Lab 2 – Volumetric Measuring and Making Solutions

Objectives
 To understand how liquid volumes are measured
 To investigate the accuracy of beakers, volumetric flasks, graduated cylinders, and pipettes
 To learn basic types of pipettes and their functions
 To understand the use and choice of pipettes for specific measurements
 To demonstrate repeatable, accurate pipette techniques
 To learn how to make solutions and perform serial dilutions

Introduction
Volumetric Measuring
Typical volumetric containers used in chemistry and biology labs include beakers, flasks, cylinders, and
pipettes. In most cases, these are made of glass and calibrated at a specific temperature. However, for very small
volume measurements, micropipettes, constructed out of plastic, with disposable tips, are used. A beaker is a simple
container for liquids, very commonly used in laboratories.
Beakers are generally cylindrical in shape, with a flat bottom. Beakers are available in a wide range of
sizes, from 10 mL up to several liters. They may be made of glass (typically Pyrex ®) or of plastic. Beakers may be
covered, perhaps by a watch glass, to prevent contamination or loss of the contents. Beakers are often graduated or
marked on the side with lines indicating the volume contained. For instance, a 250 mL beaker might be marked with
lines to indicate 50, 100, 150, 200, and 250 mL volumes. The accuracy of these marks can vary from one beaker to
another. A beaker is distinguished from a flask by having sides which are vertical rather than sloping. In the lab,
beakers are used more often than flasks.
Laboratory flasks come in several shapes and a wide range of sizes, but a common distinguishing aspect is
a wider vessel "body" and one (or sometimes more) narrower tubular sections at the top called necks which have an
opening at the top. Like beakers, laboratory flask sizes are specified by the volume they can hold. Laboratory flasks
have traditionally been made of glass but can also be made of plastic. Volumetric flasks come with a stopper or cap
for capping the opening at the top of the neck. Stoppers can be made of glass, plastic, or rubber. In general, flasks
can be used for making, holding, containing, collecting, or volumetrically measuring solutions.
A graduated cylinder is used to accurately measure out volumes of liquid chemicals. They are more
accurate and precise for this purpose than beakers or flasks. Often, the largest graduated cylinders are made of
polyethylene or other rigid plastic, making them lighter and less fragile than glass.
A pipette is a laboratory instrument used to transport an accurately measured volume of liquid. Pipettes are
commonly used in chemistry and biology. Pipettes come in several designs for various purposes with differing levels
of accuracy and precision, from single-piece flexible plastic transfer pipettes to more complex adjustable or
electronic pipettes. A pipette works by creating a vacuum above the liquid-holding chamber and selectively altering
this vacuum to draw up and dispense liquid. Pipettes that dispense between 1 and 1000 uL (1 mL) are termed
micropipettes, while macropipettes dispense greater volumes of liquid.

Exercise 1. The accuracy of 100 mL beakers

In this procedure we will determine the accuracy of typical lab beakers.
Procedure
1. Using the 200-gram top-loading balances in the lab, tare a dry 150 mL beaker. (Note: For the following
exercises if a 150 mL beaker is unavailable, use the next size up e.g. 200 or 250 mL beaker.)
2. Using a 100 mL beaker, as carefully as possible, measure 20 mL of de-ionized (DI) water
3. Pour this measured amount of water into the 150 mL beaker. Flick any remaining water in the 100 ml
beaker into the 150 mL beaker.
4. Record the weight of the water to the nearest 0.01 g in Table 1.
 5. Again, using the 100 mL beaker, measure 20 mL of water and add it to the 150 mL beaker.
6. Record the new weight in Table 1.
7. Repeat this procedure until you have 60 mL of water in the beaker on the balance (3x total).
8. Calculate the errors in Table 1.
Table 1. Weight of water volumes measured using a 100 mL glass beaker
Volume of water Calculated weight Measured weight of Error (Column 3 – Percentage Error
(mL) of water (g) water (g) Column 2) 100 x (Column 4 /
Column 2)

20 20 g
20 20 g
20 20 g

Exercise 2. The accuracy of 25 mL graduated cylinders

1. Using a 200-gram top-loading balance, tare a dry 150 mL beaker.
2. Using a 25 mL graduated cylinder, as carefully as possible, measure 20 mL of de-ionized (DI) water.
3. Pour this measured amount of water into the 150 mL beaker.
4. Record the weight of the water to the nearest 0.01 g in Table 2.
5. Tare the balance.
6. Again, using the 25 mL graduated cylinder, measure 20 mL of water and add it to the 150 mL beaker.
7. Record the new weight in Table 2.
8. Repeat this procedure until you have 60 mL of water in the beaker on the balance (3x total).
9. Calculate the error and percentage error in Table 2.

