PTT 202:
ORGANIC CHEMISTRY FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
helya@unimap.edu.my
Amino acids are organic molecules of low relative molecular mass
(approximately 100-200)
• Contain at least one carboxyl (COOH) and one amino (NH2) group.
• Essential constituents of plant and animal tissues.
There are several methods available for the quantitation of amino
acids
• Mostly only give information about the total amino acid content of the sample
regardless of whether one or several amino acids are present.
• Cannot differentiate between the individual components.
However, for detection a particular amino acid in the presence of
others , the most useful methods are employed
• This include chromatographic of electrophoretic technique for the separation of
various amino acids in the sample.
• Calorimetric or fluorimetric methods for the qualitative and quantitative
determination of each component.
Thin-layer
chromatography
Electrophoresis
Techniques/
Methods
Gas-liquid
chromatography
Amino acid
analyzer
Paper chromatography has been used successfully for many
years and is still a useful tool due to:
• Larger volumes of sample can be applied to paper, permitting the
subsequent elution of a particular amino acid for further purification and
analysis.
• This is importance in the identification of an unknown sample
constituent.
Prior to chromatography, it may be necessary to remove
interfering substances such as protein, carbohydrates and
salts.
• This may be done using an ion-exchange resin, solvent extraction,
dialysis or protein precipitation.
The identification of an amino acid is achieved by
comparison of RF values with those of reference solutions.
The use of at least three different solvent systems is
recommended.
• The nature of the amino acids is an important factor in the choice of a
solvent and different solvent will permit better resolution of acidic, basic
and neutral components (Table 1).
• In general, increasing the proportion of water in the solvent will increase
all RF values and the introduction of small amounts of ammonia will
increase the RF values of the basic amino acids.
Table 1:
Solvents for both
paper and thin-layer
chromatography
The resolving power of paper or thin-layer chromatography can
be increased by the use of two-dimensional techniques, which
involve the use of two different solvent systems.
Two-dimensional separations permit the resolution of large
numbers of amino acids present in a sample and those having a
similar mobility in one dimension will usually be separated from
each other in the second.
• This is especially useful in the detection of components that are present only in
low concentrations and might be obscured in one dimension by other amino
acids that are present in higher concentrations.
The fact that different amino acids carry different net
charges at any particular pH permits mixture to be
separated using low or high voltage electrophoresis.
• Separations at high voltages can be achieved more quickly than at low
voltages.
One of the principal advantages of the former is that salts
and other substances that may be present in the sample
affect the quality of electrophoretogram to a less extent.
• This permits the separation of amino acids in relatively crude extracts
and untreated fluids.
Although electrophoretic separations can be achieved using
buffers over a wide range of pH values, in practice the pH
values chosen are either pH 2.0 or pH 5.3.
• At pH 2.0 all amino acids will carry a positive charge and the basic amino
acids, having the highest positive charge, will migrate furthest toward the
cathode.
• At pH 5.3 migration will occur towards both electrodes depending on the
charge carried. Separations at ph 5.3 are particularly useful to determine the
acidic or basic nature of an unknown amino acid or dipeptode.
For separating similar amino acids and short peptides
• A two-dimensional technique involving initial separation by high voltage
electrophoresis at pH 2.0 followed by chromatography is a useful means.
• Does not require desalting or excessive purification of the sample.
Derivatives of amino acids are required because
• Amino acids are not themselves sufficiently volatile for gas-liquid
chromatography
• Difficulties may be encountered in the choice and method of derivatization.
Several techniques involves for derivatization are as follows:
• The derivatization of trimethylsilyl (TMS) derivatives is carried out by the addition
of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) in acetinitrile and heating for
aprrox. 2 h at 150˚C under anhydrous conditions in a sealed tube.
• The formation of n-butyl esters of the amino acids and their subsequent
trimethylsilylation. The n-butyl esters are formed by heating the amino acids for
15 min in n-butanol and HCl and these are then converted to the N-TMS-n-butyl
ester derivatives.
• Acetylation of the butyl, metyl or propyl esters of amino acids, to give
trifluorocetyl (TFA) or heptafluorobutryl (HFB) derivatives can be performed by
reacting them with either TFA or HFB anhydrides in methylene dichloride at 150˚C
for 15 min.
Spackman, Stein and Moore developed the first amino
acid analyser based on separation by ion-exchange
chromatography and quantitation of each component in
the column effluent using the ninhydrin reaction.
The instruments currently available are based on their
original design, permitting buffers of varying pH or ionic
strength to be pumped through a thermostatically
controlled resin column (Figure 1).
Figure 1:
Schematic diagram of an
amino acid analyser using
the ninhydrin reagent for
quantitation.
The separation take place in a column of sulphonated
cross-linked polystyrene resin, which is a strong cationic
exchanger.
The matrix of the resin is strongly anionic in nature
(SO3-) and at low pH used initially, the amino acids will
be positively charged and will be attracted to the
negatively charged sulphonate groups.
As the pH of the buffer passing through the column is
raised, the amino acids will be differentially eluted as
their net positive charge diminishes and they are less
strongly attracted to the sulphonate sites on the resin
matrix.
At pH 3.25, the amino acids with an extra acidic
group in their side chain will have very little
affinity for the resin and will be the first to
emerge from the column.
At the same pH value, those amino acids
whose side chain contains an extra ionizable
group capable of carrying a positive charge,
will be strongly held on the resin and will be
eluted from the column only as the pH is
raised substantially and their net positive
charge reduced.
The determination of the relative elution position of
the amino acids, not only depends on the pH of the
eluting buffer but also the cation concentration of the
buffer.
Sodium citrate buffer solutions are commonly used
and the positive sodium ions compete with the
positively charged amino acids for the sulphonic acid
on the resin.
Although the amino acids have a considerable affinity
for the resin, the sodium ions are constantly present in
a much higher concentration, and as a result, the
equilibrium of the equation is shifted to the right and
the amino acids are displaced from the resin.
Thus, the molarity of the eluting buffer affects elution
and when the ionic concentration of the buffer is
increased, the amino acids are eluted more rapidly
from the column.
The quantitative resolution of amino acid
mixtures can be achieved by varying the
composition of the buffer flowing through the
column by either:
• Increasing the pH and maintaining a constant
molarity (cationic concentration).
• Keeping the pH constant but varying the
molarity.
• Using combination of both of the above.
The colour or fluorescence produced per mole of amino acids
varies slightly for different amino acids and this must be
determined for each one to be quantitated.
• This is done by loading a mixture of amino acids containing the same
concentration of each amino acids including the chosen internal standard and
from the areas of the peaks on the recorder trace calculating each response
factor .
An internal standard should always be used for every analysis
carried out. This is an amino acid that is known to be absent from
the sample under investigation.
• If the amount of internal standard which was added to the sample is known, the
concentration of the unknown amino acid can be determined using peak area
relationships.
Figure 2: Amino acid analyser trace
THANK YOU..
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