EXPERIMENT 1

INTRODUCTION TO SPECTROPHOTOMETER

INITIAL REPORT

Day and Date of Report Submission : Tuesday, 2nd April 2019

Student Name : Joe Vincent

Partner’s Name : Restu Rosthalia

Laurensia Felia Hidayat

NIM : 23201810003

DEPARTMENT OF FOOD BUSINESS TECHNOLOGY

SCHOOL OF APPLIED SCIENCE, TECHNOLOGY, ENGINEERING, AND MATHEMATICS
UNIVERSITAS PRASETIYA MULYA
2018
I. OBJECTIVES
 To learn the basic principles of spectrophotometry and its practical
applications.

II. EXPERIMENTAL PROCEDURE (ATTACHED)

III. PRE-LAB QUESTIONS

1. Explain terms :
a. Absorbance
b. Transmittance
c. Sample
d. Blank
2. Explain the components of spectrophotometer and their function
3. How is the relationship between concentration and absorbance? Explain!
Answer:
1. a. Absorbance is the amount of light that absorbed by a solution and it is
related logarithmically to transmission.
b. Transmittance is the ratio of the light energy transmitted through the sample
(I) to the energy transmitted through the reference blank (I0). The reference
blank is defined as 100%T.
c. Sample is a cuvette that contain the compound of interest which we want to
observe the transmittance or absorbance.
d. Blank is a cuvette that contain a solution that can ‘zero out’ any light that
absorbed by anything in the solution than the compound of interest. Or we can
say that the absorbance of the blank is set at zero (A λ = 0)

2. Components and functions of spectrophotometer are :

a. Light source : as the source of light to pass through the sample.
b. Prism : splits the light into each color that has their own
individual wave length.
c. Slit <8nm width : allows only a specific wave length of light through the
blank and sample.
d. Cuvette : a container that filled with sample or blank.
e. Photocell : an exit slit that connected with galvanometer which
translates the electrical output of the activated
photocell into a specific transmittance value.
f. Meter : show the value of a specific transmittance.

3. The relationship between concentration and absorbance is expressed by the

Lambert-Beer law which states that the absorbance of a light absorbing
material is proportional to its concentration in solution.
IV. LAB NOTES

1. Material Safety Data Sheet

a. Bromothymol blue (BTB) (18.6 μM)

b. Distilled Water

2. Equation
 Transmittance
𝐼
𝑇=
𝐼𝜊
T = Transmittance
𝐼 = the ratio of the light energy transmitted through the sample
𝐼𝜊 = the ratio of the light energy transmitted through the reference blank
 % Transmittance
𝐼
%𝑇 = x 100
𝐼𝜊
 Absorbance
A= - log T
 The Lambert-Beer Law
A=εlc
ε = The extinction coefficient of substance, has units of M-1.cm-1 (unique for each
substance)
l = the sample path length measured in centimeters (i.e. the width of the cuvette—
almost always 1 cm)
c = the molar concentration of the solution (you must express concentration in
terms of molarity)
V. REFERENCES

Lewiston. (2002). Use of the Spec 20. Retrieved from

http://abacus.bates.edu/~ganderso/biology/resources/spec20.html (Accessed on
Saturday, March 30, 2019)

Hardesty,Jon H. and Bassam Attili. 2010. Spectrophotometry and the Beer-

Lambert Law: An Important Analytical Technique in Chemistry. Collin College.

Bromothymol Blue MSDS retrieved from

https://studylib.net/doc/8786555/bromothymol-blue---santa-cruz-biotechnology
(Accessed on Saturday, March 30, 2019)

Bassiri, Eby. (n.d.). Spectrophotometer. Retrieved from

https://www.sas.upenn.edu/labmanuals/biol123/Table_of_Contents_files/8b-
EnzymeKinetics-Spec.pdf (Accessed on Saturday, March 30, 2019)

Foist, Laura. (n.d.). Spectrophotometer: Definition, Uses & Parts. Retrieved from
https://study.com/academy/lesson/spectrophotometer-definition-uses-parts.html
(Accessed on Saturday, March 30, 2019)