Re: Didymium standards

You don't want a slit width less than about 5 nm, since it will require too much sensitivity.  I think you will discover that the broad peaks of the molecules you are looking at will make it very very difficult to get anything close to a good calibration.

Check out holmium perchloride as an alternative, which has good peaks in the UV.  Sigma has it pretty cheap.

On Nov 27, 2011, at 2:59 PM, Simon Quellen Field 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 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.