UV Vis spectroscopy explained lecture

Summary

This video tutorial from 'Somos Biology' delves into the fundamentals of UV-Visible spectroscopy, a technique widely used in chemistry and life sciences for determining the concentration of molecules in solutions. The script explains the setup of a spectrophotometer, the molecular mechanism behind light absorption, and how this technique can provide insights into chemical groups present in molecules. It also discusses the importance of 'lambda max' for identifying maximum absorbance and the use of the Beer-Lambert law for calculating unknown concentrations, making complex scientific concepts accessible to viewers.

Takeaways

• 🌟 UV-Visible spectroscopy is a widely used technique in both chemistry and life sciences for various experiments and analyses.
• 🔍 It is primarily used for determining the concentration of different substances or molecules in a solution, including cells in microbiology practicals.
• 📈 The technique can also provide some information about the chemical groups present in molecules, though the data is not highly reliable for differentiating between different types of groups.
• 🛠️ The setup of a UV-Visible spectrophotometer includes a light source, a monochromator to separate light wavelengths, a beam separator, and a detector to measure light intensity.
• 🌈 The UV-Visible range of the electromagnetic spectrum is from 200 to 800 nanometers, with UV ranging from 200 to 400 nm and visible light from 400 to 800 nm.
• 🔬 The molecular mechanism involves the absorption of light energy by molecules, causing electrons to jump from a ground state to a higher energy state, which is specific to certain wavelengths.
• 📊 Beer-Lambert Law is used to relate the absorbance of light to the concentration of a molecule in a solution, which is crucial for quantitative analysis.
• 📉 A plot of absorbance against concentration typically results in a non-linear curve, which can be linearized by using a logarithmic scale to facilitate easier calculation of unknown concentrations.
• 📚 Understanding the specific wavelength (lambda max) at which a molecule absorbs light most strongly is important for identifying the maximum absorbance for different chemical groups.
• 🔬 The molecular level process of UV-Visible spectroscopy involves the excitation of electrons from bonding or non-bonding orbitals to higher energy states, such as sigma or pi orbitals.
• 👍 The video concludes by emphasizing the importance of UV-Visible spectroscopy for concentration analysis and its limitations in identifying specific chemical bonds.

Q & A

What is UV-visible spectroscopy and why is it used?

UV-visible spectroscopy is a technique widely used in chemical and life sciences to measure the concentration of different substances in a solution. It is also used to identify chemical groups present in molecules, although the data is not highly reliable for differentiating between different groups.

What are the main applications of UV-visible spectroscopy?

UV-visible spectroscopy is used for determining the concentration of molecules in a solution, including in microbiology for measuring cell concentrations. It is also used in enzymatic reactions and enzyme kinetic studies to assess enzyme-associated activities.

How does the UV-visible spectrophotometry work?

UV-visible spectrophotometry works by passing light through a sample and measuring the intensity of the light that is transmitted through it. The difference in intensity before and after the sample is used to calculate the absorbance, which is related to the concentration of the molecules in the sample.

What is the role of a monochromator in UV-visible spectroscopy?

A monochromator is a device that separates the light from a source into its different wavelengths. It allows only a specific wavelength of light to pass through, which is then used to analyze the sample.

What is the significance of the wavelength in UV-visible spectroscopy?

The wavelength is significant because different molecules absorb different wavelengths of light. By knowing the specific wavelength that a molecule absorbs, one can identify the presence of certain chemical groups within the molecule.

What is the relationship between frequency, wavelength, and energy of an electromagnetic wave?

The frequency and energy of an electromagnetic wave are directly proportional; higher frequency means higher energy. The wavelength and frequency are inversely proportional; shorter wavelength means higher frequency and vice versa.

How is the concentration of a molecule in a solution determined using UV-visible spectroscopy?

The concentration is determined by measuring the absorbance of the sample at a specific wavelength. Using the Beer-Lambert Law, which states that absorbance is proportional to the concentration of the molecule, the concentration can be calculated.

What is meant by 'transmittance' in the context of UV-visible spectroscopy?

Transmittance is the ratio of the intensity of light after passing through the sample (I) to the initial intensity of light (I0). It indicates the amount of light that has been transmitted through the sample.

