Preperation of a caliberation curve of salicylic acid with the Application of statistics Lab

Biopharmaceutics Lab.1

Preperation of a caliberation curve of salicylic acid with the Application of statistics

Caliberation curve: is a curve prepared from a series of standard solution and used as a reference curve to obtain the concentration of unknown sample of the same drug.

The curve should be fitted either:

1- By eye fitting  or

2- By least square fitting (most reliable method)

The curve is based on the equation which minimize the summation of the square deviation of the observed value from the line [Σ(y- ȳ)²], where y= observed value & ȳ = calculated value.                                                                                         

Note:

The X- axis is the Independent variable (conc.)

The Y- axis is the dependent variable (absorbance)

Linear Regression Line

ȳ= c + bx

b= (Σx.y) – [[Σx.(Σy/n)]/(Σx²)-[[( Σx)²/n]]]

c=[ Σy –(b.Σx)] /n

Where:

ȳ= calculated value

c= intercept of the least square line

b= slope of the least square line

To measure the concentration of any substance, we have many methods, one of these methods is spectrophotometry method for measurement of concentration of the sample by applying certain waves either visible light or ultraviolet light.

Visible light range from 400-700 nm, ultraviolet light range from 200-400 nm.

●The Experiment:

The Apparatus:

Spectronic-20 is an instrument which read the absorption for colored solution and used for quantitative analysis to find the concentration of the sample by regression analysis(Linear Regression Line).

The Materials:

*Color Developing Reagent:

   Mercuric chloride           40 gm

   Ferric nitrate                    40 gm

   Hcl (1N)                            120 ml

   D.W.                      ad to  1000 ml

 

*Salicylic acid  

The Procedure:

1- Prepare 250 ml of stock solution of sodium salicylate containing the equivalent of 

200 mg S.A./100 ml.

 

2- From the stock solution of sodium salicylate, use volumetric procedure to accurately prepare solutions containing the equivalent of 50, 40, 30, 20 and 10 mg S.A./100 ml.

 

3- Take 1 ml of each solution and add 5 ml of color developing reagent & mix the solution.

4- Determine the absorbance against a suitable blank at 530 nm.

 

5- Plot absorbance versus concentration of salicylic acid.

6- Apply the least square method to calculate the slope and intercept.