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If the standards are being ran at the same flow rate, and have the same
peak widths as the Ribonulcease A, then I think you can exclude hardware
issues. If the hardware were at fault then its "scan
rate" would have to slow down, or your elution rate sped
up.<br><br>
Try a different separation methodology to detect multiple species.
Ion exchange with the pH close to the "pI" might separate
small charge differences that might result from proteolytic cleavage.
<br><br>
Glen<br><br>
At 08:22 PM 2/3/2006, Richard Kingston wrote:<br><br>
<blockquote type=cite class=cite cite="">Recently, when running molecular
weight standards over a size <br>
exclusion column I made a puzzling observation.<br><br>
I routinely monitor column outflow at three wavelengths, since
our <br>
chromatography system allows it (I'm using an Amersham Biosciences
<br>
Äkta). This is to ensure that an undistorted elution profile is
<br>
obtained at at least one wavelength (For concentrated, and highly
<br>
absorbing samples, the detector response may become non-linear). So
<br>
typically I would monitor the signal at 280 nm (the peak due to Trp/
Tyr/Cys) and at then on the shoulder of this peak (293 and 300nm)
for <br>
reduced sensitivity. Or when increased sensitivity is required, I
<br>
often monitor at 220 or 230 nm, on the far shoulder of the peptide
<br>
bond absorbance peak.<br><br>
In the past, when using the instrument in this way, traces
have <br>
always been identical (up to some scaling factor) when the
absorbance <br>
is < 1.0.<br><br>
But last week, when running standards over a Superdex 75 column,
one <br>
of them (Ribonuclease A) gave different apparent elution volumes at
<br>
different wavelengths (280, 293 and 300nm). The behavior was <br>
reproducible, and the shifts in the peak position were small yet
<br>
significant (far beyond the inherent uncertainty in the peak <br>
position). Apparent elution volume at 280 nm > 293 nm > 300 nm.
This <br>
also corresponds to the order in which the wavelengths are cycled
by <br>
the monochomator.<br><br>
Yet the remaining standards (Chymotrypsinogen A, Ovalbumin,
Albumin, <br>
Blue Dextran 2000, L-Tryptophan) , run before and afterwards, in an
<br>
identical fashion, did not exhibit this behavior. In these cases
the <br>
traces at different wavelengths were all equivalent, accounting for
<br>
the differing absorbance by the sample. These standards are both
<br>
smaller and larger than Ribonuclease A.<br><br>
So the behavior is both puzzling and concerning (what wavelength
<br>
should I 'believe' in the case of Ribonuclease A?) and I'm
left <br>
scratching for an explanation. I'm hoping somebody on the list
might <br>
suggest something.<br><br>
The UV Monitor on the Akta has a 1 cm path length continuous flow
<br>
cell. Light is provided by a xenon flash lamp, monochromated with
a <br>
grating, and there is a beam splitter which sends 50% pf the light
<br>
through the cell, and the remainder through a reference fiber. Up
to <br>
three wavelengths can be recorded 'simultaneously' (in practice
the <br>
wavelength is rapidly switched, with the reported switch time < 500
ms).<br><br>
Thanks for any suggestions<br><br>
Rich.<br><br>
Richard Kingston, PhD<br>
School of Biological Sciences<br>
The University of Auckland<br>
Auckland<br>
New Zealand.<br><br>
rl.kingston@auckland.ac.nz<br><br>
<br>
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Glen Ramsay, Ph.D.<br>
Chief Scientist<br>
Aviv Biomedical<br>
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(732) 370-1300, Ext. 25<br>
(732) 370-1303, FAX<br>
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www.avivbiomedical.com<br><br>
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