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Double beam UV-Vis Spectrophotometer

A. Sample picture of the instrument

Link source: https://pr.vwr.com/store/product/12243110/vwr-double-beam-uv-vis-spectrophotometer

B. Brief Introduction

A UV-Vis spectrophotometer is used to determine the absorption of light from a sample

and can be used as a detector for (High Performance Liquid Chromatography) HPLC. A
sample is placed in the UV/VIS beam and absorbance versus wavelength is measured. A
double beam spectrophotometer utilizes two beams of light: a reference beam and a
sampling beam that passes through the sample. Some double beam spectrophotometers
have two detectors that allow the two beams to be measured at one time.
https://www.shimadzu.com/an/service-support/technical-support/analysis-
basics/basic/what_is_hplc.html#:~:text=HPLC%20is%20an%20abbreviation%20for,instrument%20used%20to%20co
nduct%20chromatography.

Double beam spectrophotometer is an analytical instrument in which the light beam

coming from the light source splits into two fractions. One-fraction acts as the reference
(the reference beam) while the other fraction passes through the sample (sample beam).
As a result, the reference beam does not pass through the sample.
 Figure 02: The Pathway of Light Beam in a Double Beam Spectrophotometer

The sample beam can measure the absorbance of the sample. The reference beam can measure
the absorption (the sample beam can be compared with the reference beam). Therefore, the
absorption is the ratio between the sample beam (after passing through the sample) and a
reference beam. A spectrophotometer has a monochromator that isolates the desired
wavelengths from a light beam. The reference beam and sample beam recombine before moving
to the monochromator. Consequently, this avoids or compensate the electronic and mechanical
effects on both sample and reference beams, equally.
https://www.differencebetween.com/difference-between-single-beam-and-double-beam-spectrophotometer/

C. History

Evolution of UV-Vis Spectrophotometers

1930s
In 1935, Arnold O. Beckman founds National Technologies Laboratories—later named
Beckman Instruments.

1940s
The first commercially available UV-Vis spectrophotometers are introduced. In 1940,
Beckman and colleagues at National Technologies Laboratories make their first
laboratory spectrophotometer. Howard H. Cary, who leads the project, would later form
the Cary Instrument Company.

In 1941, Beckman introduces the DU UV-Vis spectrophotometer, which has higher

resolution and lower stray light in the ultraviolet region than any other commercial
instrument.

In 1946, Cary Instruments is founded by Howard Cary, George W. Downs, and William
C. Miller under the name Applied Physics Corporation. Previously, Howard Cary was
vice president in charge of development for Beckman Instruments.

In 1947, Applied Physics Corporation delivers the first commercially available recording
UV-Vis spectrophotometer, the Cary 11, to the Mellon Institute in Pittsburgh, PA.

1950s
1950s – 1970s Mass production reduces the cost of UV-Vis spectrophotometers. New
photodiode arrays collect all wavelengths simultaneously, reducing the time required to
scan a spectrum from minutes to seconds. In 1950, National Technologies Laboratories
changes its name to Beckman Instruments, Inc.

In 1953, Bausch & Lomb introduces the SPECTRONIC 20 UV-Vis spectrophotometer,

the first massproduced, low-cost UV-Vis spectrophotometer.
In 1954, Applied Physics Corporation launches the Cary 14 spectrophotometer, the first
commercially available double-beam spectrophotometer. The double beam design
greatly simplifies and speeds up sample analysis by simultaneously measuring sample
and solvent transmittance over the wide spectral range of ultraviolet, visible, and near
infrared wavelengths.

1960s
In 1963, JASCO introduces the ORD\UV-5 with double-beam UV-Vis capabilities.

In 1966, Applied Physics Corporation is purchased by Varian Medical Systems,

becoming the Cary Instruments division of Varian.

In 1969, Cecil Instruments introduces the CE 212, the world's first commercially
available variable wavelength detector for HPLC, allowing users to select—without
changing filters or lamps—detection wavelengths on a single detector.

1970s
In 1979, Hewlett-Packard launches the first commercially available diode-array
spectrophotometer, the 8450A. Unlike traditional scanning spectrophotometers with a
single photomultiplier tube that scans one wavelength at a time, the 8450A utilizes an
array of photodiodes to scan simultaneously the full spectrum of wavelengths in
seconds.

1980s
The proliferation of personal computers in the 1980s improves data acquisition and
instrument control. In 1980, Bausch & Lomb introduces the Spectronic 2000 UV-Vis
spectrophotometer, the first microprocessor-controlled double-beam UV-Vis
spectrophotometer. Now, instead of measuring sample and solvent transmittance
separately, which the single-beam spectrophotometers required, the double-beam
design greatly simplifies and speeds up sample analysis by simultaneously measuring
sample and solvent transmittance.

In 1987, Pye Unicam Corporation. introduces the PU-8700 UV-Vis spectrophotometer,

the first mouse-driven, graphical interface UV-Visible spectrophotometer.

In 1989, Dr. Arnold O. Beckman, now 88 years old, receives the National Medal of
Science for his leadership in analytical instrumentation development.

1990s
1990s, External software now provides PC control, onscreen spectra display, and
spectra reprocessing and storage. Fiber optics reduce instrument size, and fiber optic
sampling accessories allow sample measurement outside the UV-Vis
spectrophotometer’s sample compartment, eliminating the need to fill sample cells and
cuvettes.
In 1995, Hewlett-Packard launches the 8453A, the first small-footprint and full-featured
diode-array spectrophotometer.

