SPECTROPHOTOMETRY

3B 2023
INTRODUCTION

Invented in 1940, by Arnold J. Beckman

and his colleagues at National
Technologies Laboratories, the company
Beckman had started in 1935.
INTRODUCTION

● Ultraviolet - visible spectrophotometry widely used

for:
1. Determining species
2. Studying biochemical processes
● Makes determination of micromolar concentrations of
substances possible
- Most biochemical compounds absorb light with
wavelengths from UV to visible light
Types of Spectrophotometer

1. Single beam (used in the lab)

- Usually less expensive and less complicated
1. Double beam
2. Visible light
3. Ultraviolet light
4. Infrared Light
BEER-LAMBERT LAW (BEER’S LAW)

Beer's law states that the concentration of a chemical solution is directly

proportional to its absorption of light.

A = εcl
Where:
A - absorbance
ε - molar absorptivity / molar extinction (M-1cm-1)
c - concentration of solution (M or mol/L)
l - length of lightpath or the width of the cuvette (cm)
MATERIALS & METHODS
 Compute for
FBS
Fasting Blood Sugar
& TSC
Calibrate Total Serum Cholesterol
Spectrophotomete
r

8
USES OF SPECTROPHOTOMETER

The spectrophotometer is useful for measuring the

absorption spectrum of a compound, the absorption of
light by a solution at each wavelength.
USES OF SPECTROPHOTOMETER
A spectrophotometer is used in many areas including microbiology,
biochemistry, forensics, physics, and medical health. Specifically,
spectrophotometer can be used for:

● Detection of concentration of substances and impurities (drug

effectivity and safety, illness diagnosis)
● Structure elucidation and characterization of organic compounds,
proteins and other compounds (Qualitative Analysis)
● Determination of enzyme-catalyzed reactions
● Molecular weight determination
● Environmental applications
● Also used by wine-makers
POSSIBLE SOURCES OF ERROR

1. Presence of contaminating enzymes

2. “Stray light” or unwanted radiation
3. Light scattering
4. Environmental effects on photometer and sample,
temperature, line voltage fluctuations, vibrations,
contamination, or heating of the sample by the
photometer.
POSSIBLE SOURCES OF ERROR

5. Use of micro-cuvette causing the width of the sample

to be less than the width of the light beam.
6. Interaction between the photometer and sample such
as: Interreflections
 REFERENCES
Apel Co.(n.d.). PD-303S Catalog Retrieved from http://www.apel.co.jp/products/analytical_equipment/pd-303s.html?lang=en
November 02, 2019

Helmenstine, Anne Marie, Ph.D. (2019, June 24). Beer's Law Definition and Equation. Retrieved November 2, 2019
from https://www.thoughtco.com/beers-law-definition-and-equation-608172

Libretexts. (2019, September 30). Spectrophotometry. Retrieved from

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Th
eoretical_Chemistry)/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry November 02, 2019

Libretexts. (2019, September 30). The Beer-Lambert Law. Retrieved November 2, 2019, from
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Th
eoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law.

Some Sources of Error ad Artifacts in Spectrophotometric Measurement. (1975). Clinical Chemistry, 21, 776–777.

Reule, A. (1976). Errors in spectrophotometry and calibration procedures to avoid them. Journal of Research of the National Bureau of
Standards Section A: Physics and Chemistry, 80A(4), 609. doi: 10.6028/jres.080a.060

Rojas, F. , Bosch - Ojeda, C. , Pavon, J. (2013). Spectrophotometry: Biochemical Applications. Reference Module in
Chemistry, Molecular Sciences and Chemical Engineering. DOI: 10.1016/B978-0-12-409547-2.00501-1

Virtual Laboratories Amrita Vishwa Vidyapeetham University. ( 2011). Spectrophotometry. Retrieved November 3,
2019 from https://vlab.amrita.edu/?sub=2&brch=190&sim=338&cnt=1