The Beer-Lambert law are the laws that describe the relationship (they are the Beer law and the Lamber law combined) and they say that light absorbed is proportional to the concentration and the light absorbed is proportional to how much liquid the light travels through (the path length). When combined, the laws give the equation A = ε c l when A = absorbance, ε = extinction coefficient, c = concentration and l = path length. If you look at A = ε c l it looks like y = mx (with m being ε and l), hence fitting a straight line will work.
That is a very interesting question. It should be a straight line. The Beer-Lambert law - A = ε . c . l - looks a lot like y = mx which is the question for a straight line. So, yes, it should be a straight line (you can read more about this in an old blog post of mine - teaching.drnickmorris.com/2010/10/the-beer-lambert-law-straight-line-and.html ). However, there are times when the law breaks down, such as when you have very high levels of the material you are measuring or when you are using an 'indirect assay' (an indirect assay is when you are using the binding of one thing to another, e.g. a dye to a protein, to measure something, in this case, the protein) and the binding agent is limiting.
Thank you for your video. Our professor said that our concentration should never be higher than the greatest absorbance value of the standard curve. Your greatest absorbance value is 0.980. Do you know why this would be?
Thanks for the question. If you look at the data for the standard curve you will see that the top absorbance is 0.98 - you can see the data about 4 seconds in on the video. I then plot the data. The sample with an unknown concentration has an absorbance of 0.6 and I plot that on the graph and find the concentration (see at about 4 min). I think what your Prof was talking about is when you are determining the unknown concentration because then you cannot go outside the range of the graph. That is, you cannot go higher than the maximum absorbance in the standard curve. So, if my unknow had an absorbance of 1.2, then I couldn't determine its concentration as 1.2 is greater than the maximum absorbance of the highest standard, 0.98. Why can't we do this? Why can't we just extrapolate the graph? Why can't we just extend the line? Well, we don't know what happens 12 mM. Maybe the line continues straight (as it would expect it to under the Beer-Lambert Law) or maybe it curves? We don't know as we have not measured it. So, to be safe (and accurate) we don't extrapolate. We would say that a sample with a reading of 1,2 does not fall on the standard curve and we would repeat the experiment with the unknown diluted so its absorbance does fall on the curve. I hope that clears things up.
We have constructed the standard curve using known concentrations of a substance and measuring their absorbance and the reason we have done this is because we wish to determine the concentration of a new sample. The sample of unknown concentration has an absorbance of 0.6, so we can use the standard curve to determine the concentration. Hence, we find 0.6 and then plot the lines on graph (see from 4 minutes on) and then determine the concetration.
If you get a negative concentration, then you must have a negative absorbance. The Beer-Lambert equation is A = ε c l, where A is absorbance, ε = the extinction coefficient, c = concentration and l = path length. If c is negative, then A must also be negative as part of the Beer-Lambert law says A is proportional to c. Now, if A is negative, then this means the blank solution (the solution that contains none of the material you are measuring) was not set to zero. That is, it wasn't blanked correctly.
Finally got it . Thanks for helping
Great! Good to hear....
Have you got a video explaining if we don't know absorbance?
If you don't know the absorbance, then you cannot determine the concentration. Are you asking how you measure the absorbance?
I did this using the line equation, however I keep seeing online about needing to use the beer lambert law and im not sure if it is needed or not
The Beer-Lambert law are the laws that describe the relationship (they are the Beer law and the Lamber law combined) and they say that light absorbed is proportional to the concentration and the light absorbed is proportional to how much liquid the light travels through (the path length). When combined, the laws give the equation A = ε c l when A = absorbance, ε = extinction coefficient, c = concentration and l = path length. If you look at A = ε c l it looks like y = mx (with m being ε and l), hence fitting a straight line will work.
@Tammi Oechsle thanks but how is that relevant to line equations ?
Thank you, and I have a question, does the graph always have to be a straight line or it can be a curve?
That is a very interesting question. It should be a straight line.
The Beer-Lambert law - A = ε . c . l - looks a lot like y = mx which is the question for a straight line. So, yes, it should be a straight line (you can read more about this in an old blog post of mine - teaching.drnickmorris.com/2010/10/the-beer-lambert-law-straight-line-and.html ). However, there are times when the law breaks down, such as when you have very high levels of the material you are measuring or when you are using an 'indirect assay' (an indirect assay is when you are using the binding of one thing to another, e.g. a dye to a protein, to measure something, in this case, the protein) and the binding agent is limiting.
Thank you for your video. Our professor said that our concentration should never be higher than the greatest absorbance value of the standard curve. Your greatest absorbance value is 0.980. Do you know why this would be?
Thanks for the question.
If you look at the data for the standard curve you will see that the top absorbance is 0.98 - you can see the data about 4 seconds in on the video. I then plot the data.
The sample with an unknown concentration has an absorbance of 0.6 and I plot that on the graph and find the concentration (see at about 4 min).
I think what your Prof was talking about is when you are determining the unknown concentration because then you cannot go outside the range of the graph. That is, you cannot go higher than the maximum absorbance in the standard curve.
So, if my unknow had an absorbance of 1.2, then I couldn't determine its concentration as 1.2 is greater than the maximum absorbance of the highest standard, 0.98.
Why can't we do this? Why can't we just extrapolate the graph? Why can't we just extend the line?
Well, we don't know what happens 12 mM. Maybe the line continues straight (as it would expect it to under the Beer-Lambert Law) or maybe it curves? We don't know as we have not measured it. So, to be safe (and accurate) we don't extrapolate. We would say that a sample with a reading of 1,2 does not fall on the standard curve and we would repeat the experiment with the unknown diluted so its absorbance does fall on the curve.
I hope that clears things up.
@@NickMorrisPhD Thank you for your thoughtful response! This helps so much.
@@hannahd8375 Good to hear. Glad I was able to help.
Hello,
Why did you chose to use 0.6 to find the concentration of the sample instead of any other number?
We have constructed the standard curve using known concentrations of a substance and measuring their absorbance and the reason we have done this is because we wish to determine the concentration of a new sample. The sample of unknown concentration has an absorbance of 0.6, so we can use the standard curve to determine the concentration. Hence, we find 0.6 and then plot the lines on graph (see from 4 minutes on) and then determine the concetration.
@@NickMorrisPhD Thank you for your help! I understand much better.
@@bryannaanzueto77 Great..... good to hear.
Thanks a lot ❤️
You're welcome 😊
If I calculate the concentration of unknown and I get a negative number, what does that suggest?
If you get a negative concentration, then you must have a negative absorbance. The Beer-Lambert equation is A = ε c l, where A is absorbance, ε = the extinction coefficient, c = concentration and l = path length. If c is negative, then A must also be negative as part of the Beer-Lambert law says A is proportional to c. Now, if A is negative, then this means the blank solution (the solution that contains none of the material you are measuring) was not set to zero. That is, it wasn't blanked correctly.