What is the difference between transmittance and absorbance?

Transmittance measures the amount of light that passes through the sample, while absorbance measures the amount of light that is absorbed by the sample. They are inversely related; higher transmittance means lower absorbance and vice versa.

Why is it difficult to extrapolate data from a graph of percent transmittance versus concentration?

It is difficult because the relationship between percent transmittance and concentration is not linear, making it challenging to predict the concentration of unknown samples from such a graph.

How can a graph of absorbance versus concentration be made linear for easier data interpretation?

By plotting the logarithm of the concentration (or the reciprocal of transmittance) against absorbance, the graph becomes linear, which simplifies the process of extrapolating the concentration of unknown samples.

What is the molecular mechanism behind the absorption of light in UV-visible spectroscopy?

The molecular mechanism involves the excitation of electrons within molecules from a lower energy state (ground state) to a higher energy state when they absorb energy from light of a specific wavelength. The energy absorbed corresponds to the difference in energy levels between the molecular orbitals.

Why are UV-visible spectra not reliable for identifying specific chemical bonds?

UV-visible spectra are not reliable for identifying specific chemical bonds because many molecules with similar bonds and structures absorb light at similar wavelengths, making it difficult to distinguish between them based on UV-visible spectroscopy alone.

Outlines

00:00

🔬 Introduction to UV-Visible Spectroscopy

The video begins with an introduction to UV-Visible spectroscopy, a widely used technique in chemistry and life sciences for determining the concentration of molecules in a solution. The speaker explains that while this technique can provide some information about the chemical groups present in a molecule, it is primarily used for concentration analysis due to the reliability of its data. The video promises to delve into the setup, mechanism, and application of UV-Visible spectroscopy, including its use in colorimetry and enzymatic reactions.

05:01

🌟 Understanding the Setup of UV-Visible Spectrophotometry

This paragraph explains the basic setup of a UV-Visible spectrophotometer, starting with a light source that emits UV or visible light. A monochromator is used to separate the light into its individual wavelengths, creating a monochromatic beam. The beam separator directs this light towards two chambers, one for a control sample and one for the actual sample. The differences in light absorption between the two samples are then detected and recorded. The explanation includes the importance of wavelength and frequency in the context of the electromagnetic spectrum and how they relate to the energy of the light used in the technique.

10:03

📈 The Molecular Mechanism of UV-Visible Spectroscopy

The speaker describes the molecular mechanism behind UV-Visible spectroscopy, focusing on how molecules absorb specific wavelengths of light, causing electrons to jump from a ground state to a higher energy state. This absorption of energy results in a decrease in light intensity, which is measured by a detector. The concept of transmittance and absorbance is introduced, with the former being the ratio of light intensity after passing through a sample to the initial light intensity, and the latter being a measure of how much light is absorbed by the sample. The paragraph also touches on the importance of lambda max, the wavelength at which a molecule absorbs light most strongly.

15:04

📊 Data Interpretation in UV-Visible Spectroscopy

This section discusses how to interpret the data obtained from UV-Visible spectroscopy. The speaker explains the relationship between transmittance, absorbance, and concentration, and how a graph plotting absorbance against concentration can be used to determine the concentration of an unknown sample. The paragraph emphasizes the importance of using absorbance rather than transmittance for creating a linear graph, which facilitates the calculation of unknown concentrations. The explanation includes the Beer-Lambert Law, which quantifies the relationship between absorbance, the molar absorption coefficient, concentration, and path length.

20:07

🧬 Molecular-Level Understanding and Practical Application

The final paragraph delves into the molecular-level understanding of why molecules absorb light at specific wavelengths, discussing the energy states of different chemical bonds and how the energy from light is absorbed to excite electrons to higher energy states. The speaker also explains the limitations of UV-Visible spectroscopy in identifying specific chemical groups due to the similarities in the absorbance patterns of different molecules. The paragraph concludes with a reminder of the technique's primary use for concentration analysis and a call to action for viewers to like, share, and subscribe for more educational content.

