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Indentification of Raman and Rayleigh bands in Fluorescence Spectrum
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Fabio Mancini
science forum beginner


Joined: 22 Dec 2005
Posts: 17

PostPosted: Tue Jun 06, 2006 9:53 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

the rayleigh peak of I order is at the same wavelength (WL) of the
excitation.
the raman peak is shifted to DELTAlambda with respect to the excitation.

for example: if you have a trp containing protein, and you work
with exWL = 280 and emWL from 250 to 600, you will observe:
1. 280 nm peak (Rayleigh I)
2. 310 nm water Raman peak (280 + 30 nm; DELTAlambda = +30nm)
3. 560 nm peak (Rayleigh II) (280 x 2 = 560 nm)
4. (if any) fluorescence of trp-containing protein

The Rayleigh and Raman peaks change their position by changing the exWL,
but
the fluorescence doesn't





Quote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.




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Fabio Mancini
science forum beginner


Joined: 22 Dec 2005
Posts: 17

PostPosted: Mon Jun 05, 2006 6:44 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Quote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.


the rayleigh peak in the emission spectrum is at the same wl of the
excitation
wavelength, and also at the double value (i.e. if the excitation is 280
nm
you will observe two rayleigh peaks at 280 nm and 560 nm - of course if
you
scan for such interval). The raman peak is at a certain DELTAlambda from
the rayleigh peak, for water +30nm.
Example
excitation 280 nm
emission 250-600 nm => you will see a 280 peak (Rayleigh I), 310 nm
(water Raman),
560nm (Rayleigh II) + a fluorofor band (if any)

notice that rayleigh peaks depends on the excitation wl, and also the
raman peak,
but it is always liked by a DELTAlambda on the excitation wl.
the fluorescence band, in contrast, is located independently from the
excitation peak





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Shankar Bhattacharyya
science forum beginner


Joined: 15 Mar 2006
Posts: 7

PostPosted: Sat Jun 03, 2006 6:31 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

"Farooq W" <farooq.w@gmail.com> wrote in
news:1147835807.443798.67580@38g2000cwa.googlegroups.com:

Quote:
Some
question that come in mind reagrding people's fear of dye additives
in chillies or anything else are:

In a report that I studied said that the dye concentration should
be about 100 to 1000 ppm (mg/kg) to impart a suitable color to the
chillies. I wonder if 10 ppb should be of any use to the adulterant
and the need to spending a huge amount on buying sophisticated
instruments

10ppb of these dyes does nothing for the color and is not an
adulteration issue directly, in that nobody put that "in the sauce".
The situation is that chile peppers get ground and then used at low
levels in lots of things. The result is sauces which contain a few ppb
of an adulterant which was present at a few hundred ppm in some
component of a blend, which blend was used at a low level in some
intermediate product on the way to the sauce.

The interest in the low level detection is less regulatory than
business driven.

Some companies which have found themselves on the unfortunate end of a
situation where some regulatory authority has detected Sudan dyes at a
low level in their products have responded with a global approach
requiring anyone who sells anything to them which has any possibility
of being adulterated at any stage of the production process to
demonstrate that the product is free of Sudan and other dyes at 10ppb,
sometimes without any evidence that methodology exists to do this.

So startlingly expensive instruments are required.

Personally, as a business matter, I do not care whether it makes sense
to test at these levels. If there is a demand for the analytical
capability, we are interested in providing the service. I do recognize
that there is a certain amount of craziness here.

There is also a public relations issue. No government agency wants to
go before the public and say, "We will allow 50ppb or 100ppb or 1ppm
of these possibly carcinogenic compounds in the food you eat."
Consumers, certainly in the USA, are deeply suspiciously of the
government. They don't want any "bad stuff" in their food. Any.

Anyway, it keeps analytical chemists employed.

Quote:
You talked about Rhodamine-B,that is one of the best fluorescent
dye (high quantum effieciency) but who is adding this carcinogen to
foods? How cruel of them.

Rhodamine B is used as an adulterant in at least some food-related
contexts in India. Worse things happen. There used to be cases of
mustard oil being "made" by adding carotene and allyl iosthiocyanate
to diesel, chile peppers whose color had been improved by a
measningful amount of lead chromate, so on and so forth.

