Free Essay

# Introduction for Biochemistry Lab -Spectrophotometry

In: Science

Submitted By emra
Words 716
Pages 3
Introduction
Every chemical compound absorbs, transmit or reflect light over a certain range of wavelength. In this experiment one of the main purpose is to introduce students to the quantitative and qualitative study of spectrophotometry. Spectrometry is the measurement of light that is absorbed or transmitted by the sample solution (Vo, n.d). It is one of the most commonly used methods in various fields to study the quantitative analysis of light that passes through a solution. Meanwhile, the use of spectrophotometer to determine the extent of absorption of wavelengths of visible of light is known as colorimetry.
The intensity of light that passes through the sample as well as the concentration of the substance is measured by a spectrophotometer. The instrument consists of some basic components: Radiation light source (tungsten for visible region and deuterium lamp for ultraviolet region), wavelength selector (to isolate a desired wavelength form the source), a cuvette or container to hold the sample solution and a detector (photometer) which delivers the signal in a display device. The voltage output is determined by the difference in the amount of light absorbed by the sample. The fraction of light that passes through the solution is called transmittance, T and the proportion of light absorbed is the absorbance, Abs.

Thus, the value of transmittance can be calculated by using the following formula:
Transmittance, I = It/ I0 where, It = intensity of the incident radiation entering the medium, I0 = intensity of the transmitted radiation leaving the medium

Meanwhile transmittance, T, and absorbance, Abs, is related by the formula of absorbance:

Absorbance (Abs) = -log (T) = -log (It/I0)

The data is written in transmittance, % transmittance or absorbance. Absorbance has no unit as it is a ratio of light. However, sometimes, it is also expressed absorbance units.
On the other hand, to determining the unknown concentration of the absorbing solution, the beer lambert law is used as it is the linear relationship between transmittance and absorbance. This law states that absorbance is proportional to the amount of the concentration of analyte, the path length and the molar coefficient. Therefore,
Absorbance, A = εcl
Where,
ε= molar extinction coefficient for the absorbing material at wavelength in units of 1/(mol x cm) c= concentration of the absorbing solution (molar) l = light path in the absorbing material (l=1 cm for our purposes)

In the UV/Visible region, compounds absorb light in wavelength of (200nm-700nm). The functional which is responsible to light absorption is known as chromophore. The result obtained is plotted against wavelength which shows the absorption bands corresponding to structural groups within the molecules. The absorption spectra are caused by the transition of electrons in its excited and ground states. Increasing solvent polarity from the effect of increased solvation of lone pair and decreased conjugation results in shorter wavelength which gives rise to the blue shift or hypochromic shift (Clark, 2007). Meanwhile, the inverse, lower polarity solvent and increased conjugation gives rise to red shift that result in increased wavelength. These shifts in wavelengths and intensities are the results of the molecules structure and the interaction between solute and solvents molecules (Robinson, Frame & Frame II).

In colorimetry, calibration curve where absorbance (A) of such solutions can be plotted against the concentration (c) of the test substance producing the color. Some important highlights in this method include that it is a destructive technique in which once the sample is reacted, it cannot be recovered, the chromophore reflects the complementary color that it absorbs and the calibration curves may vary with the different batches of reagents and standards used. Colorimetry is usually used to determine the concentrations of chemicals substance which can give colours either directly or indirectly or after the addition of other chemicals. In addition, with its efficiency in producing large amount of absorbance with relatively small amount of material, it is highly used in biochemistry assay.

References

Clark, J., (2007). UV-Vis Absorption Spectroscopy: Theorical principals. Retrieved at teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm on 9th May 2014.

Robinson, J, W., Frame, E.M.s., and G.M., (2004). UV spectra and the structure of organic molecules. Undergraduate Instrumental Analysis, 6th Ed, p350.

Vo, K., (n,d). Spectrophotometry. Retrieved at chemwiki.ucdavis.edu/physical_chemistry/kinetics/Reaction_Rates/Experimental_Determination_ofkinetics/Spectrophotometry on 9th May 2014.

### Similar Documents

Free Essay

#### Biology Lab

...Course Practical 3 Laboratory manual Isolation of nucleic acid and spectrophotometry Introduction: The ability to isolate and quantify nucleic acids accurately and rapidly is a prerequisite for many of the methods used in biochemistry and molecular biology. The concentration of DNA or RNA in a sample, and its condition, are often estimated by running the sample on an agarose gel. Such concentration estimates are semiquantitative at best and are time-consuming. For a more accurate determination of the concentration of DNA or RNA in a sample, a UV spectrophotometer is commonly used. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. The absorption maximum of DNA/RNA is approx 260nm. The purity of a solution of DNA can be determined using a comparison of the optical density values of the solution at various wavelengths. For pure DNA, the observed A260/A280 ratio will be near 1.8. Elevated ratios usually indicate the presence of RNA. The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8-2.1 is indicative of highly purified RNA. The 260/280 ratio below 1.8 often signal the presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is indicated by 230/260 ratios greater than 0.5. Workflow Time 2 days before the lab session During lab session 1:30 pm Task Cell culture 2:00 pm RNA isolation ...

