Hello everyone,
I was particularly inspired by the paper by Antoine, Vlachos, & Rylander 2015 - Tunable collagen I hydrogels for engineered physiological tissue micro-environments. In it, they used turbidimetric assays to track polymerization of collagen gels and to understand polymerization kinetics. In their paper, they use readings at 405 nm. My question is how would someone decide on a wavelength to do their reading at. I imagine it would depend on the gel they are studying. I performed a spectral scanning from about 350 nm to 900 nm for my gels, and found very high OD readings at around in the 300-400 nm range, and then quickly dropping down after about 400 nm and staying pretty constant all the way through. I am not sure if anything in this scan could tell me which wavelength to use.
Could anyone tell me how they would choose a wavelength for absorbance readings when you have no reference to fall back on? Thank you.
Hello,
I would say you can choose any wavelength as you prefer! But to have an accurate and reproducible reading you should choose a wavelength with maximum absorbance.
Hello,
I would say you can choose any wavelength as you prefer! But to have an accurate and reproducible reading you should choose a wavelength with maximum absorbance.
In this case, you are using the scattered light, not the absorbed light as your signal. So you should avoid wavelengths where there are absorption peaks. I suggest choosing the wavelength that changes most during your experiment.
In this area, I find the papers by C.G. (Kees) de Kruif (active on Researchgate) very inspiring. He uses turbidity to track the aggregation of milk protein. It seems to me that the same theory underlies the work that you cite.
Don't get stuck on a range. Instead, figure out experimentally what wavelength or range of wavelengths will accomplish what you need for your specific application.
I recall writing up a method for reaction monitoring of a polyurethane hydrogel using a scanning UV/VIS spectrophotometer back in the 1980's (It was eventually published, but I do not recall where or when). I chose a wavelength and wide bandwidth where the initial gel solution gave me a good initial signal and one where all of the reaction time-point samples that followed provided a clean signal within the dynamic range of the detector all the way to the end of the reaction. It proved to be a simple way to monitor the reaction progress of the polyurethane at the pilot plant stage (when HPLC instrumentation and trained personnel were not always available).
Hi Sebastian Freeman
U can go through this article "Bonding interactions and stability
assessment of biopolymer material
prepared using type III collagen of avian
intestine and anionic polysaccharides"
Here we use 310nm as the wavelength for absorbance. based on the preliminary studies, we confirmed that, at this wavelength, maximum aggregation occurs (i.e maximum absorbance). you may repeat your spectral measurements at a very low concentration. There you can conclude the maximum absorbance at the corresponding wavelength and the concentration.
Hi
Due to beer-Lambert law:
A=log(I/I0)
I is a certain number so:
Bigger A is equivalent to:
Smaller I0 : lesser amount of light has been scattered
More amount of light has been absorbed
:)
when you are choosing the peak, you are actually choosing the state in which most of the I has been absorbed by the medium. That is strongest signal, slighter noise
Good luck
Using the maximum wavelength gives us the best results. This is because at the peak absorbance, the absorbance strength of light will be at the highest and rate of change in absorbance with wavelength will be the smallest. Measurements made at the peak absorbance will have the smallest error. Long answer: It really depends on what is the largest source of error. Taking the readings at the peak maximum is best at low absorbance, because it gives the best signal-to-noise ratio, which improves the precision of measurement. If the dominant source of noise is photon noise, the precision of absorbance measurement is theoretically best when the absorbance is near 1.0. So if the peak absorbance is below 1.0, then using the peak wavelength is best, but if the peak absorbance is well above 1.0, you might be better off using another wavelength where the absorbance is closer to 1. Another issue is calibration curve non-linearity, which can result in curve-fitting errors. The non-linearity caused by polychromatic light is minimized if you take readings at either a peak maximum or a minimum, because the absorbance change with wavelength is the smallest at those wavelengths. On the other hand, using the maximum increases the calibration curve non-linearity caused by stray light. Very high absorbances cause two problems: the precision of measurement is poor because the transmitted intensity is so low, and the calibration curve linearity is poor due to stray light. The effect of stray light can be reduced by taking the readings at awavelength where the absorbance is lower or by using a non-linear calibration curve fitting technique. Finally, if spectral interferences are a problem, the best measurement wavelength may be the one that minimizes the relative contribution of spectral interferences (which may or may not be the peak maximum). In any case, don't forget: whatever wavelength you use, you have to use the exact same wavelength for all the standards and samples. See http://terpconnect.umd.edu/~toh/models/BeersLaw.html Tom O'Haver Professor
I think the using max wavelength for different species is to obtain minimum error on the absorbance. that is why we select the wave length of known to determine the absorbance of unknown which is directly related to concentration of unknown obtained from equation of calibration curve.
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