Kristine Q.

Zabala
Biology 1012 Lab Section 33
Dr. Brumwell
6 February 2015
Lab 2: Techniques and Measurements
- OBJECTIVE: The objective of this lab was divided into a three-part symposium: the first
whose goal was to prepare stock solutions of NaCl in H2O; the second whose goal was to
measure the relative absorbance of five Coomassie Blue solutions; and the third whose goal was
to determine the concentration of unknown solutions using a standard curve.
- MATERIALS & METHODS
Experiment 1: Preparation of Stock Solution of NaCl in H2O
A weighing paper was placed on an electrical balance (rather than the suggested
trip balance) and was then tared. Then, 15.0g of NaCl was weighed out on the
weighing paper. 50mL of H2O was then added to two 100-mL graduated
cylinders. Next, A stirrer bar was placed to one of the graduated cylinders and
was then placed onto the stirring base. Then, the stirrer was started and the NaCl
was gradually added to the cylinder so as to fully dissolve. More H2O from the
other cylinder was added to the original graduated cylinder to make 100mL of the
resulting solution. Procedures for this experiment can be found on pages 3 and 7
of the Lab 2 Handout.
Experiment 2: Determination of the Concentration of an Unknown Solution of Coomassie
Blue using a Standard Curve
Five test tubes were respectively labeled with tape. With the provided stock
solution of Coomassie Blue, whose concentration is 0.1 mg/ml and H2O, a pipette
was used to prepare five 10 mL solutions with different concentrations:
0.005mg/mL, 0.010mg/mL, 0.015mg/mL, 0.020mg/mL, 0.025mg/mL. A
spectrophotometer was then calibrated at 595nm in order to determine the
Relative Absorbance of each of the five dilute solutions. Another unknown
dilution was provided by the lab instructor, and this solution was also recorded
using the spectrophotometer at the same wavelength. The solutions were placed

on a test tube rack in order to proceed onto Experiment 3. Procedures for this
experiment can be found on pages 1-6 as well as page 8 of the Lab 2 Handout.
Experiment 3: Sources and Effects of Error
With the solutions from Experiment 2, two new solutions were created. The first
was one in which 4.5 mL of the most concentrated solution was added to 0.5 mL
of the most dilute solution. Then, spectrophotometer was used to determine the
Relative Absorbance for this mixture. Then, another solution, which comprised of
4.5 mL of the most dilute solution and added 0.5mL of the most concentrated
solution, was prepared. This new mixture was also placed into the
spectrophotometer to determine its Relative Absorbance. Procedures for this
experiment can be found on pages 1-6 as well as page 9 of the Lab 2 Handout.
- RESULTS
Experiment 1: Not all of the H2O was added to the final solutionabout 4.0 mL
remained. The concentration of the NaCl/H2O solution is 150g/L. The theoretical
concentration of the solution with the remaining 4.0mL of water would have been
144g/L.
CONCENTRATION OF SALT SOLUTION
15.0
1000
150

=
100
1

CONCENTRATION OF THEORETICAL SALT SOLUTION COMPRISED OF
15.0g NaCl and 100 mL H2O
15.0
1000 144

=
100 + 4.0 2
1
1

Experiment 2: Determination of the Concentration of an Unknown Solution of Coomassie

Blue using a Standard Curve
Table 1
Tube#

Concentration

Relative Absorbance

(mg/mL)

(@595nm)

0.005

0.079

0.010

0.112

0.015

0.176

0.020

0.243

0.025

0.298

Blank

Standard Curve for Coomassie Blue

0.35

Relative Absorbency (@595nm)

0.3

0.025, 0.298
0.023, 0.258
0.02, 0.243

0.25

0.2
0.015, 0.176
0.15
0.008, 0.112
0.1

0.01, 0.112

0.007, 0.092
0.005, 0.079

0.05

0
0

0.005

0.01

0.015

0.02

Concentration (mg/mL)

0.025

0.03

Experiment 3: Sources and Effects of Error

TABLE 2
Tube #

Concentration

Concentration

Relative

(mg/mL) from

(mg/mL) from

Absorbance

Standard

Calculations

(@595nm)

Curve
Unknown #1

~0.008

.112

(Exp 2)
Unknown A

~0.023

.023

.258

~0.007

.007

.092

(Exp 3)
Unknown B
(Exp 3)

CONCENTRATION A:
.

.
.
= . + .
= .

= .

. .
=

.

CONCENTRATION B
0.025
0.005
= 0.0125 + 4.5
= 0.0225 = 0.035

0.035
0.07

=
5.0

0.5

- DISCUSSION
Experiment 1: Preparation of Stock Solution of NaCl in H2O
We found that in adding the H2O from the second cylinder, we did not add all of
the H2O from the second cylinder, since the NaCl, as well as the stir bar,
comprised of about 4.0mL of the solution. If all of the water had been added to
the solution, the concentration of the mixture would have been slightly lower (144
g/L). In addition, had we added water to the solution instead of the NaCl, most of
the salt would remain at the bottom of the graduated cylinder. Some errors include
NaCl sticking to sides (due to adhesive forces), which would result in a lower
concentration.
Experiment 2:Determination of the Concentration of an Unknown Solution of Coomassie
Blue using a Standard Curve
Concentration and relative absorbance are directly proportional. This relationship
is seen as the points are plotted in a linear trend. Some errors in this experiment
include using apparatus that was not fully cleaned as well as making minor spills
from tubes during transfer.
Experiment 3: Sources and Effects of Error
The addition of a small quantity of a dilute solution to a large quantity of
concentrated solution changes the concentration of the final solution significantly.
The same is true for the inverse. The calculated concentrations were the same as
those determined in #2. Although they produced the same results, the more
accurate measure would be the one that was calculated because it leaves a smaller
chance for experimental errors such as spillage or the improper distribution of
respective chemicals. In addition, it leaves a smaller chance for crosscontamination (for example, neglecting to rinse a pipette between transferring two
different solutions). In addition, using dirty glasses could be contaminated with
old reagents, which would then react with new ones, thus changing the
concentration.
- REFERENCES
Biology 1012 Lab 2 Handout