In 1997, Beckman Instruments, Inc. acquires Coulter Corporation, the leading

manufacturer of systems for blood and cell analysis. In 1998, the company is renamed
Beckman Coulter, Inc.

In 1999, Hewlett-Packard announces a strategic realignment to create an independent

measurement company, Agilent Technologies.

2000s
2000s, Significant progress is made in the ability to measure micro volume liquid
samples (< 1 μL) in biotechnology and pharmaceutical applications. UV-Vis
spectroscopy is applied to alternative energy R&D such as solar energy. Instrument
manufacturers start to miniaturize instruments and develop dedicated instruments for
specific applications, such as biological applications.
In 2000, Thermo Scientific introduces the GENESYS 10 instruments with out-of-plane
optics that minimize stray light and reduce noise caused by instrument optics.

In 2002, Varian Inc. releases the 6000i UV-Vis-NIR spectrophotometer. The Cary 6000i
uses an InGaAs detector that improves signal-tonoise ratio over conventional lead
sulfide detectors. Its operating range of 175 nm to 1800 nm is applicable to materials
science research.

In 2003, Thermo Scientific introduces the Evolution 300 spectrophotometer, the first
double-beam xenon lamp-based spectrophotometer. The double-beam design simplifies
and speeds up sample analysis. Xenon flash lamps provide a high-energy light source
with a shorter warm up time and longer lamp life than traditional tungsten and deuterium
lamps.

In 2004, Shimadzu introduces the SolidSpec-3700/3700DUV series of UV-Vis-NIR

spectrophotometers, the first UV-Vis-NIR spectrophotometer with three detectors—a
photomultiplier for the UVVis region, and an InGaAs detector and a cooled PbS detector
for the NIR region.

In 2005, the NanoDrop ND-1000 UV-Vis spectrophotometer (from NanoDrop

Technologies) for micro-quantitation of only 1 μl of sample enters the market. The
sample is directly pipetted onto a fiber optic measurement surface where it is held in
place by surface tension, eliminating the need for cuvettes or capillaries.

In 2006, JASCO manufactures a new range of UV-Vis-NIR spectrophotometers with

compatible accessories for life sciences, materials analysis, and semiconductor R&D.

In 2008, Shimadzu launches the UV- 1800 compact UV-Vis spectrophotometer. It

occupies a 15% smaller footprint than the model it replaces, the UV-1700.
 Also in 2008, Perkin Elmer releases the LAMBDA Bio UV-Vis spectrophotometer pre-
configured with standard methods for biological applications including protein assays,
cell density measurements, as well as DNA, RNA, and oligonucleotides concentration
and purity.

2010s
In 2010, Agilent Technologies acquires Varian Inc. and continues to offer the Cary
spectrophotometer series under the Cary name.

Also in 2010, Thermo Scientific introduces the Evolution 200 Series spectrophotometer.
Its application-focused beam geometry tailors the instrument's optical system to specific
applications for microcells, solid sampling, and fiber optics.

Also in 2010, JASCO offers the SAH-769 One Drop accessory to measure micro volume
samples of proteins and nucleic acids with UVVis spectrophotometers.

In 2011, Agilent Technologies releases the Cary 60 UV-Vis spectrophotometer with low
cost of ownership—the xenon lap typically lasts 10 years—and remote sampling options
that minimize sample handling.

Future of UV-Vis Spectrophotometers

Future improvements in UV-Vis spectrophotometers will focus on ease-of-use, portability,

and application-specific instruments. UV-Vis analysis of solid samples and materials
continues to grow in areas such as solar cell research, semiconductor products, and
coating materials. Advances in light sources will provide new developments in
conventional spectrophotometers and handheld UV-Vis instruments. Further development
in remote sensors will enable more types of samples to be measured outside the
laboratory.

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18481#:~:text=In%201953%2C%20Bausch%20%26%20Lomb%20introduces,commercially%20available%2
0double%2Dbeam%20spectrophotometer.

D. Applications, Uses & Importance

A double beam spectrophotometer is suitable for a wide range of applications including

general research, pharmaceutical, biochemical and clinical laboratory use, new material
development, and quality control.

• Standard photometrics - absorption, transmission, concentration

• Quantitative analysis and kinetics
• Multi-wavelength scan, spectrum scan
• DNA/RNA and protein analysis

The instrument can be used in stand-alone mode or in conjunction with a PC using the
supplied UV-VIS Analyst software which then delivers more powerful data processing,
expanded data collecting, and removes the limit on storage capability.
 https://pr.vwr.com/store/product/12243110/vwr-double-beam-uv-vis-spectrophotometer

⇒ The spectrophotometer is commonly used for the determination of the concentration of

colored as well as colorless compounds by measuring the optical density or its
absorbance.

⇒ It can also be used for the determination of the course of the reaction by measuring the
rate of formation and disappearance of the light absorbing compound in the range of the
visible & UV region of electromagnetic spectrum.

