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TRIONE® Ninhydrin Reagent Bulletin 12
A Prepared Reagent for Automated Post-column Derivatization
of Primary and Secondary Amines
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TRIONE Ninhydrin reagent is specially formulated for amino acid
analysis. It is so stable that it does not require refrigeration,
either in shipment, storage, or in the reservoir. Quantitation is
consistent from the first to the last mL, so there is no waste.
The high signal-to-noise ratio of TRIONE, when compared to DMSO-containing
reagents, permits detection sensitivity to be increased with minimum
increase in background noise—a feature particularly appreciated
at sample concentrations of <50 picomoles.
Two preparations are available to suit your usage and storage requirements:
T100
T200
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Combine two solutions, swirl, and use.
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Twelve-month shelf life before mixing; one
month in the reservoir.
TRIONE Ninhydrin Reagent is a proprietary formulation containing
Ninhydrin, Hydrindantin (reduced Ninhydrin), a Lithium Acetate buffer,
and Sulfolane, a water-miscible organic solvent. The solvent is
necessary to maintain the solubility of both the Hydrindantin and
the primary amine product, Ruhemann’s Purple. The buffer is
required because the reaction is pH dependent. The active ingredients—Ninhydrin
and Hydrindantin—are required for proper development of secondary
and primary amines, respectively.
Ninhydrin is a selective oxidizing agent which causes oxidative
decarboxylation of  -amino
acids, producing CO2, NH3, and an aldehyde
with one less carbon atom than the parent amino acid. The reduced
ninhydrin then reacts with the liberated Ammonia to form Ruhemann’s
Purple, a complex which maximally absorbs light at 570 nm. Secondary
amines, Proline and 4-Hydroxyproline, react via a different path
and form a yellow derivative with an optimal absorbance at 440 nm.
Since the reaction with amines is highly specific and the absorption
characteristics of the formed chromophores follow Beer’s Law,
reagents based on Ninhydrin have long been the most popular choice
for detection and quantitation of amines and amino acids.
Ninhydrin reacts slowly at room temperature. Consequently, in automated
amino acid analysis,
elevated temperatures of up to 130 °C are employed to reduce
the conversion time to about one minute. This reduction in dwell
time results in minimal band spreading with no sacrifice in reproducibility.
Detection of the derivatives can be accomplished with variable
wavelength detectors or with fixed filter pho-tometers. Background
subtraction at 690–700 nm is sometimes used to improve signal-to-noise.
TRIONE AGING
The three-month aging process for TRIONE begins when it is made.
On storage an unopened bottle of TRIONE T100 does not lose potency.
Rather, signal-to-noise improves approximately 0.1% per day. This
is due to a change in the Hydrindantin. The changed form of Hydrindantin
is less soluble and therefore is more likely to precipitate in the
heated reactor.
TRIONE T200 can be stored unopened for 12 months without
change.
TRIONE OXIDATION
Upon exposure to air (O2) TRIONE can become oxidized,
thereby losing potency for primary amines. Depending on the exposure,
part of or all of the Hydrindantin will be oxidized to Ninhydrin.
Only primary amine development requires Hydrindantin. Consequently,
as the Hydrindantin disappears, so will the primary amine signal.
To reduce the potential for oxidation it is of critical importance
not only to maintain the reservoir under N2, but also
to ensure that only air-impermeable tubing is connected between
the gas regulator and reservoir, and between the reagent pump and
reservoir. Either Saran ® or PEEK may be used, with Saran the
more cost-effective alternative. Saran has approximately 1/1000
the oxygen permeability of fluorocarbon tubing.
Secondary amine development requires no Hydrindantin, so upon oxidation
no change in signal occurs for secondary amines, e.g., Proline and
4-Hydroxyproline. However, since the background color of TRIONE
is proportional to the Hydrindantin concentration (
max = approx. 400 nm), and the secondary amine absorption is near
( max = 440
nm), the loss of primary signal makes the secondary signal appear
greater than usual for oxidized TRIONE.
STORAGE AND HANDLING
Both TRIONE T100 and T200 Ninhydrin reagents are sensitive to oxygen.
They should be stored at room temperature (20–25 °C) in
the original, unopened containers.
T100 is guaranteed to be usable for three months from the time
it is manufactured. Detailed instructions on the use of TRIONE in
an amino acid analyzer accompany each bottle, or case of four bottles.
In some automated amino acid analyzers such as the Beckman System
6300, refrigeration during storage and use of the Beckman Nin-RX
reagent is mandatory. TRIONE may be employed under refrigeration
in the System 6300 as long as it is maintained above 5 °C. If
it is necessary to keep the samples at a lower temperature, then
the Ninhydrin reservoir should be placed in the pump compartment
of the analyzer.
The bottles in which TRIONE T100 and T200 are shipped are not pressure-rated,
and must not be used as a reservoir. We recommend installing the
Pickering Reservoir Assembly, which can be pressurized safely to
0.15–0.3 bar with Nitrogen.
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