Keywords

💡UV Visible Spectroscopy

UV Visible Spectroscopy is a technique used to analyze the interaction between light in the ultraviolet and visible range with chemical compounds. It is central to the video's theme as it is the main subject being discussed. The script explains how this technique is widely used in chemistry and life sciences to determine the concentration of molecules in a solution, with examples including the measurement of protein concentration and enzymatic activity.

💡Concentration Analysis

Concentration Analysis refers to the process of determining the amount of a particular substance in a given volume of solution. It is a key concept in the video, as UV visible spectroscopy is often used for this purpose. The script mentions that this technique can measure the concentration of different molecules in a solution, including cells during microbiology practicals.

💡Molecular Mechanism

Molecular Mechanism describes the process by which molecules interact with light at the molecular level during spectroscopic analysis. The script explains that when a molecule absorbs light at a specific wavelength, its electrons are excited from a lower energy state to a higher one, which is fundamental to understanding how UV visible spectroscopy works.

💡Monochromator

A Monochromator is a device that separates light of different wavelengths into a single beam of a specific wavelength. In the context of the video, it is part of the spectrophotometer setup, which is crucial for UV visible spectroscopy. The script describes how the monochromator uses a prism to separate light into its different colors and allows only one specific wavelength to pass through at a time.

💡Beam Separator

A Beam Separator is a component in a spectrophotometer that directs the monochromatic light into separate paths for control and sample measurements. The script mentions the beam separator in the context of the spectrophotometer setup, explaining that it guides the light to two different chambers, one for the control and one for the sample.

💡Detector

A Detector in the context of spectroscopy is a device that measures the intensity of light after it has passed through the sample. The script explains the role of the detector in recording the data that ultimately provides information about the concentration of molecules in the sample.

💡Transmittance

Transmittance is the ratio of the intensity of light that passes through a sample to the intensity of incident light. It is a key concept in the script, as it is used to calculate the absorbance and thus determine the concentration of the sample. The script explains that transmittance is inversely related to absorbance and is measured by the detector.

💡Absorbance

Absorbance is a measure of how much light is absorbed by a molecule at a specific wavelength. It is directly related to the concentration of the molecules in the sample. The script discusses how absorbance is calculated from transmittance and used to determine the concentration of unknown samples by comparing it to a standard curve.

💡Lambda Max (λmax)

Lambda Max (λmax) is the wavelength at which a molecule absorbs light most strongly. The script explains that each type of chemical group has a specific λmax, which can be used to identify the presence of certain groups in a molecule, although it is not a definitive method for determining specific chemical bonds.

💡Molar Absorption Coefficient (Epsilon)

The Molar Absorption Coefficient, often denoted as epsilon (ε), is a constant that relates the absorbance of light by a molecule to its concentration and the path length of the light through the sample. The script discusses how this coefficient is used in the Beer-Lambert Law to calculate the concentration of an unknown sample from its absorbance.

💡Beer-Lambert Law

The Beer-Lambert Law, also known as Beer's Law, describes the relationship between the absorbance of a solution and the concentration of the absorbing molecules. The script explains that this law is fundamental to UV visible spectroscopy, allowing for the calculation of the concentration of unknown samples based on their absorbance.

Highlights

UV visible spectroscopy is widely used in chemical and life sciences for various experiments.

The technique helps in determining the concentration of different molecules in a solution.

UV visible spectroscopy can also measure the concentration of cells in microbiology practicals.

The technique provides limited information on the chemical groups present in molecules.

The video explains the setup of UV visible spectrometry, starting with a light source.

A monochromator is used to separate different wavelengths of light into a single beam.

The role of a beam separator in directing light to control and sample chambers is discussed.

The detector measures the intensity of transmitted light, which is key to determining concentration.

The molecular mechanism involves electrons absorbing energy and jumping to higher energy states.

Sigma and pi bonds require different amounts of energy for electron excitation.

The video explains the concept of transmittance and absorbance in the context of spectroscopy.

Lambda Max is identified as the maximum wavelength for which absorbance is the highest.

The Beer-Lambert Law is used to relate the concentration of a molecule to its absorbance.

A graph of absorbance versus concentration can help determine the concentration of unknown samples.

The video demonstrates the process of creating a calibration curve for concentration analysis.

UV visible spectroscopy is not used for identifying specific chemical bonds due to limitations.