However, I do not know much about what is adulterated with what, by
whom or why. I am a narrow, specialist, analytical chemist. It is hard
enough to stay up with that.

However, India has developed a large enough middle class with lots of
education and wide access to information. They want their food safe
and know about this sort of thing and the government has responded
with regulation and oversight. At present no production lots of chiles
are allowed to be exported from India without first being tested by a
government lab. They have a couple of LC/MS-MS instruments and do good
work.

Quote:
Would it work to selectively solvent extract the dyes from chillies
or still solvent extraction process shows a dirty matrix?

We extract with acetonitrile. Other people extract with methanol. Some
people extract with methanol containing a little ammonia, which
presumably helps to extract the Sudan dyes, which are phenols. All
produce dirty extracts. There are SPE cleanups available but there are
already too many dyes of too wide a range of chemical and
chromatographic properties for any existing SPE cleanup to work for
all of them in one shot.

We are now working with 19 dyes, including the natural bixin. A major
stakeholder in this business wants something like six more dyes to be
included in the analysis starting July 1, 2006.

A practical method needs to be able to deal with these very dirty
matrices. The volume of work which needs to be done is too large for
elaborate preliminary cleanups. Also, the continuous escalation in
number prevents us from settling on a cleanup method. We would have to
revisit it every time anyone adds a dye.

Quote:
BTW, do you know which dye is used to color rice orange (if you
have heard the name zarda - the sweet dish of rice colored orange
boiled in sugar). The dye is availbale is at really dirt cheap rate
and I suspect this is nothing but a textile dye.

Sorry, no idea. I imagine "saffron" would not be the right answer.

- Shankar
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Steven Arnold
science forum beginner


Joined: 17 May 2006
Posts: 1

PostPosted: Wed May 17, 2006 4:48 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Farooq W wrote:
Quote:
Shankar Bhattacharyya wrote:

"Farooq W" <farooq.w@gmail.com> wrote in
news:1145932279.817722.279440@y43g2000cwc.googlegroups.com:


David Stone wrote:

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me
and was worried about a intense chopped peak i.e a flat top. The
excitation spectrum was checked by comparing its absorption
spectrum. Both of them match. However the literature is "silent" of
such an intense peak in the emission spectrum but they mention a
very small peak nearby it, whose value matches with the literature
value.

As fluorescence goes in analytical work, the fluorescence of capsaicin
is not something people would describe as small. Fluorescence is the
preferred way to measure capsaicinoid concentrations by hplc. We
measure the capsaicinoid content of chile peppers and related products
pretty much every day and think of it as an easy detection.

Of course, "small peak" depends on context.

- Shankar


The person was not interested in capsaicin concentration, rather he was
trying to a develop method for the detection artificial dyes (Sudan
dyes) by spectrofluorometry, so the interference of capsaicin was being
checked.

Is HPLC, the standard method of detecting dye additives?


I'm glad you identified the real cultprit. Done that more times than
I'd like to admit. Just a little more to add to the discussion. On
sensitive modern instruments, you can indeed see RAMAN bands from many
solvents. Granted they are weak, and in fact many instrument
manufacturers specify the S/N ratio of the RAMAN peak of water in their
sensitivity specs. They can be significant if your sample fluorescence
is weak. Also recall that they will appear as broader bands than in a
RAMAN instrument because you are scanning them with much wider slit
widths in fluorescence instruments.

They can also affect fluorescence lifetime measurements, because
scattering is instantaneous.

I can't actually say I've ever used an original Aminco-Bowman, but I've
got one with no detection system. I was tinkering around with it, with
the intention of cobbling something together when something better came
along. Once while digging around in some lab drawers I found an old
advertisement for them. There was a testimonial from one of my old
profs! Wish I'd kept it, but I let it get thrown out somehow.
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Farooq W
science forum Guru Wannabe


Joined: 25 Mar 2005
Posts: 240

PostPosted: Wed May 17, 2006 3:16 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Shankar Bhattacharyya wrote:
Quote:
"Farooq W" <farooq.w@gmail.com> wrote in
news:1147660972.955923.179740@y43g2000cwc.googlegroups.com:

Shankar Bhattacharyya wrote:
As fluorescence goes in analytical work, the fluorescence of
capsaicin is not something people would describe as small.
Fluorescence is the preferred way to measure capsaicinoid
concentrations by hplc. We measure the capsaicinoid content of
chile peppers and related products pretty much every day and think
of it as an easy detection.

