Dear Shuming
You are right; do not try to dry the picric acid. I still have a copy of the
procedure as used at Torry Research Station and the section on preparation
of reagents states:
"Approximately 2% picric acid stock solution. Picric acid is normally stored
under water and is unstable when dry. Remove some crystals from the bottle
and dry as much as possible and dry as much as possible by pressing between
filter paper. Weigh out 3g of the damp crystals and add to 100ml of toluene.
Shake to dissolve."
The weight of damp crystals to take agrees more or less with that
recommended by Wekell, and the concentration of picric acid is not critical
anyway. Be careful how you dispose of any unused crystals and the filter
paper; down the sink with plenty of water.
There are a couple of other points with regard to the picrate procedure
which are in my opinion not adequately dealt with in descriptions of
procedure.
The TMA in extract after making alkaline is extracted into toluene and the
calibration curve is prepared by similarly extracting standard solutions of
TMA into toluene. You should note that the extraction of TMA into the
toluene phase is not complete. The proportion is high, around 90% depending
on the concentration of alkali used, and is affected by the total solids in
solution - a salting out effect. The fish extract is prepared in a TCA or
perchloric acid solution whereas the procedures describe the TMA standards
being prepared in dilute hydrochloric. I recommend that the working TMA
standards be diluted from the stock using TCA or perchloric acid of
approximately the same concentration as in the extract. Similarly extracts
should be diluted with this solution if they are of high concentration and
need to be diluted into the working range. This brings me to the other
consideration.
Typical demersal fish muscle contains about 80% water. A common extraction
ratio for TMA determination is 1:3 fish muscle:extractant. Starting with,
say, 100g of fish the final volume of the aqueous phase is 380ml. In my
opinion the TMA in the 100g is dissolved in 380 ml and the amount in the
aliquot taken for analysis should be scaled up to this volume to give the
concentration in the muscle. This is the procedure always used in the
laboratory I worked in, but some published procedures, including the AOAC
procedure, scale up to the total mass of the extraction blend, 400g in the
case of my example. This latter procedure will overestimate the
concentration by around 5% compared with the former. The procedure as
described by Dyer scales up to total mass, (using 200ml of TCA solution in
this case, i.e scaling up to 300 rather than 280). Most papers on TMA
contents do not declare the how final concentration are calculated or
adequately cite the reference for the procedure used. These comments apply
also to determination of TVB by distillation of extracts.
Another consideration for reproducible results is to standardise how the
sample is taken from the fish. The TMA concentration within a fillet as the
fish spoils and the TMA increases varies throughout the fillet. The highest
concentration as one might expect are around the belly cavity and lowest
near the tail. In the case of a small fish perhaps the whole fillet can be
taken, in a large fish perhaps standardise by taking a section above the
belly cavity, but not including the belly flaps.
Peter Howgate
----- Original Message -----
From: "Shuming Ke" <shumingk@foodsci.umass.edu>
To: "Seafood research and extension information exchange"
<seafood@ucdavis.edu>
Sent: Friday, October 05, 2007 4:53 PM
Subject: how to measure TMA in fish tissue by Dyer method
> hi,
> I plan to use picrate method described by Dyer (j fish res bd can,
> 1945:351-
> 358) to determine TMA content in marine fish tissue. The basic concept is
> to
> react TMA extracted in toluene with 0.02% picric acid and measure
> absorbance
> at 410nm. In Dyer' method, dry picric acid is disolved in toluene to
> prepare
> 2% picric acid stock solution in toluene. Since dry picric acid is highly
> explosive and shock sensitive, this raises the question-- how to prepare
> picric acid solution in toluene? Do I just order dry (moisture free)
> picric
> acid solid and disolve in toluene? Any one has practical ideas on how to
> carry
> on the analysis? Many thanks.
> --
> Shuming Ke, Ph.D.
> Research Associate
> University of Massachusetts Marine Station
> P.O.Box 7128
> 932 Washington Street
> Gloucester, MA 01930
> shumingk@foodsci.umass.edu
>
> Office: 978-281-1930 x. 2
> Fax: 978-281-2618
>
>
>
>
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