⇒ By spectrophotometer, a compound can be identified by determining the absorption

spectrum in the visible region of the light spectrum as well as the UV region of the
electromagnetic spectrum.

https://paramedicsworld.com/biochemistry-practicals/demonstration-of-spectrophotometer-principle-
components-working-applications/medical-paramedical-studynotes

E. Parts and functions

https://www.researchgate.net/figure/Schematic-representation-of-the-dual-beam-UV-VIS-
spectrometer_fig2_336637497

There are 7 essential parts of a spectrophotometer

a. Light source – In spectrophotometer three different sources of light are commonly

used to produce light of different wavelength. The most common source of light
used in the spectrophotometer for the visible spectrum is a tungsten lamp. For
Ultraviolet radiation, commonly used sources of are the hydrogen lamp and the
deuterium lamp.

b. Monochromator – To select the particular wavelength, prism or diffraction grating

is used to split the light from the light source.
 c. Sample holder – Test tube or Cuvettes are used to hold the colored solutions. They
are made up of glass at a visible wavelength.

d. Beam splitter – It is present only in double beam spectrophotometer. It is used to

split the single beam of light coming from the light source into two beams.

e. Mirror – It is also present only and double beam spectrophotometer. It is used to

the right direction to the splitted light from the beam splitter.

f. Photodetector system – When light falls on the detector system, an electric current
is generated that reflects the galvanometer reading.

g. Measuring device – The current from the detector is fed to the measuring device
– the galvanometer. The meter reading is directly proportional to the intensity of
light.

https://paramedicsworld.com/biochemistry-practicals/demonstration-of-spectrophotometer-principle-
components-working-applications/medical-paramedical-studynotes

F. How to operate the instrument

https://youtu.be/kN3JQ_w1-cI
https://youtu.be/sVc-XIysw0c

G. Proper Handling of the instrument (maintenance, do’s & don’ts)

1. CLEANING THE INSTRUMENT

The spectrophotometer case and sample compartment should be kept clean. Cleaning
should be done with a soft cloth slightly dampened with water or a solution of water and a
mild detergent. Do not use an excessively damp cloth that liquid can drip into the
spectrophotometer.

Materials
Soft lint-free cloth, Water, Mild detergent
Procedure
1. Turn off instrument.
2. Clean instrument case and sample compartment with a soft cloth slightly dampened
with water or a solution of water and a mild detergent.

2. EXCHANGING THE DEUTERIUM OR TUNGSTEN LAMP

You exchange the deuterium or the tungsten lamp when the intensity test, which is
executed through your computer software, falls below the specified level or when one of
the lamps no longer ignites
Materials
Deuterium or tungsten Lamp, Pozidriv screwdriver, optical tissue, isopropanol

Procedure
1. Turn off the spectrophotometer and disconnect the power cord. If you have been using
the spectrophotometer, wait at least five minutes for the lamps to cool down.
2. Open the plastic lamp door at the right side of the instrument.
3. Open the metal door behind the plastic door by sliding the lock mechanism down and
simultaneously pull the door out. Slide the metal door out to have access to the lamps.
4. Disconnect the lamp cables by pressing the plug which connects the lamps to the
printed circuit together on both sides and lift it up.
5. Use a Pozidriv screwdriver to open the two screws that hold together each lamp and
take it out holding it at the lamp ring. Never touch the quartz envelope of the deuterium
lamp with your fingers. If this happens, use isopropanol to wipe the fingerprint off.
6. Place the lamp(s) on a clean optical tissue or another place where it cannot collect dust.
7. Holding the lamp(s) at the lamp ring, carefully slide it into the lamp housing. The
tungsten
lamp ring has a flat part which has to show towards the deuterium lamp location. The
deuterium lamp has a recessment for a location pin on the lamp housing.
8. Take a pozidriv screwdriver and fix the two screws which fix each lamp.
9. Connect the plug(s) of the lamp(s) to the electronic board. There is only one way to get
the plug(s) in.
10. Slide in the metal door and close so that it is locked. Close the plastic door.
11. Reconnect the power line and turn on the instrument. Initiate a self-test from the
computer software. Make sure the spectrophotometer passes its self-test, this means that
the green light on the front panel comes on and that the instrument is ready for a
measurement.
12. Record this maintenance on the UV-Visible maintenance log. If self-test fails after all
of the above steps have been taken, then consult technical support.

CLEANING THE LENSES

Cleaning the lenses which are accessible from the sample compartment is recommended
at one-year intervals or more frequently when you operate the spectrophotometer in a
particularly dirty environment. An indication of a dirty lens is when after exchanging the
lamps the intensity test executed by the operating software still falls below the specified
level.

Materials
Lint-free cotton swab, Isopropanol, Oil-free air canister

Procedure
1. The source lens is a lens system which has one optical surface accessible from inside
the lamp housing. The other optical surface is behind the shutter, thus accessible from the
sample compartment.
2. Turn-off the spectrophotometer and disconnect the power cord.
3. Remove any cuvette holder from the sample compartment.
4. Dampen a lint-free cotton swab with isopropanol and gently swab the surface of the
source lens. Repeat as needed using new lint-free cotton swab each time for each lens.
5. Use an oil-free air canister to further clean the source and spectrograph lenses.
6. Reconnect the power line and turn on the instrument. Initiate a self-test from the
computer software. Make sure the spectrophotometer passes its self-test, this means that
the green light on the front panel comes on and that the instrument is ready for a
measurement.
7. Record this maintenance on the UV-Visible maintenance log. If self-test fails after all
of the above steps have been taken, then consult technical support.
______________________________________________________________________

Keeping your spectrometer clean and free of contamination is the key to its successful
operation. Spectrophotometers are generally low maintenance, but not zero maintenance.
The following outlines steps you can take to keep your instrument performing up to spec.

