Instructor: Dr.

Gautam Dhar

Pipeting and Dilution Lab

Name_____________________________ Subject__________________ Date____________

Concentration:
Units of concentration:
A. Percent concentration: units of solute dissolved in 100 unit of solution.
% (m/m) = g solute x 100 / g solution
% (m/v) = g solute x 100 / mL solution
% (v/v) = mL solute x 100 / mL solution
B. Molarity: Molarity is defined as the number of moles of solute in 1L of solution
M= moles/L solution
Dilution
In laboratory we often need to dilute solutions from a concentrated stock. If C1 and V1 be the
concentration and volume of the stock and C2 and V2 are the concentration of the diluted solution
respectively then,
C1V1 = C2V2
C1/C2 = V2/V1 = dilution factor (DF)
If a laboratory uses 100 mM NaCl for its experiment but the stock is 1 M then the stock is 10X
concentrated. To make a working solution one has to take 1 vol of the stock and add 9 volume of water.
Or in other word, make the total solution volume 10 times to that of the volume of the stock taken.
Serial Dilution
Often in laboratory we use chemical that are very potent and need to be diluted many times (1000-
100000 fold). In those cases, instead of doing 1 step dilution we do serial dilution. For example, a stock
solution of a chemical is 10000X concentrated. To make a working solution we need to dilute it 10000
times. If we start with 1 mL of stock we need to prepare a 10L solution which is rather a wastage.
Instead. We can perform a serial dilution as shown below. In each step we will dilute the solution 10
times
1. Take 1 mL stock and water to a total volume of 10 mL. This will generate a 1000X stock
2. Take 1 mL 1000X solution and water to a total volume of 10 mL. This will generate 100X stock.
3. Repeat two more times and you will get a 10 mL 1X stock for your work.
Each of these sub-stocks can be labeled and stored for future use.
In general
Final DF = (D1) x (D2) x (D3)……
D1, D2, D3…. represents the fold dilution done in each step.
 Instructor: Dr. Gautam Dhar

Pipeting and Dilution Lab

Name_____________________________ Subject__________________ Date____________

Prelab:

1. 1 mL of a stock solution of Coomassie Blue reagent with a concentration of 34 mg/mL is poured into a
test tube. Then 4 mL of H2O is added to the test tube.

a. What is the final concentration of Coomassie Blue?

b. What is the dilution factor for this 1-step process?

2. You performed a 1:25 dilution to a stock solution of Bovine Serum Albumin, BSA (2.5 g/mL).

a. What is the final concentration of BSA?

b. If you poured 1 part stock BSA into a test tube to perform this dilution, how many parts of H2O would
you need to dilute this to its proper concentration?

c. If you poured 2.4 mL of stock BSA into a test tube to perform this dilution, how much H2O would you
need to add to dilute this to its proper concentration?
 Instructor: Dr. Gautam Dhar

3. You performed a 4-step serial dilution to a stock solution (2.5 g/L). The sequence of dilution factors
is as follows: 1st (2X), 2nd (10X), 3rd (5X), 4th (150X)

a. What is the final concentration of the solution after the 3rd serial dilution?

b. On the 4th step serial dilution, you perform a 150X. If your final volume is 3 mL after the dilution, what
is your initial volume (in L) would you need of your previous dilution to correctly perform this last step?

c. What is the overall dilution factor of your serial dilution from steps 1 through 4?

4. You are given a test tube containing 10 mL of a solution with 8.4 x 107 cells/mL. You are to produce a
solution that contains 84 cells/mL. You will perform a 3-step serial dilution in your laboratory.

Draw test tubes and properly label concentrations after each step of your serial dilution. Also indicate the
dilution factors for each step along with the volumes of solution and H2O needed to dilute each step.
 Instructor: Dr. Gautam Dhar

Procedure:

Part 1: Checking the precision and accuracy of micro pipette.

1. Take the pipettes and measure out the volume of water indicated in each table. Use an analytical
balance (3-4 decimal) for this experiment.
2. Plot graph as directed and give the equation and R2 for the best fit line

Part 2: Preparation of Standard curve and dilution techniques

Record all masses to the nearest milligram (0.001 g).

