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Cation exchangers bind to proteins with negative charges.
Cation exchangers bind to proteins with negative charges.
False
Anion exchangers bind to proteins with positive charges.
Anion exchangers bind to proteins with positive charges.
False
The compounds adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) can be separated using DEAE cellulose.
The compounds adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) can be separated using DEAE cellulose.
True
The eluting buffer used in the experiment has a pH of 7.0.
The eluting buffer used in the experiment has a pH of 7.0.
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One advantage of this chromatography technique is that it can resolve molecules with small differences in charge.
One advantage of this chromatography technique is that it can resolve molecules with small differences in charge.
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The experimental procedure calls for 10 ml solutions of eluting buffer to be prepared ranging from 0.25M to 0.05M.
The experimental procedure calls for 10 ml solutions of eluting buffer to be prepared ranging from 0.25M to 0.05M.
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It is required that turbidity in the solution is below 10 ppm for this experiment.
It is required that turbidity in the solution is below 10 ppm for this experiment.
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The final height of DEAE-cellulose in the column should be between 5 to 6 cm.
The final height of DEAE-cellulose in the column should be between 5 to 6 cm.
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Ion exchange chromatography is used to separate molecules based on their charge.
Ion exchange chromatography is used to separate molecules based on their charge.
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Cation exchange chromatography attracts positively charged molecules to a negatively charged solid support.
Cation exchange chromatography attracts positively charged molecules to a negatively charged solid support.
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Anion exchange chromatography attracts negatively charged molecules to a negatively charged solid support.
Anion exchange chromatography attracts negatively charged molecules to a negatively charged solid support.
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Salt ions can be used to release bound molecules from the ion exchange column.
Salt ions can be used to release bound molecules from the ion exchange column.
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Proteins with higher binding affinities for the ion exchange resin will elute faster than those with lower affinities.
Proteins with higher binding affinities for the ion exchange resin will elute faster than those with lower affinities.
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The stationary phase in ion exchange chromatography consists of a mobile phase containing salt.
The stationary phase in ion exchange chromatography consists of a mobile phase containing salt.
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S-resin and CM-resins are commonly used for cation exchange chromatography.
S-resin and CM-resins are commonly used for cation exchange chromatography.
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Anion exchangers are used to bind positively charged molecules.
Anion exchangers are used to bind positively charged molecules.
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Study Notes
Ion Exchange Chromatography
- Ion exchange chromatography is a separation technique used to purify or analyze molecules based on their charge.
- It separates charged molecules from uncharged ones, or molecules with different charges.
- Ionizable chemical groups are fixed on a solid support (e.g., cellulose or agarose). This support forms a column.
- Oppositely charged molecules bind to the column through electrostatic interactions. Uncharged molecules pass through.
- Molecules bound to the column are released by adding salt (e.g., NaCl), as salt ions compete for binding sites.
- This process is referred to as "ion exchange".
- The method preserves analyte molecules on the column due to ionic interactions.
- Mobile phase (buffer, pH, and salt concentration) affects solute ions that are attracted to the stationary phase.
- Stationary phase (resin) covalently attaches anions or cations.
Types of Ion Exchange Chromatography
- The resin name describes the molecules being exchanged, not the bound molecule.
- Anion exchangers: bind cations (positively charged)
- Cation exchangers: bind anions (negatively charged)
Cation Exchange Chromatography
- Positively charged molecules are attracted to negatively charged solid support.
- Common cation exchange resins: S-resin, sulfate derivatives, and CM resins, carboxylate derivatives.
Anion Exchange Chromatography
- Negatively charged molecules are attracted to positively charged support.
- Common anion exchange resins: Q-resin (quaternary amine), and DEAE resin (diethylaminoethyl).
Protein Separation using Ion Exchange
- Proteins bind to ion exchangers with varying affinities.
- Proteins with lower affinities elute (leave) the column faster.
- Stronger binding affinity results in slower elution.
- Proteins are eluted by altering the elution buffer (higher salt concentration or different pH).
- Cation exchangers bind to positively charged proteins.
- Anion exchangers bind to negatively charged proteins.
Separating AMP and ATP using Ion Exchange
- Experiment involves separating Adenosine 5'-monophosphate (AMP) and Adenosine 5'-triphosphate (ATP) using diethylaminoethyl (DEAE) cellulose chromatography.
- Mixture loaded onto column, eluted with NH4Cl/NH3 gradient.
- Reagents include AMP and ATP, 0.25M NH4Cl/NH3 (pH 9.0) buffer.
Experimental Procedure
- Column preparation with DEAE-cellulose.
- Preparation of eluting buffers (0.05M to 0.25M in increments of 0.05M).
- Column equilibration and compound loading.
- Effluent collection in test tubes.
- Absorbance measurement at 260 nm and 280nm.
- Data analysis includes tabulation and graphing absorbance vs. fraction number, and chemical structure drawing of separated compounds with correct ionic charges.
Advantages of Ion Exchange Chromatography
- Non-denaturing technique.
- Useful at various purification stages and scales.
- Can act as a concentrating step.
- High selectivity for resolving molecules with small charge differences.
Disadvantages of Ion Exchange Chromatography
- Costly equipment and chemicals
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Description
This quiz delves into the principles and techniques of ion exchange chromatography, focusing on its applications in molecule purification and analysis. It covers the separation of charged and uncharged molecules, the role of stationary and mobile phases, and the mechanisms involved in binding and releasing analytes. Test your understanding of this important separation technique.
