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BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE

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Offline Michael

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PostPosted: Thu Oct 30, 2008 3:50 pm   Post subject: BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE   

INHIBITION OF BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE


By Erina J. M. Kent,1 M.Sc.; Douglas A. Elliot,1,2 Ph.D.; and Gordon M. Miskelly,1 Ph.D.:

JOURNAL OF FORENSIC SCIENCES


Quote:
ABSTRACT: The luminol chemiluminescence presumptive test for blood is based on the mild peroxidase activity of hemoglobin in basic peroxide
solution. However, this test is subject to interference by strong oxidants, certain transition metal ions, and true peroxidases. This paper reports
methods for reducing the interference caused by hypochlorite-containing bleaches. Amines such as 1,2-diaminoethane react rapidly with hypochlorite
without interfering significantly with the hemoglobin-catalyzed oxidation. Thus, addition of 0.1 mol/L 1,2-diaminoethane to a standard luminol-
peroxide spray lead to almost complete inhibition of hypochlorite-induced chemiluminescence while satisfactory chemiluminescence was still
observed from bloodstains. If time allows, an alternative method for reducing interference from hypochlorite bleach is to wait several days until the
bloodstains have dried thoroughly, by which time the hypochlorite will have decomposed.
KEYWORDS: forensic science, criminalistics, chemiluminescence, bloodstains, luminol

The luminol-hydrogen-peroxide test for blood can be subject to
interferences from peroxidases, metal ions, or oxidants (1). The
most likely oxidants to be found at a crime scene are hypochloritebased
bleaches. The presence of these on surfaces being sprayed
leads to bright flashes of chemiluminescence, as opposed to the
more gradual development of chemiluminescence by blood. Although
an experienced forensic scientist can usually differentiate
between blood and bleach-induced chemiluminescence this will
not always be possible (personal observation, D. Elliott, (2,3)). In
addition, the time-averaging implicit in long-exposure photography
can lead to the two sources of chemiluminescence appearing
identical. A recent paper has reported that the spectra of the bleachinduced
and blood-induced chemiluminescence are slightly different
(4), but the small shift in emission wavelength combined with
the broadness of the emission band mean that spectral filtering is
unlikely to be able to separate the two causes.

We have investigated chemical methods for reducing the effect
of hypochlorite-containing bleaches on luminol chemiluminescence.
Antioxidants such as ascorbic acid or polyphenols (5–7)
cannot be used because they also inhibit the blood-catalyzed luminol-
peroxide chemiluminescence. Therefore we sought to target
the chlorine donor properties of hypochlorous acid (the conjugate
acid of hypochlorite), since this is a different mode of oxidation
from the mechanism occurring with peroxide. The reaction of
amines with hypochlorous acid to form chloramines (Eq 1) is a
well-known process (8,9).

RRNH  HOCl → RRNCl  H2O R, R  alkyl or H (1)

Indeed, analytical procedures for amines have been reported
based on the inhibition of the chemiluminescence observed when
hypochlorite oxidizes luminol (10). Other studies have shown that
although primary and secondary amines inhibit oxidative chemiluminescence,
tertiary amines actually increase the chemiluminescence,
presumably via catalytic formation of the N-chlorotrialkylammonium
ions that act as reactive oxidants (11,12). We have
investigated whether primary and secondary amines can inhibit the
chemiluminescence due to hypochlorite under the conditions typical
of forensic luminol sprays, and whether the presence of amines
has an effect on the heme-catalyzed luminescence of luminol.

The choice of amines was guided by studies by Margerum (8,9)
and Antelo (13,14), who showed that the reaction rate between
hypochlorite and amines is pH dependent and depends on the basicity
of the amine. The observed pH dependence, Fig. 1, occurs
because the reaction involves specifically the amine (RRNH) reacting
with hypochlorous acid (HOCl) in the reaction, as shown in
Eq 1, whereas the protonated amine (RRNH
2) and the deprotonated
hypochlorite (OCl) are much less reactive (9). The reactivity
of many with hypochlorous acid amines can be fitted by a curve
given by Eq 3 (13),
Rate  k[HOCl][RRNHn] (2)
k  (2.9  0.5)  (0.48  0.05)
pKa(RRNH2
(n1)) L mol1 s1 (3)
where pKa(RRNH2
(n1)) is the negative logarithm of the acid dissociation
constant of the conjugate acid of the amine. The only
amine that was reported to deviate significantly from this relationship
was ammonia, which had a rate nine times lower than would
be predicted by this equation (9).

