CHMI-2227EL Lecture Notes - Lecture 5: Myoglobin, Ionic Strength, Stainless Steel
27 Jun 2018
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Biochemistry - Day 5 2018.01.26.
Separation and detection of amino acids Continued
Ion Exchange Chromatography (IEC)
-Resins carrying positive charges, binds negatively
charged amino acids (anion exchangers)
-Resins carrying negative charges binds positively
charged amino acids (cation exchangers)
-A common way to separate amino acids on such
columns is to elute them with buffers of differing
pH
-By knowing the pI of the amino acid and pH of the
buffer, you can predict whether it will have a net
negative, positive, or neutral charge
-pH < pI = amino acid is positive
-pH > pI = amino acid is negative
-pH = pI = amino acid neutral
-Example
-Suppose we have a column containing carrying negative charge
(cation exchangers). We want to separate a mixture of 3 kinds of
molecules (histidine (pI = 7.6), serine (pI=5.7), aspartic acid (pI=3))
-This mixture is in a buffer (pH = 3.25)
-At this pH = 3.25:
-The aspartic acid will have a negative charge (no binding to the ion
exchanger) and will be eluted quickly through the column
-The serine is electrically neutral, it will be eluted after the Asp
-The histidine has a net positive charge, therefore it will be stuck to the
column so we have to increase the Ph of the buffer (elution)
-Practice
-If we have a mixture of pH = 11 of
-Phe (pI = 5.5)
-Thr (pI = 6.5)
-Asp (pI = 3)
-His (pI = 7.6)
-Lys (pI = 9.8)
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-And we load the mixture on an anion exchange column and then elute
with a pH gradient of lowering pH, in what order will the amino acids
elute?
-At pH 11, all amino acids are negatively charged because all the pI’s
are less then 11, therefore the amino acids will elute in order as their
pI’s are reached:
-Lys (9,8) , His (7.6), Thr, Phe, Asp
-So the binding affinity of an amino acid for an anion or cation resin
exchanger depends on
-A) The net charge of the amino acid in question
-B) The pH of the solvent
-C) The ionic strength of the solvent
-The amino acid bound to the resin exchanger is eluted by either
-1) Varying the pH of the solvent or
-2) by increasing the salt concentration
High Performance Liquid Chromatography (HPLC)
-The matrix of HPLC consists of beads that are smaller and more tightly
packed then the beads used in regular chromatographic columns
-High pressure is applied to amino acid mixture and solvent through the
column
-Columns are made of stainless steel so they can withstand pressures of
several pounds per square inch
-The speed, sensitivity, and resolution is very high
-The separation is still based on the same principles that govern ion
exchange or other types of chromatography
Protein-Biological Functions
-Definition: The term Protein is derived from a Greek word proteios,
meaning “of the first rank”
-Proteins play several roles in human and other organisms
-***Some proteins function as
-1) Structural material which provide structure and support like collagen
(fibrous connective tissue), ribosomal proteins (RNA ribosomes)
-2) Catalytic like hydrolysis of peptide bonds by trypsin or DNA
polymerase (DNA synthesis)
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Document Summary
Resins carrying positive charges, binds negatively charged amino acids (anion exchangers) Resins carrying negative charges binds positively charged amino acids (cation exchangers) A common way to separate amino acids on such columns is to elute them with buffers of differing ph. By knowing the pi of the amino acid and ph of the buffer, you can predict whether it will have a net negative, positive, or neutral charge. Ph < pi = amino acid is positive. Ph > pi = amino acid is negative. Ph = pi = amino acid neutral. Suppose we have a column containing carrying negative charge (cation exchangers). We want to separate a mixture of 3 kinds of molecules (histidine (pi = 7. 6), serine (pi=5. 7), aspartic acid (pi=3)) This mixture is in a buffer (ph = 3. 25) The aspartic acid will have a negative charge (no binding to the ion exchanger) and will be eluted quickly through the column.