A Determining Mass % of Known Sugar Solutions

1. Obtain a clean, dry small bottle or beaker, and label it “A”. Tare the bottle or beaker on the
balance. Be sure to complete steps 2-3 before preparing solutions “B” and “C”! It is best to
prepare the solutions one at a time so you do not lose the “tare” on your balance.
2. To make solution “A,” place about 2.5 g of sugar into the bottle. Record the actual mass to the
nearest milligram (0.001 g) for the amount of sugar added in Table 1.
3. Using a graduated cylinder, transfer approximately 48 mL of distilled water to the bottle. Record
the total mass of the solution in Table 1.
4. Cap the bottle, and shake thoroughly or stir until all the sugar has dissolved.
5. Repeat the procedure above by using the other sugar solutions: Solution “B” will have about 5.0 g
of sugar; Solution “C” will have about 7.5 g of sugar. Solution “D” will have about 20.0 g of sugar
6. For each solution, calculate the mass percent sugar according to Equation 2. Record in Table 1.
7. Solution “E” is pure water. Pour about 50 mL of distilled water into a small bottle. Label the bottle
“E.” This solution is your 0.00% sugar solution.
B Determining the Densities of Known Sugar Solutions
8. Tare a clean dry 50-mL beaker.
9. Measure out 1.000 mL of solution E and obtain the mass in an analytical balance.
10. Repeat the procedure above at least two more times, so that there are 3 determinations that
agree within the limits specified by your laboratory instructor. Tare the balance before each step.
11. Repeat steps 8-12, using Solutions A, B, C and D.
12. For each solution (A-E), calculate the Average Mass of 1.000 mL of Solution and record in Data
Table 1.
13. For each solution (A-E), calculate the Average Density of the Solution and record in Data Table 1.
14. Graph your results by plotting the Average Density of the Solution vs. Mass % of Sugar using
Microsoft Excel or similar program. Keep in mind the independent variable (x-axis) is the Mass %
and dependent (y-axis) is Average Density. Do not start your y-axis at 0. Instead, select y-axis
scale units so that the lowest value is slight below the Average Density of Solution D, and the
highest value is slightly above the Average Density of Solution C. Your x-axis should run from
0.00 to 20 Mass % Sugar. Perform a linear regression analysis for your graph. Record the
equation for the resulting trend line and correlation value on your Data Sheet.

C Dilution of Sugar solutions and determination of densities

17. Make the following dilutions and label them as indicated in Table 2. Measure out 1.000 mL of
each dilutions and obtain the masses in analytical balance and complete the table.
 Instructor: Dr. Gautam Dhar

Data Sheet:
Part 1:
A. 2-20 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
2

10

15

20

Equation:
R2 =

B. 20-200 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
25

50

75

100

150

200

Equation:
R2 =
 Instructor: Dr. Gautam Dhar

C. 200-1000 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
250

500

750

1000

Equation:
R2 =

Data Sheet Part 2:

Data Table 1. Determining Mass Percent & Average Densities of Known Sugar Solutions
Mass of Mass of Mass Mass of 1.000 mL Average Average
Sugar Solution Percent of Solution (g) Mass of Density of
Solution
(g) (g) Sugar Determination Solution Solution
(%) 1 2 3 (g) (g/mL)
A

E 0.000 g 0.00%

Graph:
1. Using Microsoft excel or similar program, input the data from Mass Percent Sugar (%) and Average
Density of Solution (g/mL). Input the data so that the Mass % Sugar is the independent variable (x-axis)
and the Average Density of each solution as the dependent variable (y-axis).
2. Be sure to label the axes and title the graph appropriately.
3. Perform a linear regression analysis. Include the equation of the trend line and correlation value.

density, g/mL = (_______________) (mass percent sugar, %) + ________________

slope
y-intercept

correlation value: _________________

 Instructor: Dr. Gautam Dhar

Data Table 2. Dilution of solution D and determination of densities

Vol of Calc. Average Average Expected %

Mass of 1.000 mL
Vol of Water Mass Mass of Density density Error
of Solution (g)
Soln soln Percent Solution of from
Determination
Sugar (g) Solution standard
1 2 3
(%) (g/mL) curve
2 ml 8 mL
D1
D
4 mL 6 mL
D2
D
5 mL 5 mL
D3
D
5 mL 5 mL
D4
D3
5 mL 5 mL
D5
D4

Post Lab Questions:

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

2. What are the dilution factors of D1, D2, D3, D4 and D5 in Part2 table 2?

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

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