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Forum index » Science and Technology » Chem » Analytical
Effect of Flow Rate on Area Count
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David Stranz
science forum beginner


Joined: 15 Jul 2005
Posts: 30

PostPosted: Mon Jul 10, 2006 3:26 pm    Post subject: Re: Effect of Flow Rate on Area Count Reply with quote

David Stone <no.email@domain.invalid> wrote in
news:no.email-0F9724.10462110072006@news1.chem.utoronto.ca:

Quote:
In article <Xns97F852B8C95DFdavidstranz@216.196.97.131>,
David Stranz <david_stranz@remove_this_to_reply_MassSpec.com
wrote:

denise.pattavina@gmail.com wrote in
news:1152196327.544418.197140@a14g2000cwb.googlegroups.com:

Can someone please help me explain to my boss (because he
won't except that this is just the way it is) why and how
flow rate has an effect on peak areas?

Thanks!



I presume you are referring to liquid chromatographic peak
detection, with digital conversion of the detector signal by
some sort of computerized acquisition system?

In theory, there should be no dependence of peak area on flow
rate, assuming a perfect world where the detector faithfully
sees and integrates everything coming through the column and
there is no noise, background, or interference by other
coelutants.

Actually, I don't believe that is true.

* the higher the flow-rate, the faster each sample zone is
eluted
from the column into the detector

* peak area is in units of [signal*time]

=> all other factors being equal, peak area varies with
flow-rate


I don't understand how this can be true in the ideal case. If the
detector is operating under Beer's law conditions, you have an
infinitely fast sampling rate, and all of the injected material
passes through the column (and thus through the detector), then it
shouldn't matter how fast or slow the elution rate is, eventually -
all- of the analyte should be seen and integrated by the detector.
[signal * time] = constant.

Sample zones, theoretical plates, and the other elements of
chromatographic separation theory concern the resolving power of
the column; the rate at which zones elute should have no effect on
the integrated signal seen by the detector, but does determine the
instantaneous signal level and the duration of the signal due to an
analyte. Those will determine peak height and width, but total
peak area is a function of the amount of analyte and its optical
properties.

Quote:
=> all other factors being equal, peak area varies with
flow-rate

Peak -height- varies; peak -area- should be invariant, presuming
peak endpoints are accurately determined under all flow rate
conditions.

Quote:
Whether you see a difference for a given compound, column,
mobile phase, and flow-rate will depend on where on the Van
Deemter curve you start out, since this can result in either (a)
a decrease in H (less broadening so area even lower), (b)
negligible change in H (some decrease in area due to faster peak
migration), or (c) an increase in H (increased broadening vs
faster elution)

Obviously, it also depends on the magnitude of the change in
flow rate, which the original poster didn't specify...
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David Stone
science forum beginner


Joined: 27 Mar 2006
Posts: 14

PostPosted: Mon Jul 10, 2006 2:46 pm    Post subject: Re: Effect of Flow Rate on Area Count Reply with quote

In article <Xns97F852B8C95DFdavidstranz@216.196.97.131>,
David Stranz <david_stranz@remove_this_to_reply_MassSpec.com> wrote:

Quote:
denise.pattavina@gmail.com wrote in
news:1152196327.544418.197140@a14g2000cwb.googlegroups.com:

Can someone please help me explain to my boss (because he won't
except that this is just the way it is) why and how flow rate
has an effect on peak areas?

Thanks!



I presume you are referring to liquid chromatographic peak
detection, with digital conversion of the detector signal by some
sort of computerized acquisition system?

In theory, there should be no dependence of peak area on flow rate,
assuming a perfect world where the detector faithfully sees and
integrates everything coming through the column and there is no
noise, background, or interference by other coelutants.

Actually, I don't believe that is true.

* the higher the flow-rate, the faster each sample zone is eluted
from the column into the detector

* peak area is in units of [signal*time]

=> all other factors being equal, peak area varies with flow-rate

Whether you see a difference for a given compound, column, mobile
phase, and flow-rate will depend on where on the Van Deemter curve
you start out, since this can result in either (a) a decrease in
H (less broadening so area even lower), (b) negligible change in
H (some decrease in area due to faster peak migration), or (c)
an increase in H (increased broadening vs faster elution)

Obviously, it also depends on the magnitude of the change in
flow rate, which the original poster didn't specify...
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David Stranz
science forum beginner


Joined: 15 Jul 2005
Posts: 30

PostPosted: Thu Jul 06, 2006 3:07 pm    Post subject: Re: Effect of Flow Rate on Area Count Reply with quote

denise.pattavina@gmail.com wrote in
news:1152196327.544418.197140@a14g2000cwb.googlegroups.com:

Quote:
Can someone please help me explain to my boss (because he won't
except that this is just the way it is) why and how flow rate
has an effect on peak areas?

Thanks!



I presume you are referring to liquid chromatographic peak
detection, with digital conversion of the detector signal by some
sort of computerized acquisition system?

In theory, there should be no dependence of peak area on flow rate,
assuming a perfect world where the detector faithfully sees and
integrates everything coming through the column and there is no
noise, background, or interference by other coelutants.

Once you start sampling the signal with a digitizer, this perfect
world starts to show its warts. What the analog detector sees as a
smoothly varying signal, the digitizer sees as discrete points
sampled at specific time intervals.

The software attempts to reconstruct the smooth curve by summing
these time samples over the peak. The more points sampled over the
time the peak is seen by the detector, the better the approximation
of this discrete integration to the actual peak area.

If you keep the sampling rate constant but increase flow rate, the
peak spends less time in view of the detector, so fewer points are
sampled over the peak. Thus, peak areas as calculated by the data
system will almost certainly be different for slow vs. fast
elution, all other things being equal.

In addition to this, determination of peak limits (start and end)
will be somewhat affected by the sampling rate, since most software
probably requires that the peak endpoints correspond to data
sampling points. Fewer points over the peak mean that the
endpoints are less well-determined, and different integration
limits may be used.

A good rule of thumb is a minimum of 20 points over the peak. For
LC-only systems with a fixed wavelength detector, sampling rates
can generally be increased to match whatever chromatographic
conditions require. For LC/MS or LC/DAD systems on the other hand,
there are limits to how fast the detector can sample and still
acquire useful spectroscopic data, and you can suffer a severe loss
in quantitative accuracy by attempting to push a sample through the
system too quickly.

Hope this is what you were looking for.

David
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denise.pattavina@gmail.co
science forum beginner


Joined: 06 Jul 2006
Posts: 2

PostPosted: Thu Jul 06, 2006 2:32 pm    Post subject: Effect of Flow Rate on Area Count Reply with quote

Can someone please help me explain to my boss (because he won't except
that this is just the way it is) why and how flow rate has an effect on
peak areas?

Thanks!
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