Table 2. Weight of water volumes measured using a 25 mL graduated cylinder

Volume of water Calculated weight Measured weight of Error (Column 3 – Percentage Error
(mL) of water (g) water (g) Column 2) 100 x (Column 4 /
Column 2)
20 20 g
20 20 g
20 20 g

Exercise 3. The accuracy of 100 mL graduated cylinders

Procedure
1. Using a 200-gram top-loading balance, tare a dry 150 mL beaker.
2. Using a 100 mL graduated cylinder, as carefully as possible, measure 20 mL of de-ionized (DI) water.
3. Pour this measured amount of water into the 150 mL beaker.
4. Record the weight of the water to the nearest 0.01 g in Table 3.
5. Tare the balance.
6. Again, using the 100 mL graduated cylinder, measure 20 mL of water and add it to the 150 mL beaker.
7. Record the new weight in Table 3.
8. Repeat this procedure until you have a total of 60 mL of water in the beaker on the balance (3x total).
9. Calculate the error and percentage error in Table 3.

Table 3. Weight of water volumes measured using a 100 mL graduated cylinder

 Volume of water Calculated weight Measured weight of Error (Column 3 – Percentage Error
(mL) of water (g) water (g) Column 2) 100 x (Column 4 /
Column 2)
20 20 g
20 20 g
20 20 g

Exercise 4. Accuracy of Glass/Disposable Pipette Measurements

Glass pipettes come in several sizes. For our lab today, we will use disposable 10 mL pipette tubes for this exercise.
In addition, these glass pipettes are clearly marked TD. TD stands for “to deliver”. TD pipettes, such as the one you
will use today, are meant to be blown out “to deliver” the entire volume. Thus, to deliver 10 mL, you will load the
pipette to 10 mL and then force all the liquid out.
The pipettes are wrapped individually and are sterile until opened. It is important to remember several things when
using pipettes:
 Do not open the wrapper until you are ready to use the pipette.
 Try not to make contact with any part of the pipette, especially the tip. If the tip touches you or anything
else, consider it contaminated and use a new pipette.
 Do not cross contaminate solutions by using the same pipette. Always use a sterile pipette when
transferring liquid to different solutions.
Procedure
1. Using a 200-gram top-loading balance, tare a dry 150 mL beaker.
2. Using a 10 mL disposable pipette tube and green pipette pump draw 10 mL of DI water into the pipette
tube.
3. Transfer this measured amount of water into the 150 mL beaker.
4. Record the weight of the water to the nearest 0.01 g in Table 4.
5. Tare the balance.
6. Again, using the 10 mL pipette, transfer 10 mL of DI water into the beaker.
7. Record the new weight in Table 5.
8. Repeat this procedure until you have 30 mL of water in the beaker on the balance.
9. Calculate the error and percentage error in Table 4.
Table 4. Weight of water volumes measured using a 10 mL glass pipette
Volume of water Calculated weight Measured weight of Error (Column 3 – Percentage Error
(mL) of water (g) water (g) Column 2) 100 x (Column 4 /
Column 2)
10
10
10