The video concludes by summarizing the practical applications and limitations of UV visible spectroscopy.

Transcripts

[Music]

welcome back friends welcome to another

video tutorial from somos biology and in

this video tutorial we want to talk

about UV visible spectroscopy I've got a

request from many of my subscribers to

make a video on that previously made

video on IR spectroscopy and extra NMR

spectroscopy so if you want to know

about NMR and IR spectroscopy you can

look at my channel and you will get

videos on that so let's begin with UV

visible spectroscopy UV visible

spectroscopy is very widely used

technique in not only chemical or

chemistry labs but also in life sciences

many different experiments to rely on

the idea of UV visible spectroscopy but

the first question is why we need this

technique if it's a technique then what

this technique is giving us the answer

to that is in many situation we need to

find concentrations of different

substances concentration of different

molecules in a solution and that we can

measure perfectly with UV visible

spectroscopy and in some cases also

concentration of cells during the

microbiology practicals we can also use

UV visible spectroscopy to measure so it

can give us a good data about the

concentration of any molecule that is

present in a solution but it also can

tell us a little bit about the chemical

group that is present in the molecule

but the data that we get is not very

much reliable because for the different

types of group the UV visible

spectroscopy can give us very similar

data so you generally use it for only

concentration analysis but how exactly

we will get to know the concentration

from the UV visible spectrophotometry

let's look at that in this video to

understand using visible spectrometry we

also tagged it with known as the

colorimetry which is linked something

with the different colors of the color

substances which can help us to identify

the concentration of the colored

compound that is present in there and we

use it extensively in enzymatic

reactions to find out enzyme

associated activities an enzyme kinetic

studies so the first thing that will

tell you is what easy visible

spectroscopy and how the whole technique

is oriented like how the mechanic the

mechanism of the technique so all these

instruments that are present there to

make this technique possible then I'll

tell you the molecular mechanism with

which this whole technique works so

let's begin with the actual setup of

this UV visible spectrometry and that

gives us start with a source that you've

definitely a light source because as we

see UV visible spectrometry utilizes UV

or visible range of light as the energy

source primary energy source to to find

out the important feature of

concentration in case of is spectrometry

we know that infrared is the range of

the light that they've used but in this

case we use UV or visible range and you

know UV and visible range like

electromagnetic range you should have a

little idea about the electromagnetic

wavelength that we see completely and

some basic idea before talking into the

details is if you look at the

electromagnetic spectrum you will find

out different wavelengths it's ranging

from like gamma rays to ultimately

ending there in the microwave as well as

infrared waves till the end so what

those different waves are telling us the

important feature you always need to

know is the idea of two things one is

the wavelength and the frequency of that

electromagnetic wave the more the

frequency of a wave the higher energetic

that wave is that energy is that's idea

more frequency more energy and if you

have more frequency the wavelength will

be long I mean so the less sorry more

frequency less wavelength higher energy

and less energy means less frequency and

longer wavelength so very long

wavelength that will give you less

frequency and less energy higher like

shorter wavelength more frequency more

energy so in this case if we divide this

UV visible spectrum into the small part

and fractions what we'll find here is

simply ranging me from

200 to 800 that's kind of the range

although we have we can extend it till

10 20 nanometer in the wavelength but

from 200 nanometer to 800 nanometer that

will be the range from 200 to 400

approximately taken as a UV range and

from 400 to 800 approximately taken here

for the visible light range that we

usually use for the UV visible

spectrometry but normally actually UV

range ranges even back less wavelength

less number of nanometer of the

wavelength till this part as well so

what we know here by looking at the

spectrum is that as we are going close

to UV the wavelength is shorter so the

frequency is higher as we going this

side the frequency is low so this right

hand side we find more like a like a

microwave IR or infrared that means long

wavelengths less energy less frequency

till we get towards UB x-ray gamma ray

that means very short wavelength and

frequency is very very high energy also

very very high this is the scheme now

from this scheme you only take the 200

nanometer to 800 nanometer range now in

this case whenever we go to 400 to 800

nanometer we have the visible range of

the light that is visible colors that we

know the big G or the color violet

ultraviolet that from the from the

violet till the red that is a different

7 separate colors are ranging from this

part of the visible range now what we do

now is simply if you look at the no

instruments it goes like the source okay

