The other day I was going to prepare my spectrophotometer to analyze the absorbance (OD600 nm) of a series of test tubes containing LB liquid media. Therefore, I calibrated my spectrophotometer using a test tube containing clean, sterile LB liquid media (blank). After doing that, the spectrophotometer showed an absorbance of 0.000 showing that it was properly calibrated. Then, for the sake of science, I introduced a test tube without any liquid inside (just empty air) and it produced a higher absorbance than the blank used to calibrate the equipment (0.014, I don't quite remember the numbers). I was extremely puzzled by this finding and I was wondering if anyone could explain why did I observe a higher absorbance when measuring an empty test tube. Thank you very much in advance.
Hello, Actually, the blank should not show any absorbance at all, as the only intensity losses due to the blank should be caused by reflectance at its different interfaces, otherwise I would not call it a blank (actually there are reference samples where the concentration is non-zero).
https://doi.org/10.1002/cphc.201600114
Yes, because of the difference in refractive index and consequently how much light reaches the detector
Paul Milham, precisely that is my concern. The blank I used contained LB media which has a slightly yellowish colour. To my understanding, the LB media should absorb more light than thin air. Therefore, shouldn't an empty test tube read a lower absorbance rather than a higher one? Or does it have to do with the refractory ability of air being greater than liquid LB media? Is there some physics concept that I'm missing? Thank you for your time.
The refraction in water is greater than in air. The amount of reflected light reaching the detector may also be related to the difference in refractive index between air and water. Lastly, the colour is unimportant if you are not measuring absorbance at the corresponding wavelength. That exhausts my knowledge!
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Could be the total internal reflection is higher for the tube with air so more light ibounces around and goes out the sides away from the detector eye like a fiber optic cable where when filled with liquid less light is internally reflected between surfaces less light bounces around due to the refractive index being higher.
Opps pressed answer was not sure it worked sorry about the multiple times
What you actually measure is not absorbance, but transmittance. Therefore, not only the fraction of light that is absorbed does not reach the detector, but also the part that is reflected. Reflection takes place whenever there is a difference in refractive index between two media and it is the stronger the larger the difference is. Since the difference between glass and air is larger than the one between glass and water, reflection is also stronger. Alec Feinberg Transmittance is usually measured with normal incidence. This means that there is no internal reflection and also no portion of light leaving through the sides of the tube, but you certainly get multiple reflection if this is what you mean.
In normal incidence snells law indicates no bendung of light. So how can light have any refraction??? There are two possibilities i can think of. The first is the light is not perfectly normal. The second is the test tube may be say qyartz which is non isotropic so due to polarization there are two waves the ordinary wave does not refract and the extraordinary wave does so snells law refraction may occur loss of transmission no absorption.
Alec Feinberg Indeed you are right - there is no bending in case of normal incidence, but why is this important and what has this to do with reflection? Concerning Quartz, there is no way that either the ordinary wave or the extraordinary wave in the visible spectral range could be refracted while the other is not - the optical constants of both are just to similar.
Alec Feinberg No. When a wave impinges on an interface between two media, be the second anisotropic or not, the tangential components of the wavevector are preserved. In other words, if they are zero in the incidence medium (= normal incidence), they are necessarily zero in the second medium (= normal to the interface). Have a look at chapter 6 of my lecture notes and the references therein: Preprint Wave optics in Infrared Spectroscopy
By the way, the SiO2 cuvette material is not Quartz, but vitreous silica.
When a beam of unpolarized light enters a birefringent crystal at normal incidence (but not along the optic axis), two light beams emerge. Quartz is birefringent. the other possibility is the test tube which is rounded makes it difficult for true normal incidence. Hope that helps.
Certainly, at normal incidence, two light beams with different speeds emerge in general in the anisotropic medium, but they still travel along the same path perpendicular to the interface, at least in this universe... ;-)
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