Exercise 5. Accuracy of Micropipette Measurements

A micropipette is used to measure and transfer small volumes of liquid. The volume of air space in a barrel is
adjusted by screwing the plunger farther in or out of the piston, and the volume is displayed on a digital readout.
Depressing the plunger displaces the specified volume of fluid from the tip. The withdrawn fluid is then expelled by
depressing the plunger again.
 The volume range for a micropipette can vary. The micropipettes we will use are adjustable within a
specified range. A small volume pipette, such as a P10 has a range from 0.5-10 µL. The P20 has a range from 1-20
µL, the P200 from 20-200 µL, and the P1000 from 100-1000 µL. The precision of these pipettes vary depending on
the quality of the pipette and the manufacturer as well as internal calibration.
Take the following precautions when using a micropipette.
 NEVER rotate the volume adjuster beyond the upper or lower range of the pipette
 NEVER invert or lay the micropipettor down with a filled tip; fluid can run back into the piston leading to
future contamination
 NEVER let the plunger snap back after withdrawing or expelling fluid; this could damage the piston
 NEVER immerse the barrel of the micropipettor in fluid.
 NEVER flame the tip of the micropipettor.
 NEVER reuse a tip that has been used to measure a different reagent.
Pipette tips come in individually packed, sterilized containers. It is essential that the correct tip is used for the
selected micropipette.
Procedure
1. Obtain a weigh boat.
2. Place the weigh boat on the fine balance and zero the balance.
3. Obtain a micropipette as specified in table 5.
4. Note the mechanism that allows you to set the volume you wish to deliver. Set the pipette to the indicated
volume in table 5.
5. Install the proper pipette tip on the micropipette body.
6. Press the button on the top of the pipette. Notice that there are two stops. The first stop is for withdrawing
(sucking up) your sample, while the second stop is for dispensing.
7. Place the tip about 1-3 mm into a beaker of DI water.
8. Press down to the first stop and then release the plunger slowly. The volume of water will be drawn into the
tip.
9. Place the tip to the weigh boat and press down to the second stop. The water will be transferred into the
weigh boat.
10. Record the weight of the water in Table 5. Repeat this procedure 3 times.
Table 5. Average error of micropipette measurements.
Micropipette Volume Expected Weight Actual Weight % error Average error
P20 (low volume) 2 µl 0.002 g
2 µl 0.002 g
2 µl 0.002 g 
P20 (high volume) 18 µl 0.018 g
18 µl 0.018 g
18 µl 0.018 g 
P200 (low volume) 30 µl 0.030 g
30 µl 0.030 g
30 µl 0.030 g 
P200 (high volume) 185 µl 0.185 g
185 µl 0.185 g
185 µl 0.185 g 
P1000 (low volume) 250 µl 0.250 g
 250 µl 0.250 g
250 µl 0.250 g 
P1000 (high volume) 850 µl 0.850 g
850 µl 0.850 g
850 µl 0.850 g 

Making Solutions
Purpose and Background
In this activity, you will learn how to calculate and make copper sulfate (CuSO4) solutions of differing
mass/volume concentrations. Any metric mass in any metric volume is possible, but the most common units of
mass/volume concentrations are as follows:
g/mL grams per milliliter
g/L grams per liter
mg/mL milligrams per milliliter
µg/mL micrograms per milliliter
µg/µL micrograms per microliter
ng/mL nanograms per milliliter
ng/µL nanograms per microliter
Although concentrations can be reported in any mass/volume units, these 7 mass/volume units are the most common
in biotechnology applications. The prefix “nano-“ means one-billionth. A nanogram is equal to 0.001 µg, and there
are 1000 ng in 1 µg. To determine how to prepare a certain volume of a solution at a certain mass volume
concentration, use the equation that follows.

Note:
- the units in the numerator & denominator should cancel if the equation is set up properly.
- you must make sure that the units used in the calculation can be cancelled, and you may need to convert units
within the metric system if necessary.
Example: You need to make 20 mL of a 40 mg/mL solution of glucose. How many grams of glucose will you need
to measure to make this solution?

Notice how the mL units cancel out during multiplication to leave the answer in mg to be weighed out. Since the
balances measure in grams, it was necessary to convert mg to g.
To make this solution, 0.8 g of glucose is weighed and put into a graduated 50 mL tube. Approximately 15 mL (~75
% of final volume) of solvent (deionized water or buffer) is added to the tube, and the contents are mixed using the
vortex. Then, additional solvent is added to reach a total volume of 20 mL. The reason that not all 20 mL of solvent
is added initially is that the solute may take up some space when it dissolves in the solvent. Thus, if we had added
all 20 mL to the solute at once, the final volume may have in fact been greater than 20 mL.

