Reference no: EM133853606

Question

Cation exchange column (SP (sulphopropyl)-Sepharose) was used to separate cytochrome c (from horse) and ovalbumin. More specifically, a 10mL SP-sepharose column equilibrated with 20mM sodium phosphate, pH 6.0 was used. After applying the sample to the column, 20mL of 20mM sodium phosphate, pH 6.0 was applied to the column and 2mL fractions (1- 10) were collected as indicated in the table below. Next, 25mL of 20mM sodium phosphate buffer (pH 6.0), containing 2.5M NaCl was applied to the column and fractions 11- 19 were collected as indicated in the table below. The absorbance of all fractions at a wavelength of 280nm and 400nm was measured as presented in the table below. The column was re-equilibrated with the 20mM sodium phosphate buffer, pH 6.0. Fraction # Elution Volume (Ve) (mL) Absorbance at 280nm Absorbance at 400nm 1 2 0.079 0.019 2 4 0.026 0.008 3 6 0.184 0.004 4 8 0.269 0.003 5 10 0.115 0.002 6 12 0.038 0.002 7 14 0.014 0.002 8 16 0.009 0.002 9 18 0.007 0.002 10 20 0.007 0.002 11 22 0.007 0.002 12 24 0.01 0.003 13 26 0.341 1.113 14 28 1.093 3.38 15 30 0.317 1.052 16 32 0.075 0.236 17 34 0.025 0.064 18 36 0.012 0.02 19 38 0.01 0.01 a. (5 points) Before measuring the absorbance of fractions 1 - 10, what should be used to "Blank" or zero the spectrophotometer? b. (5 points) Before measuring the absorbance of fractions 11 - 19, what should be used to "Blank" or zero the spectrophotometer?