The document discusses UV-VIS spectroscopy. It introduces the technique, including principles such as the Beer-Lambert law. It describes the components of a spectrophotometer and various modes of analysis including quantitative analysis, kinetics measurements, and multi-component analysis. It also covers topics like method development and validation, including calibration procedures to control absorbance, limit stray light, and ensure proper resolution. The document provides an overview of the fundamentals and applications of UV-VIS spectroscopy.

1. ““UV-VISSpectroscopy”
PresentedBy – Dr. KULDEEPPATEL

2. I. IntroductionofSpectrometricAnalysis
The study how the sample interacts with different wavelength
in a given region of electromagnetic radiation is called
spectroscopy or spectrochemical analysis.
The collection of measurement signals (absorbance) as a
function of electromagnetic radiation is called a spectrum.

3. • Units for UV-VIS Spectroscopy
1.Wavelength: It is the distance between the two adjacent
crests (C-C) or troughs (T-T) in a particular wave. It is
denoted by the letter lambda.
1A0 =10-8
2. Wave number : It is the reciprocal of wavelength and it is
expressed in per centimeter.
3. Frequency: It is defined as the number of waves which can
pass through a point is one second.

4. SPECTROSCOPY
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)

5. Spectroscopic Techniques and Common Uses
UV-vis UV-vis region
Quantitative
analysis/Beer’s Law
Atomic Absorption UV-vis region
Quantitative analysis
Beer’s Law
FT-IR IR/Microwave Functional Group Analysis
Raman IR/UV
Functional Group
Analysis/quant
FT-NMR Radio waves Structure determination
X-Ray Spectroscopy X-rays Elemental Analysis
X-ray Crystallography X-rays 3-D structure Anaylysis

6. Why UV Visible Spectroscopyshould be used
• Non-Destructive
• Simple operation
• Economic
Low Instrument cost
AR grade solvent is used
Less solvent consumption
More samples can be analysed in a day
• Rapid
Less sample preparation time
Less sampling time.
• Rugged Instrument
• Spectrophotometric method complies with all validation parameters as
per USFDA .

7. PRINCIPLE
When UV- Visible radiation is passed through a substance
under examination the absorption of energy result in the
promotion of the electron from the ground electronic state to
excited electronic state. During the process of absorption a
large no. of collision may occur but only those collision will
cause absorption of energy in which the photon match the
energy difference between ground and excited electronic state
of the molecule.

8. BEER LAMBERT LAW
Glass cellfilled with
concentration of solution (C)
I
I
Light
0
As the cell thickness increases, the intensity of I
(transmitted intensity of light ) decreases.

9. R- Transmittance
R = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path
through the solution).
I
I0
I
I0
I0
I
1
T
I
I0

10. A  CL = KCL by definition and it is called the Beer
Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of solute
per liter of solution
A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution

11. UV-VIS SPECTROSCOPY
•Spectrophotometer - an instrument which can measure
the optical density of a sample at any wavelength.
Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits

12. Light Sources
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp

13. Lens: By elimating lenses, chromatic aberration
are eliminated.
Slit: In practical spetrophometry the
monochromator module is not capable of
isolating a single wavelength of radiation from
the continous spectrum emitted by the source.
Rather a definite band of radiation is passed by
the monochromator.

14. DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM
Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Prism - spray out the spectrum and choose the certain wavelength
(l) that you want by slit.

15. • Detectors
1. Photomultiplier tubes
2. PDA
3. UV
4. Refractive Index
5. Fluorescence

17. Advantages of Diode Array
• Scanning instrument
• High spectral resolution
• Long data acquisition time (several minutes)
• Low throughput
• Diode array
• Fast acquisition time (a couple of seconds), compatible
with on-line separations
• High throughput (no slits)
• Low resolution (2 nm)

19. SPECTROPHOTOMETR READINGS

20. CHROMOPHORIC STRUCTURE
(Woodward-Fiesher Rules)
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261

21. Shimadzu 1700 UV-Vis spectrometer
• Specifications
• Wavelength range-190 to 1100nm
• Spectral bandwidth1nm (190 to 1100nm)
• Wavelength display0.1-nm increments
• Slit width:1nm
• Wavelength accuracy±0.1nm @ 656.1nm D2
±0.3nm (190 to 1100nm)
• Wavelength repeatability±0.1nmStray lightless than 0.02% NaI @ 220nm, NaNO2 @
340nm less than 1.0% KCl @ 198nm
• Photometric system: Double Beam Photometric
• No moving parts (except shutter)
• High wavelength reproducibility and accuracy
• 21 CFR Part 11 compliant
• Good Laboratory Practice