Of course, "small peak" depends on context.

The person was not interested in capsaicin concentration, rather he
was trying to a develop method for the detection artificial dyes
(Sudan dyes) by spectrofluorometry, so the interference of
capsaicin was being checked.

I don't think most of the Sudan dyes are fluorescent to any meaningful
extent. The basic structural unit in the Sudan dyes is a 2-napthol
unit, with an aryl diazo substitution at position 1. I don't see
anything there to encourage the idea of fluorescence as a detection
handle. However, I don't know that for a fact by any means.

Is HPLC, the standard method of detecting dye additives?

The answer to that question is rather context-dependent. Right now the
major interest in this area is for the Sudan dyes. They can be
detected at a detection limit of about 1ppm in spices by hplc with
diode-array detection. An Agilent 1100 series DAD, which has rather
good signal to noise, gives a quite nice set of peaks for a 20ul
injection of a 100ppb solution of the common Sudan dyes. You really
need a DAD to do the analysis, however, since the commonly affected
spices, chile peppers and paprika, have lots of red compounds in them
and these come off all over any realistic hplc run. You have to be
able to compare peak spectra to have even reasonable success. Our lab
was involved in a collaborative study under the American Spice Trade
Association (ASTA) umbrella, to validate the method, which is now
officially ASTA 28.0. That covered 8 dyes, maybe nine. My memory on
the exact number is a little fuzzy, even though I participated in the
process. "Eight or nine dyes" feels like a long time ago.

The food industry is demanding much lower detection limits, by the
way. There is considerable demand for a 10ppb detection limit across
the board. We are now involved in a collaborative study involving labs
in the US, Europe and China, to examine LC/MS-MS detection of 18 dyes
with a target detection limit of 10ppb on sample basis. I think a
decent, at least mid-range triple quad is pretty much indispensible
for this purpose. The matrix also wrecks mass spectrometric response
rather easily.

Not all of the dyes are Sudan dyes, by the way. One such dye is
Rhodamine B, which is the only one of the eighteen which is obviously
fluorescent (it is obvious if you look at a solution) but even that is
not sufficient signal to see at 10ppb on sample basis over the
background. Spices provide an extremely dirty matrix. There is also
interest in detecting Bixin, which is a natural carotenoid present in
annato and is a common adulterant in paprika. Bixin also provides
rather low mass-spectrometric signal.

As of today there are about ten more dyes in which people are
variously interested, some Sudan dyes, some not. We may address that
in the medium term. I think we have enough on our hands for the
present.

Our initial intention is to develop a set of criteria for
demonstration of capability in this area. Right now there are claims
of detection limits being made with an abandon which makes one wonder
what people are smoking.

As part of the same study we are working to develop a standardized
approach to testing for these dyes and to determine what detection
limits are possible with current practical triple quads. Tomorrow's
triple quad will make that irrelevant but we hope that a set of agreed
criteria will help to produce comparable performance measures.

Incidentally, there was a recent collaborative study in Europe, under
ISO, to develop a reverse-phase hplc method for detection of
adulteration of saffron with artificial dyes. I don't think any of
those were Sudan dyes. What I saw of the method looked quite good.

Dyes cover a huge range of chemical features. The Sudan dyes are
mostly quite non-polar and elute quite late from a C-18 column. There
are also highly polar dyes, with sulfonic acid groups on the dyes,
quarternary ammonium moities and so on. It is quite hard to imagine
all of them under one class. There is clearly interest in hplc
methods. After all, you have to separate the dyes from the natural
pigments, unless you can find highly selective tools such as unique
fluorescence signals.

There are also a variety of tlc methods to detect various artificial
dyes in foods, including a rather nifty one using C-18 SPE separation
of medium-polarity dyes from less polar natural pigments.

Sorry to ramble on. This is something of a major preoccupation for me
now. In a few months there might even be something useful to say.

- Shankar

This was not a ramble, of course, but very interesting. Some question
that come in mind reagrding people's fear of dye additives in chillies
or anything else are:

In a report that I studied said that the dye concentration should be
about 100 to 1000 ppm (mg/kg) to impart a suitable color to the
chillies. I wonder if 10 ppb should be of any use to the adulterant and
the need to spending a huge amount on buying sophisticated instruments

I don't know whether Sudan dyes are highly fluorescent or not but
someone told me that there was a patented spectrofluorometric method
for Sudan dyes. You might search on that.