Cleaning the instrument

It is important to wipe down the surfaces of the spectrophotometer with a cloth moistened
with a mild cleaning agent. Harsh cleaning agents can damage the instrument surface.
You can also clean the cuvette itself. The cuvette shaft should only be cleaned with a lint-
free cotton swab that has been dampened with ethanol or isopropanol. This prevents liquid
from entering the cuvette shaft. If you must dampen the shaft with water to remove
contamination, use a cotton swab dampened with ethanol or isopropanol to accelerate
drying.

The cuvette shaft cover may also be cleaned. However, it is important not to soak the
cover in cleaning agent. Instead, lift the cuvette shaft cover with one hand. With the other
hand, hold the cover at the height of the locking pin and pull the cover to the right at a 90°
angle until the locking pin has been removed. Clean the cover with a cloth or lint-free
cotton swab dampened with a mild cleaning agent. To reassemble the cuvette, slide the
locking pin back into the housing as far as it will go—until the locking pin has completely
disappeared into the housing. Furthermore, when the spectrophotometer is not being
used, close the cuvette shaft using the cuvette shaft cover to protect it from dust and other
contaminants.

Disinfection and decontamination

If the spectrophotometer becomes contaminated with microorganisms, perform the
following steps for disinfection and decontamination. First, clean the device with a mild
cleaning agent before disinfection. It is important to choose a disinfection method that
corresponds to the legal regulations and guidelines in place for your particular application
and organization. Second, wipe the surfaces with a cloth you have moisturized with the
chosen disinfectant, which is usually an alcohol-based solution. Next, wipe with a cloth
you have moistened with the disinfectant. You should also disassemble and clean the
cuvette shaft as described above.

Checking the components

Another aspect of spectrophotometer maintenance is checking the unit’s photometric
accuracy. Eppendorf offers a filter kit (the BioSpectrometer reference filter kit) for
 assessing photometric accuracy and systematic wavelength error. The kit contains one
blank filter, three filters for checking photometric accuracy and three filters for checking
wavelength systematic error. The absorbance value for each filter is measured against the
blank filter. The kit not only enables the user to obtain information on the accuracy of his
or her measurements, but also information on their precision, including average value and
variation coefficient, which are calculated from 15 measurements per wavelength.

To measure accuracy and precision, place the blank filters and sample filters into the
cuvette shaft. The output absorbance values measured for the test filters should then be
compared to the permitted range of values. For individual filters, the limit values for the
permitted area are printed in a table on the lid of the filter box.

It is also necessary to check the spectrometer unit’s wavelength systematic error as part
of routine maintenance. To check the wavelength system, measure the absorbance of the
three test filters at a corresponding wavelength (260 nm, 280 nm, 800 nm) to determine
the coefficient of variation for the entire set at each wavelength.

Finally, many spectrophotometers—including all Eppendorf instruments—come with a

special feature, the self-test function. Eppendorf recommends users run the self-test at
least once a week, but the frequency of the automatic self-test can be set as desired. The
self-test checks several of the instrument’s components. It verifies the detector by
measuring the random error across the available spectral wavelengths. It also verifies the
light source by checking maximum available energy, random error, signal at the reference
sensor and light intensity. Finally, the self-test determines systematic and random error of
the wavelength by looking at the precision of the position of an intensity peak in the
ultraviolet range of the spectrum.

Follow these suggestions, and your spectrophotometer should provide trouble-free

performance well into the future.

H. Analytical signal (parameter used in the instrument)

I. How DBUVS works

WORKING OF THE SPECTROPHOTOMETER

⇒ When using a Spectrophotometer, it requires being calibrated first which is done by

using the standard solutions of the known concentration of the solute that has to be
determined in the test solution. For this, the standard solutions are filled in the Cuvettes
and placed in the Cuvette holder in the spectrophotometer that is similar to the colorimeter.

⇒ There is a ray of light with a certain wavelength that is specific for the assay is directed
towards the solution. Before reaching the solution the ray of light passes through a series
of the diffraction grating, prism, and mirrors. These mirrors are used for navigation of the
 light in the spectrophotometer and the prism splits the beam of light into different
wavelength and the diffraction grating allows the required wavelength to pass through it
and reaches the Cuvette containing the standard or Test solutions. It analyzes the
reflected light and compares with a predetermined standard solution.

⇒ When the monochromatic light (light of one wavelength) reaches the Cuvette some of
the light is reflected, some part of the light is absorbed by the solution and the remaining
part is transmitted through the solution which falls on the photodetector system. The
photodetector system measures the intensity of transmitted light and converts it into the
electrical signals that are sent to the galvanometer.

⇒ The galvanometer measures the electrical signals and displays it in the digital form.
That digital representation of the electrical signals is the absorbance or optical density of
the solution analyzed.

⇒ If the absorption of the solution is higher, there will be more light absorbed by the
solution and if the absorption of the solution is low, then more lights will be transmitted
through the solution which affects the galvanometer reading and corresponds to the
concentration of the solute in the solution. By putting all the values in the formula given in
the below section one can easily determine the concentration of the solution.

