Question

I need to prepare a standard calibration curve for gamma globulin. absorbances on Y and mg of standard protein per assy on X. used .125mg/ml gamma stock for tubes 2-6. (Water (ml), gamma (ml), Abosrbance)--> (.9, .1, .587) (.8, .2, .672) (.6, .4, .602) (.4, .6, .895) (.2, .8, .987) - I"m not getting a straight line. I know in class we did a 1:10 dilution. What is the math and how do you get the standard curve?

Answer 1

Did you dilute the gamma globulin solution at the beginning of your experiment? If that is the case, then we can look at the solution below.

So, we start with a 1:10 dilution, which I am assuming means that you took 1 ml of gamma globulin and diluted 10 times, so the concentration that you are working with is really 125 mg/mL * 1/10 = 12.5 mg/mL.

So, let use find out the concentrations in mg/mL in the stock solutions (test tubes 2-6) that you have prepared.

Test tube	Water (mL)	Globulin (mL)	Concentration (mg/mL)	Absorbance
2	0.9	0.1	1.25	0.587
3	0.8	0.2	2.50	0.672
4	0.6	0.4	5.00	0.602
5	0.4	0.6	7.50	0.895
6	0.2	0.8	10.00	0.987

We use the Beer’s-Lambert law as

A = Ɛ.C.l where Ɛ = absorptivity co-efficient, C is the concentration and l is the path length of the solution. The Plot of A vs C should be a straight line. We obtain the following plot.

Obviously, the data points do not fall on a straight line. This is really a best fit line. The best way to improve on the plot is to re-measure the values to check that you are really getting these values. In case, that doesn’t help, try to take in more measurements by using further dilutions, like say 0.3, 0.5, 0.7 mL, etc. You can calculate the absorptivity co-efficient from the slope of the plot. R² value is usually a good indication of how good or bad a plot is and for good plots, the R² value should be atlest 0.95+.