Written report

In
Technical Analysis

Spectrophotometric method

Prepared By:
Riza M. Andulte - BsChemistry IV

Prepared To:
MA. Lourdes C. alvarez, PhD

Spectrophotometric Method
 Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation)
over a certain range of wavelength. Spectrophotometry is a measurement of how much a
chemical substance absorbs or transmits. It is widely used for quantitative analysis in various
areas such as: chemistry, physics, biology, biochemistry, material and chemical engineering,
chemical applications, and industrial applications.

Objectives:
 Definition of Spectrophotometry
 History of spectrophotometry
 Theory of Spectrophotometry
 Components of Spectrophotometry
 Applications of Spectrophotometry
 Video Presentation

Spectrophotometry
 The quantitative measurement of the reflection or transmission properties of a material as
a function of a wavelength. 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.
 It uses photometers, known as spectrophotometers (commonly used for the measurement
of transmittance or reflectance of solutions, transparent or opaque solids, such as polished
glass, or gases) that can measure a light beam’s intensity as a function of its color
(wavelength). Important features of spectrophotometers are spectral bandwidth (the range
of colors it can transmit through the test sample), the percentage of sample-transmission,
the logarithmic range of sample-absorption, and sometimes a percentage of reflectance
measurement.

History of Spectrophotometry
 By 1940 several spectrophotometers were available on the market, but early models
could not work in the ultraviolet. Arnold O. Beckman developed an improved version at the
National Technical Laboratories Company, later the Beckman Instrument Company and
Ultimately Beckman Coulter. Models A, B, and C were developed (three units of model C
were produced), then the model D which became DU. All the electronics were contained
within the instrument case, and it had a new hydrogen lamp with ultraviolet continuum, and a
better monochromator. This instrument was produced from 1941 until 1976 with essentialy
the same design.

Theory of Spectrophotometry

Single wavelength spectrophotometer

Older Spectrophotometers must be calibrated by a procedure known as "zeroing",
to balance the null current output of the two beams at the detector. The transmission of a
reference substance is a set as a baseline value, so the transmission of all other substances
are recorded relative to the initial "zeroed" substance.
Spectrophotometry is an important technique used in many biochemical
experiments that involved DNA, RNA, and protein isolation, enzyme kinetics and
biochemical analyses.
Comparing the absorbency of a blank sample that does not a 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 or by an
enzymatic reaction as seen between β-galactosidase and ONPG (turns sample yellow).
Spectrophotometry is a helpful procedure for protein purification and can also be
used as a method to create optical assays of a compound.
Spectrophotometer measures the wavelength of a compound through its color, a
dye binding substance can be added so that it ca undergo a color change and be
measured. To determine various relationships between transmittance and concentration,
and absorbance and concentration, here’s the equation used.
Spectrophotometers use a monochromator containing a diffraction grating to
produce the analytical spectrum. The grating can either be movable or fixed. If a single
 detector, such as a photomultiplier tube or photodiode is used, the grating can be scanned
stepwise to the so that the detector can measure the light intensity at each wavelength.

Components of Spectrophotometers

Two Major Classes of Devices:

1. Single beam spectrophotometer- A double beam spectrophotometer compares
the light intensity between two light paths, one path containing a reference sample
and the other the test sample.
2. Double beam spectrophotometer- A single-beam spectrophotometer measures
the relative light intensity of the beam before and after a test sample is inserted.
These measurements from double-beam are easier and more stable, single-beam
instruments can have a larger dynamic range and are optically simpler and more
compact.
 UV-visible spectrophotometry
o Ultraviolet-visible (UV-vis) spectroscopy involves energy levels that excite
electronic transitions. The energy of light at 00 nm is ~250KJoules/mol
(~60kcal/mol), which is of the order of the energy of chemical bonds. Absorption
of UV-vis light excites electrons that are ground-state orbitals to their excited-
state molecular orbitals.
o Visible region 400-700 nm spectrophotometry is used extensively in colorimetry
science. It is a known fact that it operates best at the range of 0.2-0.8 O.D.
Traditional visible region spectrophotometers cannot detect if a colorant or the
base materials has fluorescence. This can make it difficult to manage color issues
if for example one or more of the printing inks is fluorescent. Where a colorant
contains fluorescence, a bi-spectral fluorescent spectrophotometer is used. There
are two major setups for visual spectrum spectrophotometers, d/8 (spherical) and
0/45. The names are due to the geometry of the light source, observer and interior
of the measurement chamber.
o If the compound is more concentrated more light will be absorbed by the sample;
within small ranges, the Beer-Lambert Law holds and the absorbance between
samples vary with concentration linearly.
o Samples are usually prepared in cuvettes ; depending on the region of interest,
they may be constructed of glass, plastic (visible spectrum), or quartz (far UV
spectrum).

Beer-Lambert Equation:
A=−log 10 T= ɛc ∨¿OD
  IR spectrophotometry
o Spectrophotometers designed for the infrared region are quite different because of
the technical requirements of measurement in that region. One major factor is the
type of photosensors that are available for different spectral regions, but infrared
measurement is also challenging because virtually everything emits IR ligt as
thermal radiation, especially at wavelength beyond about 5µm.

 Spectroradiometers
o Spectroradiometers, which operate almost like the visible region
spectrophotometers, are designed to measure the spectral density of illuminants.
Applications may include evaluation and categorization of lighting for sales by
the manufacturer.

Applications:
 The light source shines onto or through the sample.
 The sample transmits or reflects light.
 The detector detects how much light was reflected from or transmitted
through the sample.
 The detector then converts how much light the sample transmitted or
reflected into a number.
 Estimating dissolved organic carbon concentrated
 Specific Ultraviolet Absorption for metric of aromaticity
 Bial’s Test for concentration of pentoses
 The light source is shone into a monochromator, diffracted into a rainbow,
and split into two beams. It is then scanned through the sample and the
reference solutions
 Fractions of the incident wavelengths are transmitted through, or reflected
from, the sample and reference.
 The resultant light strikes the photodetector device, which compares the
relative intensity of the two beams.
 Electronic circuits convert the relative currents into linear transmission
percentages and/or absorbance/concentration values.
References:

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textboo

_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetics
Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry
https://aai.solutions/concepts/spectrophotometer

https://buck-scientific-norwalk.myshopify.com/collections/aa-systems/products/220
graphite-furnace-system

https://buck-scientific-norwalk.myshopify.com/collections/aa-systems/products/205
atomic-absorption-spectrophotometer