Words: 842 - Pages: 4

Free Essay

#### Biology

...ULTRAVIOLET/VISIBLE SPECTROSCOPY PURDUE UNIVERSITY INSTRUMENT VAN PROJECT ANALYSIS OF PLANT PIGMENTS USING PAPER CHROMATOGRAPHY AND VISIBLE AND/OR UV SPECTROSCOPY (1-31-96) INTRODUCTION We have seen that all cells must constantly consume fuel molecules to maintain themselves, grow, and reproduce. Fuel molecules such as glucose constitute an immediate source of energy for biological work that can be released by catabolic cell processes. However it is necessary that life on earth have a constant source of energy that can be harvested and used to generate complex fuel molecules from simple starting materials. The ultimate energy source upon which all life forms depend is visible light from the sun. Light energy must first be transformed into chemical(bond) energy before it can be utilized by the living cell. This transformation is achieved only in the cells of green plants and certain bacteria. In green plants it is coupled with a transformation of matter in which relatively low-energy compounds, carbon dioxide and water, are converted into high energy chemical molecules that become subunits of carbohydrates. There are four different pigment groups present in leaves of photosynthesizing plants. Studies indicate that only the chlorophyll IS involved in the actual absorption of light energy and later conversion to chemical energy of living cells. The other pigments also absorb light energy, but it is transferred to the chlorophyll for conversion to chemical energy. Biochemists......

Words: 2526 - Pages: 11

Free Essay

#### Biotech

...Summer Project Report Study of MDA (malondialdehyde) as abiotic stress marker in CSV-17 variety of Sorghum bicolor. Submitted in partial fullfilement of the requirement for B.Tech. Biotechnology Semester VII AMITY INSTITUTE OF BIOTECHNOLOGY AMITY UNIVERSITY RAJASTHAN JAIPUR 2011 Supervised by: Dr Ajit Kumar Sr. Research Officer S.P. Institute of Biotechnology, Jaipur Submitted by: Ravi Pareek DECLARATION I hereby declare that the project report entitled “Study of MDA (malondialdehyde) as abiotic stress marker in CSV-17 variety of Sorghum bicolor” is a record of the work compiled by me under the supervision and guidance of Dr. Ajit Kumar, S.P. Institute of Biotechnology, Jaipur as a part of my 45 days summer training. Ravi Pareek (B.TECH-BIOTECHNOLOGY) (AUR0821094) ACKNOWLEDGEMENT First of all with due regard to my respective god with whose kindness and blessing we could be able to accomplish the task of training. Mr. Sourabh Pareek, for his kind permission to allow me to undergo my major project at S. P. Institute of Biotechnology, Jaipur. I am overwhelmed with rejoice to take this opportunity to evince my profound sense of reverence and gratitude to my esteemed supervisor respective Dr. Ajit Kumar, for giving his regular advice and excellent suggestion which have helped us for completing the study. His regular assistance and guidance really helped me to bring formidable task in successful manner. Sincere thanks to Dr. Sonali Jana and Dr. Neha Upreti for their......

Words: 12095 - Pages: 49

#### Thesis: Malunggay Cupcake as Supplementary Food

... help from friends, and support from my family. We would like to express my deepest gratitude to my advisor, Mrs. Modie Flores, for her excellent guidance, caring, patience, and providing us with an excellent atmosphere for doing research. We would like to thank our Parents, who let us experience the research of Malunggay Cupcake in the field and practical issues beyond the textbooks, internet, patiently corrected our writing and financially supported our research. We would also like to thank Menchie Hermones, and Ludy Balagosa, for guiding our research for the past several weeks and helping us to develop our background in biochemistry. We would like to thank our Classmates, who as a good friend, was always willing to help and give their best suggestions. It would have been a lonely lab without them. We would also like to thank our parents, two elder sisters, and elder brother. They were always supporting us and encouraging us with their best wishes. We give thanks to God almighty for giving us the understanding, knowledge and wisdom during the course of our study. Finally, we would like to thank our classmate Auntie, Sally Barral. She was always there cheering us up and stood by us through the good times and bad. Dedication We dedicate my dissertation work to our family and many friends. A special feeling of gratitude to our loving......

