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Demonstrate the extraction and qualitative and quantitative estimation of secondary metabolites




               Table 1: Qualitative tests for detection of Phenolics and Flavonoids



Quantitative Analysis



 Folin Ciocalteu (FC Reagent) method:  (Phenolic compounds determination)


  • For the assay, aliquots (100µl) of extracts of different concentrations (10, 20, 30, 40 and 50 µg/ml) were taken in test tubes and the volume was made up to 1 ml with distilled water.


  • Then 0.5 ml of FC phenol reagent (1:1with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube.


  • Soon after vortexing the reaction mixture, test tubes were placed in dark for 40 minutes and the absorbance was recorded at 725nm against the reagent blank.


  • The analysis was performed in triplicate and the results were expressed as mg of gallic acid (GAE) per gram of dry weight of the extract.



Aluminium chloride method: (Flavonoid compounds determination)


  • For the assay different concentrations (10, 20, 30, 40 and 50 µg/ml) were prepared for the extract.


  • In a test tube, 0.5 ml of the extract was taken and volume was adjusted to 1 ml. At the start of the experiment, 0.15 ml of 5% NaNO2 was added.


  • After 5 minutes, 0.15 ml of 10% Alcl3 was added. At 6 minutes, 1 ml of 1 M NaOH was added to the mixture. Finally the total volume was made to 3 ml. The absorbance of the mixture was determined at 510 nm vs the prepared blank.


  • Total flavonoid content was expressed as mg of Querectin (Que) per gram of dry weight of the extract. 




Quantitative analysis of unknown compounds by HPLC


The measurement of the amount of a compound in a unknown sample can be calculated from the obtained chromatogram in two ways:


  • Determination of the peak height of a chromatographic peak from the baseline
  • Determination of the peak area.




                    Figure 1: Typical Mechanism of an HPLC separation



  • In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. In many cases there is a linear relationship between the height or area and the amount of sample.




                                      Figure 2: A chromatogram derived from an HPLC


  • In chromatography an internal standard represents a compound which is added to sample in known concentration. It is used to do the quantitative determination of the sample components. The concentration of the internal standard remains constant. For each measurement, the concentration and the peak area of the analyte are compared with those of the internal standard.


  • The size of the isolated peak is proportional to the concentration of the analyte. If we measure the peak, we can evaluate the concentration of the analyte.


  • There are several measurements used to determine the size of the peak. They include height, width and area. However, height and width are effected by how fast the analyte moves through the detector. An analyte that is moved slowly will produce a short, broad peak. If we speed up the process, the analyte will produce a tall, thin peak. Therefore, the preferred measurement is the area of the peak.




                                Figure 3: Peak Detection- Integration events




  • Before we can quantitate our analyte, we must know something about the relationship between peak area and concentration. The simplest method is to determine a response factor. The response factor (RF) is the proportionality constant for the analyte. Each analyte will have a unique RF under given instrumental conditions. (Our job is to keep those conditions constant). Equation 1 shows us that RF is simply the concentration (C) divided by the area (A). [M=W/w]


  • If we prepare a sample of a known concentration (called a standard) and evaluate it, we can measure the peak area and determine the RF. This process is referred to as a calibration. (The standards used for calibrating an instrument are simply referred to as calibration standards).


  • Once we know the RF for our target analyte we can determine the concentration of that analyte in our sample by running it on the instrument and getting the peak area. Concentration is; [C=RFxA]


  • This method works fairly well provided the concentration in our calibration standard is close to the concentration in our sample. If the two differ greatly, then we lose accuracy. However, since we don't know what the concentration is in the sample then it is difficult to test what concentration calibration of standard to use. We can solve this problem and increase our accuracy by constructing a calibration curve.


  • A calibration curve is simply a graph where concentration is plotted along the x-axis and area is plotted along the y-axis. (Response, absorbance, intensity, peak height, etc.)  can also be used depending on the instrument.


  • The user will make several calibration standards at different concentrations. After running each one on the instrument and getting an area, the points are then plotted on the graph. The points are then connected with a line. That line represents the calibration curve.


  • Note that every different analyte will produce a different calibration curve. We must construct a calibration curve for each analyte we are interested in. This can be clearly explained by the example given below:


  • E.g. A compound was diluted in an organic or polar solvent and injected into the HPLC column. The concentrations were made to 5µg, 10 µg, 15 µg, 20 µg, 25 µg/ml).  Each concentration gave a peak area of (5000, 10000, 15000, 20000 and 25000). Find the concentration of the analyte.


   Solution:  First we have to plot a graph of peak area versus the concentration




Figure 4. Standard Calibration Curve


  • Once we have constructed our curve, we can analyze our sample. We simply determine the peak area for the analyte in our sample, and then draw a line on the graph at that area (note the blue line).


  • When the calibration curve is reached, we drop a line down to the x-axis. That will give us the concentration of the analyte in our sample. The curve in the figure is a linear equation. Not all analytes will give a linear response for all ranges of concentrations.


  • However, most analytes are linear for certain ranges. This range of concentrations is referred to as the linear dynamic range. If we analyze our sample in the linear dynamic range, we can calculate a regression line equation and use it to solve for concentration.


  • Note: This is one of the methods to calculate the concentration of an analyte.




Extraction of flavonoids from Tea


The protocol for extraction is given below:




Figure 5. Flowchart of extraction of flavonoids from Tea




Extraction of flavonoids from Maize leaves by using Acetone and Chloroform


The detailed protocol for anthocyanin separation is described below:




Figure 6. Flowchart of extraction of flavonoids from Maize




Extraction of flavonoids from Petunia flowers by using Acidified Methanol


The detailed protocol is described below:




Figure 7. Flowchart of extraction of flavonoids from Petunia







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