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<BLOCKQUOTE><FONT COLOR="#800000">Hi Bala<BR>
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The upper limit <B>so far as detection goes</B> is not so much the concentration as the level of absorbance at the wavelength used. You definitely do not have to stick to 280 nm in the XL-A - its monochromator is pretty good both downwards and upwards in the spectrum. A simple trick to cope with 'too much OD at 280 nm' is to go down to whatever minimum you can find (via a wavelength scan) around 260 nm. You will usually get close to a 2-fold gain that way. Alternatively, you can take the wavelength up (say to 295-300 nm) until absorption is within Beer's Law range, and keep your fingers crossed that the monochromator stays stable from scan to scan (which we find it usually does - but do not under any circumstance try to scan at more than one wavelength). Above 300 nm you are mostly into turbidity rather than true absorption, and I would avoid doing that (unless of course you have a protein with a chromophore up there).<BR>
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BUT - being able to log the data is one thing, interpreting it is another. As I pointed out in a recent mail, getting interaction terms sorted out is not simple, and at (say) 5-8 mg/ml they just cannot be neglected. But that is another story, and not what your query is about. Bottom line - if you can find a wavelength at which your absorbance is within the Beer's Law limit (best not to go much above 1.0) then <I>there is no upper limit on the concentration which you can study</I>. Well - unless you get steep gradients and 'schlieren' effects, but they tend to visually screech at you.<BR>
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Regards<BR>
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Arthur<BR>
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</FONT></BLOCKQUOTE><FONT COLOR="#800000">--<BR>
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Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
NCMH Business Centre<BR>
University of Nottingham<BR>
School of Biosciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD UK<BR>
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Tel: +44 (0)115 951 6156<BR>
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<BLOCKQUOTE><FONT FACE="Times New Roman">Hi Ultraspinners,</FONT> <BR>
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<FONT FACE="Times New Roman">I want to know the reason behind the upper limit of concentration in the absorbance measurements using XL-A.</FONT> <BR>
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<FONT FACE="Times New Roman">Why a higher concentration (like 5-8 mg/ml) can not be measured using UV absorbance? <BR>
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<FONT FACE="Times New Roman">Is it to do with the linearity of the detector OR it is due the break down of Lambert-Beer law at high concentration?</FONT> <BR>
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<FONT FACE="Times New Roman">Cheers,</FONT> <BR>
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<FONT FACE="Times New Roman">Bala</FONT> <BR>
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<FONT FACE="Times New Roman">IMCB</FONT> <BR>
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<FONT FACE="Times New Roman">Singapore</FONT> <BR>
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