Words: 8495 - Pages: 34

Free Essay

#### Labs

...INSTRUCTOR GUIDE Human Anatomy & Physiology Laboratory Manual MAIN VERSION, Eighth Edition Update CAT VERSION, Ninth Edition Update FETAL PIG VERSION, Ninth Edition Update ELAINE N. MARIEB, R.N., Ph.D Holyoke Community College SUSAN T. BAXLEY, M.A. Troy University, Montgomery Campus NANCY G. KINCAID, Ph.D Troy University, Montgomery Campus PhysioEx™ Exercises authored by Peter Z. Zao, North Idaho College Timothy Stabler, Indiana University Northwest Lori Smith, American River College Greta Peterson, Middlesex Community College Andrew Lokuta, University of Wisconsin—Madison San Francisco • Boston • New York Cape Town • Hong Kong • London • Madrid • Mexico City Montreal • Munich • Paris • Singapore • Sydney • Tokyo • Toronto Editor-in-Chief: Serina Beauparlant Project Editor: Sabrina Larson PhysioEx Project Editor: Erik Fortier Editorial Assistant: Nicole Graziano Managing Editor: Wendy Earl Production Editor: Leslie Austin Composition: Cecelia G. Morales Cover Design: Riezebos Holzbaur Design Group Senior Manufacturing Buyer: Stacey Weinberger Marketing Manager: Gordon Lee Copyright © 2009 Pearson Education, Inc., publishing as Pearson Benjamin Cummings, 1301 Sansome St., San Francisco, CA 94111. All rights reserved. Manufactured in the United States of America. This publication is protected by Copyright and permission should be obtained from the publisher prior to any prohibited reproduction, storage in a retrieval system, or transmission in any form or by any......

Words: 120457 - Pages: 482

Free Essay

#### Analytical Chem

...Chemistry Modern Analytical Chemistry David Harvey DePauw University Boston Burr Ridge, IL Dubuque, IA Madison, WI New York San Francisco St. Louis Bangkok Bogotá Caracas Lisbon London Madrid Mexico City Milan New Delhi Seoul Singapore Sydney Taipei Toronto McGraw-Hill Higher Education A Division of The McGraw-Hill Companies MODERN ANALYTICAL CHEMISTRY Copyright © 2000 by The McGraw-Hill Companies, Inc. All rights reserved. Printed in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a data base or retrieval system, without the prior written permission of the publisher. This book is printed on acid-free paper. 1 2 3 4 5 6 7 8 9 0 KGP/KGP 0 9 8 7 6 5 4 3 2 1 0 ISBN 0–07–237547–7 Vice president and editorial director: Kevin T. Kane Publisher: James M. Smith Sponsoring editor: Kent A. Peterson Editorial assistant: Jennifer L. Bensink Developmental editor: Shirley R. Oberbroeckling Senior marketing manager: Martin J. Lange Senior project manager: Jayne Klein Production supervisor: Laura Fuller Coordinator of freelance design: Michelle D. Whitaker Senior photo research coordinator: Lori Hancock Senior supplement coordinator: Audrey A. Reiter Compositor: Shepherd, Inc. Typeface: 10/12 Minion Printer: Quebecor Printing Book Group/Kingsport Freelance cover/interior designer: Elise Lansdon Cover image: © George......

Words: 88362 - Pages: 354

#### Biochemistry Test Bank Questions

...Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18 Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Introduction to Biochemistry Water Amino Acids and the Primary Structures of Proteins Proteins: Three-Dimensional Structure and Function Properties of Enzymes Mechanisms of Enzymes Coenzymes and Vitamins Carbohydrates Lipids and Membranes Introduction to Metabolism Glycolysis Gluconeogenesis, The Pentose Phosphate Pathway, and Glycogen Metabolism The Citric Acid Cycle Electron Transport and Oxidative Phosphorylation Photosynthesis Lipid Metabolism Amino Acid Metabolism Nucleotide Metabolism Nucleic Acids DNA Replication, Repair, and Recombination Transcription and RNA Processing Protein Synthesis Recombinant DNA Technology 1 10 27 46 65 85 104 119 137 153 169 185 199 213 227 241 256 269 284 300 315 330 348 Chapter 1 Introduction to Biochemistry 1) Which elements account for more than 97% of the weight of most organisms? A) C, H, N, Mg, O, S B) C, H, N, O, P, S C) C, H, N D) Fe, C, H, O, P E) Ca2+ , K+ , Na+ , Mg 2+ , ClAnswer: B Page Ref: Section 2 2) Proteins in biological membranes may be A) porous. B) attached to the membrane surface. C) span the membrane. D) All of the above E) B and C only Answer: D Page Ref: Section 3 3) Which......

Words: 70772 - Pages: 284