1.

Spectrophotometry

Figure 1: Spectrophotometer set-up

The spectrophotometer is a routinely used instrument in scientific research.

Spectrophotometry is the quantitative measurement of how much a chemical substance absorbs
light by passing a beam of light through the sample using a spectrophotometer. In this video, basic
concepts in spectrophotometry, including transmittance, absorbance and the Beer-Lambert Law
are reviewed in addition to the components of the spectrophotometer. These concepts provide a
foundation for how to determine the concentration of a solute in solution that is capable of absorbing
light in the ultraviolet and visible range. Furthermore, a procedure for how to operate the
spectrophotometer is demonstrated, including instructions on how to blank and measure the
absorbance of a sample at the desired wavelength. The video also covers how to make a standard
curve for determination of analyte concentration. Several applications of the spectrophotometer in
biological research are discussed, such as measurement of cell density and determination of
chemical reaction rates. Finally, the microvolume spectrophotometer is introduced, as well as its
advantage in measuring the quality and quantity of protein and nucleic acids
In chemistry, spectrophotometry is the quantitative measurement of the reflection or
transmission properties of a material as a function of wavelength (Allen, D., Cooksey, C., & Tsai,
B. 2010) It is more specific than the general term electromagnetic spectroscopy in that
spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover
time-resolved spectroscopic techniques.
A spectrophotometer is commonly used for the measurement of transmittance or
reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. Although
many biochemicals are colored, as in, they absorb visible light and therefore can be measured by
colorimetric procedures, even colorless biochemicals can often be converted to colored
compounds suitable for chromogenic color-forming reactions to yield compounds suitable for
colorimetric analysis (Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee, 2010). However, they
can also be designed to measure the diffusivity on any of the listed light ranges that usually cover
around 200 nm - 2500 nm using different controls and calibrations ((Allen, D., Cooksey, C., & Tsai,
B. 2010). According to Schwedt, Georg (1997) within these ranges of light, calibrations are needed
on the machine using standards that vary in type depending on the wavelength of the photometric
determination.
According to Trumbo et al (2013) spectrophotometry is an important technique used in
many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and
biochemical analyses. A brief explanation of the procedure of spectrophotometry includes
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comparing the absorbency of a blank sample that does not contain a colored compound to a sample
that contains a colored compound. This coloring can be accomplished by either a dye such as
Coomasie Brilliant Blue G-250 dye measured at 595 nm or by an enzymatic reaction as seen
between β-galactosidase and ONPG measured at 420 nm (Ninfa,2009). The spectrophotometer is
used to measure colored compounds in the visible region of light ,(Ninfa, Alexander J.; Ballou,
David P.; Benore, Marilee, 2010) thus it can be used to find more information about the substance
being studied. In biochemical experiments, a chemical and/or physical property is chosen and the
procedure that is used is specific to that property in order to derive more information about the
sample, such as the quantity, purity, enzyme activity, etc. Spectrophotometry can be used for a
number of techniques such as determining optimal wavelength absorbance of samples,
determining optimal pH for absorbance of samples, determining concentrations of unknown
samples, and determining the pKa of various samples.] Spectrophotometry is also a helpful process
for protein purification (Cortez, C.; Szepaniuk, A.; Gomes da Silva, L., 2010) and can also be used
as a method to create optical assays of a compound. Spectrophotometric data can also be used in
conjunction with the Beer-Lambert Equation, A= -log10T=εcl=OD, in order to determine various
relationships between transmittance and concentration, and absorbance and concentration.
Because a spectrophotometer measures the wavelength of a compound through its color, a dye
binding substance can be added so that it can undergo a color change and be measured (Garrett,
Reginald H.; Grisham, Charles M., 2010). It is possible to know the concentrations of a two
component mixture using the absorption spectra of the standard solutions of each component. To
do this, it is necessary to know the extinction coefficient of this mixture at two wave lengths and the
extinction coefficients of solutions that contain the known weights of the two components.
Spectrophotometers have been developed and improved over decades and have been widely used
among chemists. Additionally, Spectrophotometers are specialized to measure either UV or Visible
light wavelength absorbance values. It is considered to be a highly accurate instrument that is also
very sensitive and therefore extremely precise, especially in determining color change
(Mavrodineanu, Radu; Schultz, J. I.; Menis, Oscar 1973). This method is also convenient for use
in laboratory experiments because it is an inexpensive and relatively simple process.
2. Electrophoresis
Chromatography is a laboratory technique for the separation of a mixture. The mixture is
dissolved in a fluid called the mobile phase, which carries it through a structure holding another
material called the stationary phase. The various constituents of the mixture travel at different
speeds, causing them to separate. The separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus affect the separation.McMurry, John (2011)