so let any kind of light source a

filament in there this is the light

source and after the light source it has

a separate unit known as a monochromator

let me draw this unit this is mono

chromatic what motor chromatid does is

normally you know the source light as it

will have visible and you arrange it

have this this basic light that we know

the law

coming from the lightbulb and a

headlight of your scooter so dancing 27

separate all these different wavelengths

so monochromator separates all those

different wavelengths of the light into

single beam that's why called mono

chromatic because if we make single beam

from all this mixture of this light and

after the monochromator what else we

have we have the beam separator and the

beam separator almost like like almost

what we can say simple norm which can

separate the beam and guide it into two

separate directions and in this two

separate direction we have two separate

chambers one is for the control and the

sample chamber and then right after both

this chamber both the chambers are

connected to a detector this is the

simplest drawing of any individual

spectrophotometer and it begins with the

with the source and the source light

comes in and here we have small small

gap slits through which the light passes

after passing there there will be this

this mirrors will reflect it but

ultimately there is a prism inside this

monochromator so this is prism so I have

this prism this prism will separate

those that light into all these

different types all these different

wavelength of the light different color

so only one specific wavelength will be

allowed to pass through this ultimate

chain now you can alter this like

anytime you can pass a red light you can

pass blue it can pass orange except a

scalar depends on in which angle are the

the channel is present through which if

we go to this beam separator and the

beam separator will separate the same

monochromatic light send it to two

chambers the Chamber's with the control

and the Chamber's with the sample and

then from both this they will get the

data

detector detection by the detector and

then finally they will record the data

and give you the answer to it now the

question is in the simplest structure

that we see why this kind of structure

is paper and what exactly our role in

this case the idea of UV visible

spectrometry is simply whenever the

light hits a specific wavelength of

light hits a molecule that molecule get

excited and the electron once excited is

jumped up from a lower or we can say

ground state to a higher energy state

and whenever the electron jumps out what

it does is absorb that particular light

energy because the electron utilizes

that energy to move from the ground

state to the top state right because you

know one thing all your light is that

energy cannot be created it cannot be

destroyed the only thing possible is

transforming energy from one form to the

other so in this case getting the energy

so energy is little bit absorbed at that

point energy is utilized at this point

is in a more scientific way so what we

know if the energy is utilized then what

we know the intensity of that light that

received is lost a little bit right so

what we know if we put a blank a little

cubic qubit are small chambers where we

add samples to date so generally they

are made a bit glass so this Gloves

cubed is present here and in this qubit

we only put water so there is no

molecules present there or nothing is

very completely basic water so what

happens that light beam passing through

water and water there is no molecule to

finally absorb any light or utilizing

the energy from the light so what it

does simply it usually passing almost

the same intensity that ultimately

reaches here so what we get here there

are two separate intensity that a

detector always can measure in both the

sku which we put water then what will

happen

same like that become so normally the

light that generally come if there is no

absorption known as the intensity of the

light starts with i0 this is the start

point of the intensity of the light

then if you put the sample then we get

another intensity depending upon the

molecule that is present in that sample

but here same so we also call it I

because that's the intensity of the

sample but in this case we know if you

put water in both the chambers then we

will get the value of i0 will be equal

to I will be seen so the intensity of

start and intensity at the end are same

that means we're dealing with same

solution and literally this solution is

not taking any intensity that is first

transmitted through this beam separator

but now imagine in the sample tube in

the sample chamber we put a qubit and

here what we put a solution let the

protein let's say put protein solution

in there now proteins mainly there are

bulky rings in the proteins and all

those aromatic amino acids those amino

acids will receive then we energy at a

specific wavelength and if you also put

DNA they will also be absorb a specific

wavelength of light compared to the

other like and that is also due to the

bases that are present in the DNA so

usually for proteins that we know that

it will absorb the 260 nanometers of the

wavelength so once you see the 250

nanometer of the wavelength here and

that causes absorption of that

wavelength absorption of few of this

energy by the number of protein

molecules that are present there the

amount of protein the concentration of

protein that is present there the more

