This is the best video about Spectrophometer ever. I really learned alot. Thank you so much for the time you took to make it so simple. Kudos. Daniel from USA
I did some research and found that the incident light is not white light. It’s a certain color of light frequency split by a prism that is directed through the sample in a spectrophotometer. Then you can also measure each of the other light frequencies from the prism by directing them through the sample. This is what gives a unique set of readings that identifies the substance. This is why spectrophotometer are more expensive than colorimeter which uses a set of R,G,B filters. Wouldn’t this mean that Tommy’s device is really a colorimeter and not a spectrophotometer as he states in his subject title?
Big thankful of your video. I watched your video for my knowlodge. and this is the best video of spectrophotometers. but I think my brain diden't want to know all of this things lol..
Hi there - glad you enjoyed the video. No - the two things are separate methods. Beers law describes a linear relationship between absorbance and concentration for molecules that absorb light *directly* - if you plotted the graph it would be a straight line. This works for molecules like chlorophyll and NADH which absorb light directly and are relatively unaffected by temperature/pH etc, hence you can mathematically predict the concentration with Beers law for these molecules. The calibration curve method is used for measuring molecules that *indirectly* absorb light eg via a chemical reaction. The classic example for the curve method would be using Bradfords reagent to measure protein concentration - the reaction between Bradfords and the protein causes the Bradfords reagent to change colour which you can measure on the spectrophotometer. This is a non-linear relationship that will depend on the temperature/pH/buffer, which is why you need to construct the calibration curve every time you want to make measurements. Hope that helps!
A bit confusing to use percentage to illustrate I0 and I, as it’d be an absolute quantity of irradiance rather than a fraction to anything. Would make more sense to illustrate the initial fraction via transmittance, then show absorbance as the anti logarithm to the base 10 of that.
If your molecule absorbs light directly it will be a linear curve, if you measuring indirectly via an assay it will probably be curved. H2O2 absorbs directly in the UV range at 240, or you can measure indirectly via xylenol orange assay, which would need a standard curve making. Hope that helps!
No - wavelength uses the lambda symbol. Epsilon is a measure of how inherently absorbent a particular molecule is at a particular wavelength, so acts as a calibration constant
My assignment has asked me to calculate the concentration of several solutions using beer's law - we calculated their absorbance and I'm assuming L is 1cm - so by converting the equation C = A/EL - but it hasn't given us an epsilom symbol, @@katharinehubbard5043
I should have probably done this explanation the other way around. For molecules that absorb light directly (eg NADH, chlorophyll, DNA) the relationship will definitely be linear. However for some spectrophotometer assays that measure concentration indirectly there will be saturation. The obvious example here is a Bradford assay to measure protein - the Bradford reagent is only sensitive to low/medium protein concentrations, and saturates at high concentration. The relationship between protein concentration and absorbance is therefore non linear, so the standard curve method is used. Hope that makes things clearer!