You talked about Rhodamine-B,that is one of the best fluorescent dye
(high quantum effieciency) but who is adding this carcinogen to foods?
How cruel of them. Just yesterday I was washing some fennel seeds
(saunf) for pickles, and all water turned green, there was so much of a
green dye to make them look fresh. The ture color was then becomae
brown I couldn't use them.

Would it work to selectively solvent extract the dyes from chillies or
still solvent extraction process shows a dirty matrix?

BTW, do you know which dye is used to color rice orange (if you have
heard the name zarda - the sweet dish of rice colored orange boiled in
sugar). The dye is availbale is at really dirt cheap rate and I suspect
this is nothing but a textile dye.
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Shankar Bhattacharyya
science forum beginner


Joined: 15 Mar 2006
Posts: 7

PostPosted: Wed May 17, 2006 2:20 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

"Farooq W" <farooq.w@gmail.com> wrote in
news:1147660972.955923.179740@y43g2000cwc.googlegroups.com:

Quote:
Shankar Bhattacharyya wrote:
As fluorescence goes in analytical work, the fluorescence of
capsaicin is not something people would describe as small.
Fluorescence is the preferred way to measure capsaicinoid
concentrations by hplc. We measure the capsaicinoid content of
chile peppers and related products pretty much every day and think
of it as an easy detection.

Of course, "small peak" depends on context.

The person was not interested in capsaicin concentration, rather he
was trying to a develop method for the detection artificial dyes
(Sudan dyes) by spectrofluorometry, so the interference of
capsaicin was being checked.

I don't think most of the Sudan dyes are fluorescent to any meaningful
extent. The basic structural unit in the Sudan dyes is a 2-napthol
unit, with an aryl diazo substitution at position 1. I don't see
anything there to encourage the idea of fluorescence as a detection
handle. However, I don't know that for a fact by any means.

Quote:
Is HPLC, the standard method of detecting dye additives?

The answer to that question is rather context-dependent. Right now the
major interest in this area is for the Sudan dyes. They can be
detected at a detection limit of about 1ppm in spices by hplc with
diode-array detection. An Agilent 1100 series DAD, which has rather
good signal to noise, gives a quite nice set of peaks for a 20ul
injection of a 100ppb solution of the common Sudan dyes. You really
need a DAD to do the analysis, however, since the commonly affected
spices, chile peppers and paprika, have lots of red compounds in them
and these come off all over any realistic hplc run. You have to be
able to compare peak spectra to have even reasonable success. Our lab
was involved in a collaborative study under the American Spice Trade
Association (ASTA) umbrella, to validate the method, which is now
officially ASTA 28.0. That covered 8 dyes, maybe nine. My memory on
the exact number is a little fuzzy, even though I participated in the
process. "Eight or nine dyes" feels like a long time ago.

The food industry is demanding much lower detection limits, by the
way. There is considerable demand for a 10ppb detection limit across
the board. We are now involved in a collaborative study involving labs
in the US, Europe and China, to examine LC/MS-MS detection of 18 dyes
with a target detection limit of 10ppb on sample basis. I think a
decent, at least mid-range triple quad is pretty much indispensible
for this purpose. The matrix also wrecks mass spectrometric response
rather easily.

Not all of the dyes are Sudan dyes, by the way. One such dye is
Rhodamine B, which is the only one of the eighteen which is obviously
fluorescent (it is obvious if you look at a solution) but even that is
not sufficient signal to see at 10ppb on sample basis over the
background. Spices provide an extremely dirty matrix. There is also
interest in detecting Bixin, which is a natural carotenoid present in
annato and is a common adulterant in paprika. Bixin also provides
rather low mass-spectrometric signal.

As of today there are about ten more dyes in which people are
variously interested, some Sudan dyes, some not. We may address that
in the medium term. I think we have enough on our hands for the
present.

Our initial intention is to develop a set of criteria for
demonstration of capability in this area. Right now there are claims
of detection limits being made with an abandon which makes one wonder
what people are smoking.

As part of the same study we are working to develop a standardized
approach to testing for these dyes and to determine what detection
limits are possible with current practical triple quads. Tomorrow's
triple quad will make that irrelevant but we hope that a set of agreed
criteria will help to produce comparable performance measures.

