I performed typical temperature-dependent UV-VIS measurements at a fixed wavelength (260 nm) of a DNA probe (Tm experiments). For the samples very diluted the absorbance does not remain constant over the time and fluctuate largely compared with concentrated samples with a much better stability. What could cause this effect?
I mention that measurements were performed in quartz cuvette (1 cm pathlength) with a Cary 100 Spectrophotometer in single beam mode.
The ramp temperature was 1 °C/min.
Dear Marius,
as answered for previous questions of researchers, please give the following infos because it is very important for good help and answer!!
- Typ of spectrometer (single , dualbeam or diode arrays ).
- Typ of cuvette (Commonly 3 kinds of quarz cuvettes) (Quartz /Colour of the label)
- Cut off of the label (fused quartz : below 380 nm p.e.)
- Have you stopped your cuvettes (if Dualbeam)?
- Infos about solvent and buffer for DNA (pH...)
VERY IMPORTANT!!
- Please note the following hint concerning the dilution of your samples:
- In order to obtain reliable data, the peak of absorbance for a drug needs to be ca. three times higher in intensity than the background noise of the instrument (After Andrew R. Barron). Please test this aspect.
Good luck!
JRG
I totally agree with Professor Jean Grezes he has given a good breakdown and a reliable direction
Dear Mr. Grezes,
I will kindly appreciate if you can provide me some reference information of Andrew R. Barron. Thanks for your answer!
Dear Marius,
No problem! Here the wished " Barron link ":
http://cnx.org/content/m34525/1.1/
Another hint : Have you tested the source (lamps) of your spectrometer prior measurement of DNA samples. How old is the Cary 100?.
Old light source (deuterium/Tungsten) can generate trouble besides very low concentration.
Please give me some infos concerning the sample holder and the temperature gradient controlling thermostat .
On other part, I send you another link about isolation, spectrometric analysis and melting of onion DNA
:
http://chem247.files.wordpress.com/2007/09/chem-247-dna-lab.pdf
and another link (very useful too) :
http://homepages.gac.edu/~cellab/chpts/chpt14/ex14-5.html
Good luck
JRG
Hi Marius,
after the warm-up time of the instrument (about 30 minutes for your type of spectrometer) the photometric noise should reach a constant value. It can be evaluated e.g. from the baseline of a spectral scan. The bigger a measured OD value (OD = optical density, or absorbance value) the smaller is the contribution of this noise and the more stable the signal.
As an example:
spectrometer baseline noise: ±0.001
large measured OD: 1.2 => CV% = 0.08%
small measured OD: 0.05 => CV% = 2%
very small measured OD: 0.01 => CV% = 10%
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