⇒ In double beam spectrophotometers, the beam splitters are present which splits the
monochromatic light into two beams one for the standard solution and the other for test
solution. In this, the absorbance of Standard and the Test solution can be measured at
the same time and any no. of test solutions can be analyzed against one standard. It gives
more accurate and precise results, eliminates the errors which occur due to the
fluctuations in the light output and the sensitivity of the detector.

J. Difference from Single Beam

The key difference between single beam and double beam spectrophotometer is that in
single beam spectrophotometer, all the light waves pass through the sample whereas, in
double beam spectrophotometer, the light beam splits into two parts and only one-part
passes through the sample. Spectrophotometers are analytical instruments used to
quantify the analytes in a given sample using a light beam. Therefore, this technique
measures the absorption of light by the sample.
https://www.differencebetween.com/difference-between-single-beam-and-double-beam-spectrophotometer/

______________________________________________________________________________________

A double beam UV visible spectrophotometer works on the same principle as the single beam
spectrophotometer and double beam spectrophotometer works. However, some additional phenomena of light
rays do happen in the UV vis spectrophotometers. It makes them unique from the conventional ones and
single and double beam spectrophotometers.

The main working principles are as follows:

1. Dispersion of Light Rays

Dispersion of light rays is well seen in nature, just after the rain. The droplets diffract the sun’s light rays by
creating a spectrum of color. Here, the spectrum is called rainbow. The same happens in the double beam
Uv visible spectrophotometer where the diffraction of light happens through the specimens. The refraction of
light makes the process of spectrophotometry more reproducible. In this instrument, the diffraction angles
depend on the wavelength of the main incident light. The disks are present in the instrument which helps in
forming the angles of diffraction. This gives rise to better measurements of the biochemical entities present
in the sample.

2. Absorbance
Absorbance is usually defined as the measurement of the ratio of incident light to transmitted radiant light rays
through a biological sample during the spectrophotometry process. The cells present in the samples reflect
the light and also absorb the same that gets transmitted to the detectors present in the instrument. In the case
of biological samples, which scatter light, absorbance is defined as the ability of the sample to absorb more
and more light passing through it. The absorbance term is used in many technical areas to quantify the results
obtained from the experimental measurement. In a double beam Uv vis spectrophotometer, the absorbance
is seen which is further used for the quantification of the results.

The term absorption is associated with the physical process of absorbing light in the spectrophotometry
process, while absorbance is the ability of a sample to absorb the light. It may also measure the attenuation
of the lens used to the instrument, which is caused by absorption, in both single and double beam
spectrophotometers.

3. The Visible spectrum Scattering of Light Rays

In the spectrophotometric analysis in a double beam UV vis device, when white light passes through the
sample and is reflected by a colored substance, a portion of the mixed wavelengths is absorbed. The
remaining light beam will be assumed the complementary color to the wavelength of light absorbed. This fact
is demonstrated by the color spectrum.

After traveling through every part of the specimen cell or sample cell the light beams which does not get
absorbed, most of them are directed onto the photo transducer or light detector in the double beam UV vis
spectrophotometer. This part of the component converts the arrival of photons in the light rays into an electrical
signal. This conversion is made through the computer screen. In this way, the light paths through the
spectrophotometer need not be in a straight line. It can be disarrayed also. As it is seen that the light beam of
rays can be redirected using mirrors in the device and can also be scattered through the help of a collimator.

Conclusion
The double beam Uv visible spectrophotometer helps to analyze the various biological specimens for good
results. The electromagnetic spectrum is also used in the process of making measurements out of samples.
The dispersion, reflection, refraction all processes happen in the spectrophotometry devices which further
helps in making analytical measurements. The spectrophotometer is one such good and the precise analytical
optical instrument used for measuring the detections gained by biological specimens. You can check the UV
spectrometer price online as well as offline medium.
Major Differences Between Single Beam Spectrophotometer And Double Beam Uv visible
Spectrophotometer

In a double beam Uv visible spectrophotometer, the beam from the light source is
split into two parts. While in the single beam spectrophotometer, there is only one
beam strike from a light source, which radiates the specimens at the holder region.

The double beam Uv vis spectrophotometer, the two beam of light has different
parts to illuminate and radiates. The first part illuminates the reference standard
and the second part radiates the whole of the samples. The light beams after
excitation may be recombined before they reach a single monochromator device
attached to the optical instrument. In some conditions of spectrophotometry, two
monochromators are used. Here, in this case, the splitting of the light. With such
specifications, double beam UV spectrophotometers have become popular.

While in the single beam spectrophotometers, the light beam is single from a light
source which work is to illuminate the specimen as well as the reference
points. The monochromator selects the wavelength of light used here.

In the case of a single beam instrument, it measures the concentration of a

radiated analyte in a sample. This is done by measuring the number of light beams
absorbed by that element in the specimen. Here, the Beer-Lambert Law comes
into operation for measuring the light rays. According to this law, the concentration
of an analyte in the sample is directly proportional to the absorbance factor in the
samples.

When it comes to a double beam Uv visible spectrophotometer, it can measure the

absorbance of all light beams in the biological sample. The reference beam of light
can measure the absorption rate within the sample holders of the
instrument. Here, the specimen can be compared with the reference point used in
the device. Hence, it is said that the absorption value is the ratio between the light
beams and the light absorbed in the instrument. The light beams or rays are
measured after passing through the biological sample and a reference beam of
light. a light beam. reference beams of light sand sample beams of light recombine
before moving to the monochromator.