Incidentally, there was a recent collaborative study in Europe, under
ISO, to develop a reverse-phase hplc method for detection of
adulteration of saffron with artificial dyes. I don't think any of
those were Sudan dyes. What I saw of the method looked quite good.

Dyes cover a huge range of chemical features. The Sudan dyes are
mostly quite non-polar and elute quite late from a C-18 column. There
are also highly polar dyes, with sulfonic acid groups on the dyes,
quarternary ammonium moities and so on. It is quite hard to imagine
all of them under one class. There is clearly interest in hplc
methods. After all, you have to separate the dyes from the natural
pigments, unless you can find highly selective tools such as unique
fluorescence signals.

There are also a variety of tlc methods to detect various artificial
dyes in foods, including a rather nifty one using C-18 SPE separation
of medium-polarity dyes from less polar natural pigments.

Sorry to ramble on. This is something of a major preoccupation for me
now. In a few months there might even be something useful to say.

- Shankar
Back to top
Farooq W
science forum Guru Wannabe


Joined: 25 Mar 2005
Posts: 240

PostPosted: Mon May 15, 2006 2:42 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Shankar Bhattacharyya wrote:
Quote:
"Farooq W" <farooq.w@gmail.com> wrote in
news:1145932279.817722.279440@y43g2000cwc.googlegroups.com:


David Stone wrote:
IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me
and was worried about a intense chopped peak i.e a flat top. The
excitation spectrum was checked by comparing its absorption
spectrum. Both of them match. However the literature is "silent" of
such an intense peak in the emission spectrum but they mention a
very small peak nearby it, whose value matches with the literature
value.

As fluorescence goes in analytical work, the fluorescence of capsaicin
is not something people would describe as small. Fluorescence is the
preferred way to measure capsaicinoid concentrations by hplc. We
measure the capsaicinoid content of chile peppers and related products
pretty much every day and think of it as an easy detection.

Of course, "small peak" depends on context.

- Shankar

The person was not interested in capsaicin concentration, rather he was
trying to a develop method for the detection artificial dyes (Sudan
dyes) by spectrofluorometry, so the interference of capsaicin was being
checked.

Is HPLC, the standard method of detecting dye additives?
Back to top
Shankar Bhattacharyya
science forum beginner


Joined: 15 Mar 2006
Posts: 7

PostPosted: Sun May 14, 2006 11:48 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

"Farooq W" <farooq.w@gmail.com> wrote in
news:1145932279.817722.279440@y43g2000cwc.googlegroups.com:

Quote:

David Stone wrote:
IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me
and was worried about a intense chopped peak i.e a flat top. The
excitation spectrum was checked by comparing its absorption
spectrum. Both of them match. However the literature is "silent" of
such an intense peak in the emission spectrum but they mention a
very small peak nearby it, whose value matches with the literature
value.

As fluorescence goes in analytical work, the fluorescence of capsaicin
is not something people would describe as small. Fluorescence is the
preferred way to measure capsaicinoid concentrations by hplc. We
measure the capsaicinoid content of chile peppers and related products
pretty much every day and think of it as an easy detection.

Of course, "small peak" depends on context.

- Shankar
Back to top
Marvin
science forum Guru Wannabe


Joined: 24 Mar 2005
Posts: 224

PostPosted: Wed Apr 26, 2006 3:53 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Farooq W wrote:
Quote:
Marvin wrote:

Farooq W wrote:

David Stone wrote:


In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:



Farooq W wrote:


Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.


Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.


Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.

So I thought these could be instrument's artefact or other components
of the fluorescence spectrum. The following were tried:

(i) Running a empty cuvette - no peaks
(ii) Very pure water - One very intense peak (not the same as
excitation wavelength) and another small peak neabry
(iii) Pure methanol - One very intense peak (not the same as
excitation wavelength) and a small peak nearby.

The wavelengths of water and methanol are slightly different.

The spectrofluorometer is quite sensitive. What could be the origin of
these? Should I advise to reduced the voltage at the PMT?

For example what should one do if those artefacts are closely the
actual analyte peak?


