Re: Didymium standards

On Sun, Nov 27, 2011 at 2:59 PM, Simon Quellen Field <sfield@scitoys.com> wrote:
> Nice.
> Didymium glass is easy to come by for those who don't need an NIST
> traceable calibration source.
> But at 5 nm resolution, it is not as good as the peaks from a fluorescent
> light
> bulb (I get sub-nanometer resolution in my $20 spectrograph). On the other
> hand,
> it has lots of nice peaks all through the visible spectrum.
> For DNA quantification, we would be looking for something with a peak
> absorbance at 260 nanometers. To assess DNA contamination of protein
> samples, we would want something with peak absorbance at 280 nanometers.
> It turns out that sub-nanometer accuracy is useful for the 280 nanometer
> reading (much less so for the 260). The values for the five interesting

your link doesn't work

> nucleic
> acids given on that page might also be useful calibration targets. If a
> solution
> of guanine shows a 1.15 ratio, and a solution of adenine shows a 4.50 ratio,
> then we can be pretty happy with whatever results we get with our sample,
> for
> the 260/280 ratio test.
> According to that web page, two different equally accurate (to 1 nm)
> spectrometers
> may disagree by as much as 0.4 on the 260/280 ratio reading of the same
> sample.
> So presumably, the accuracy of a 260/280 test is no better than that, and so
> half
> a significant digit in the ratio is adequate for publication:
> Accuracy of test at 1 nanometer resolution:
>                           low       mid     high
> Guanine:  0.75 1.15 1.55
> Adenine:  4.10 4.50 4.90
> Cytosine: 1.11 1.51 1.91
> Uracil:   4.60 4.00 4.40
> Thymine:  1.07 1.47 1.87
> For those building a DIY spectrophotometer for this test alone, the focus
> problems
> go away and you don't have to tilt the sensor, or even use a 2D sensor. The
> peak
> at 260 is a plateau anyway, so if it is out of focus, we don't care. We need
> sub-nanometer
> resolution only at 280 nanometers, so we focus on that spectral line, and
> everything
> is copacetic. Such a scope can be built in about 15 minutes for around $20,
> using a
> digital camera for the sensor.
> And it should be easy to make calibration cuvettes from pure nucleic acids.
> You don't care much about how concentrated the sample is -- you just care
> about the ratio of absorption at 260 and 280 nm. But pure samples of nucleic
> acids
> are easy to come by, and can be used for the non-ratiometric calibrations we
> were
> discussing earlier.
>
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>
>
> On Sun, Nov 27, 2011 at 10:07 AM, Tom Knight <tk@csail.mit.edu> wrote:
>>
>> You want a didymium perchlorate solution as your wavelength standard.
>>  Check out the sharp peaks in its spectrum:
>> http://www.starnacells.com/d_ref/d_wl/DL.html
>>
>> You could check out some of their other reference standards on the left.
>>
>>
>>
>>
>>
>

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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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