22. Shimadzu UV1700 Spectrophotometer Basic Startup Guide
• Turning the machine on. First, raise the LCD screen on the top of the
machine so that you can see what the machine is doing. On the left
hand side toward the back of the instrument is the ON switch.The
spectrophotometer then goes through a fairly long startup process
(about 5 minutes, so be patient) before it can be used. Wait until the
stars have all been highlighted.
• Select 1. Photometric by pressing 1 on the numeric key pad below
the screen. This will take you to the basic absorbance mode.
• Select the desired wavelength where absorbance will be measured.
On the keypad, select GOTO WL (go to wavelength), enter the
desired wavelength (l) in nanometers, and press ENTER.

23. • Make sure the instrument is reading absorbance (ABS). If it is reading %
transmittance, press the F1 key on the control panel.
• This machine uses plastic, glass, or quartz square containers called
cuvettes to hold samples. You will need at least 2 cuvettes to use this
instrument. The plastic cuvettes we are using hold 4 mL of sample, max.
After putting your samples into the cuvettes, carefully wipe the sides of the
cuvette to remove dust and fingerprints with a kimwipe. Light will pass
through the cuvette going left to right, so make SURE those two sides are
transparent and clean!
• These spectrophotometers are dual beam instruments, meaning that your
sample is being directly compared to a blank, or negative control, sample.
Place your blank in the REAR cuvette holder (underneath the cover on the
left hand, top side of the instrument) and close the lid.
• Some of the light in the control sample is now being absorbed, and your
reading should be negative. To bring this value to zero, press the AUTO
ZERO button on the control panel.

24. • After wiping the cuvette clean, place your sample to be analyzed in the
front cuvette holder and close the lid. Allow the reading to stabilize for a
few seconds, and then record the absorbance for your lab report.
•Remove the sample cuvette (leaving the blank where it is!) and place
your next sample into the machine. Repeat until you have all of the
required measurements.
•Clean all used cuvettes using distilled water, and dry them inverted on a
paper towel. Make sure that they are clean so that the next person will
get results (you might be the next person!).

25. UV-1700
UV-VIS SPECTROPHOTOMETER
Wavelength repeatability and other automatic test
functions are built-in, providing easy compliance with
GLP and ISO-9000 requirements.
Photometrics PC Control
Measures absorbance or transmittance at a The built-in RS-232C allows connection between
wavelength of your choice. By connecting an the UV-1700 and your PC, permitting full control
optional multicell sample compartment, up 16 from the PC, automatic system configuration,
samples can be measured continuously (when data exchange, and an expanded array of data
using MMC-1600/1600C and 16-cell micro processing functions.
multicell). And, data may be conveniently stored on IC
cards.
Maintenance
The maintenance functions include an indication of the
date and time of the previous baseline correction,
confirmation of lamp usage time, and unit validation.
IC Cards
Special IC cards greatly extend the functionality of the
UV-1700 system. IC card utilities allow
Parameter Recall data pack copying, IC card initialization, and
The measurement mode and its parameters can other functions.
be stored in memory or on IC cards, available for recall
and setting at any time. Operation is easy and there is
less possibility of making setting errors.
5

26. UV-Vis spectrophotometer : Modes
Spectrum
A samples spectrum is recorded using wavelength scanning. Repeat scans let you
follow sample changes over time. Zoom in on the finished spectrum for a better
view, and then use the peak/valley pick function to select maxima and minima and
perform a wide verity of data processing functions.
Kinetics
.
time, and obtain the enzymatic activity value. Select among single wavelength,
multicell, double wavelength and rate measurement methods. Using the MMC-
1600/1600C (8/16 cells) or the CPS-240A Cell Positioner (6 cells), multiple samples
can be measured at the same .
Multi-Component Quantitation
Quantitates up to 8 components mixed in a single sample. The calibration equation
is determined using pure or mixed components with known values.

