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Describe a common buffer system and elution method in ion exchange chromatography. What are the salt ions in the elution buffer good for?

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Lelia Lubowitz
Lelia LubowitzLv2
28 Sep 2019
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Related questions

In an Affinity Chromatography lab experiment, during the equilibrate step of the protocol, you accidentally use the elution buffer instead of the affinity binding buffer. How would this affect the purification process?

---Please describe what would happen, what will result in the purification process, and what the results will look like from this error.

BUFFERS:

(Final concentration for 1x is 20 mM Tris; 500 mM NaCl; 1 mM CaCl2; 1 mM MgCl2; pH 7.4)

5x ConA Buffer, (1L)

• 700 ml dH2O, set stirring and add:

• 100 ml 1M Tris, pH 7.2-7.4

• 5 ml 1M MgCl2

• 5 ml 1M CaCl2

• 146 g NaCl

• Check pH is between 7.2 and 7.4

• To 1000 ml with dH2O

Elution Buffer A: ~0.2M AMM: 0.1 g alpha methyl mannopyranoside in 2 mls ConA buffer

Elution Buffer B: ~0.5M AMM: 0.2 g alpha methyl mannopyranoside in 2 mls ConA buffer

Elution Buffer C: ~1 M AMM: Add 0.4 g alpha methyl mannopyranoside in 2 mls ConA buffer (you may need more of this)

Formula weight for alpha methyl mannopyranoside is 194.2 g.

Questions:

Buffers: Calculate the concentrations of Tris, CaCl2 and MgCl2 in the 5x ConA Buffer stock

Tris:

CaCl2:

MgCl2:

For Buffer B: What is the exact concentration of the AMM to three significant figures

Which elution order you might expect in CZE for the following components: benzene, benzoic acid, aniline, and phenobarbital if 5% of heaxadecyltrimethylammonium chloride is added to the buffer and system was kept under high voltage (EOF on) for 24 hours?

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