PIPETING AND DILUTION LAB

BIOCHEMISTRY
LABORATORY EXPERIMENT NO. 1

BY

MIA CLAIRE ANDERSON

SUBMITTED TO

Dr. Gautam Dar

LOS ANGELES TRADE TECHNICAL COLLEGE

February 26, 2019

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ABSTRACT
Oftentimes, a lab experiment procedure requires that the concentration of a solution be changed
by adding or subtracting the amount of a solvent. Dilution is the process of adding the solvent
while the process of concertation involves the removal of the solvent. This in turn increases the
concentration of the solute. In this experiment, serial dilution will be employed to obtain the
necessary sample.

The objectives of this experiment are as follows:

 To check checking the precision and accuracy of a micropipette

 To prepare Standard curve
a. To determine Mass % of a known sugar solution
b. To determine the densities of the known sugar solution
c. To dilute a Sugar solution and determine its

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INTRODUCTION
Biochemistry, the chemistry of life, depends on specific molecules that interact with one another.
Successful interaction of these molecules usually occurs while they are dissolved in water. A
substance dissolves in water make up as a solution. These dissolves substances are known as
solutes while the liquid that contains the dissolved substances is known as the solution. An
important property of solutions is their concentration. Concentrations refer to the amount of a
dissolved substance in a solution expressed in percentages. In this experiment calculating the
percent concentration and the molarity of a solution is obtained.

Oftentimes, a lab experiment procedure requires that the concentration of a solution be changed
by adding or subtracting the amount of a solvent. Dilution is the process of adding the solvent
while the process of concertation involves the removal of the solvent. This in turn increases the
concentration of the solute. In this experiment, serial dilution will be employed to obtain the
necessary sample.

It is important to note that the amount of the solute stays the same. It is the amount of the solvent
that changes. Various methods of computing for the concentrations will be explored in this
experiment including computing the percent concentration using the grams of the solute and the
volume of the solute. In order to measure the amount of liquid obtained with precision, the
technique of using a pipette is used.

The pipette is indeed a reliable instrument. It has been used for many years. Just like all other
forms of instrumentation though it is only as good as the technique of the operator. In the
laboratory, especially with making drugs, optimal pipette technique is of great importance. Both
systematic and observer contribution to errors are controlled in order to ensure accuracy and
precision. To Summarize, this experiment will involve the following:

1. Checking the precision and accuracy of a micropipette

2. Preparation of Standard curve and dilution technique
d. Determination of the Mass % of an a known sugar solution
e. Determination of the densities of the known sugar solution
f. Dilution of Sugar solution and determination of densities

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PROCEDURE
A. CHCKING THE PRECISION AND ACCURACY OF THE MICROPIPETTE
1. Use the pipette to measure out the volume of water indicated in each table.
2. Use the analytical balance to measure its weight in grams.
3. Plot the graph. Compute the equation of the line and R2.

B. PREPARATION OF STANDARD CURVE AND DILUTION TECHNIQUES

DETERMINING THE MASS OF % OF THE KNOWN SUGAR SOLUTION
1. Get a clean beaker. Label this A. Tare the beaker on the balance.
2. Make solution A by placing 2.5 g of sugar into the bottle. Record the actual mass to the
nearest milligram for the amount of sugar added int able 1.
3. Use a graduated cylinder, transfer 48 mL of distilled water to the bottle. Record the total
mass of the solution in table 1.
4. Cap the bottle. Shake thoroughly and stir. Ensure that all the sugar has dissolved.
5. Repeat the procedure above by using the other sugar solution.
6. Calculate the mass percent sugar according to equation 2. Record this in table 1.
7. Mark a container with pure water as Solution E. Pour about 50 mL of distilled water into
a small bottle. This serves as the control.
DETERMINIG THE DENSITITIES OF KNOWN SUGAR SOLUTIONS
1. Tare another clean dry 50 ml beaker
2. Measure 1.000 mL of solution E and obtain the mass using an analytical balance.
3. Repeat the procedure at least 2 more times. Tare the balance between each step.
4. Repeat steps 1-4 using Solution, A, B, C and D.
5. For each solution (A-E) Calculate the average density of the solution and record in Data
Table 1.
6. Graph results by plotting the Average Density of the Solution vs. Mass % of Sugar using
Microsoft Excel.
DILUTION OF SUGAR SOLUTIONS AND DETERMINATION OF DENSITIES
1. Make the following dilutions and label them as indicated in Table 2. Measure out 1.000
mL of each dilution and obtain the masses in analytical balance and complete the table.