27. UV-Vis spectrophotometer : Modes
Multi Wavelength Measurement
Measure the absorbance and transmittance at up to eight designated wavelengths. For
absorbance measurements, calculation of data for up to four wavelengths, including
the difference or ratio between two wavelengths, is possible.
Quantitation
.
Generates a calibration curve from measurement of standards, and then calculates the
concentrations of unknowns. Allows various combinations of wavelength number (1 to
3curves wavelengths peak area values) calibration curves (K factor, 1st to 3rd order).
Time Course Scan
Measures the changes in absorbance, transmittance or energy as a function of time.
Using the MMC-1600/1600C (8/16 cells) or the CPS-240A Cell Positioner (6 cells),
multiple samples can be measured at the same time.

28. UV-Vis spectrophotometer : Modes
Optional Application Programs
In seconds the easy –to-inset program packs reconfigure the uv-1700 for
specific laboratory protocols.
Instrument Conditions
.
The basic instrument conditions can be set to accommodate the method of use for even
greater convenience. And, built-in self-performance check functions ensure the
continued reliability of measurement data.

29. From transparent samples to samples with suspended and
components, they can all be measured and quantitated. Even
volume and enzyme activity measurements are possible.
1.Micro Volume Sample Measurement
3uL capillary cells and 50µL ultramicro cells are available for measurement of
minute volumes of sample. The spectra in the first figure were obtained from
measurement of DNA in herring sperm in phosphate buffer solution. (A 3µr
capillary cell was used.) Even though measurement was conducted in the
ultraviolet region, the light beam was condensed sufficiently to provide a
spectrum with a high signal-to-noise ratio. The second figure shows the
linearity of the calibration curve.
2.Multicell Kinetic Measurement
Multicell kinetics measurement can be performed using a temperature
controlled cell positioner. At right, the enzyme activity of cholinesterase
in blood serum was measured. Enzyme reactions can be measured in up
to 16 cells at one time.
3.Multi Wavelength Measurement
Measures the absorbance or transmittance for a sample at up to eight selected
wavelengths. When absorbance is measured, results can be displayed or
output by calculating data using up to four wavelengths.
6

30. From transparent samples to samples with suspended and
components, they can all be measured and quantitated. Even
volume and enzyme activity measurements are possible.
4. Multi-component Analysis
.
6
Quantitative analysis can be performed for up to 8 components mixed in a
single sample. At right, aspirin, caffeine and salicylic acid were present in a
sample in ratios of 2 : 1 : 1, as confirmed in the separated quantitation result.
The second figure shows the spectral characteristics of the three ingredients and
the mixture. Note the distinctly different spectral characteristics, allowing the
various compounds to be distinguished.
5. Instrument Validation Functions
These functions can conduct performance checks (measurement, pass/fail
evaluation and printing) for nine items, including wavelength accuracy.
Validation is divided into fully automatic checks, where all procedures from
measurement to evaluation are automatically completed, and semi-automatic
checks, where the insertion and removal of inspection jigs are required.
The semi-automatic checks are performed interactively. The user can conduct
the validation by inserting or removing inspection jigs as instructed by
messages on the screen.

31. Concentration of the unknown sample can
be determined by extrapolation method.

32. In this graph, values above A=1.0 are not linear. If we
use readings above A=1.0, graph isn’t accurate. Most
acceptable working absorbance range is : 0.1-1.0.

33. Standard Practice
• Prepare standards of known concentration
• Measure absorbance at l max
• Plot A vs. concentration
• Obtain slope
• Use slope (and intercept) to determine the
concentration of the analyte in the unknown

34. METHOD DEVELOPMENT AND VALIDATION

35. METHODDEVELOPMENTANDVALIDATION
1. Solvent effect:
• Solubility
• Stability
• Spectral character
2. Wavelength selection
3. Method selection
• Single point
• Multipoint calibration method
• K-factor method

41. Calibration of UV Visible Spectroscopy :
Proper calibration of the instrument is essential for obtaining accurate analysis.
Calibration of UV-Visible spectroscopy includes:
1. Control of absorbance
2. Limit of stray light
3. Resolution
4. Control of wavelength

42. 1. Control of absorbance:
 For control of absorbance the solution of potassium dichromate in 0.005 M H2SO4
is prepared.
 Use the potassium dichromate solution as sample and 0.005 M H2SO4 as a blank.
When we take absorbance then the exact
value of E1% for each wavelength and
tolerance limit is as follows:
E1% = Measured absorbance X 10000
Wt. of K2Cr2O7 (mg)
S.N. Wavelength (nm) E1% std. Tolerance limit
1 235 124.5 122.9 to 126.2
2 257 144.0 142.4 to 145.7
3 313 48.6 47.0 to 50.3
4 350 106.6 104.9 to 108.2

