Question

Blue Dextran 2000 was eluted during gel filtration in a volume of 36.4mL from a 40 * 2cm (length diameter) column of Sephadex G-50, which fractionates molecules in the molecular mass range 1500-30000.

(a) At what retention volume would hemoglobin (molecular mass 64000 ) be expected?

(b) Suppose that radioactive${}^{\mathbf{22}}\mathbf{NaCI}$, which is not adsorbed on the column, is eluted in a volume of 109.8 mL. What would be the retention volume of a molecule with K_av= 0.65?

Short answer

a)The Da value of given stationary phase Sephadex- G 50 and molar mass of the Blue Dextran is 2000 so it does not elute from this column

b) Column Retention volume of NaCl is 84.1mL

Step-by-step solution

Find retention volume would hemoglobin

Interpretation:

The separation of Blue Dextran has 2000 in gel filtration column using should be explained.

Concept introduction:

Gel filtration in open columns:

In the gel filtration column, the pore size of the stationary phase is chosen depends on the molecular mass of the molecules to be separated.

The fractionation range for globular proteins (Da) are used as gel in gel filtration columns and more size of gel are available based on it is size so the stationary phase size (Da) is depends on molar mass of the compound, which is going to separate.

The (Da) value of Sephadex-G 50 is 1500-30000 but Blue Dextran has 2000 molar mass so it is not eluted from this column.

Explanation of Solution

To explain the separation of Blue Dextran 2000 in gel filtration column (Sephadex- G 50 is 1500-30000 ). In the gel-filtration column, the (Da) value of Sephadex (stationary phase) is directly proportional to the molar mass of compound, which is going to separate.

Therefore, the Da value of given stationary phase Sephadex- G 50 and molar mass of the Blue Dextran is 2000 so it does not elute from this column.

Find retention volume of a molecule

(b) Interpretation Introduction

Interpretation:

The retention volume of a molecule in gel-filtration column should be determined.

Concept introduction:

The volume of column is given by the mathematical expression it is,

${\mathrm{V}}_{\mathrm{t}}={\mathrm{\pi r}}^2\mathrm{l}$

Where,

V_tis volume of column

$\mathrm{\pi }$is 3.14

R is radius of the column

L is length of the column

The Elution Equation :

${\mathrm{K}}_{\mathrm{av}}=\frac{{\mathrm{V}}_{\mathrm{r}}-{\mathrm{V}}_0}{{\mathrm{V}}_{\mathrm{m}}-{\mathrm{V}}_0}$

Retention volume of NaCl is 84.1 mL

Explanation of Solution

To determine the retention volume of a molecule in gel-filtration column should be determined.

Given,

The initial volume is 36.4mL

The total volume of mobile phase is 36.4 mL

${\mathrm{K}}_{\mathrm{av}}=0.65$

Therefore, retention volume of NaCl is,

$$\begin{gathered} {\mathrm{K}}_{\mathrm{uv}}=\frac{{\mathrm{V}}_{\mathrm{r}}-36.4}{109.8-36.4} \\ 0.65=\frac{{\mathrm{V}}_{\mathrm{r}}-36.4}{109.8-36.4} \\ {\mathrm{V}}_{\mathrm{r}}-36.4=0.65\times 109.8-36.4 \\ {\mathrm{V}}_{\mathrm{r}}=84.1\mathrm{mL} \end{gathered}$$

The given volume and ${\mathrm{K}}_{\mathrm{av}}$ are plugged in above equation to give a retention volume of NaCl given column Retention volume of NaCl is 84.1mL