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DATA

PART 1 2-20 uL pippette

TRIAL AVERAGE
VOLUME (UL) TRIAL 1 2 TRIAL 3 (g) SD
2 0.0018 0.0022 0.00206 0.00202 0.000166
5 0.0043 0.005 0.0044 0.004567 0.000309
10 0.0093 0.0101 0.0101 0.009833 0.000377
15 0.0144 0.0151 0.0146 0.0147 0.000294
20 0.0204 0.0197 0.0197 0.019933 0.00033

In part 1, a pipette that measured between 2-20 uL was used. For a volume of 2 uL, the average for 3
trials was 0.00202 g. The standard deviation was 0.00017. For 5 uL the average after 3 trials was
0.004567g. The standard deviation was 0.000309 g. For the 10uL, the average of 3 trials was 0.009833g.
The standard deviation was 0.0038 g. For a volume of 15 uL, the average of 3 trials was 0.0147 g with a
standard deviation of 0.00029g. Lastly for a volume of 20 uL, the average of 3 trials was 0.0199g with a
standard deviation of 0.0003g. The average standard deviation was 0.000295g indicating that the
measurements obtained were close to the computed average. Since the number obtained for the
standard deviation was close to 0, it can be concluded that the measurements were precise.

B: 20-200 uL PIPPETTE
VOLUME AVERAGE
(uT) TRIAL 1 TRIAL 2 TRIAL 3 (g) SD
25 0.0257 0.0255 0.0259 0.0257 0.000163
50 0.04947 0.0506 0.05 0.050023 0.000462
75 0.0741 0.0742 0.0742 0.074167 4.71E-05
100 0.0989 0.0991 0.0987 0.0989 0.000163
150 0.1478 0.1475 0.1475 0.1476 0.000141
200 0.1963 0.1965 0.1966 0.196467 0.000125

In this table for a volume of 25uL of the sucrose solution an average of 0.0257 g was obtained with a
standard deviation of 0.000163. For a volume of 50 uL, an average of 0.050 was obtained with a standard
deviation of 0.000462. For a volume of 75 uL an average of 0.074167 was obtained with a standard
deviation of 0.000047g. For 100 uL, an average of 0.09898 g was obtained with a standard deviation of
0.0002g. For a volume of 150 uL, an average of 0.1476 g was obtained with a standard deviation of 0.0001
g. Lastly, for a volume of 200uL, an average of 0.1965 g was obtained with a standard deviation of
0.000125. The standard deviation for all trials was less than 1, specifically 0.0002. This means that the
data obtained were close to the computed average. This also indicates that the measurements were
precise since the number obtained is close to 0.

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 C: 200-1000 ML PIPPETTE

VOLUME AVERAGE
(uL) TRIAL 1 TRIAL 2 TRIAL 3 (g) SD (g)
250 0.2475 0.2500 0.2499 0.249133 0.001156
500 0.498 0.4976 0.4958 0.497133 0.000957
750 0.7455 0.7424 0.7412 0.743033 0.001812
1000 0.9954 0.986 0.99283 0.99141 0.003967

For this table, for a volume of 250uL, the average mass was 0.2491g with a standard deviation of 0.0012g.
For the volume of 500uL the average mass was 0.4971g with a standard deviation of 0.001g. For a volume
of 750uL the average mass was 0.7430g with a standard deviation of 0.0018g. Lastly for a volume of 750
uL the average was 0.9914g with a standard deviation of 0.004 g. The average standard deviation was
0.00197g. This indicates that the measurements obtained were close to the average and that the
measurement was precise since the obtained value is close to 0.

PART 2
DATA TABLE 1: DETERMINING MASS PERCENT AND AVERAGE DENSITIES OF KNOWN SUGAR SOLUTIONS

AVE. MASS
MASS OF MASS OF
MASS OF SOLUTION PERCENT OF MASS OF 1.000Ml OF SOLUTION SOLUTION AVERAGE DENSITY OF
SOLN. SUGAR (G) (G) SUGAR (%) (G) DETERMINATION (G) SOLUTION (G/Ml)

1 2 3
A 2.5 48.961 5.106105 1.015 1.017 1.017 1.016 1.016
B 5 51.447 9.71874 1.035 1.035 1.036 1.035 1.035
C 7.5 53.536 14.00926 1.059 1.057 1.051 1.056 1.056
D 20 63.955 31.27199 1.15 1.093 1.115 1.119 1.119
E 0 48.579 0 0.996 0.995 0.998 0.996 0.996

For the 2.5 g sugar, the mass percent was 5.106 %. The average density of the solution was
1.016g/ml. For solution B, 5 grams of sugar was computed to produce a mass percent of 9.712%
The average density of the solution is 1.03 g/mL. Solution C which was 7.5 g, was computed to
have a mass percent of 14.00% with an average density of 1.056g/mL. Solution D with 20 grams
of sugar had a percent mass of 31.27%. The average density and mass of the solution 1.119 g/mL.
Solution E had no sugar in it. It had a density of 0.996g.