43. 2. LIMITS OF STRAY LIGHT:
In limit of stray light 1.2 % w/v solution of potassium chloride in distilled water to
be prepare. Then measurement of stray light is perform by using the 1.2 % w/v
potassium chloride solution as sample and distilled water as a blank.
MEASUREMENT CONFIGURATION:
Measuring mode - abs
Recording range - 0.0 to 3.0
Wavelength range (nm) - 250 to 190
Slit width (nm) - 1.0
Scan speed - slow
Acceptance criteria: – Absorbance of 1.2 % w/v potassium chloride is ≥ 2
at 200nm.

44. 3. RESOLUTION:
In resolution 0.02% v/v solution of toluene in hexane is prepare. Then measurement
of resolution is performing by using the 0.02 % v/v toluene in hexane solution as
sample and hexane as a blank.
Acceptance criteria: – The ratio of absorbance of 0.02 % v/v solution
of toluene in hexane at 269 and 266 is > 1.

45. UV-Visible Spectroscopy Applications

53. OTHERS APPLICATION
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

54. Troubleshooting
When your UV-visible Spectroscopy System does not come up
or does not work properly, you have to apply troubleshooting
procedures.
When the spectrophotometer is turned on, it runs through a self
test sequence.
The different stages of the self test sequence are indicated by
the
• colour of front panel LED,
• speed of fan,
• movement of shutter.
To troubleshoot your instrument, switch the instrument off. Then
push the
power button of the spectrophotometer to switch it on again and
check the
starting sequence of the instrument as shown in Figure 03

55. Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

56. Troubleshooting
Checking the Keyboard
When the handheld controller is used very frequently, the keys might show
aging effects and do not work properly any more. To check the performance
of the keys of your keyboard individually, apply the following procedure:
1. Choose a task (e.g Single WL).
2. Press the F5-key (System).
3. Press the F3-key (Tests), select Diagnostics & Maintenance and press
Enter.
4. In the Tests screen press the F5-key (Keyboard).
5. Press all keys of the keyboard successively. When a key is working
properly, the symbol on the screen representing the key will turn dark.
6. To exit the keyboard test, press Escape twice in a row.

57. Troubleshooting Your Handheld Controller
Resetting the Software
If your handheld controller does not work correctly, disconnect the handheld
controller CAN connector from the rear of the spectrophotometer and
reconnect it. This will restart the software of the handheld controller.
If the problem still remains, then
1 power off the spectrophotometer and wait 1 minute, then power the
spectrophotometer on again.
2 check if the LED at the front of the spectrophotometer turned Green after
the boot-up sequence. In case the LED stays Orange or Red, call your service
representative.
PC-card not Recognized by the Controller
When your PC-card is not recognized by the handheld controller, either the
card is defective or the card has not been inserted when the UV
UV-vis Spectroscopy System was turned on.
To resolve the problem apply the following procedure:
1 Disconnect the handheld controller from the spectrophotometer by
unplugging the Can connector.
2 Insert the PC-card.
3 Reconnect the handheld controller to the spectrophotometer by plugging in
the Can connector in the right Can interface.
If the problem still remains, then
4 insert another PC-card and redo step 1 through 3 to check if your PC-cardwas
defective.
If the problem still remains, call your service representative.

58. Checklist for Best Results
Cell:
✔ Cell is made of quartz or glass
✔ Cell windows are free of fingerprints and other contamination
✔ Flow cell used instead of an apertured standard cell
Measurements:
✔ Transparent surface of Cell should face the light source.
Solution in cell is free of floating particles
✔ Solution in cell and cell walls are free of bubbles
✔ Solution in cell is mixed homogeneously
✔ Blank measured on same solvent as sample
✔ Blank measurement shows a flat baseline
Figure 1 : Example of a Blank on Water Showing a Good Baseline
Figure 2 : Example of a Blank on Water with Bubbles Causing a Poor Baseline

59. Cont…
Cell orientation of blank and sample measurements is the same
✔ Ideally the cell is not removed between the measurements,
which means the
cell is filled/rinsed using a pipette or a flow cell is used
✔ Time between blank and sample measurement should be
short
Always analyses dilute sample first followed by Samples of
higher
concentrations.
Always put silica bag when instrument is closed.

60. Thanks