6.2-2
Section 6
ApplicAtions
Pinnacle Operators Manual
Pickering Laboratories Inc.
An alternative reagent system based on o-phthalaldehyde (OPA) can be used for high-sensitivity detection of
primary amino acids. OPA reacts rapidly with primary amines and Thiofluor
™
(N,N-dimethyl-2-
mercaptoethylamine) under mild basic conditions to produce a strongly fluorescent isoindole derivative
(Figure 6.2-B). OPA does not react with secondary amines or aryl amines, so fails to detect Proline and other
secondary amino acids.
However, it is possible to detect secondary amino acids by using a two-step reaction in which they are first
oxidized and then reacted with OPA. This technique has some disadvantages, and is not often used. Contact
Pickering for details.
The Pickering Pinnacle PCX derivatization instrument for fluorescent detection of amino acids is similarly
designed to the ninhydrin instrument, except that it contains a 150 µl reactor and the reacton is carried out
at 45°C.
Basic Sample Preparation
The following is a general sample preparation for Physiologic Fluid and Protein Hydrolysate samples. There are
many more types of samples that can be used for amino acid analysis. For details, consult the AOAC methods
and laboratory procedures.
Native amino acids are those found “free” in samples such as serum, urine and other physiological fluids, plant
extracts, foods and beverages. Although preparation of these samples for amino acid analysis is much simpler
and less time-consuming than protein hydrolysis, control of pH and normality, and removal of soluble protein
are critical factors which can affect the chromatography.
OO
-
OPA
H
NH
3
Fluorescent Isoindole
R
+
Thiofluor
HS
COOH
1
0
Amino Acid
pH>9, ambient T
CHO
CHO
S
H
N
CH
3
CH
3
N
R
N
CH
3
CH
3
Figure 6.2-B
To Next Page
Loading...