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DATA TABLE 2: DILUTION OF SOLUTION D AND DETERMINATION OF DENSITIIES

CALC. AVE. AVERAGE EXPECTED

VOL. MASS MASS DENSITIY DENSITY
OF PERCENT MASS OF 1.000 Ml OF OF OF FROM
VOL. OF WATER SUGAR SOLUTION (G) SOLN. SOLUTION STANDARD %
SOLN. SOLN (mL) (%) DETERMINATION (G) (G/ML) CURVE ERROR
1 2 3
D1 2 ML D 8 6.2544 1.0143 1.0159 1.0154 1.0152 1.0152 1.0213 0.5965
D2 4 ML D 6 12.5088 1.039 1.0378 1.0364 1.0377 1.0377 1.0457 0.7635
D3 5 ML D 5 15.6360 1.0529 1.0504 1.0511 1.0515 1.0515 1.0579 0.6031
D4 5 ML D3 5 7.8180 1.0207 1.0185 1.0213 1.0202 1.0202 1.0274 0.6998
D5 D ML D4 5 3.1272 1.00651 1.0073 1.0082 1.0073 1.0073 1.0091 0.1780

Solution D1 had a mass percent of sugar of 6.2544%. The average density of the solution is 1.0152 g/ml.
This had a 0.5965 % error from the expected density. Solution D2 had a calculated mass percentage of
sugar of 12.5088%. The average density of the solution was 1.0377 g/ml. This had a percent error of 0.7635
% from the expected density. Solution D3 had a calculated mass percent of 15.6360%. The average density
of the solution was 1.0515 g/mL . This had a 0.6031 % error from the expected density. Solution D4 had a
calculated mass percent of 7.8180% of sugar. The average mass of the solution was 1.0202 g/mL. This
resulted in a 0.6998 % error. Lastly, solution D5, had a calculated mass percent of sugar of3.1272%. The
average density of the solution was 1.0073 g/mL. This resulted in a 0.1080% error from the expected
density.

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INTERPRETATION

PART 1 2-20 uL pippette

MASS (g) VS. VOLUME (uL)y = 998.36x + 0.206
R² = 0.9994
25

20 20
Volume (uL)

15 15

10 10

5 5

2
0
0 0.005 0.01 0.015 0.02 0.025
Mass (g)

For this table, the R2 is 0.9994. This means that 99.94% of the variation in the volume can be attributed
to the changes in the mass of grams. An R2 of less than 1 means that the data fits the model and can
account for all variances. R2 measured the strength of the relationship of the model and the dependent
variable. The equation of the line is y= 998.36X +0.0206 where the slope indicates the density of the sugar
solution.

B: 20-200 uL PIPPETTE

TABLE 2: Mass (g) vs. VOLUME (uL)

250

y = 1024.4x - 1.2201
200 200
R² = 1
Volume (uL)

150 150

100 100
75
50 50
25
0
0 0.05 0.1 0.15 0.2 0.25
Mass (g)

R2 is a statistic that will give some information about the goodness of fit of a model. In regression,
the R2 coefficient of determination is a statistical measure of how well the regression predictions
approximate the real data points. An R2 of 1 indicates that the regression predictions perfectly fit the
data. The equation of the line is y=1024.4x = 1.2201 where the slope indicates the density of the
solution

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 C: 200-1000 ML PIPPETTE

Table 3: Mass (g) vs. Volume (uL)

1200

y = 1011x - 2.0148
1000 1000
R² = 1

800
750
Volume (uL)

600
500
400

250
200

0
0 0.2 0.4 0.6 0.8 1 1.2
Mass (g)

An R2 of 1 indicates that the predictions perfectly fit the data and that the changes in the volume are
affected by the changes in the mass of the sucrose solution. The equation of the line is y= 1011x-2.0148
where the slope indicates the density of solution.

AVERAGE DENSITY OF SOLUTION (g/mL)

1.14
y = 0.0039x + 0.9969
1.12 1.119
R² = 0.998
Average Density of Soln. 9g/mL)

1.1

1.08

1.06
1.056
1.04
1.035
1.02
1.016
1
0.996
Mass % of Sugar
0.98
0 5 10 15 20 25 30 35

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 The equation of the line for this graph is y=0.0039x-0.9969. The R2 value is less than 1 with a value of
99.8%. This indicates that an R2 of less than 1 means that the data fits the model and can account for all
variance.