I had a similar situation in the '50s, with an early model
Aminco-Bowman spectroflourometer, though the wavelengths
were very different. It turned out to be a combination of
two things. The "emission bands" were scattered excitation
light, which showed up in the second order of the grating,
looking like it was near twice the excitation wavelength.
But it wasn't exactly twice because the wavelength scales on
the excitation and emission monochromators were out of
calibration. I'm not sayimng that you have just the same
situation in your example, but just an indication of why:
1: Don't trust any instrument until it is thoroughly checked.
2: A combination of factors may be at work.


History repeats itself Smile. This spectrofluorometer is also of Aminco
Bowman. I guess they were the first one make a commercial
spectrofluorometer.

It was. It was built by Robert Bowman, at NIH in Washington.

Some people at NBS in Washington told me that they also
took part in building it.
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Farooq W
science forum Guru Wannabe


Joined: 25 Mar 2005
Posts: 240

PostPosted: Wed Apr 26, 2006 2:59 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Marvin wrote:
Quote:
Farooq W wrote:
David Stone wrote:

In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:


Farooq W wrote:

Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.


Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.


Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.

So I thought these could be instrument's artefact or other components
of the fluorescence spectrum. The following were tried:

(i) Running a empty cuvette - no peaks
(ii) Very pure water - One very intense peak (not the same as
excitation wavelength) and another small peak neabry
(iii) Pure methanol - One very intense peak (not the same as
excitation wavelength) and a small peak nearby.

The wavelengths of water and methanol are slightly different.

The spectrofluorometer is quite sensitive. What could be the origin of
these? Should I advise to reduced the voltage at the PMT?

For example what should one do if those artefacts are closely the
actual analyte peak?

I had a similar situation in the '50s, with an early model
Aminco-Bowman spectroflourometer, though the wavelengths
were very different. It turned out to be a combination of
two things. The "emission bands" were scattered excitation
light, which showed up in the second order of the grating,
looking like it was near twice the excitation wavelength.
But it wasn't exactly twice because the wavelength scales on
the excitation and emission monochromators were out of
calibration. I'm not sayimng that you have just the same
situation in your example, but just an indication of why:
1: Don't trust any instrument until it is thoroughly checked.
2: A combination of factors may be at work.

History repeats itself Smile. This spectrofluorometer is also of Aminco
Bowman. I guess they were the first one make a commercial
spectrofluorometer.
Back to top
Marvin
science forum Guru Wannabe


Joined: 24 Mar 2005
Posts: 224

PostPosted: Tue Apr 25, 2006 7:43 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

Farooq W wrote:
Quote:
David Stone wrote:

In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:


Farooq W wrote:

Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.


Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.


Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.

So I thought these could be instrument's artefact or other components
of the fluorescence spectrum. The following were tried:

(i) Running a empty cuvette - no peaks
(ii) Very pure water - One very intense peak (not the same as
excitation wavelength) and another small peak neabry
(iii) Pure methanol - One very intense peak (not the same as
excitation wavelength) and a small peak nearby.

The wavelengths of water and methanol are slightly different.

The spectrofluorometer is quite sensitive. What could be the origin of
these? Should I advise to reduced the voltage at the PMT?

For example what should one do if those artefacts are closely the
actual analyte peak?

I had a similar situation in the '50s, with an early model

Aminco-Bowman spectroflourometer, though the wavelengths
were very different. It turned out to be a combination of
two things. The "emission bands" were scattered excitation
light, which showed up in the second order of the grating,
looking like it was near twice the excitation wavelength.
But it wasn't exactly twice because the wavelength scales on
the excitation and emission monochromators were out of
calibration. I'm not sayimng that you have just the same
situation in your example, but just an indication of why:
1: Don't trust any instrument until it is thoroughly checked.
2: A combination of factors may be at work.
Back to top
Farooq W
science forum Guru Wannabe


Joined: 25 Mar 2005
Posts: 240

PostPosted: Tue Apr 25, 2006 1:37 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

David Stone wrote:
Quote:
In article <1145932279.817722.279440@y43g2000cwc.googlegroups.com>,
"Farooq W" <farooq.w@gmail.com> wrote:

David Stone wrote:
In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:

Farooq W wrote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.

Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.


Ok, as soon as I see an intense "flat-topped" peak, I refer to the
second part of my post (which, unfortunately, you trimmed!) Take
a good, hard look at the scan settings for the errant spectrum.
It sounds very much like someone has overlapped the emission and
excitation wavelengths, resulting in an intense scatter peak when
the two coincide. If that isn't the case, then something else is
going on. Check the emission and excitation ranges first, though!

