KOH + chloroform/methanol=precipitate?
BiGt
Part of my work involves tissue digestion using 12M KOH initially. The next
step involves the addition of chloroform/methanol (2:1) to de-lipidise the
tissues. However, I am left with a white precipiate/flocculate which is
incredibly difficult to remove. Does anybody have any idea what this
precipitate may be and how to remove it?
I also use acetone in the procedure, will this be causing the problem?
Thanks
TOne.
mike crimmins
in the presence of 12 M KOH. I am reaching here but the chloroform might react
with concentrated base to form a dichlocarbene which would insert into whatever
it wants to.
Where does the acetone come in? I would eliminate that variable first.
mike crimmins
Uncle Al
>
> Hi,
>
> Part of my work involves tissue digestion using 12M KOH initially. The next
> step involves the addition of chloroform/methanol (2:1) to de-lipidise the
> tissues. However, I am left with a white precipiate/flocculate which is
> incredibly difficult to remove. Does anybody have any idea what this
> precipitate may be and how to remove it?
>
> I also use acetone in the procedure, will this be causing the problem?
12 M KOH and CHCl3 will deprotonate the chloroform. CCl3(-) will
alpha-eliminate to dichlorcarbene (:CCl2) and you will get addition to
double bonds to dichlorocyclopropanes (aromatic systems go to
dichloronorcaradienes), as well as insertion into sigma bonds.
12 M KOH will aldol condense/dimerize acetone into acetonyl alcohol. 12
M KOH plus CHCl3 plus acetone will deprotonate the chloroform, the anion
will attack the carbonyl, and you will get 2,2,2-trichloro-t-butanol.
None of this gives you a flocculent white ppt difficult to handle. Try
ether in place of chloroform and see if you get the same problem. How
mucb calcium and phosphate are in the tissue?
--
Uncle Al Schwartz
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Laura Moen
the carbohydrates which modify them. Many proteins/glycoproteins are insoluble
in organic solvents, so it can be a useful method for eliminating things you
don't want during a purification process.
BiGt wrote:
> Hi,
>
> Part of my work involves tissue digestion using 12M KOH initially. The next
> step involves the addition of chloroform/methanol (2:1) to de-lipidise the
> tissues. However, I am left with a white precipiate/flocculate which is
> incredibly difficult to remove. Does anybody have any idea what this
> precipitate may be and how to remove it?
>
> I also use acetone in the procedure, will this be causing the problem?
>
> Thanks
>
> TOne.
Dr. Steve Simpson
>
> Acetone should form a condensation product that would likely end up as a gunk
> in the presence of 12 M KOH. I am reaching here but the chloroform might react
> with concentrated base to form a dichlocarbene which would insert into whatever
> it wants to.
> Where does the acetone come in? I would eliminate that variable first.
> mike crimmins
>
>
> > Hi,
> >
> > Part of my work involves tissue digestion using 12M KOH initially. The next
> > step involves the addition of chloroform/methanol (2:1) to de-lipidise the
> > tissues. However, I am left with a white precipiate/flocculate which is
> > incredibly difficult to remove. Does anybody have any idea what this
> > precipitate may be and how to remove it?
> >
> > I also use acetone in the procedure, will this be causing the problem?
> >
> > Thanks
> >
> > TOne.
base - there is a very high risk of a runaway free radical reaction
which could ignite or explode. The dichlorocarbene produced from
base+chloroform is very reactive towards acetone.
The precipitate from acetone and base may be diacetone alcohol (mp. 164
deg C). It is soluble in water.
--
Dr. Stephen J. Simpson Senior Admissions Tutor
Department of Chemistry Senior Lecturer in Chemistry
University of Salford Tel: (44) 161 295 5978
Salford. M5 4WT Fax: (44) 161 295 5111
United Kingdom.
E-mail: S.J.S...@chemistry.salford.ac.uk
Jeff E. Janes
>
> Hi,
>
> Part of my work involves tissue digestion using 12M KOH initially. The next
> step involves the addition of chloroform/methanol (2:1) to de-lipidise the
> tissues. However, I am left with a white precipiate/flocculate which is
> incredibly difficult to remove. Does anybody have any idea what this
> precipitate may be and how to remove it?
>
> I also use acetone in the procedure, will this be causing the problem?
>
What is the condition of your sample at this point? Is it biphasic, or does
methanol serve as a bridge to make the water and chloroform miscible? If
it is biphasic, where is the ppt., in the aqueous or organic layer? As others
have pointed out, chloroform and KOH are not a particularly happy combination.
Is this a published protocol you are following?
In extracting DNA solutions with chloroform, it is normal to
get white gunk between the chlororm and water, which is contaminating protein. We
often do extractions with phenol(T.E. equilibrated)/chloroform and water, because
protein is more soluble in phenol/chloroform than chloroform alone. However, I
wouldn't recommend adding phenol to your witched brew above without checking it out
safety-wise first. Sometimes I just spin the solution for a long time in eppendorfs,
which compacts the interphase to a managable layer.
Bye
Jeff
Fred Flintstone
>
> Hi,
>
> Part of my work involves tissue digestion using 12M KOH initially. The next
> step involves the addition of chloroform/methanol (2:1) to de-lipidise the
> tissues. However, I am left with a white precipiate/flocculate which is
> incredibly difficult to remove. Does anybody have any idea what this
> precipitate may be and how to remove it?
>
> I also use acetone in the procedure, will this be causing the problem?
>
>
> TOne.
Could this possibly be finely divided KCl??
Fred
c.p
in the MeOH/CHCl3 or aqueous layer?