Separation And Determination Of The Phenolic Antioxidants Biology Essay

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Antioxidants are one of the most common active ingredients of nutritionally functional foods which can play an important role in the prevention of oxidation and cellular damage inhibiting or delaying the oxidative processes. [1] Recently there has been an increase in scientific interest surrounding the function and application of antioxidants in the body. This is due to the fact that they are being considered for medical treatment as a potential anticarcinogenic.

Within antioxidants, phenolic compounds are an important sub category, commonly present in a wide variety of plant food materials. Their correct determination is crucial nowadays and involves their extraction from the material, analytical separation, identification, quantification and interpretation of the data.

Figure 2. A simplified classification of phenolic compounds and the basic structures belonging to benzoic acids, hydroxycinnamic acids, flavones, isoflavones, flavanones, flavonols, flavanols, anthocyanins and tannins.

Hydroxycinnamic acids are a class of phenolic compounds with a basic C6-C3 skeleton. These compounds are hydroxy derivatives of cinnamic acid.

Figure 1. [2] Structures of different hydroxycinnamic acids

Caffeic acid and ferulic acid are two specific types of hydroxycinnamic acids. Their structures and properties are shown below.

Molecular Structure and Physical Properties of Caffeic Acid. [3] 

Linear Molecular Formula


Molar Mass g/mol-1


Melting Point /°C

211-213 °C 


Yellow powder

Molecular Structure and Physical Properties of Ferulic Acid.

Linear Molecular Formula


Molar Mass g/mol-1


Melting Point /°C

168-172 °C


Yellow crystalline

There are varying methods used to separate, detect and quantify the amount of hydroxycinnamic acids in plant food materials. The results collected from this are crucial for the quantification of the antioxidants within the specific sample. The most readily used analytical technique for identifying the hydroxycinnamic acids is High Performance Liquid Chromatography (HPLC). The method of this will be examined in conjunction with Gas Chromatography (GC) being used as the analytical technique.

This review will focus on data and results from the following papers:

1. 'Separation, Characterization, and Quantitation of Benzoic and Phenolic Antioxidants in American Cranberry Fruit by GC-MS.' Y. Zuo et al. [4] 

2. 'Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography.' H. Chena et al. [5] 

'Separation, Characterization, and Quantitation of Benzoic and Phenolic Antioxidants in American Cranberry Fruit by GC-MS.' Y. Zuo et al.

This journal examines the GC-MS method for the separation and characterization of benzoic and phenolic acids in cranberries. Varieties of phenolic acids were identified on the basis of their GC retention times and simultaneously recorded mass spectra.

Sample Preparation

Extraction of Free Phenolic Acids in Cranberry Juice. Natural cranberry fruit (60 g) was thoroughly ground in distilled-deionized water (200 mL) with an electrical high-speed blender. A 10--mL aliquot of filtrate was acidified by adding 1 N HCl to maintain pH at 2 and extracted with 10 mL of ether twice. The ethereal phase contained free phenolic acids and was extracted with 10 mL of 5% NaHCO3 twice. In this way, the phenolic acids were substantially separated from the other phenolic moieties. The alkali aqueous solution was acidified with 1 N HCl to maintain pH at 2 and extracted again with 10 mL of ether twice. The ethereal extract was dried over anhydrous MgSO4 and evaporated to dryness on a rotary evaporator under reduced pressure at 35 °C. The dry residue was dissolved in 100 μL of freshly distilled pyridine, and 10 μL of the pyridine solution was used for derivatization before GC analysis. For major aromatic components, such as benzoic acid, dilution was made before derivatization.

The samples were prepared via enzymatic extraction. The detailed method involved

adding the sample (0.2-1.5 g) to hydrochloric acid (25 ml, 0.1 M) and autoclaving for

30 minutes at 121 °C. Sample was then cooled and adjusted to pH 4.0 with 4 M

sodium acetate, takadiastase (100 mg) was added and solution incubated at 45 °C

for 18 hours. This was followed by dilution to an appropriate concentration with 0.01

M hydrochloric acid and finally the solution was filtered through a 0.2 μm filter prior to being injected into the HPLC system[5].

Analytical Method

The HPLC system used consisted of a reversed phase column (Supelcosil LC-18-

DB, see table 2) with an isocratic solution of methanol : buffer (35:65) as the mobile

phase. The buffer was adjusted to pH 3.3 using sodium heptasulfonate (12.7 mM),

tetraethyl ammonium chloride (0.1 %) and potassium dihydrogenphosphate (50 mM).

Table 2: Supelcosil LC-18-DB Reversed Phase Column[9]

Column length x internal diameter / mm 250 x 4.6

Particle size / μm 5.0

Pore size / Å 120.0

Matrix Silica gel

Matrix active group Octadecyl phase

The presence of Riboflavin in the eluent was detected by a fluorescent detector

using excitation and emission wavelengths of 468 nm and 520 nm respectively.

A flow rate of 1 mL / minute was used to elute the compounds.


Figure 2: Chromatogram of whole milk

(a) and of a sample containing known

amounts of FMN and Riboflavin (b)[5]

Rt of Riboflavin in the known

sample was found to be 6.592

minutes. Rt of Riboflavin in whole

milk was found to be 6.552

minutes. It can therefore be

confidently assumed that Riboflavin

is present in whole milk, and the

amount present is proportional to

the peak intensity.

The quantitation limit of Riboflavin

was found to be 0.001 mg of

Riboflavin per 100 g of sample.

The whole milk sample whose chromatogram is shown above was found to contain

1.48 ± 0.02 mg or Riboflavin per 100 g of sample.

Conclusions and Comparison of Analytical Methods