Figure 2: Simple Chromatography

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 According to Hostettmann, K,(1998) chromatography may be preparative or analytical. The
purpose of preparative chromatography is to separate the components of a mixture for later use,
and is thus a form of purification. Analytical chromatography is done normally with smaller amounts
of material and is for establishing the presence or measuring the relative proportions of analytes in
a mixture. The two are not mutually exclusive.
According to Kumar, (2012) here are different kinds of chromatographic techniques and
these are classified according to the shape of bed, physical state of mobile phase, separation
mechanisms.
The twelve types are:
(1) Column Chromatography
(2) Paper Chromatography
(3) Thin Layer Chromatography
(4) Gas Chromatography
(5) High Performance Liquid Chromatography
(6) Fast Protein Liquid Chromatography
(7) Supercritical Fluid Chromatography
(8) Affinity Chromatography
(9) Reversed Phase Chromatography
(10) Two Dimensional Chromatography
(11) Pyrolysis Gas Chromatography
(12) Counter Current Chromatography.
The spectrophotometer is a routinely used instrument in scientific research.
Spectrophotometry is the quantitative measurement of how much a chemical substance absorbs
light by passing a beam of light through the sample using a spectrophotometer. In this video, basic
concepts in spectrophotometry, including transmittance, absorbance and the Beer-Lambert Law
are reviewed in addition to the components of the spectrophotometer. These concepts provide a
foundation for how to determine the concentration of a solute in solution that is capable of absorbing
light in the ultraviolet and visible range. Furthermore, a procedure for how to operate the
spectrophotometer is demonstrated, including instructions on how to blank and measure the
absorbance of a sample at the desired wavelength. The video also covers how to make a standard
curve for determination of analyte concentration. Several applications of the spectrophotometer in
biological research are discussed, such as measurement of cell density and determination of
chemical reaction rates. Finally, the microvolume spectrophotometer is introduced, as well as its
advantage in measuring the quality and quantity of protein and nucleic acids.
The use of spectrophotometers spans various scientific fields, such as physics, materials
science, chemistry, biochemistry, and molecular biology (Rendina, 2005). They are widely used in
many industries including semiconductors, laser and optical manufacturing, printing and forensic
examination, as well in laboratories for the study of chemical substances. Spectrophotometry is
often used in measurements of enzyme activities, determinations of protein concentrations,
determinations of enzymatic kinetic constants, and measurements of ligand binding reactions
(Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2010). Ultimately, a spectrophotometer is
able to determine, depending on the control or calibration, what substances are present in a target
and exactly how much through calculations of observed wavelengths.

3. Refractometer
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 Figure 3: Refractometer

Refractometry is the analytical method of measuring substances' refractive index in

order to, for example, assess their composition or purity. A refractometer is the instrument used
to measure refractive index ("RI"). Although refractometers are best known for measuring
liquids, they are also used to measure gases and solids; such as glass and gemstones.
Refractometers are instruments to measure substances dissolved in water and certain
oils. The refractometer works using the principle of light refraction through liquids. As light
passes from air into a liquid it slows down. This phenomenon is what gives a "bent" look to
objects that are partially submerged in water. To put it simply, the more dissovled solids water
contains, the slower light travels through it, and the more pronouced the "bending" effect on
light. Refractometers use this prinicple to determine the amount of dissolved solids in liquids
by passing light through a sample and showing the refracted angle on a scale.
The scale most commonly used is referred to as the Brix scale. The Brix scale is
defined as: the number of grams of pure cane sugar dissolved in 100 grams of pure water
(grams sugar/100 grams H20). Other scales have been developed to measure salt, serum
proteins (albumen) and urine specific gravity.
Light travels at different speeds through different media, and when a ray of light
crosses the interface between two substances it changes direction. This phenomenon is known
as refraction. This paper describes the principles of refraction in detail, and the functions and
applications of refractometers in general. Focusing on the various types of refractometers
available, the document describes the Bellingham+Stanley refractometer in detail. It also
includes empirical analysis of the principles of refractometry in order to be able to appreciate
the application of different kinds of refractometers.
The instrument used for the determination of the refractive index is known as the
refractometer. There are different refractometers used for the determination of the refractive
index. They are as follows:
Traditional handheld refractometer: The main principle involved in this refractometer
is the measurement of the critical angle. It is comprised of the lenses and prisms to project the
black line on the glass when the sample is placed between the measuring prism and the plate.
Digital hand held refractometer: The principle is the same as the traditional handheld
refractometer. The main difference is that the light from the LED light source is focused on the
prism. This creates the black line due to the refection of the photodiode arrays.
Abbes refractometer: This is a benchtop refractometer, designed by Ernst Abbe,
which provides high accuracy. In this refractometer, the sample is held between the illuminating
prism and the refracting prism. A light source is allowed through the illuminating prism and the
detector is placed behind the refracting prism.
Inline process refractometer: This is mainly used for the continuous measurement
of a fluid flowing through a pipe. This refractometer consists of a sensor placed in the flow of
the fluid. This is connected to the control box which gives the digital readout.
FACTORS AFFECTING THE REFRACTIVE INDEX
Temperature: It is inversely proportional to the refractive index.
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 Viscosity: It is inversely proportional to the refractive index.
Wavelength: The sodium D-line at 595 nm is the appropriate wavelength for the
determination of the refractive index.

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