OTHER REFERENCE
Conclusion

In the biological world, the double beam UV visible spectrophotometer is used for
the analytical measurements over the single beam spectrophotometer. The single
and double beam spectrophotometers are used for getting results of high
precision and accuracy.

A single beam spectrophotometer has only one beam of light, while a double beam
spectrophotometer has two beams of light, one passing through a reference solution and one
passing through the sample. In single-beam instruments, because there is only one light path which
passes through the sample, it therefore requires manually switching a reference cuvette with the
sample cuvette for calibration. Double beam spectrophotometers operate faster and provide more
reproducible results because they perform an automatic correction for the loss of light intensity as the
beam passes through the sample and reference solution.
Spectrophotometers come in both single beam and double beam configurations. A single beam
spectrophotometer uses a reference standard to standardize or blank the instrument before taking
measurements. A double beam spectrophotometer splits the beam of light into two different paths, one of
which passes through the sample while the other passes through a reference standard. Double beam
spectrophotometers measure the ratio of light intensities and, therefore, are not as sensitive to fluctuations
in the light source or detector. However, single beam spectrophotometers are usually more compact and
have a higher dynamic range.

What is the major difference between single beam and double beam UV spectrophotometer?

The difference between single beam and double beam spectrophotometer is that, in single
beam spectrophotometer, all the light waves pass through the sample whereas, in double
beam spectrophotometer, the light beam splits into two parts and only one part passes
through the sample.
As an example of the difference in time stability between the configurations,
the fluctuations in measurement values over time (drift) were compare d using
a single-beam instrument (blue line) and a double -beam instrument (red line).
The top figure shows the results from placing the single beam and double
beam instruments in the same room and using each to obtain time -course
measurements for one hour at 5-second intervals. The double -beam instrument
had less time variability than the single -beam instrument.
This means the double -beam system provides more stable measurement values
than the single-beam system. The single -beam system requires waiting unti l
the light source and detector stabilize, performing frequent blank corrections
to minimize such time variability. A summary of single -beam and double -beam
characteristics is shown in the table at bottom.

https://www.smacgigworld.com/blog/difference-single-beam-spectrophotometer-and-double-beam-uv-vis-
spectrophotometer.php

Possible questions

1. Source of visible radiation in UV Visible double beam spectrophotometer

- Often, the radiation source is a Tungsten filament (300–2500 nm), a deuterium arc lamp, which is
continuous over the ultraviolet region (190–400 nm), Xenon arc lamp, which is continuous from 160
to 2,000 nm; or more recently, light emitting diodes (LED) for the visible wavelengths.

2. Which of the following type of detector is most often used in a UV-Vis spectrophotometer?
- The most common type of light detector in UV/Vis spectrophotometers is
the photomultiplier tube (PMT).

3. What are the limitations of UV visible Spectroscopy?

- The main disadvantage of using a UV-VIS spectrometer is the time it takes to
prepare to use one. With UV-VIS spectrometers, setup is key. You must clear the
area of any outside light, electronic noise, or other outside contaminants that could
interfere with the spectrometer's reading.

4. UV /Visible Spectrophotometer
 - A spectrophotometer is an instrument for measuring the transmittance or
absorbance of a sample as a function of the wavelength of electromagnetic
radiation. Used mostly for quantitative analysis of molecular or ionic species in
solution.

Single-Beam Spectrophotometers
Single-beam spectrophotometers perform blank correction and sample
measurements using only the sample beam. First the instrument records the
intensity of the sample beam during blank correction (S B l a n k ). Then, during sample
measurement, the instrument records the intensity of the sample beam (S M e a s ).
Measurement results are then calculated using S B l a n k and S M e a s . For example,
transmittance (%T) is calculated using the following formula. However, if the
intensity of the light source varies between the blank correction and sample
measurement, it can appear as variations in S B l a n k and S M e a s values. In other words,
if the light source intensity varies between the time the blank correction and
sample measurement are performed, inaccurate data may be acquired. Thus, in
single-beam systems, fluctuations in the light source intensity ca n have a major
effect on measurement results. Therefore, single -beam systems require waiting
until the system stabilizes before starting measurements. As a general rule, they
require waiting about one to two hours after switching the power ON.

Double-Beam Spectrophotometers
Double-beam spectrophotometers perform blank correction and measure
samples using both sample and reference beams. During blank correction, the
instrument records the S B l a n k /R B l a n k ratio, based on the sample beam intensity
 (S B l a n k ) and the reference beam intensity (R B l a n k ). Next, during sample
measurement, the instrument records the S M e a s /R M e a s ratio, based on the sample
beam intensity (S M e a s ) and the reference beam intensity (R M e a s ). Measurement
results are then calculated using S B l a n k /R B l a n k and S M e a s /R M e a s . Unlike single-beam
systems, double-beam systems determine measurement values using the ratio
of sample beam intensity to reference beam intensity during both sample
measurement blank correction. As shown, fluctuations in the light in tensity do
not affect measurement results. Essentially, using the ratio of sample and
reference beam intensities in double -beam systems cancels out any fluctuations
in the intensity of the light source and reduces the potential of such fluctuations
affecting measurement values.

