Analytical Dairy Chemistry manual

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reservoir containing 500 ml of 0.1 mol/ litre HCl. Adjust the height of the reservoir to give a
flow rate of about 1 ml/ min and collect a total of forty 2 ml fractions. Test five of the tubes
at a time for the presence of amino acids by spotting a sample from each tube on to a filter
paper: dip this in the acetone solution of ninhydrin and heat in an oven at 105�c. If amino
acids are present, they will show up as blue spots on the filter paper. When the first amino
acid has been eluted, remove the reservoir of 0.1 mol/ litre HCl and allow the level of acid
to fall to just above the resin. Run 2 ml of 0.2 mol/ litre tris- HCl buffer (pH 8.5) on to the
column, then connect to a reservoir of this buffer and continue with the elution until the
third amino acid is removed from the column.
Detection of amino acids: Adjust the pH of each tube to 5 by the addition of a few drops of
acid or alkali. Add 2ml of the buffered ninhydrin reagent and heat in a boiling water bath for
15 min. cool the tubes to room temperature, add 3 ml of 50 percent v/v ethanol and read
the extinction at 5570 nm after allowing the tubes to stand for 10 min. set up the appropriate
blanks and standards, and plot the amount of amino acid in each fraction against the volume
eluted.
Applications:
Ion exchange has proved to be one the major methods of fractionation of labile
biological substances. It is used most widely for protein and peptide separations, enzyme
purification, hormone preparation, studies, carbohydrate and lipids separations and vitamin
and co enzyme work etc. the special characteristic of ion exchange is that the separation is
based on charge, so that when it is combined with techniques which use other criteria for
separation, such as gel filtration (size) or affinity chromatography (bio selectivity), it is a very
powerful analytical and preparative tool.
97
EXERCISE 28
AFFINITY CHROMATOGRAPHY
Introduction:
Affinity chromatography is a unique separation technique that does not rely on
differences in the physical properties of the molecules to be separated. Instead, it exploits
the unique property of extremely specific biological interactions to achieve separation and
purification. As a consequence, affinity chromatography is theoretically capable of giving
absolute purification, even from complex mixtures, in a single process. The technique was
originally developed for the purification of enzyme, but it has been extended to nucleotides,
nucleic acids, immunoglobulin, membrane receptors and even whole cells and cell fragments.
The technique requires that the material to be isolated is capable of reversible
binding to a specific ligand which is attached to an insoluble matrix.
M + L k + 1 ML
Macro ligand complex
Molecule (attached to k - 1
Matrix)
Under the correct experimental conditions when a complex mixture containing the specific
compound to be purified is added to the insolubilized ligand, generally contained in a
conventional chromatography column, only that compound will bind to the ligand. All other
compounds can therefore be washed away and the compound subsequently recovered by
displacement from the ligand (fig 28.1).
Practical procedure:
The procedure for affinity chromatography is similar to that used in other forms of
liquid chromatography. The ligand treated matrix is placed into column in the normal way
for the particular type of support. A buffer is used which will encourage the binding
macromolecule to be strongly bound to the ligand. The buffer generally has a high ionic
strength to minimize non specific adsorption of polyelectrolyte ligand. The buffer must
also contain any co factors such as metal ions necessary for ligand macromolecule
interaction. Once the sample has been applied and the macromolecule bound, the column is
eluted with more buffers to remove non specifically bound contaminants. The purified
compound is finally recovered by either specific or non specific elution. Non specific
elution may be achieved by a change in either pH or ionic strength. pH shift elution using
dilute acetic acid or ammonium hydroxide results from a change in the DNA by a similar
procedure. The method has also been used to isolate whole genes by hybridizing practically
single stranded DNA with the complementary mercurated mRNA which is then reacted
with a thioleted matrix.
98
EXERCISE 29
GAS LIQUID CHROMATOGRAPHY
Introduction:
Gas liquid chromatography has also been to as vapour phase chromatography, gas
liquid partition chromatography, gas partition chromatography, gas chromatography,
vapour fractometry and by several similar designations.
This technique, which is based upon the distribution of compounds between a liquid
and a gas phase, is a widely used method for the qualitative and quantitave analysis of a large
number of compounds size it has high sensitivity, reproducibility and speed of resolution. It
has proved to be most valuable for the separation of compounds of relatively low polarity. A
stationary phase of liquid material such as silicone grease is supported on an inert granular
solid. This material is packed into a narrow coiled glass or steel column 1 3m long and 2
4mm internal diameter through which is passed an inert gas (the mobile phase) such as
nitrogen or argon. The column is maintained in an oven at an elevated temperature which
volatilizes the compounds to be analyzed. The basis for the separation of compound being
analyzed is the difference in the partition coefficient of the volatilized compounds as they are
carried through the column by the gas. As the compounds leave the column they pass [ Pobierz całość w formacie PDF ]

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