Protein
A protein is a biological polymer comprising a number of different a-amino acids joined together by peptide bonds. The peptide bond is formed by the condensation of the carboxyl group of one amino acid with the amino group of a second amino acid. The condensation of a few amino acids (10-20) produce small biopolymers which are called peptides; the condensation of peptides in the same manner yield polypeptides and the condensation of polypeptides yield proteins that can have extremely high molecular weights. There are 20 a-amino acids associated with mammalian proteins. The groups attached to the peptide bond may be dispersive (cf London’s dispersive forces or hydrophobic), polar (or hydrophilic) or ionic. If dispersive groups dominate in the protein’ then the overall property of the protein will be dispersive in character (hydrophobic) and interact readily with dispersive substances. If polar groups dominate in the protein molecule it will be polar in character (hydrophilic) and interact readily with polar substances. Proteins are separated by liquid chromatography using short chain dispersive bonded phases as the stationary phase. The use of short chains is usually necessary as longer chains can have excessively strong interaction with the dispersive parts of the protein which can cause protein denaturation.
Author: RPW Scott
Book:Principles and Practice of Chromatography
Section:Principles Available-Stationary-Phase Chiral
, irrespective of the type of support material, the
amount of stationary phase in an LC column is primarily determined by its
surface area. In addition, the amount of available stationary phase on a bonded
phase can be modified by adjusting the molecular size (chain length) of the
bonded material.
The chain
length of the bonded material can be critical when separating proteins as
dispersive interactions between the alkane chains and the dispersive
(hydrophobic) groups of the protein can be strong enough to cause structural
deconformation; (i.e., the protein becomes denatured). Reducing the
chain length of the bonded material, the dispersive forces can be reduced
significantly and the deconformation diminished. In practice, carbon chains
only two or four carbon atoms long are among those most commonly used for
separating labile proteins.
Stationary Phase Limitation by Chiral Selectivity.
The extent to
which an enantiomer can interact with the
Principles Available-Stationary-Phase Chiral
Author: RPW Scott
Book:The Mechanism of Chromatographic Retention
Section:Retention Chiral-Chromatography Chiral-Polysiloxane-Stationary-Phases
Bayer and his
coworkers (19), synthesized a stationary phase that consisted of a chiral agent
attached by an amide linkage to a carboxyl group of a polymer matrix of
dimethylsiloxane or (2-carboxypropyl)-methylsiloxane. This combined the chiral
selectivity of L-valine-t-butylamide with the high thermal
stability and low volatility of the polysiloxanes. This stationary phase could
be used over the temperature range of 30˚C to 230˚C. and would allow
the separation of all the racemic protein amino acids to be separated in one
chromatogram taking only about 30 min. An example of the separation of the N-(O,S)-pentafluoro-propanoyl-isopropylesters
of the amino acids, employing this phase system, is shown in figure 32. It is
seen that the column bleed is quite manageable at 185˚C and that a clean
separation of all the enantiomers is achieved. Chiral polysiloxanes can also be
prepared in a number of ways. For example, the cyano groups of OV-225 can
be hydrolyzed and the
Retention Chiral-Chromatography Chiral-Polysiloxane-Stationary-Phases
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Applications
to provide adequate peak capacity (see
Plate Theory and Extensions).
The particle diameter of the packing was 6 mm and thus, at the
optimum velocity, an efficiency of about 25000 theoretical plates
should be produced. It is seen in figure 61 that the dead volume time is about
19 minutes and a flow-rate of 1.0 ml/min this would be equivalent to a dead
volume of 19.5 ml.
A more typical
application of micro-reticulate resins in exclusion chromatography is shown by
the separation of a standard protein mixture depicted in figure 62.
Courtesy of Asahipak Inc.
Figure 62.
The Separation of a Standard Protein Mixture by Exclusion Chromatography on a
Vinyl Alcohol-Styrene Co-Polymer Hard Gel
HPLC Applications
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC HPLC-Mobile-Phases
Due to their
being stable to extremes of pH, their use for the separation of peptide and
proteins at both high and low pH has been well established. An example of a
macro-reticulated resin phase used as an exclusion medium in the separation of
a crude protein extract is shown in figure 39. The column, 9TSKgel QC-PAK GFC
399GL0, was 15 cm long and 8 mm in diameter and the separation was carried out
at a flow rate of 1 ml/min. The mobile phase consisted of 0.5M NaCl in 0.05M
sodium phosphate buffer at pH 7.0. The sample was 5ml of a rat liver extract. An excellent separation based on
molecular size is obtained from which the molecular weight range of the mixture
and even that of the individual components could be estimated.
LC Mobile
HPLC HPLC-Mobile-Phases
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Macroporous-Polymers
with almost any desired pore size, ranging from 20Ĺ to 5,000Ĺ.
Underivatized, they exhibit strong dispersive type interactions with solvents
and solutes together with some induced polarizability arising from the aromatic nuclei
in the polymer if strongly polar solutes are being separated. Consequently, the untreated resin has
found some use as an alternative to the C8 and C18 reverse phase columns based
on silica.
Courtesy of TOSOHAAS Inc.
Figure 39.
The Separation of a Crude Protein Extract by Exclusion on a Micro-Reticulated
Resin Column
HPLC Macroporous-Polymers
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC HPLC-Mobile-Phases Aqueous-Solvent-Mixtures
gradient
will compensate for the strongly concave form of the unassociated
methanol concentration profile shown in figure 47 which will be the strongest
eluting component of the mobile phase. The strong association of methanol with
water could also account for the fact that proteins can tolerate a significant
amount of methanol in the mobile phase before they become denatured. It is
clear that this is because there is virtually no unassociated methanol present
in the mixture which could cause protein denaturation since all the methanol is
in a deactivated state by association with water.
Katz,
Lochmüller and Scott also examined acetonitrile/water, and
tetrahydrofuran(THF)/water mixtures in the same way and showed that there was
significant association between the water and both solvents but not to the same
extent as methanol/water. At the point of maximum association for methanol, the
solvent mixture contained nearly sixty percent of the methanol/water associate.
In contrast
HPLC HPLC-Mobile-Phases Aqueous-Solvent-Mixtures