Technical Report on the p-Anisidine Test for Measuring Secondary Oxidation in Fats and Oils
Abstract
This report evaluates the p-anisidine test as a method for assessing secondary oxidation in fats and oils. A comparative analysis between the traditional AOCS Cd 18-90 method and the CDR FoodLab® AnV test is presented. The chemical mechanism of p-anisidine reacting with aldehydes is explored, along with the challenges posed by highly pigmented oils. Additionally, spectrophotometric factors affecting absorbance in traditional and LED-based spectrophotometers are discussed. The study concludes with a comparative table summarizing reagent use, equipment requirements, toxicity, analysis time, and reliability.
Introduction
Oxidation in fats and oils significantly impacts their quality, shelf life, and safety. Primary oxidation leads to hydroperoxide formation, while secondary oxidation results in aldehydes and ketones, which contribute to rancidity and off-flavors. The p-anisidine test measures secondary oxidation by quantifying aldehyde content, making it a key quality control tool. However, the traditional AOCS Cd 18-90 method encounters limitations with highly pigmented oils, necessitating alternative approaches such as the CDR FoodLab® AnV test.
Chemical Mechanism of p-Anisidine Reaction
The reaction of p-anisidine with fats or oils is primarily used to evaluate the presence of secondary oxidation products, such as 2-alkenals and 2,4-dienals, which are degradation products of polyunsaturated fatty acids. This chemical reaction is crucial for analyzing the quality and stability of edible oils and fats.
1. Formation of Aldehydes in Fats and Oils:
1.1. During the oxidation of fats and oils, hydroperoxides are formed as primary oxidation products.
1.2. These hydroperoxides decompose to form secondary oxidation products, including aldehydes (e.g., 2-alkenals and 2,4-dienals).
Example:
Hydroperoxide → Aldehyde (e.g., 2,4-dienal) + Other products Hydroperoxide → Aldehyde (e.g., 2,4-dienal) + Other products
2. Reaction of p-Anisidine with Aldehydes:
2.1. p-Anisidine reacts with the aldehyde group (–CHO) of the secondary oxidation products to form a Schiff base (imine).
2.2. The reaction involves nucleophilic attack by the amino group (–NH₂) of p-anisidine on the carbonyl carbon of the aldehyde, followed by the elimination of water.
3. Mechanism:
p-Anisidine (4-methoxybenzenamine) + Aldehyde (R-CHO) → Schiff base (R-CH=N-Ar) + H2O
Where:
3.1. RR = alkyl or alkenyl group (e.g., from 2-alkenals or 2,4-dienals).
3.2. Ar = p-anisidine aromatic ring (4-methoxyphenyl).
The Schiff base is formed with a pattern of alternating single and double bonds that absorbs light at 350 nm, allowing for spectrophotometric quantification.
Comparison of Traditional vs. CDR FoodLab® AnV Method
The traditional p-anisidine method (AOCS Cd 18-90) employs acetic acid and Isooctane as a solvents and measures absorbance at 350 nm. Its accuracy is compromised in dark-colored oils due to reagent interactions with pigments.
Procedure for Anisidine Value (AnV) Determination
Sample Preparation:
Dissolve a known amount of fat or oil in an organic solvent (e.g., iso-octane).
Reaction with p-Anisidine:
Add a solution of p-anisidine in acetic acid to the sample.
Allow the reaction to proceed for a specific time (e.g., 10 minutes) to ensure complete formation of the Schiff base.
Spectrophotometric Measurement:
Measure the absorbance of the solution at 350 nm using a spectrophotometer.
The absorbance is proportional to the concentration of aldehydes in the sample.
Calculation of AnV:
The Anisidine Value is calculated using the formula:
Where:
As= absorbance of the sample after reaction with p-anisidine.
Ab = absorbance of the sample before reaction with p-anisidine.
m = mass of the sample (in grams).
Procedure for Anisidine Value (AnV) Determination on CDR FoodLab®
1. Warm up the reagent inside the analyzer
2. Add 20µL of your sample
3. Mix
4. Read in 2 minutes
Why is p-Anisidine Hydrochloride More Accurate for AnV Testing?
