SOPs: UV- sprectroscopy

1. UV VISIBLE
SPECTROSCOPY
Priyanka Sharma
Pre Ph.D, 04
Delhi Pharmaceutical Science And Research University
Delhi Pharmaceutical Sciences and Research University.

2. Spectroscopy
• It is the branch of science that deals with the study of interaction of matter
with light.
OR
• It is the branch of science that deals with the study of interaction of
electromagnetic radiation with matter

3. Why we use UV spectroscopy
• Detection of functional groups.
• 2. Detection of impurities.
• 3. Qualitative analysis.
• 4. Quantitative analysis.
• 5. Single compound without chromophore.
• 6. Drugs with chromophoric reagent

4. Electromagnetic Radiation
• Electromagnetic radiation consist of discrete packages of energy which are
called as photons.
Frequency (ν):
• It is defined as the number of times electrical field radiation oscillates in one
second.
• The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second
• Wavelength (λ):
It is the distance between two nearest parts of the wave in the same phase
i.e. distance between two nearest crest or troughs.

5. • The relationship between wavelength & frequency can be written as:
c = ν λ
• As photon is subjected to energy, so
E = h ν = h c / λ

6. Absorbance Laws
BEER’S LAW
• “ The intensity of a beam of monochromatic light decrease exponentially
with the increase in concentration of the absorbing substance” .
Arithmetically;
- dI/ dc ᾱ I
I= Io. eˉkc ------- eq (1)

7. LAMBERT’S LAW
• When a beam of light is allowed to pass through a transparent medium, the rate of decrease
of intensity with the thickness of medium is directly proportional to the intensity of the
light”
mathematically;
-dI/ dt ᾱ I
-In . I = kt+b -------------- eq(2)
the combination of eq 1 & 2 we will get
A= Kct
A= ℇct (K=ℇ)

8. Chromophore…
• The part of a molecule responsible for imparting color, are called as
chromospheres.
OR
• The functional groups containing multiple bonds capable of absorbing
radiations above 200 nm due to n→ π* & π → π* transitions.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

9. Continues…
• e.g. Acetone which has λmax = 279 nm
and that cyclohexane has λmax = 291 nm.
When double bonds are conjugated in a compound λmax is shifted to longer
wavelength.

10. Auxochrome…
• The functional groups attached to a chromophore which modifies the ability
of the chromophore to absorb light , altering the wavelength or intensity of
absorption.
OR
• The functional group with non-bonding electrons that does not absorb
radiation in near UV region but when attached to a chromophore alters the
wavelength & intensity of absorption.

11. Principle of UV Visible
Spectroscopy
• The UV radiation region extends from 10 nm to 400 nm and the visible
radiation region extends from 400nm to 800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
• Far UV spectroscopy is studied under vacuum condition.
• The common solvent used for preparing sample to be analyzed is either
ethyl alcohol or hexane.

12. Ultraviolet absorption spectra arise from transition of electron with in
a molecule from a lower level to a higher level.
A molecule absorb ultraviolet radiation of frequency (𝜗), the electron
in that molecule undergo transition
from lower to higher energy level.
The energy can be calculated by the equation,
E=h𝜗 erg

13. E₁-Eₒ= h𝜗
Etotal = Eelectronic + Evibrational + Erotational
The energies decreases in the following order:
Electronic ⪢ Vibrational ⪢ Rotational

14. Electronic Transition
The possible electronic transitions are
• 1- σ → σ* transition
• 2- π → π* transition
• 3- n → σ* transition
• 4- n → π* transition

15. Absorption& Intensity Shifts
1- Bathochromic Shift (Red Shift)
2- Hypsochromic Shift (Blue Shift)
3- Hyperchromic Effect
4- Hypochromic Effect

17. Bathochromic Shift (Red Shift)
• When absorption maxima (λmax) of a compound shifts to
longer wavelength, it is known as bathochromic shift or red
shift.
• The effect is due to presence of an auxochrome or by the
change of solvent.
• e.g. An auxochrome group like –OH, -OCH3 causes
absorption of compound at longer wavelength.

18. Hypsochromic Shift (Blue Shift)
• When absorption maxima (λmax) of a compound shifts
to shorter wavelength, it is known as hypsochromic shift
or blue shift.
• The effect is due to presence of an group causes removal
of conjugation or by the change of solvent.

19. Hyperchromic Effect
• When absorption intensity (ε) of a compound is increased, it is
known as hyperchromic shift.
• If auxochrome introduces to the compound, the intensity of
absorption increases.

20. Hypochromic Effect
• When absorption intensity (ε) of a compound is
decreased, it is known as hypochromic shift.

21. Applications
Qualitative & Quantitative Analysis:
◦ It is used for characterizing aromatic compounds and conjugated
olefins.
◦ It can be used to find out molar concentration of the solute under
study.
Detection of impurities:
◦ It is one of the important method to detect impurities in organic
solvents.
Detection of isomers are possible
Determination of molecular weight using Beer’s law

22. 1. Detection of Impurities.
• UV absorption spectroscopy is one of the best methods
for determination of impurities in organic molecules.
• Additional peaks can be observed due to impurities in the
sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific
wavelength, the impurities can be detected.