Thanks I guess you have pin-pointed the problem , the emission
monochromator setting overlaps with the excitation monochromator.
Actually, it is difficult to find out the published spectrum of
capsaicin and according to the person who brought the spectra to me
that told me that as he decreases the concentration of the analyte the
intensity of the "flat topped" peak is decreases. I was unable to trace
the origin of this peak. I must ask to check the emission monochromator
settings of his instrument.
Back to top
David Stone
science forum beginner


Joined: 27 Mar 2006
Posts: 14

PostPosted: Tue Apr 25, 2006 11:32 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

In article <1145932279.817722.279440@y43g2000cwc.googlegroups.com>,
"Farooq W" <farooq.w@gmail.com> wrote:

Quote:
David Stone wrote:
In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:

Farooq W wrote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.

Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.


Ok, as soon as I see an intense "flat-topped" peak, I refer to the
second part of my post (which, unfortunately, you trimmed!) Take
a good, hard look at the scan settings for the errant spectrum.
It sounds very much like someone has overlapped the emission and
excitation wavelengths, resulting in an intense scatter peak when
the two coincide. If that isn't the case, then something else is
going on. Check the emission and excitation ranges first, though!
Back to top
Farooq W
science forum Guru Wannabe


Joined: 25 Mar 2005
Posts: 240

PostPosted: Tue Apr 25, 2006 2:31 am    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

David Stone wrote:
Quote:
In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:

Farooq W wrote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.

Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Someone brought the the emission spectrum of pure capsaicin to me and
was worried about a intense chopped peak i.e a flat top. The excitation
spectrum was checked by comparing its absorption spectrum. Both of them
match. However the literature is "silent" of such an intense peak in
the emission spectrum but they mention a very small peak nearby it,
whose value matches with the literature value.

So I thought these could be instrument's artefact or other components
of the fluorescence spectrum. The following were tried:

(i) Running a empty cuvette - no peaks
(ii) Very pure water - One very intense peak (not the same as
excitation wavelength) and another small peak neabry
(iii) Pure methanol - One very intense peak (not the same as
excitation wavelength) and a small peak nearby.

The wavelengths of water and methanol are slightly different.

The spectrofluorometer is quite sensitive. What could be the origin of
these? Should I advise to reduced the voltage at the PMT?

For example what should one do if those artefacts are closely the
actual analyte peak?
Back to top
David Stone
science forum beginner


Joined: 27 Mar 2006
Posts: 14

PostPosted: Mon Apr 24, 2006 6:44 pm    Post subject: Re: Indentification of Raman and Rayleigh bands in Fluorescence Spectrum Reply with quote

In article <124q44fdn33l2a2@corp.supernews.com>,
Marvin <physchem@cloud9.net> wrote:

Quote:
Farooq W wrote:
Could anybody guide me as to what is standard procedure of
"identifying" Raman or Rayleigh scatter peaks in the fluorescence
spectrum? If HPLC grade water and methanol is run on a
spectrofluorometer, their emission spectrum shows two emission peaks in
the 350 to 400 nm range. Methanol shows an intense (out of range since
the peak shape is like a "chopped peak") at 351 and a close by small
peak at 392 nm. If I assume the solvents are very pure and the peaks
are due to the scattering processes, how those peaks can be eliminated
in the emission scan of an analyte?

Thanks.

Raman scattering is such a weak phenomenon that it is
unlikely to be seen in an ordinary spectrophotometer. And
Raman lines are sharp, while fluorescnce emission is broad.

IIRC you can see at least one Raman band for pure water in our
PE spectrofluorimeters. To identify any Rayleigh peak, simply
look at what your excitation (for emission spectrum) or emission
(for excitation) wavelengths are.

Quote:
If the emission is the same for both solvents, it is likely
to be from something besides the solvent. Tomas Hirschfeld
used to say "Everything fluoresces." And it may be an
instrument artifact. Do the emission peaks shift when you
change the excitation wavelength?

In one of our instrumentation labs, I have the students set
an emission wavelength of 450 nm, and scan the full excitation
range of the instrument (i.e. across the emission wavelength).
Then, I ask them to explain what they see...
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