The p-Anisidine Hydrochloride, used in the CDR FoodLab® AnV method, offers superior accuracy and reliability compared to standard p-anisidine. This hydrochloride form enhances solubility in aqueous and buffered environments, reducing matrix interferences from lipophilic components in complex oil samples.
p-Anisidine hydrochloride is more selective toward aldehydes than the free base form of p-anisidine because:
The protonated amino group (–NH₃⁺) is less nucleophilic, reducing reactivity with less electrophilic carbonyl compounds like ketones and carboxylic acids this will avoid the risk of false positives from ketones and carboxylic acids that could lead to overestimation of oxidation levels.
The acidic environment further suppresses side reactions, enhancing selectivity for aldehydes, in this case the CDR reagent contains HCl.
This selectivity is crucial for accurate quantification of aldehydes in methods like the Anisidine Value (AnV) test, ensuring reliable assessment of oxidative rancidity in fats and oils.
Spectrophotometric Challenges in Dark Samples
Darkly colored oils and fats are a significant challenge in accurately measuring anisidine value (AnV) using traditional spectrophotometric methods. These oils and fats absorb light in the same wavelength range (around 350 nm) as the Schiff base formed by the reaction between p-anisidine and aldehydes. This overlap in absorbance spectra leads to inaccurate readings, as the high background absorbance from the oil itself makes it difficult to isolate the absorbance specifically due to the reaction.
This interference is further exacerbated using traditional spectrophotometers with filament lamps, which have a broad spectral output, increasing the amount of light absorbed by the oil. Consequently, the measured absorbance at 350 nm is lower than the true value, leading to an underestimation of the AnV.
Influence of Spectrophotometer Technology
The type of light source used in the spectrophotometer can also affect the accuracy of AnV measurements in colored samples.
Filament Lamps: Traditional spectrophotometers often use filament lamps, which have a broad spectral output. This can increase background absorbance and exacerbate the challenges posed by sample color.
LEDs: Modern spectrophotometers equipped with LEDs offer a narrower spectral bandwidth, reducing background interference and improving the accuracy of absorbance measurements, especially in challenging samples.
Conclusion & Recommendations
The p-anisidine test remains a crucial tool for evaluating the oxidative state of fats and oils. However, traditional methods, while widely used, are susceptible to interference from sample color, particularly in dark and pigmented oils and fats. This limitation highlights the need for modern alternatives like the CDR FoodLab® AnV test, which utilizes LED-based photometry and refined reagents to overcome these challenges. By offering improved accuracy, reduced toxicity, and faster analysis, the CDR FoodLab® AnV test presents a compelling solution for industries seeking reliable assessment of lipid oxidation, especially in dark-colored samples a validation assay might be required according to how dark is the sample. Adopting such advanced methods is essential for ensuring product quality and safety in an increasingly demanding market.
Scientific References
AOCS Official Method Cd 18-90: p-Anisidine Value. Available at: AOCS
Enhancing oxidative stability and shelf life of frying oils with antioxidants. Available at: AOCS. (n.d.)
CDR FoodLab®: p-Anisidine Value (AnV) Test in Fats and Oils. Available at: CDR FOODLAB® line website
Check Oxidation in Fats & Oils by Testing p-Anisidine Value. Available at: CDR FOODLAB® line website
p-Anisidine Value (AnV) Test in Fats and Oils with CDR FoodLab® Range Analysis Systems. Available at: YouTube
CDR PalmOilTester: p-Anisidine Value (AnV) Test in Palm Oil. Available at: CDR FOODLAB® line website
Choe, E., & Min, D. B. (2006). "Mechanisms and factors for edible oil oxidation." Comprehensive Reviews in Food Science and Food Safety, 5(4), 169–186.
Shahidi, F., & Zhong, Y. (2010). "Lipid oxidation and improving the oxidative stability." Chemical Society Reviews, 39(11), 4067–4079.
IUPAC. (1992). "Standard Methods for the Analysis of Oils, Fats and Derivatives." 7th Edition.
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