proteins present there the more

absorption and the less is this

intensity of the incident and intensity

of the of the reflected eye because you

know light is passing reflected and

finally reaching the detector so the

more absorption there is I value will be

decreased so in that case we know there

is a net change in intensity and what we

can find about the net change is this

this is the net change in the intensity

is it it so what we know here as as

known as a transmittance transmittance

known as the value for so we call it

transmittance or capital T

the value for I by i0 that is known as a

transmittance so the more light is

refracted the more light is removed

that means the more transmission

transmission take place and the less

light is removed which is the value of I

will be less the less transmittance is

there so if we know the value of

transmittance now what is about

absorbance transmittance and absorbance

are opposite to each other

different to each other so transmittance

is I by I 0 in that case absorbance will

become a lot of value of i0 by I that's

how you measure absorbance so more

transmittance less absorbance less

transmittance more absorbance and that's

kind of a true and it depends on the

concentration of the molecules that is

present in the sample now what kind of

molecule that is present there dictates

also what kind of group chemical group

is present also dictates at which

wavelength they will receive the energy

that is also very very important and for

every single type of chemical groups

there is a specific wavelength for which

the absorbance is maximum known as a

lambda Max or lambda maximum lambda max

is the maximum wavelength for which they

will give you most absorbance and the

readings they're known as lambda max we

can also get the value of lambda max for

different types of chemical groups now

the question is why we they absorb this

light and how this process works

molecular level we'll talk about that in

a moment but before going in there let

me first tell you how exactly we

interpret this data because you know

detector detects two things v-0 and I

the difference between the two will tell

you whether they will absorb some light

or they transmitted more light so for

that what we can find in this particular

case is simply we can plot a data with

the the absorbance and the difference in

the absorbance by that percent like the

difference of the absorbance along with

the concentration so if you know one if

you know if you know the absorbance we

can also find the concentration

there but how exactly because we know

that the transmittance if we look

normally in this data they are not going

to give us absorbance as a value because

the detector detects the transmittance

right because the absorbance is there in

the sample and detector only detects how

much is transmitted how much transfers

through this Q weight to the detector so

always the value that we get will be in

the transmittance form so simply what we

can draw from here we can draw a graph

and in that graph what we say the

percent transmittance and also

concentration of the solution of the

molecule so transmittance will be almost

the value of 10 to the power minus

concentration into the length of the

qubit that is the length of the qubit

dictates also how and how many molecules

of that molecule is present in that

qubit so according to beer and Lombard

they told us that P our transmittance

can be measured in the form of 10 to the

power minus concentration into the

length but transmittance is not going to

give us much data because if we put the

data in the terms of transmitted wave

let's say the concentration is very less

transmission should be maximum right

absorbance in that case will be very

less so transmittance will be highest

then let's elevate them a little bit

more and more and more so what kind of

graph we get we get a graph like this

slope like this this is not a straight

line this is not a linear drop now the

problem with this graph is although we

are getting the data directly from the

detector as observed percent

transmittance and the concentration it

will be very difficult for us to

extrapolate the data let's say we know

samples and we utilize some case samples

of the concentration which we know and

we plotted the plot with it then what we

do then we work an unknown and it

tasting that unknown and somehow the

unknowingly here here she becomes very

difficult for us to finally calculate

what exactly the concentration of that

unknown it becomes really difficult but

if the if the data is always straight

line

it becomes very easy to predict the

concentration of any unknown sample and

that's exactly what we do normally in

lab if you are checking up a protein

concentration we first prepare standard

concentration of proteins that the

concentration of which we already know

and then what we do then we plot a data

with it a straight line and then the

unknown sample whenever we get the

absorbance by putting in the data we can

simply extrapolate the data to the

concentration to find the actual

concentration of that protein but how

exactly we'd get a straight line in the

curve that is a big problem and the only

way is possible if we get the data in

absorbance scaled because knowing

absorbance scale the data should be

something like that we'll start from

local sensation low absorbance high

concentration high absorbance and linear

relation but the transmittance is not a

linear relation so in this case to make