If the behavior of this reaction rate is examined as a function
of total hypochlorite and total amine species Eq 2 can be rewritten
in the form
Rate  k[HOCl]total[RRNH]total (2a)
k  k 
  (4)
where k would be the observed rate constant, [HOCl]total 
[HOCl]  [OCl], [RRNH]total  [RRNHn]  [RRNH2
(n1)],
and Ka(HOCl)( 3.6  108) is the acid dissociation constant of
hypochlorous acid. A plot of k vs. pH, Fig. 1, shows the variation
in the rate of this reaction with pH and with the basicity of a given
amine. It is seen that amines with low pKas (e.g., tris, pKa(trisH)
 8.1) have a high observed rate constant at their pH optimum, but
this rate decreases rapidly (10-fold per pH unit) at higher pH. In
contrast, basic amines have a lower maximum observed rate constant
but this value is maintained over a greater pH range. At any
given pH Eq 4 predicts that the optimum amine will be that for
which pKa  pH 0.03 i.e., pKa  pH. Therefore strongly basic
amines will be the most effective competitors for hypochlorite under
the conditions of common forensic sprays (those reported by
Grodsky and Weber both lead to pH  10). Luminol itself can react
with hypochlorous acid, (15,16) and this is indicated in Fig. 1.
The observed rate constant for the direct reaction of luminol with
HOCl is predicted to be comparatively low at high pH based on the
above analysis. This may be incorrect, because luminol has a second
deprotonation with pKa  15 (17), and the basic form may
show higher reactivity towards HOCl. Indeed, one literature report
shows that the predicted behavior in Fig. 1 is followed until pH 
11.5, where an increase in rate was observed (15). Figure 1 also
shows a rate constant-pH curve for hydrogen peroxide (pKa 11.65),
based on literature data. It has the same shape as for amines, but the
rate constant for the reaction of HO2
 and HOCl is 4.4  107 L
mol/L s1,(18) i.e., seven-fold lower than for an amine of similar
basicity.

Methods

Bovine blood obtained from a local abattoir was preserved with
Ka (amineH(n1))

[H3O]  Ka (amineH(n1))
[H3O]

[H3O]  Ka (HOCl)
0.2% w/v EDTA and stored at 4°C. Domestic strength sodium
hypochlorite bleach was standardized by titration with standardized
thiosulfate solution using starch-iodine indicator.
Diethylamine, 1,2-diaminoethane (ethylenediamine), ethylamine,
taurine, tris(hydroxymethyl)methylamine (TRIS), and triethylamine
were used as received.

The luminol sprays tested were Grodsky: (a solution containing
1 g/L luminol and 50 g/L Na2CO3 mixed with an equal volume of
7 g/L sodium perborate solution) and Weber: (25 mL of water containing
0.72 g/L luminol and 2 g/L NaOH was mixed with 25 mL
0.6% H2O2 and 175 mL H2O). In both cases the mixing of solutions
was performed as soon as possible before a measurement was to be
made.

Visual observations were conducted in a darkened room after the
investigator was dark-acclimatized for at least 5 min. Spraying of
luminol solutions was performed using a calibrated spray-gun to
ensure uniform delivery of reagent. Substrates were vinyl flooring
tiles and unbleached cotton. The tiles were used as received,
whereas the cotton was washed with clothes detergent, then rinsed
in tap water and finally boiled in Milli-Q deionized water and then
dried.

Supporting experiments examining the effect of amines on
bleach-induced chemiluminescence were performed using an
Ocean Optics S2000 fibre optic diode array spectrophotometer
with the reagent feed being controlled by a peristaltic pump.
Hypochlorite solution (1:20 dilution) adjusted to pH 12.1 was
mixed with luminol-H2O2-perborate solution at pH 12.1 at a rate of
10 mL/min (each reagent stream) in a 2 mm diameter T-junction
cell with two right-angle turns 1 cm after the initial junction to increase
mixing. Light emission in the 2 cm of tubing immediately
after the mixing point was monitored under constant flow conditions.
The high flow rate was needed to avoid the build up of gas
bubbles in the cell. Studies of the effect of amines on the blood-induced
chemiluminescence used the same spectrophotometer
equipped with a 1 cm cuvette holder.

Safety: All initial experiments involving spraying of luminol solutions
were performed in chemical fumehoods. When the sprays
were used outside fumehoods appropriate personal protection including
goggles, respirator, and gloves were worn. If amines are
added to the luminol solution these precautions are especially important,
and the room should be thoroughly ventilated after the
chemiluminescence has been observed.