To summarize, data is not affected by light source fluctuations because the

reference beam continuously compensates for any fluctuations in the intensity
of the light source in real time. In other words, double -beam spectrophotometers
accurately measure samples over long time periods by cancelling out lamp
variations via ratio of the sample and reference beams. This means double -beam
systems offer superior time stability.

What is a Spectrophotometer?
A spectrophotometer is an instrument for measuring the transmittance or absorbance of a
sample as a function of the wavelength of electromagnetic radiation. The components of a
spectrophotometer include:

• a source that generates a broad band of electromagnetic radiation,

• a dispersion device that selects from the broadband radiation of the source a particular
wavelength,
• a sample area,
• one or more detectors to measure the intensity of radiation.

What are the sources of Radiation used in UV-Visible Spectrometers

Two sources are commonly used in UV-Visible spectrophotometers:
• The deuterium arc lamp. Provides a good intensity continuum in the UV region and useful
intensity in the visible region. A half-life of the lamp (the time required for the intensity to fall to
half of its initial value) is approximately 1,000 hours.
• The tungsten-halogen lamp. Yields good intensity over part of the UV spectrum and over the
entire visible range. Has useful life of 10,000 hours.

How to choose a sample-cell (cuvete) for my application

• Fused quartz cells are transparent down to 210 nm (190 nm .– the best cells)
• Glass cells are not suitable for measurements below 320 nm
• Plastic cells (polystyrene, methacrylate), lowest in cost, disposable absorb strongly below 300
nm (see Table). These cells are not resistant to all solvents.

Cell Types?
• The most frequently used cell is the open-topped rectangular cell with path length of 10 mm
(quartz cells of 3.0 ml capacity).
• Micro (0.5 ml capacity) or semimicro (1.0 ml capacity) cells are used when sample volume is
limited.

How to Choose Solvent for my Application?

The ideal solvent for the preparation of sample solutions:

• dissolves all types of compound,

• completely transparent at all wavelengths,
• nonflammable and nontoxic.

Distilled water would be ideal for for spectrophotometric measurements but is not suitable for
nonpolar organic compounds. With volatile organic solvents, such as acetone, methylene
chloride, use a stopper cell in order to eliminate evaporation (results in changes in
concentration). See table for properties of some common solvents.
 DETECTOR TYPES?
Spectrophotometers usually contain either of the following detectors (function of the detector to
convert a light signal into an electrical signal): photomultiplier tube or photodiode detector.

What would be the result when double beam UV-Vis spectrophotometer is used in
spectrometry?
In the spectrophotometric analysis in a double beam Uv vis device, when white light
passes through the sample and is reflected by a colored substance, a portion of the
mixed wavelengths is absorbed. The remaining light beam will be assumed the
complementary color to the wavelength of light absorbed.

What are some possible factors that can interfere with UV-Vis absorbance
measurement?
List of factors affecting UV-Vis Spectroscopy
• Effect of sample temperature.
• Effect of sample concentration.
• Effect of sample pH.
• Effect of solvent.
• Effect of steric hindrance.
• Effect of conjugation.

UV-Vis spectroscopy is a quantitative laboratory technique used for the

measurement of the absorbance of light by a chemical compound. It is performed
through measurement of the intensity of light, which passes through a sample with
respect to the intensity of light through a reference. It is used particularly in
ultraviolet and visible regions. The obtained light intensity versus wavelength is
called the spectrum.

Sometimes, the spectrum obtained from the UV-Vis spectrophotometer need not
be the same every time. The result may be erroneous. Reproducibility is missing if
any parameters like temperature, pH, concentration are changed. Hence, it is
essential to study the factors influencing UV-Vis spectroscopy, so that a clear
picture will be obtained for careful measurement of the spectrum. This blog takes
you to the study of variables, which affect the spectrum obtained from UV-Vis
spectroscopy.

List of factors affecting UV-Vis Spectroscopy

 For measurement of absorbance and studies, there are numerous factors, which
impact the results in ultraviolet visible spectroscopy. The factors are as follows:

• Effect of sample temperature

• Effect of sample concentration
• Effect of sample pH
• Effect of solvent
• Effect of steric hindrance
• Effect of conjugation

Effect of Sample Temperature

The change in temperature of the sample plays a role in the spectrum.

• With the decrease in temperature, the sharpness of absorption bands increases.

• With the decrease in temperature, the position of the peak (absorption maximum)
moves very little towards the longer wavelength side.
• However, total absorption intensity is independent of the temperature.
• Simple thermal expansion of the solution can change the intensity of the absorption
band.
• Rotational and vibrational energy states depend on the temperature.
• When the temperature is decreased, rotational and vibrational energy states of the
molecules also get lowered.
• Fine absorption bands will be produced when absorption occurs at lower temperatures
due to the smaller distribution of excited states..
• The position of the band maximum does not shift much with a decrease in
temperature.
• Hence, for obtaining more accurate results, the spectrum needs to be taken at a
constant or particular temperature.

Effect of Sample Concentration

The concentration of sample present is directly proportional to the intensity of light

absorption, thus influencing the spectrum.

