Spectroscopy || Beer- Lambert's Law

#biologyanimation #biophysics #spectroscopy #spectrophotometer


Today we are going to learn about the spectroscopy and beer lambert’s law.

What is spectroscopy?

It is the study of the interaction between electromagnetic radiation and matter.
This matter can be any atoms, molecules or ions.
The electromagnetic spectrum ranges from very short wavelengths like gamma rays to long wavelengths such as radio waves.
The visible range is approx. 400-700 nanometer.
Now, coming to the types of spectroscopy.
When electromagnetic radiation meets the matter, the matter may absorb emit or scatter the radiation.
Depend on which there are three types of spectroscopy-absorption, emission and scattering.
Today we will talk about only absorption spectroscopy.
The basic principle of absorption spectroscopy is when a beam of monochromatic light passes through a solution or matter, some of its radiation may be absorbed.
This absorbance is measured with the help of an instrument known as a spectrophotometer.
Beer-Lambert’s law is based on two different laws.
The lambert’s law states that, when a monochromatic light passes through a transparent medium, the intensity of transmitted light decreases exponentially as the thickness of absorbing material increases.
Beer’s law states that the intensity of transmitted monochromatic light decreases exponentially as the concentration of the absorbing substance increases.
This Beer-Lambert’s law can be expressed mathematically by the formula
A=log I_o/I=εcl
Where A refers to the absorbance, I_o & I is the intensity of incident & transmitted light, respectively.
ε is the excitation coefficient which is constant for a particular matter.
C is the concentration of the sample & l is the path length.
T refers to the transmittance which is expressed by the ratio of I and I_o.
If we derive the relationship of absorbance with the percent transmittance from the above formula, we get absorbance is equal to 2-〖log〗_10%T
So, in spectrophotometer, you can get both the reading of absorbance & transmittance.
Now, see if we increase the sample concentration, the absorbance increases while the transmittance decreases.
However, if we change the path length, the absorbance increases and the transmittance decreases further.
Now, coming to the applications of the spectrophotometer, for example, we are taking protein estimation.
Let you have a sample of unknown protein concentration with you and you want to estimate it’s concentration.
First, you have to take a series of known concentrations of protein along with a blank.
Now you will add reagents to all the tubes.
The protein will give color after reacting with reagents.
The color intensity is directly proportional to the protein concentrations.
Now you have to measure the absorbance for each known concentration of the proteins along with your sample.
When you plot those readings in a graph, it will give you a standard curve.
From the standard curve, the value of 1 absorbance is calculated.
Now using the formula, you can calculate the protein concentration of the unknown sample.
Apart from protein estimation, spectroscopy is used to measure DNA/RNA concentration in enzymatic assays, in ELISA or measuring any concentrations of molecules which give any color of the UV-Visible range when in a solution.

[humahuma]

Thanks. I wish our PhD teachers in the university had only one tenth of your teaching talent

[danishacademy]

Wonderfully explained...Quiet thankful for your help and answering the questions arising in my mind..

[nibeditabag]

Thank you Mam. Khub sundor explaination. Many many thanks.

[MokhtarMuhammed-pc]

Wow, your tutorials is very interesting me so much and your sound also make me understand what you speak morre

[yassineb]

Thank you very much, I'm french I revise this chapter intituled : "Beer Lambert Law" or in French : "La loi de Beer Lambert" and in order to develop in English level and enhance it, I would to look this video. It is very good explicated ! Thank you !

[makhtaruzzaman]

Many many thanks for your nice and effective

[clairelovespandas]

great video! subscribed :D this helped me learn stuff for science olympiad. very thankful for this vid

[Sjb]

Thank you ma'am for an best explanation video 😊🙏🏻.

[funstudy]

Epsilon is the extinction coefficient..
(Not excitation ) 😁🙏🏻

[knowledgeisthesuccesspath]

you just grabbed my strong attention...awesome

[RoboChemist-nt]

Thank you so much Mam❤
Itni mehnat ki app ne hamare liye 😢
Agr app Sir Hoti to main likhta ..Chuma u 😘

[ScarfaceEugene]

Oh my God this video is so informative thanks I wish you could do more on spectroscopic methods please

[jyotirmayjana]

Very informative video ❤ it helps me a lot. Thank you ma'am😊.

[curryfromscratch]

Most underrated biology channel
You are doing a great job mam😇😍

[RoboChemist-nt]

One question: -
Lambert law states that if thickness increases, intensity light decreases .
But question is that how can we increase thickness?

[ChinmoyGhoshFilm]

Mam its so nice explanation 😊 it help me a lot❤

[monaallam]

Hello, thank you so much for your informative video, i am interested in measure absorbance for polluted water or water from river and lakes, can you pls tell me 1- how to convert reflectance to absorbance 2- how to correct absorbance and remove bad value, thanks again so much great teacher.

[anas_syed-YT]

Ma'am the epsilon is the extinction coefficient not the excitation coefficient

[monaallam]

Hello, can you pls preapare video how to correct Specta using filters

[AmanMishra-zkxq]

Thankyou teacher thanks alot for this great explanation I was fed up of my teacher's teaching
But I love it and for the first time I'm feeling confident that now I can explain and wite it
Please make more and more videos
UV spectrophotometer
IR spectrophotometer
Fluorimetry and colorimetry and many more topics please
I have likes and shared to all my classmates and also subscribed
Thankyou sooo much teacher
Thanks alot