How does a spectrophotometer work?like how do you conclude from the absorbence results? for instance you are investigating pigment formation by polyphenol oxidase.

Question

anonymous · Answer

I'm a bit fuzzy on the details, but I'll share what I know.

A spec works by shining a beam of light through a cuvette filled with a sample of substance X. A detector detects the intensity of light that is transmitted through (not absorbed by the sample), and then quantifies that data through a formula: log(Intensity of light before it hits the cuvette / Intensity of light after it goes through), which is called your Absorbance.

Now how is this useful? In (at least) two ways. The first is it allows you to identify your substance. Each substance has, in general, a characteristic absorbance curve....for example, if you have a sample showing a strong peak at 280 nm, you could reasonably be sure that there is some tryptophan in there. The second is it allows you to gauge the concentration of said substance. *Absorbance levels are proportional to the concentration of the absorbing solute*. The more tryptophan you have, the more light gets absorbed, and you can perform calculations to arrive at your amount of tryptophan.

anonymous · Answer

Absorbance is also equal to (molar extinction coefficient)*(concentration of absorbing species)*(path length of light). This is all summarized neatly and explained much better here: http://en.wikipedia.org/wiki/Beer-Lambert_law

mphetang · Answer

does this mean that the higher the reading of absorbence in the spectrophotometer, the more light is being transmitted through, hence less product (in the sample in the curvette)?

anonymous · Answer

More absorbance means actually the opposite - less light is getting through. More product is there in the sample to "soak up" the light. 

Let's take a quick look at our formula for Absorbance again:
log(Intensity of light before it hits the cuvette / Intensity of light after it goes through)

The more light that gets through, the closer the fraction will be to 1. Log(1) as you know is 0: in other words, no light was absorbed. Which makes sense - there was no decrease in the intenseity of the light, right? 

Conversely, if a bunch of light is absorbed, you'll have a tiny value for intensity of light after it goes through. The fraction will accordingly be much bigger; taking the log of that will result in higher absorbanc.e

mphetang · Answer

thank you very much kma230. u really helped clear the mist....i now get it crystal clear.thank you once more.