it a linear relation of any curved line

remember that in your lifetime you will

always do a log

so the log value of concentration in two

lengths that can give you the value of

absorbance that will give you the value

of observers and the log value that we

utilize here is simply utilizing as a

constant that is known as molar

absorption coefficient or epsilon into

concentration into length we also can

write it as epsilon concentration is

written as C length is length of the

cubed known as L so the formula comes

down to epsilon C l equals absorbance

this is the net absorbance formula ecl

derived from lambert bier equation and

from this equation what you can also

tell if you know absorbance because in

the experiments we can find absorbance

so if you know absorbance you can find

concentration so for a concentration it

will be absorbance divided by epsilon

into

and this is how we can calculate a

concentration of an unknown molecule

unknown molecule that can be protein din

or any other molecule out there by this

fashion what we can create we can create

a straight line of the graph we can

trial of easily predict the

concentration let's say this is the

straight line of the graph and we know

the unknown protein is sommore somehow

represented and present someplace like

here so the concentration is simply drag

and we find it here and in that case

instead of transmittance EDB absorbance

so that's how you get the concentration

value from the graph as well okay so

this in a sense is the mechanism of how

we use UV visible spectrometry and to

get the data out of it but the second

part is also left why and how it's

working in the molecular level remember

for all this type of molecules I told

you it for all these different varieties

of molecules there is only a specific

wavelength for which that molecule will

get some energy and absorb the energy

and do some transmittance of the energy

now what is that mechanism to understand

that let me do some of this

so what we know at this particular point

is all those different different

molecules of chemical groups like

different bondings inside so for any

molecule if we look at here it's simply

we can divide in their energy states and

energy state with that like the ground

states like the nonbonding state or N

and then we have pi PI star Sigma star

Sigma this is almost like the simplest

idea of how the energy states are looks

like an atom in this particular case we

have the Sigma Phi n which is a

nonbonding and then five-spice turn and

Sigma star these are also nonbonding but

these are the bonding so the idea is we

have carbon carbon this is a single bond

present between carbon and carbon this

is a PI bond so what happens normally

here in this case of carbon

sorry carbonyl carbon this is Sigma

very sorry so have it's a sigma bond

strong very much strong Sigma bond and

Sigma bonds require more energy to

create and break so here Sigma bond is

there so so the electron that is present

let's say here in this orbital here and

the electron whenever the receives a

specific energy it can jump up to the

Sigma star so it will jump from one

orbital to the other this is a ground

state to the higher energy state but the

problem is in the higher energy state

the bond will not be very much stable so

again you need to remove from that so

this is how the electrons jump from a

ground state to a higher energy state

for a sigma bond directly from Sigma to

Sigma star now they can also jump to

this nonbonding orbital but if it takes

only less energy

similarly if it's C double bond o in

that case we know this is a PI bond so

for a pipe on the journey will begin

from this PI and it will go to the PI

star and from the PI to the in now it

depends on how much energy available at

what is the exact wavelength of light

that's sitting that's why they rely on a

specific wavelength for excitation for

UV visible spectrum matrix always the

excitation of that molecule in response

to the energy that they receive in

response to the wavelength of light

that's hitting now if the wavelength of

light is shorter that means very much

high frequency high energy for a sigma

bond it will directly jump to the Sigma

star but for a PI bond if it's work for

a very like a comparatively less energy

it cannot push the electron to the PI

from PI to PI star instead it will push

from PI to the nonbonding orbital and

that's how the whole thing's worked and

based on that the absorbance also vary

because the energy taken for the

electron to jump from the ground state

to the energy state is lost that's the

energy which is absorbed rest of the

energy is transmitted out that's how we

find the transmittance okay so in a turn

this is the idea from looking at this

idea you can tell

we kind of can bein able to get an idea

about what kind of bonds that are

present and what kind of chemical groups

that are present in a molecule but in

reality there are so many molecules with

similar types of bonds and the same

length same formula of those molecules

you will be you cannot recognize we

cannot recognize the exact group by

looking at this technique that's why we

don't use it to get an idea about the

specific chemical bonds that are present

which we use for NMR which we'll use for

the a magnetic resonance spectroscopy as

well as in case of is spectroscopy but

not in case of UV visible you only keep

you be visible or the process of

concentration analysis of molecules okay

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