• At a high concentration of solvent, molecular interactions occur, which

causes changes in the shape and position of absorption bands. For
 qualitative work, this effect needs to be identified and taken into
consideration.
• The type of solvent used also affects the fineness of the absorption band in
the UV spectra. Polar solvents provide broader bands, but non-polar solvents
give better resolution. Removing the solvent gives the best resolution. These
effects are all due to solvent-solute interactions.
• There are stronger solvent-solute interactions if the dielectric constant of the
solvent is high.
• Water and ethanol, which are polar solvents, exhibit a stronger binding to
solute through induced dipole-dipole interactions or hydrogen bonding.
• Through London interactions in non-polar solvents, ground state and
excited state will change and the frequency of absorbed photons can be
changed. This leads to overlap of different transition energies in spectra,
which broaden the absorption band.

Effect of Sample pH

The change of pH of the solution and the aquatic environment has an influence on
the spectrum obtained from UV-Vis spectroscopy.

• If the pH of the solution is changed, absorption spectra of aromatic compounds like

amines and phenols also change. Upon addition of a base, acidic compounds like
phenols and substituted phenols undergo a change in absorption spectra. On removal
of the phenolic proton, phenoxide ion is obtained, which increases the conjugation.
This leads to a decrease in energy difference between LUMO and HOMO orbitals,
resulting in a shift to a longer wavelength along with an increase in absorption
intensity.
• If an aromatic amine gets protonated in an acidic medium, there is a disturbance of
the conjugation system. The shift of peak towards shorter wavelength happens and a
decrease in intensity also occurs.
• The acid-base indicators have an application due to their absorptions in the visible
region of the UV-Vis spectrum.
• A minute change in the chemical structure of the indicator causes a change in the
chromophore, which absorbs wavelength maximum at different values resulting in
color change at different pH.
• An example is a phenolphthalein. It is a weak acid, dissociating in water to give anions,
which adds a negative charge to the oxygen atom, contributing a shift of absorption
maximum to a longer wavelength. The anion of phenolphthalein is orange, while non-
ionized phenolphthalein is colorless. At neutral and acidic pH, the equilibrium shifts
 towards the left and there will be a lesser concentration of anions and pink color is
not observed. But at basic pH, equilibrium shifts towards the right, leading to a higher
concentration of anions, and pink color is observed.
• Hence, to maintain pH at a constant value, the UV-Vis spectrum should be measured
in an appropriate buffer solution. Over the wavelength range of measurement, the
buffer requires it to be transparent. The absorbance value is higher if the buffer
solution also absorbs the light.

Effect of Solvent

To an extent, the choice of solvent also affects the spectrum.

• The absorption spectrum is also dependent on the solvent in which the absorption
molecule gets dissolved. The option of choosing a solvent can shift the absorption
peak to longer or shorter wavelengths. It is based on the interaction of solvent with
the chromophore of the desired molecule.
• When compared to hexane solution, ethanol gives absorption maximum at longer
wavelengths.
• Alcohols and water can form hydrogen bonding with the substance, which shifts the
absorption bands of polar molecules. As the polarities of ground and excited states of
chromophore are different, a change in solvent polarity causes a change in the energy
gap between the two states.
• Highly pure and non-polar solvents do not interact with the solute molecules either in
the ground state or excited state. However, polar solvents impact the molecular
orbitals at the ground state of the excited state.
• Hence, the spectrum recorded in non-polar solvent differs from that one recorded in
the polar solvent.
Effect of Steric Hindrance

The configuration of molecules also has a say in the spectrum.

• When a molecule is planar in conjugation, electronic conjugation works well. The

position of the absorption peak is dependent on the effectiveness and length of the
conjugative system. If autochrome is there, it prevents the molecule to exist in a planar
configuration and shift towards longer or shorter wavelengths depending on the
distortion. Distortion of chromophore causes the absorption peak to shift, due to loss
of conjugation.
• Steric hindrance can also be seen in geometric isomerism. Trans isomers exhibit
absorption peaks at longer wavelengths and molar absorptivity is higher than the cis
counterpart. Due to the steric effect, trans-stilbene absorbs with greater intensity at
longer wavelengths.
Effect of Conjugation

Molecular conjugation plays a role in determination of spectrum.

• The absorption peak is shifted to a shorter frequency or longer wavelength when two
or more chromophores are conjugated. Conjugation enhances the energy of the
highest occupied molecular orbital and mitigates the energy of the lowest unoccupied
molecular orbital. Hence, lesser energy is needed for an electronic transition to take
place in a conjugated system. If the number of conjugated bands increases, the value
of the absorption peak also increases. An increase in the double bonds of a
conjugation leads to lesser energy needed for electronic transition. Conjugation of
two chromophores also leads to an increase in molar absorptivity and intensity.
• An increase in the number of conjugated bonds leads to absorption of visible light
and compounds will be colored. An example is beta carotene, which is a precursor
compound of vitamin A. It has 11 conjugated bonds and the absorption peak is shifted
from the UV region to the visible region (blue), giving it an orange color.
Conclusion

There are so many factors that impact the absorbance studies in UV-Visible
spectroscopy. Choosing a proper solvent and buffer will make the reading accurate.
At the same time, optimal temperature and concentration also provide the
absorbance value with precision. Similarly, solvent pH should be optimized before
taking the reading.

What type of samples can be measured by UV-Vis?

By far the most common application in UV/Vis spectrophotometry is the measurement of liquids
in cuvettes. However, high performance UV/Vis and UV/Vis/NIR instruments are more
commonly used for material's characterization, which is an area dominated by solid samples.