I am doing CD studies on amino acids by measuring the CD intensity (quantative) for various concentrations of amino acids both when they are pure and when they have been used as precursors for nanoparticles. However, I'm having problems with different results on different days. Any suggestions?
Arezoo Setayesh
Apparently you are using IR-, CD spectroscopy. I was looking for such a device in Russia and could not find it. You are well equipped and should say thank you to your management.
At low concentrations of amino acids, you should have small errors in the intensity determination. At high concentrations and in the presence of precursors and nanoparticles, the manifestation of the quantum nuclear effect is possible. A quantum object is created in a solution, the behavior of which is impossible to predict. For example, you cannot pinpoint the coordinates of an electron in a hydrogen atom. We can only talk about the probability of finding an electron in a certain volume of space. In your case, it is necessary to apply statistics, which I can write about in a personal letter for now.
Arezoo Setayesh
To locate the source of your variability (I'm not calling it 'errors') then construct an uncertainty budget on the 3M (Machine, Method, Material) principle:
- Machine - the inherent variability of the device you're using. Usually (by far) the smallest of all the variables and assessed by using a known, certified standard
- Method - the variability accrued by factors such as detection limit, concentration dependence, purity of reagents, temperature, pH, and so on. This can be the most stringent to investigate and is probably the source of your variation
- Material - the inherent variability in your material e.g. caused by (non-)representative sampling, segregation, inadequate dissolution etc
Also consider what is acceptable variability (1%, 5%, 10% jne.) as this will have an effect on what you're doing. Are you over-specifying? What is 'fit for purpose' and just good enough' in line with the proposed use of the materials? These last 2 questions may be the hardest to answer (As an example consider the particle size of aspirin. It can vary as much as 400% without having an effect on the dissolution and thus bioavailability. It may cause issues in compressing the tablet. What would you think of a specification of +/- 200% on the variation of particle size for aspirin?).
Dear Arezoo Setayesh,
It is likely your sample degradation from time to time. Always use fresh made samples for your experiments to work out difference among them.
In addition, you used precursors for nanoparticles which for sure are not stable if not protected from air or moisture or light.
@Yuri Mirgorod
Thank you for your answer. Do you have any suggestion how we can reduce these variations in CD intensity?
Arezoo Setayesh
Send me the names of acids, concentrations and intensities, your device. Only after that, I will be able to give specific advice on error statistics.
Arezoo Setayesh
>However, I'm having problems with different results on different days. Any suggestions?
Sometimes such a situation can happen. But usually, nobody can find a real reason except yourself. You can find an explanation from analysis of the results of several test experiments. It would help if you organized them according to Alan F Rawle system.
Between all possible sources of variability, it seems for me that first of all, you should check your samples.
For example, do they depend on oxidation in the air? For such test, you prepare two completely identical samples and put one of them into a vacuum hermetic container or even into some completely filled bottle, so there will be no air, another one put into your usual container or bottle and compare their intensity after one day, one week, etc.
How are the results dependent on dissolving liquid? Take different ones and check their variability.
How is the result dependent on the time between sample preparation and measurements? You should perform several careful measurements after, for example, 10 min., 20 min., 0.5 hours, 2 hours, 5 hours, 10 hours, etc.
To check your nanoparticles' segregation, sedimentation, etc., from day to day, record their intensity according to usual procedure, but then carefully sonificate them and compare the intensity before and after.
To check your equipment, you should use a kind of optical reference source, like, for example, a LED connected to the stabilized power source. Then see if the intensity measured in different days is changed or not?
This list of the possible reasons is not complete. You know your procedures better, so you should extend them to find the actual origin of your issue.
Good luck with your research!
Arezoo Setayesh
You have still not answered the key question in relation to (expected) standard error:
What is the acceptable variability to you (1%, 5%, 10% or other)?
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