decrease in absorbance is directly proportional to the antioxidant capacity of the sample.
Procedure of the FRAP Assay
The FRAP assay is typically performed in a 96-well microplate format. The following steps are involved:
- Preparation of FRAP reagent: A freshly prepared FRAP reagent is made by mixing acetate buffer (pH 3.6), TPTZ solution, and ferric chloride solution in specific proportions.
- Sample preparation: Samples are diluted appropriately with distilled water or buffer to ensure that the absorbance readings fall within the linear range of the assay.
- Incubation: A known volume of FRAP reagent is added to each well containing the sample. The mixture is incubated at 37°C for 30 minutes to allow the reaction to reach equilibrium.
- Measurement: The absorbance of each well is measured at 593 nm using a microplate reader.
- Calculation: The antioxidant capacity of the sample is calculated using a standard curve generated from known concentrations of a standard antioxidant, such as Trolox (a water-soluble vitamin E analog).
Advantages of the FRAP Assay
- Simplicity: The FRAP assay is relatively simple to perform and requires minimal equipment.
- Sensitivity: It is a sensitive method that can detect even low levels of antioxidants.
- Reproducibility: The assay is highly reproducible, providing consistent results.
- Versatility: It can be used to measure the antioxidant capacity of a wide range of samples, including food, beverages, and biological fluids.
Limitations of the FRAP Assay
- Specificity: The FRAP assay measures the total reducing capacity of a sample, not the activity of specific antioxidants.
- pH dependence: The assay is pH-dependent, and the results may vary depending on the pH of the sample.
- Interference: Certain compounds, such as Metal ions and reducing sugars, can interfere with the assay.
Applications of the FRAP Assay
The FRAP assay has numerous applications in various fields, including:
- Food science: Assessing the antioxidant capacity of fruits, vegetables, and other food products.
- Nutrition: Evaluating the antioxidant content of dietary supplements and functional foods.
- Pharmacology: Screening for potential antioxidant compounds in drug discovery.
- Clinical research: Measuring the antioxidant status of patients with various diseases.
- Environmental science: Determining the antioxidant capacity of environmental samples, such as water and Soil.
Factors Affecting FRAP Values
Several factors can influence the FRAP values obtained, including:
- Sample preparation: The method of sample preparation, such as extraction and dilution, can affect the results.
- Temperature: The incubation temperature can influence the reaction rate and, consequently, the FRAP values.
- pH: The pH of the sample and the FRAP reagent can affect the reaction kinetics.
- Presence of interfering compounds: Certain compounds, such as metal ions and reducing sugars, can interfere with the assay.
Table 1: Comparison of FRAP with Other Antioxidant Assays
| Assay | Principle | Advantages | Disadvantages |
|---|---|---|---|
| FRAP | Ferric reducing antioxidant power | Simple, sensitive, reproducible | Measures total reducing capacity, pH-dependent, susceptible to interference |
| DPPH | Free radical scavenging activity | Simple, widely used | Not specific to certain antioxidants, affected by pH and solvent |
| ABTS | Antioxidant capacity based on radical scavenging | Sensitive, versatile | Can be affected by pH and solvent |
| ORAC | Oxygen radical absorbance capacity | Measures antioxidant capacity against peroxyl radicals | Complex procedure, requires specialized equipment |
Table 2: FRAP Values of Common Foods
| Food | FRAP Value (µmol Trolox equivalents/100 g) |
|---|---|
| Blueberries | 1200 |
| Strawberries | 900 |
| Blackberries | 800 |
| Raspberries | 700 |
| Spinach | 600 |
| Broccoli | 500 |
| Apples | 400 |
| Oranges | 300 |
| Bananas | 200 |
Frequently Asked Questions (FAQs)
Q: What is the difference between FRAP and ORAC?
A: FRAP measures the ability of antioxidants to reduce ferric ions, while ORAC measures the ability of antioxidants to scavenge peroxyl radicals. Both assays assess antioxidant capacity but use different mechanisms.
Q: What are the units of FRAP values?
A: FRAP values are typically expressed in µmol Trolox equivalents per 100 g of sample. Trolox is a water-soluble vitamin E analog used as a standard in the assay.
Q: How can I improve the accuracy of the FRAP assay?
A: To improve accuracy, ensure proper sample preparation, use fresh reagents, control the incubation temperature, and minimize the presence of interfering compounds.
Q: What are some limitations of the FRAP assay?
A: The FRAP assay measures total reducing capacity, not the activity of specific antioxidants. It is also pH-dependent and susceptible to interference from certain compounds.
Q: What are some alternative methods for measuring antioxidant capacity?
A: Other methods include DPPH, ABTS, and ORAC assays. Each method has its own advantages and disadvantages, and the choice of method depends on the specific application.
Q: How can I interpret the FRAP values obtained?
A: Higher FRAP values indicate higher antioxidant capacity. However, it is important to compare FRAP values of different samples within the same context, considering factors such as sample type, preparation method, and assay conditions.