BIURET EXPERIMENTAL SETUP (columns 1-6)a;   DATA TO PLOT (columns 7-9)


1
2
3
4
5
6
7
8
9
Tube
No.
mL albumin
stock soln.
(2.5 mg/mL)e
mL KCl
solution
(0.5 M)
mL 
biuret
reagentc
mL 
unknown 
No. 1e
mL
unknown 
No. 2e
albumin 
concentr. (mg/mL)d
Absor-
bance
%T
1b
0.0
5.0
3.0
0.0
0.0
0.0
0
100
2
1.0
4.0
3.0
0.0
0.0
0.3125
    
3
2.0
3.0
3.0
0.0
0.0
      
4
3.0
2.0
3.0
0.0
0.0
      
5
4.0
1.0
3.0
0.0
0.0
      
6
5.0
0.0
3.0
0.0
0.0
      
7
0.0
0.0
3.0
5.0
0.0
*
    
8
0.0
0.0
3.0
0.0
5.0
*
    
a Use a different pipet for each solution; pipet as accurately as possible to minimize error.
b Solution number 1 is the "blank;" it contains everything except albumin. So, using it to set 100 %T, (after setting 0 %T with the chamber empty) will correct readings for the solutions in tubes #2-8 for any light absorption due to glass, water, KCl, or the biuret reagent. DO NOT PLOT tube #1 as part of the standard curve.
c Contains strong NaOH, an alkali (base) - caustic, poisonous. Handle carefully.
d Volume is 8.0 mL in every tube (add col. 2 through 6). Calculate the concentrations for #3, 4, 5, 6: (column 2 volume X 2.5 mg/mL) / 8 mL. Make sure the units come out right. Four decimal places are shown for tube #2 only for you to check your calculations; round to 2 decimal places for plotting data. Plot absorbance vs. albumin concentration for tubes #2 - #6, but not #1, and fit the best straight line to that set of 5 points to produce the standard curve.
e The protein, albumin, is dissolved in 0.5 M KCl in the stock solution and both "unknown" solutions. So, as you pipet different volumes of stock solution into tubes (column 2), you compensate with 0.5 M KCl (column 3) so that the total volume for every tube is 8.0 mL after the 3.0 mL of biuret reagent is added.
* Here you enter the values taken from the standard curve. Then you calculate backward to find the albumin concentrations in the bottles, i.e. before you added the 3.0 mL of biuret reagent.