[RASMB] XL-A

[Arthur Rowe]

Hi Bala

The upper limit so far as detection goes 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).

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 there is no upper limit on the
concentration which you can study. Well - unless you get steep gradients and
'schlieren' effects, but they tend to visually screech at you.

Regards

Arthur


--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Tel:        +44 (0)115 951 6156
           +44 (0)116 271 4502
Fax:        +44 (0)115 951 6157
email:      arthur.rowe at nottingham.ac.uk
Web:        www.nottingham.ac.uk/ncmh/business
*******************************************************




Hi Ultraspinners, 

 

I want to know the reason behind the upper limit of concentration in the
absorbance measurements using XL-A.

 

Why a higher concentration (like 5-8 mg/ml) can not be measured using UV
absorbance? 

 

Is it to do with the linearity of the detector OR it is due the break down
of Lambert-Beer law at high concentration?

 

Cheers, 

Bala 

IMCB 

Singapore 

 

 


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