Results and Discussion

Inhibition of Hypochlorite Bleach-Induced Chemiluminescence

Preliminary experiments showed that the observed chemiluminescence
of luminol upon reaction with either blood or bleach was
pH dependent, and that the addition of amines to solutions could
change the pH. Therefore the pH was adjusted to pH 12.1  0.1 for
the solution studies described in this paper.

Preliminary experiments showed that the observed chemiluminescence
of luminol upon reaction with either blood or bleach was
pH dependent, and that the addition of amines to solutions could
change the pH. Therefore the pH was adjusted to pH 12.1  0.1 for
the solution studies described in this paper.

The ability of amines to inhibit bleach-induced chemiluminescence
from the Grodsky formulation of luminol solution was evaluated
using a steady-state flow cell attached via fibre optics to a
diode array spectrophotometer. The amines were added at levels of
0, 0.008, 0.02, 0.04, and 0.08 mol/L. The results, Table 1, showed
that the inhibition by amines increased with the basicity of the
amines and was most marked for 1,2-diaminoethane, with 90% inhibition
at 0.08 mol/L. Ammonia caused little or no inhibition, in
accord with the reported slower rate of reaction of ammonia with
hypochlorous acid (9). Triethylamine caused increases in the observed
chemiluminescence, in agreement with literature observa-
tions on tertiary amines (11,12). The metal chelating agent EDTA,
which is also a tertiary amine, had no effect on the chemiluminescence
at all concentrations tried (0–0.08 mol/L).
Similar results to these were obtained by visual examination of
bleach spots sprayed with amine-containing luminol sprays. Depending
on the concentration of the bleach used and the length of
time it had been on the substrate, concentrations of 0.02 mol/L or
0.1 mol/L 1,2-diaminoethane in Grodsky’s luminol spray were sufficient
to reduce bleach-induced chemiluminescence to a negligible
level. In addition, it was noted that if bleach spots were left for periods
of greater than a day under conditions where they could dry
that no significant chemiluminescence was observed when they
were sprayed with standard forensic luminol-peroxide sprays
(Grodsky or Weber formulations).

Effect of Amines on Heme-catalyzed Chemiluminescence

The effect of the amine 1,2-diaminoethane on the blood-catalyzed
luminol chemiluminescence was evaluated both spectrophotometrically
in solution and visually with bloodstains on
substrates. In solution studies the addition of 0.1 mol/L 1,2-diaminoethane
at pH 12.1 caused a slight decrease in chemiluminescence
over the first 1 min, but was then identical to the chemiluminescence
of the control, Fig. 2. This experiment was performed
with aged blood, so the initial rapid change may represent activity
due to degraded hemoglobin.

Visual comparison of 1,2-diaminoethane effects were performed
using vinyl and cotton substrates with 1 h, 3 h, and 1-day-old blood
stains, and the appearance and duration of the chemiluminescence
was noted. On a given day repeated measurements showed good repeatability,
and comparisons between different conditions were always
made within the same experimental run. It was observed that
the strength and duration of chemiluminescence due to blood were
decreased by a factor of 2–3 in the presence of high concentrations
of 1,2-diaminoethane (0.1 mol/L), Table 2, although the exact level
of inhibition was also dependent on the solution pH. However,
even though the 1,2-diaminoethane causes slight inhibition in the
chemiluminescence due to blood the results obtained are still satisfactory
for use at a crime scene. Furthermore, at a crime scene repeated
spray applications can be used if the chemiluminescence duration
is too short.

The addition of 1,2-diaminoethane to Weber’s luminol formulation
was not as successful at reducing the chemiluminescence due
to bleach. Our results suggest that the presence of carbonate in the
solution plays a significant role in the observed reactivity.

A simulation of a crime scene where bloodstains on carpet,
vinyl, and cloth were washed with bleach at recommended dilutions
showed that addition of 0.1 M 1,2-diaminoethane to Grodsky’s
luminol formulation reduced the bleach interference on all
substrates and only slightly affected the blood chemiluminescence.
This solution is now included in the ESR protocol for luminol use
if there is evidence that bleach may have been used at a crime
scene. Appropriate precautions, including the use of safety glasses
and respirator masks, are taken when spraying either Grodsky’s luminol
solution or the solution including 1,2-diaminoethane. Alternatively,
if the crime scene is such that it can be left to dry for a few
days (e.g., a car interior) then this delay will reduce or remove the
bleach interference.