DATA TABLE 2: DILUTION OF SOLUTION D AND DETERMINATION OF DENSITIIES

CALC. AVE. AVERAGE EXPECTED
VOL. MASS MASS DENSITIY DENSITY
OF PERCENT MASS OF 1.000 Ml OF OF OF FROM
VOL. OF WATER SUGAR SOLUTION (G) SOLN. SOLUTION STANDARD %
SOLN. SOLN (mL) (%) DETERMINATION (G) (G/ML) CURVE ERROR
1 2 3
D1 2 ML D 8 6.2544 1.0143 1.0159 1.0154 1.0152 1.0152 1.0213 0.5965
D2 4 ML D 6 12.5088 1.039 1.0378 1.0364 1.0377 1.0377 1.0457 0.7635
D3 5 ML D 5 15.6360 1.0529 1.0504 1.0511 1.0515 1.0515 1.0579 0.6031
D4 5 ML D3 5 7.8180 1.0207 1.0185 1.0213 1.0202 1.0202 1.0274 0.6998
D5 D ML D4 5 3.1272 1.00651 1.0073 1.0082 1.0073 1.0073 1.0091 0.1780

Solution D1 had a mass percent of sugar of 6.2544%. The average density of the solution is 1.0152 g/ml.
This had a 0.5965 % error from the expected density. Solution D2 had a calculated mass percentage of
sugar of 12.5088%. The average density of the solution was 1.0377 g/ml. This had a percent error of 0.7635
% from the expected density. Solution D3 had a calculated mass percent of 15.6360%. The average density
of the solution was 1.0515 g/mL . This had a 0.6031 % error from the expected density. Solution D4 had a
calculated mass percent of 7.8180% of sugar. The average mass of the solution was 1.0202 g/mL. This
resulted in a 0.6998 % error. Lastly, solution D5, had a calculated mass percent of sugar of3.1272%. The
average density of the solution was 1.0073 g/mL. This resulted in a 0.1080% error from the expected
density

ANALYSIS

In conclusion, using the pipette is a precise and accurate method of measuring since the percent error
observed on average was less than 1% using the expected density from the standard curve. Since the R2
that were obtained also 1 or less that 1, it is safe to assume that the dependent variables were affected
by the independent variables. Due to the low standard deviation obtained (a range of 0.0001-0.0002g), it
can also be safe to say that the experimenters were also precise in their measurement.

While all experimenters strive to perform the most precise and accurate measurements, errors sometimes
cannot be avoided. The following sources of errors could have possibly lead to the accounted % error:

1. The pipette needed to be more precisely calibrated

2. The analytical balance need to be precisely calibrated
3. The weighing of the sugar sample needed to be more accurate
4. During the mixing, it is possible that undissolved sugar particles were remained in the bottle.

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The experimenters will take note of these sources of error and try to control these variables in future
experiments.

REFERENCE
 https://en.wikipedia.org/wiki/Coefficient_of_determination
 http://statisticsbyjim.com/regression/interpret-r-squared-regression/
 https://www.wikihow.com/Calculate-Mass-Percent
 https://search.yahoo.com/yhs/search?hspart=iba&hsimp=yhs-
1&type=c5pr_5056_CHW_US&p=computing%20mass%20percentage%20of%20sugar%20solution
 https://www.google.com/search?biw=1280&bih=625&tbm=isch&sa=1&ei=NkB3XLDCOZHGsAWX56
64Bw&q=density+calibration+curve+of+sugar+in+water+5%25+10%25+15+%25+&oq=dens
 y+calibration+curve+of+sugar+in+water+5%25+10%25+15+%25+&gs_l=img.3...40898.54089..54535.
..11.0..1.272.6890.7j53j1......1....1..gws-wiz-
mg.......35i39j0i67j0j0i5i30j0i8i30j0i24j0i30.wtIClSLh5r8#imgrc=_4vNhG8_ns7C_M:

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POST LAB QUESTIONS

1. What information do you get from the slope of the lie in part A for each graph? Is it the same for
all the curve? If so why?

Once the microliter is converted to mL, this data will yield density since the formula for density
is g/ml (mass / volume )

2. What are the dilution factors of D1, D2, D3, D4 and D5 in part 2 table 2?

3. What will be the density of a solution at infinite dilution??

The density of the solution will be the same. Infinite dilution is “defined as a solution that
contains so much solvent that when you add more liquid, there is no change in
concentration”,. This means that no matter how much solvent is added to the solution, the
properties of the solute -- the substrate particles -- and the system will not change. A study
published in the journal Fluid Phase Equilibria defined infinite dilution as a solution in which
each molecule of the solute is surround by solvent molecules only. This means that the
solute only interacts with the liquid and has no contact with molecules of its own kind.
https://ourpastimes.com/definition-of-infinite-dilution-12219054.html

4. What is the concentration of solution D in molarity?

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