Vitamin C (VC) Content Assay Kit (Ferrozine, Colorimetric Method)

Cat. No.: V1515894

GRADE & PURITY BioReagent

Overview

  Vitamin C, also known as L-ascorbic acid, is an essential nutrient for higher primates and a few other organisms. It is an acidic hexose derivative and a lactone of an enediol hexanoic acid. In organisms, it acts as an antioxidant, scavenging free radicals and protecting against oxidative damage, and also serves as a coenzyme in various metabolic reactions. It is widely found in fresh fruits and vegetables. Vitamin C exists as L- and D-isomers; only the L-form has physiological functions, but both the reduced and oxidized forms are physiologically active.

Assay Principle

  Under acidic conditions, Vitamin C reduces ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). The ferrous ions then form a stable red chelate with Ferrozine. The absorbance is measured at 534 nm. When the sample concentration is within the range of 0.5～35 μg/ml, the absorbance shows a good linear relationship with the Vitamin C content, allowing for quantitative determination.

  This kit is primarily intended for the detection of Vitamin C (ascorbic acid) in plant tissues. It features a stable reaction (color does not fade easily), simple operation, good specificity (no interference from reducing sugars and common reducing substances), and high sensitivity.

  Note: For research use only. Not for use in diagnostic procedures or other purposes.

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 534 nm)

• Mortar and pestle or homogenizer, Centrifuge, Incubator or water bath

• Centrifuge tubes or test tubes, Cuvettes (1 cm path length)

• Distilled water, Anhydrous ethanol

Assay Procedure (For Reference Only)

1. Dilution of Tissue Homogenate

Dilute the Tissue Homogenate (5×) by mixing it with distilled water at a ratio of 1:4 (e.g., 1 part homogenate to 4 parts water) to obtain 1× Tissue Homogenate.

2. Sample Preparation

• Take the plant material to be tested (e.g., green vegetables, fruits, pine needles). Wash and dry them. Accurately weigh 2-3 g of the sample.

• Place the sample in a mortar or grinder. Add a small amount of 1× Tissue Homogenate and grind thoroughly. Collect the supernatant.

• Grind the residue again with more 1× Tissue Homogenate.

• Combine all extracts and transfer into a 50 mL centrifuge tube. Add 1× Tissue Homogenate to bring the total volume to 22 mL. Mix well.

• Centrifuge at 4000g for 5 minutes. The resulting supernatant is the Test Solution.

3. Preparation of Vitamin C Standards

• Dissolve the 25 mg Vitamin C Standard in 1 mL of 1× Tissue Homogenate to obtain a Vitamin C Standard (25 mg/mL). This stock can be stored at -20°C for short-term preservation.

• Dilute this stock with 1× Tissue Homogenate to prepare a Vitamin C Standard (50 μg/mL).

• Use clean centrifuge tubes or test tubes to prepare a series of working standards according to the table below:

Additive (μL)	1	2	3	4	5	6
Vitamin C Std (50 μg/mL)	40	100	160	200	400	600
1× Tissue Homogenate	960	900	840	800	600	400
Equivalent Vitamin C (μg)	2	5	8	10	20	30

4. Assay Setup

Set up Blank, Standard, and Test tubes according to the table below. Add solutions in the order listed, avoiding bubbles. If the Vitamin C content in the sample is expected to be very high, reduce the volume of the Test Solution used or dilute the Test Solution appropriately. It is recommended to run samples in duplicate and calculate the average. 

Additive (mL)	Blank Tube	Standard Tube	Test Tube
1× Tissue Homogenate	1.0	—	—
Series Vitamin C Standard (1-6)	—	1.0	—
Test Solution	—	—	1.0
Anhydrous Ethanol	0.5	0.5	0.5

Mix well.

Acidic Buffer	0.25	0.25	0.25
Ferrozine Color Reagent	0.5	0.5	0.5
Vitamin C Assay Buffer	0.25	0.25	0.25

5. Measurement

• Mix well. Incubate at 30°C for 60 minutes.

• Zero the spectrophotometer with the Blank Tube at 534 nm using a 1 cm path length cuvette.

• Measure the absorbance of all Standard Tubes and Test Tubes.

6. Calculation of Results

• Plot a standard curve with the Vitamin C mass (μg) of the series standards (0, 2, 5, 8, 10, 20, 30 μg) on the x-axis and their corresponding absorbance values on the y-axis. Obtain the regression equation.

• Substitute the absorbance value of the Test Tube into the regression equation to obtain the mass of Vitamin C in the tested sample aliquot (m₀, in μg).

Calculate the Vitamin C content in the original sample using the following formula:

Vitamin C Content (mg/100g) = (m₀ × V_T × 100) / (m₁ × V_S × 1000) 

Parameter Explanation: 

m₀: Mass of Vitamin C in the tested sample aliquot obtained from the standard curve (μg). 

V_T: Total volume of the Test Solution (mL). (According to the procedure, this is 22 mL). 

m₁: Mass of the sample used for extraction (g). (e.g., 2-3 g). 

V_S: Volume of the Test Solution used in the assay (mL). (According to the procedure, this is 1.0 mL). 

100: Conversion factor for content per 100g of sample. 

1000: Conversion factor for micrograms (μg) to milligrams (mg). 

Notes 

1. Avoid repeated freezing and thawing of reagents stored at room temperature ("RT") to prevent loss of activity or efficiency. 
2. The Tissue Homogenate is corrosive. Handle with care and avoid direct contact. 
3. The reaction is not complete immediately after adding the reagents. Do not measure right away. The reaction is complete within 60 minutes. It is recommended to complete the measurement within 60-120 minutes after incubation. 
4. If the Test Solution cannot be assayed immediately, it can be stored at 2-8°C for up to 3 days. 
5. If the sample concentration is too high (absorbance reading above the highest standard), dilute the Test Solution with distilled water and repeat the assay. Multiply the final result by the dilution factor. 
6. Use the reagents as soon as possible after opening to ensure optimal performance. 
7. For your safety and health, please wear laboratory appropriate attire and disposable gloves while handling reagents.

Storage and Shipping

Storage

Protected from light,Room temperature

Shipped In

Normal

Stability And Storage

Store at room temperature long term (6 months). Store in the dark.

Contents & Storage

V1515894	Component	100T	Storage
V1515894A	Vitamin C Standard	25 mg	RT. Store in the dark.
V1515894B	Tissue Homogenate (5×)	500 mL	RT. Store in the dark.
V1515894C	Acidic Buffer	25 mL	RT.
V1515894D	Vitamin C Assay Buffer	25 mL	RT.
V1515894E	Ferrozine Color Reagent	50 mL	RT. Store in the dark.