Lab 13 Quantification of Total Phenolic Compounds and Primary Oxidation Products

Question 1

Discuss polyphenols in food.

Answer 1

• They are naturally present in plants
• Increased interest in high polyphenol foods due to their antioxidant properties
• Consumption of foods high in polyphenols has been shown to reduce risk of several diseases including CVD, diabetes, and cancer

Question 2

Describe the Folin-Ciocalteu Assay.

Answer 2

• Phenolic compounds reduce FC reagent (mixture of phosphomolybdic/phosphotungstic acid complexes) in an alkaline medium (Na2CO3), producing blue chromophores that absorb light at 765 nm.
• Absorbance directly related to phenolic content in sample
• Gallic acid used for standard curve - results reported as mg of gallic acid equivalents per L or g of food

Question 3

What is are phosphomolybdic/phosphotungstic acid complexes?

Answer 3

• Phosphomolybdic acid (PMA) and phosphotungstic acid (PTA) are two different heteropolyacids that are used as catalysts in various chemical reactions.
• They are also used in the Folin-Ciocalteu assay to measure total phenolic content.

These compounds are part of the FC reagent. The FC assay relies on the transfer of electrons in an alkaline medium from phenolic compounds to PMA/PTA complexes. This transfer of electrons results in the formation of a blue chromophore that can be measured spectroscopically at 765 nm. The intensity of the blue color is proportional to the total phenolic content of the sample.

Question 4

What is the FC assay susceptible to interference by?

Answer 4

• Other compounds that can reduce FC reagent (e.g., citric acid, ascorbic aid, glucose, frutose, sodium sulfite)

Question 5

What is PVPP?

Answer 5

• Polyvinylpolypyrrolidone (PVPP) is added to sample blanks in the FC Assay to bind phenols, thus the absorbance of the sample blanks is due to other reducing compounds in the beverages

Question 6

What is the peroxide value?

Answer 6

Quantification of the number of peroxide groups in an oxidized lipid sample (determined using titrimetry); it is a better indicator of quality of recently oxidized food than the TBARS assay.

Question 7

Why is the peroxide value a better indicator of recently oxidized foods than the TBARS assay?

Answer 7

• Lipid peroxides are primary oxidation products as opposed to malondialdehyde (MDA) measured in the TBARS assay which is a secondary oxidation product.

Question 8

What are the steps in an Iodometric titration? [5]

Answer 8

• Potassium iodide added to oil sample; peroxide groups react with I- to form I2 and 2K+
• Starch indicator is added which binds to I2 and produces a dark blue colour.
• I2 is titrated with sodium thiosulfate to produce NaI and Na2S2O6
• When all I2 has been released (titrated) from the starch, the solution will turn clear.
• Titration determines the amount of I2 which is directly proportional to the number of peroxide groups in the oil sample.

Question 9

Why must phenols be isolated prior to TPC analysis?

Answer 9

• It is susceptible to interference by many compounds that are capable of reducing the FC reagent. In food, this includes but is not limited to reductive sugars (such as fructose and glucose), organic acids (such as ascorbic, citric, and tartaric acids), ferrous sulfate, and sodium sulfite. Such interference can lead to an overestimation of TPC in foods. Therefore, phenols must be isolated from foods prior to the foods being analyzed for TPC.
• Common extraction methods utilize methanol, acetone, solid phase extraction columns, or polyvinylpolyprrolidone (PVPP) to isolate phenolic compounds. We will use PVPP in this lab and it is believed to adsorb phenolic compounds by forming hydrogen bonds as well as hydrophobic and Van der Waals interactions.

Question 10

What is sodium carbonate used for in an FC assay?

Answer 10

To create the basic/alkaline conditions required for the reaction to occur

Question 11

What is used for the standard in an FC assay?

Answer 11

Gallic acid

Question 12

In a POV titration, what is being directly measured?

Answer 12

I2 (iodine)

Question 13

What will the endpoint of a POV titration look like?

Answer 13

Clear

Question 14

What is the indicator used in a POV titration?

Answer 14

Starch solution

Question 15

What is the ideal time to incubate the FC reaction?

Answer 15

2 hours

Question 16

What is the purpose of the acetic acid-chloroform solution in the POV titration?

Answer 16

Dissolve the oil sample.

Question 17

How is POV determined?

Answer 17

• To determine the POV of fat or oil, excess potassium iodide is added to a sample which reacts with the peroxides in the sample.
• The iodine (I2) liberated is then titrated with standardized sodium thiosulfate using a starch indicator.
• Starch forms a dark blue complex with iodine.
• The end point in iodometry corresponds to a sudden disappearance of the blue colour, indicating that all iodine has been released from the starch.
• The amount of sodium thiosulfate titrant needed is then used to determine the amount of iodine liberated which equals the amount of peroxides in the sample

Question 18

When are rancid flavours evident?

Answer 18

• Oils with a POV less than 10 meq/kg are considered fresh.
• Rancidity flavours become evident when the POV reaches 20 to 40 meq/kg.

Question 19

Why is it important to begin titrating before adding the starch solution when determining the peroxide value of an oil sample?

Answer 19

It is important to begin titrating before adding the starch solution as starch-iodide complexes are not very soluble in water and thus the iodide will not get converted to iodine to be titrated if the starch is added too early on.

Question 20

The longer the incubation, the more accurate the results of the FC assay.
True or False?

Answer 20

This is true up to 2 hours.

Question 21

Does a low PV mean the oil/fat is fresh?

Answer 21

Not necessarily.
If there are rancid flavours/odours present, that could mean the fats have advanced to secondary oxidation products (e.g., MDA).