In most applications of UV-vis spectrophotometry, extinction coefficients are us

Question

In most applications of UV-vis spectrophotometry, extinction coefficients are usually expressed in units of M^-1cm^-1. For proteins and nucleic acids, however, units of mass concentrations are often used to express extinction coefficients. A somewhat dated (but still frequently used) convention is to report protein extinction coefficients as E_280^1%, which is defined as the expected absorbance of a 1% (mass/volume) solution of the protein in a 1-cm cuvette. It is also often convenient to use units of mg/mL for the extinction coefficients of proteins. using the value of 4.4 times 10^4 M^-1cm^-1 for the molar extinction coefficient for BSA, calculate E_280^1% for this protein. Although values of E_280^1% are usually not expressed with units, they probably should be. What are the proper units? Calculate the extinction coefficient for BSA in units of (mg/mL)^-1 cm^-1.

Explanation / Answer

A) From the equation below, we are going to calculate the required values:

E1%280 = c L

-where E1%280 is the absorbance of the solution (measured at 280nm for protein), is the extinction coefficient, c is the protein concentration, and L is the path length in cm.

c = E1%280 /     since, (L= 1cm when using 1cm cuvettes)


For bovine serum albumin (BSA), the mentioned extinction coefficient is 4.4 * 104 M1cm1. Using this coefficient in the equation above will yield a protein concentration in moles/L (M).

Also, the 280nm absorbance of a 1% solution is equivalent to 10mg/mL of BSA. Since 1% is equal to 10mg/mL, we end up with this equation:

c mg/mL = (E1%280 / percent)10

Now, the relationship between percent and molar is given as under,

percent =    ( molar) (10)    
                  molar mass


percent BSA =    (44,000) (10)    = 6.62     
   66,463

Now, we know that,

                                           percent c L / 10 = E1%280

Therefore, E1%280 = 6.62

B) Since absorptivity is a ratio term, therefore it is generally not represented with units. But if required, unit can be taken as follows..

Assume length in cm and concentration in Mol./L

Thus unit is Mol-1 L cm-1

C) To convert M-1cm-1 to (mg/ml)-1 cm-1 , we need to divide it with its molecular weight.

Therefore, extinction coefficient = (4.4 x 104)/ 66463

= 0.662 (mg/ml)-1 cm-1

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