Conclusion

The false positive response observed when luminol-peroxide solutions
are sprayed onto an area that has been cleaned with
hypochlorite-containing bleaches can be prevented by adding
amines such as 1,2-diaminoethane to the luminol solution. The
amines react rapidly with the hypochlorite ions, and the chlor-
amines thus formed do not cause visible chemiluminescence to occur.
The amines do slightly reduce the chemiluminescence observed
from blood, but the treatment still has sufficient intensity
and longevity of light emission that it is useful in a forensic context.
During this study it was also noted that the positive interference
by bleach is diminished if the area to be sprayed is left for several
days. Therefore, where possible a delay before spraying with
luminol-peroxide solution can significantly reduce positive interference
by bleach. Both of the procedures have been included in the
ESR protocols for luminol use.


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Offline JGW


Joined: Sat Dec 26, 2009 4:25 pm

Posts: 5

Location: Florida, USA

PostPosted: Sat Dec 26, 2009 5:04 pm   Post subject: Re: BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE   

Although very technical, this article is very informative if you have adequate background. One must be very careful when applying the information therein to the terrible events in Perugia.

Some questions for which I do not know the answers come to mind.
- Were the Italian investigators using the same modified luminol formulation described by the author? If not, it is not relevant to the investigation.
- Did the Italian investigators use the identical protocol described in the article? If not, no valid inferences can be made about what the luminol revealed at the crime scene.

The author's discussion is valid only under strict laboratory conditions where variables are limited and known. The number and existence of variables existent at a given crime scene are likely unknown and unique to that scene. One who attempts to directly apply results obtained under laboratory conditions to results obtained in the field is making a quantum leap of highly dubious validity.
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Offline Fly by Night


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PostPosted: Sat Dec 26, 2009 5:38 pm   Post subject: Re: BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE   

JGW wrote:
Although very technical, this article is very informative if you have adequate background. One must be very careful when applying the information therein to the terrible events in Perugia.


Some of the detail regarding how the investigation was conducted will likely be released in the summary of findings for the trial of Raffaele Sollecito and Amanda Knox. There was lenghty testimony and cross-examination covering the foresics aspects of this case during the trial, most of which was never covered in any reporting. The Rome-based forensics team came out of it looking like full professionals, however, having withstood many hours of technical questioning from the defense teams who were apparently unable to establish anything beyond the hypothetical possibility of contamination and the potential for investigtors to make mistakes. One comment from the stand that was reported, however, was Dr. Stefanoni's statement that the investigators were highly experienced and were familiar with and could readily identify the distinct glow of luminol when interacting with blood, as opposed to other substances.
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Offline Michael

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PostPosted: Sat Dec 26, 2009 5:48 pm   Post subject: Re: BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE   

And of course, there was no bleach in the house (none of the girls used products that contained bleach anyway) and even if there were any hyperthetical bleach, it would have dissipated by the time luminol was laid down, it being the last forensic task to be performed. Bleach is not responsible for any of the luminol blood detections in the cottage.

_________________
"The truth is incontrovertible. Malice may attack it and ignorance may deride it, but in the end, there it is." ~ Winston Churchill mike


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Offline JGW


Joined: Sat Dec 26, 2009 4:25 pm

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Location: Florida, USA

PostPosted: Sat Dec 26, 2009 7:37 pm   Post subject: Re: BLEACH-INDUCED LUMINOL CHEMILUMINESCENCE   

FBN, Michael:

Thank you for taking the time to respond to and comment on my post.

I was not questioning the forensic investigators. Rather I was attempting to verbalize that it is not necessarily wise to directly extrapolate laboratory findings to situations outside a laboratory setting. To validly do so takes much work "in the field" to ensure the results from the lab are empirically shown to be reproducible where the variables can not be controlled as closely.

Although arguments are constructed using individual points to build upon, at the end an argument is valid only if it stands on its collective merits. It is always possible to "attack" one or so of the points in an argument, but doing so does not invalidate the argument in toto. For example, I have read that in Dr. Stefanoni's lab results she made notes that indicated that a DNA sample was too low and yet later testified that the result was valid for identification. It would not be accurate to dismiss completely the work of her group based on the validity of that single point which may be seen as arguable. It would be rare for all points of any argument to be equally strong. It is obviously dangerous to be selective and choose only those points that support a preconceived notion.

I am here because I just do not know the truth beyond the horrific fate of Ms. Kercher. It seems that the information I have been able to get from sources in the US and the UK has been slanted one way or another and far from objective. It is my hope that in the end the truth will out regardless of what that truth is. At this time, I do not know what that truth is. Further, I do not care if Knox and Sollecito are guilty or innocent. The only value of that truth is justice for Meredith Kercher.

My background and education is that of a scientist (medical laboratory pathology) and an accountant. Please consider that my thinking and reasoning is that which is, is. It is not going to be what I want it to be. The facts will determine the answer.
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