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Chapter 26: Q18P (page 747)

Would molecular exclusion, affinity, or hydrophobic interaction chromatography be most appropriate for each of the following applications?

  1. Purifying and concentrating a crude mixture of an antibody.
  2. Desalting a solution containing a 30kDa protein.
  3. Finding the molecular mass distribution of polystyrene with 15kDa average mass.
  4. Separation of cytochrome c (12 400 Da) and ribonuclease A (12 600 Da). Cytochrome c has lower surface hydrophobicity than ribonuclease A.

Short Answer

Expert verified

The explanations to the above questions are:

  1. Affinity
  2. Molecular exclusion
  3. Molecular exclusion
  4. Hydrophobic interaction

Step by step solution

01

Explanations to the answers:

  1. Purifying and concentrating a crude mixture of an antibody 鈥揂ffinity
  2. Desalting a solution containing a 30 kDa protein 鈥� Molecular exclusion
  3. Finding the molecular mass distribution of polystyrene with 15 kDa average mass 鈥揗olecular exclusion
  4. Separation of cytochrome c (12 400 Da). Cytochrome c has lower surface hydrophobicity than ribonuclease A 鈥� Hydrophobic Interaction.
02

Definitions:

  • Affinity chromatography 鈥� a method based on macromolecular binding interactions, used for separating biomolecules (like proteins) from a solution.
  • Molecular exclusion 鈥� also called Gel Exclusion Chromatography, a method used for separating molecules in a solution based on their size and in sometimes on their molecular weight.
  • Hydrophobic interaction chromatography 鈥� a method used for separating molecules based on their hydrophilicity.

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Most popular questions from this chapter

A polystyrene resin molecular exclusion HPLC column has a diameter of 7.8 mm and a length of 30 cm. The solid portions of the gel particles occupy 20% of the volume, the pores occupy 40%, and the volume between particles occupies 40%. (a) At what volume would totally excluded molecules be expected to emerge? (b) At what volume would the smallest molecules be expected? (c) A mixture of polyethylene glycols of various molecular masses is eluted between 23 and 27 ml. What does this imply about the retention mechanism for these solutes on the column?

(a) The interstitial volume, V0, in Figure 26-15 is the volume at which the curves rise vertically at the left. FindV0for the 25-nm-pore-size column. To the nearest order of magnitude, what is the smallest molecular mass of molecules excluded from this column? (b) What is the molecular mass of molecules eluted at 9.7 mL from the 12.5-nm column? (c)Vmis the volume at which the curves drop vertically at the right. Find the largest molecular mass that can freely enter the 45-nm pores.

Norepinephrine (NE) in human urine can be assayed by ion-pair chromatography by using an octadecylsilane stationary phase and sodium octyl sulfate as the mobile-phase additive. Electrochemical detection (oxidation at 0.65 V versus Ag|AgCl) is used, with 2,3-dihydroxybenzylamine (DHBA) as internal standard.

  1. Explain the physical mechanism by which an ion-pair separation works.
  2. A urine sample containing an unknown amount of NE and a fixed, added concentration of DHBA gave a detector peak height ratio NE/DHAB = 0.298. Then small standard additions of NE were made, with the following results:

  3. Added concentration of NE ( ng/mL )

    Peak height ratio NE/DHBA

    12

    0.414

    24

    0.554

    36

    0.664

    48

    0.792

  • Use the graphical treatment in Section 5-3, find the original concentration of NE in the specimen.

Consider the separation of inorganic and organic anions in Figure.

(a) What is the probable charge (Xn-)of pyruvate (peak 10), 2-oxovalerate(peak 16), and maleate (peak 28)? (Hint: Look at the ions around the peaks in question.)

(b) Iodide (peak 44) is a-1ion. Explain its strong retention.

What is electroosmosis?
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