23. U.V. Spectra of Paracetamol (PCM)

24. 2. Structure elucidation of organic compounds.
• UV spectroscopy is useful in the structure elucidation
of organic molecules, the presence or absence of
unsaturation, the presence of hetero atoms.
• From the location of peaks and combination of peaks,
it can be concluded that whether the compound is
saturated or unsaturated, hetero atoms are present or
not etc.

25. 3. Quantitative analysis
• UV absorption spectroscopy can be used for the
quantitative determination of compounds that absorb
UV radiation. This determination is based on Beer’s law
which is as follows.
• A = log I0 / It = log 1/ T = – log T = abc = εbc
Where : ε -is extinction co-efficient,
c- is concentration,
b- is the length of the cell that is used in UV
spectrophotometer.

26. 4. Qualitative analysis.
UV absorption spectroscopy can characterize those
types of compounds which absorbs UV radiation.
Identification is done by comparing the absorption
spectrum with the spectra of known compounds.

27. U.V. Spectra's of Ibuprofen

28. 5. Chemical kinetics
Kinetics of reaction can also be studied using
UV spectroscopy. The UV radiation is passed
through the reaction cell and the absorbance
changes can be observed.

29. 6. Detection of Functional Groups
• This technique is used to detect the presence or
absence of functional group in the compound
• Absence of a band at particular wavelength
regarded as an evidence for absence of particular
group

30. Toluene

31. 7. Quantitative analysis of pharmaceutical
substances
• Many drugs are either in the form of raw material or in
the form of formulation. They can be assayed by
making a suitable solution of the drug in a solvent and
measuring the absorbance at specific wavelength.
• Diazepam tablet can be analyzed by 0.5% H2SO4 in
methanol at the wavelength 284 nm.

32. 8. Molecular
weight determination
• Molecular weights of compounds can be
measured spectrophotometrically by preparing
the suitable derivatives of these compounds.
9. As HPLC Detector
• A UV/Vis spectrophotometer may be used as a
detector for HPLC.

33. Standard Operating Procedure SOPs
Starting Procedure:
1. Turn on the machine with the switch on the front, bottom left corner.
2. Make sure that the proper sampling accessory is loaded.
Open the top door and remove the white box on front.
Typically, the cuvette holder should be in the machine. With this you can do
single- or dual-beam experiments with one sample at room temperature.
To take out the accessory, use a screwdriver to unscrew it from its brace and
remove it.

34. Performing Experiments:
Note: There are a great number of experiments this machine can do, but for this procedure I will
explain basic ones using the standard cuvette holder. Please ask CBIC staff for help if there’s a
different experiment you want to perform, or if you have any
Questions.
1. All of the different modes are separate programs, which are located on the
desktop
in the “Cary WinUV” folder. I will walk you through the most commonly used
programs, which are “Simple Reads” and “Scan”.
2. “Simple Reads” measures the absorbance for a single wavelength at a particular
point in time. To use “Simple Reads”:

35. Continues.....
1. Start the program.
2. Place a blank cuvette (empty or only solvent) into the holder and close the
top door. Click “Zero” on the left side of the screen. The absorbance should go
to 0.
3. Place your sample inside. There should now be an absorbance at that
particular wavelength.
4. To change the parameters, click “Setup”. Here you can change the
wavelength, as well as whether the output is absorbance, transmission, etc.

36. 3. “Scan” collects a spectrum at a variety of wavelengths. To use “Scan”:
a. Start the program.
b. Click “Setup” to change the parameters.
- In the “Cary” tab, you can customize the range of wavelengths as well what you want
the output to be (absorbance, transmission, etc.). You can also adjust the averaging
time and/or the data interval, but the defaults
should be fine unless you have a reason to adjust these parameters.
- Note: if you want to scan in the ultraviolet range, make sure you use quartz cuvettes
as glass and plastic will absorb UV.
- If you have a different accessory in the machine, there may be additional options in
the “Accessory” tabs.

37. Continues....
c. If you want to collect a baseline, click the “Baseline” tab and select an option
(in this case, “Zero/baseline correction”).
-To do this correction, place the blank sample inside and click “baseline”. It will
collect the 100% ransmission. Then, to collect the 0% transmission, block the
beam on the left side with the plate.
d. Place your sample inside and click “Start” at the top.

38. Continue....
e. It will ask you about where to save the data it collects.
- If you don’t have a folder, make one with your username.
- Write a filename for the data to be collected, and choose your folder as the save
location. Regarding filetype, “*.CSV” is the easiest to deal with so you may want to
save it as such.
-In the next “Sample name” window, you can name your sample, which allows for
multiple samples in a single file.
f. You can also save your data after it was collected by clicking “File” —> “Save
Data As” and filling out the filename, file type, location, etc

39. Continue....
4. Other programs that are available include: “Thermal”, which monitors a single
wavelength as a function of temperature; “Kinetics”, which monitors a single
wavelength as a function of time; and several others for more complicated
experiments.
`Shutting Off:
1. Close the window.
2. Make sure the temp-control water supply is off if it was
used.
3. Take out your sample.
4. Turn off the spectrophotometer.

40. References
1. Introduction to Spectroscopy, Donald A. Pavia
2. Sharma. Y.R. Elementary Organic Spectroscopy. First edition,
S.Chand Publisher; 2010.
3. Chatwal G.R. Instrumental methods of chemical analysis,
First edition. Himalaya Publisher; 2010.
4. www.chem.agilent.com/Library/applications/uv31.pdf.
5. www.uvitron.com/Applications.html.