Exercise 6 – Making Solutions of Differing Mass/Volume Concentrations

Procedure
1. Label all tubes with the sample name and concentration, and your group’s initials.
2. Review the use of the balance and weigh boats before beginning.
3. Do the calculations necessary to prepare the solutions in Table 6 for tube numbers 1 through 3. Use the
Mass/Volume equation to determine the mass of CuSO4 to measure in order to give the correct
concentration at the volume desired for each sample.
4. Prepare the solutions in labeled 15 mL test tubes, using deionized (DI) water as the solvent. Use the vortex
to help dissolve the CuSO4. You may need to let the more concentrated samples sit in the warm water bath
for several minutes in order for the CuSO4 to dissolve fully.
5. Is the difference in concentration of the tubes obvious in one tube versus another? Compare your tubes’
colors and volumes to the standard “key” solutions prepared by the instructor. If any of the volumes or
colors are obviously wrong, try to identify where you may have made an error.
Table 6. CuSO4 mass/volume solution preparation
Tube Total Concentration
CuSO4 Solution Calculation of Mass Needed (g)
# Volume (mg/ml)
300 𝑚𝑔
1 5.0 ml of 300 mg/mL 5.0 mL 300 mg/mL 5.0 𝑚𝐿 × = 1500 𝑚𝑔 = 1.5 𝑔
𝑚𝐿
2 4.0 ml of 75 mg/mL

3 3.0 ml of 18.75 mg/mL

Exercise 7 – Making solutions of differing % mass/volume concentrations

In this activity, you will learn how to prepare solutions of specific % mass/volume concentrations. You
will prepare several % mass/volume solutions and use some of them as testing reagents. The underlying rule for %
mass/volume solutions is that a 1% solution contains 1 g of solute in a total volume of 100 mL.

Procedure:
You will do the calculations for and prepare the following solutions:
• 5 mL of 5% CuSO4 solution
• 5 mL of 3% CuSO4 solution
As an example, I will do the calculation for the preparation of 5 mL of 10% NaOH solution.
Step 1: Convert 10% to a decimal by dividing by 100: (10%) / 100 = 0.1 g/mL
Step 2:

Do all the necessary calculations. Then, record the amounts of the solutes needed in Table 7 to serve as a guide
when preparing your solutions. Prepare the solutions in Table 7 and compare to the key solutions prepared by the
instructor.
Table 7. Preparation of % Mass/Volume CuSO4 Solutions
Amount of Volume
Tube label Calculations
solution (g) (ml)

5.0 % CuSO4

3.0% CuSO4

Exercise 8 – Making solutions of differing molarity concentrations.

In this activity you will be introduced to the concentration measurement of molarity, and you will learn
how to do the necessary calculations to prepare molar solutions, or solutions of a specific molarity.
The concentration of many solutions is reported as moles/liter (mol/L or M; the M is spoken “molar”) or
some fraction of those units. This concentration measurement is called molarity. Molarity is sometimes a
challenging concept to understand. However, with your recently acquired solution preparation skills, you will see
that making molar solutions requires only one extra calculation. To understand how to make a solution of a given
molarity, you must know what a “mole” is. A mole of a compound is equal to 6.02 x 10^23 molecules, but that is
not really a very useful number. So, in biotech, it is easier to use this definition: The unit “1 mole” is the mass, in
grams, equal to the molecular weight (MW), also called “formula weight” (FW) of the substance. The FW can be
determined by using a Periodic Table and adding the atomic weights of the atoms in the molecule. An easy way,
though, is to just read the label of a chemical reagent bottle, which lists the “MW” or “FW.” The molecular weight
of NaCl is 58.5 atomic mass units (amu), since the Na atom weighs 23 amu, and a Cl atom weighs 35.5 amu.
Molarity concentrations are reported as the number of moles per liter (mol/L or M). The unit “M” is used as a base
unit, just like meters, liters, or grams, so you can have “millimolar” (mmol/L, or mM) and “micromolar” (µmol/L, or
µM) concentrations, both of which are used frequently in biotech labs. If you wanted a 1 M NaCl solution, you
would measure out 1 mole of NaCl (58.5 g) and dissolve it in water to a total volume of 1 L. This gives you 1 mole
of NaCl per liter of solution, or 1 M NaCl. A liter of solution is a large volume for most purposes in biotechnology
labs. Instead, mL or µL quantities are usually used. To determine how to mix up a smaller volume of solution,
follow the equation below.

A few notes about calculations for molar solutions:

- As before, the units in the numerator & denominator should cancel if the equation is set up properly, and you
should be left with the units desired.
- You must make sure that the units used in the calculation can be cancelled, and you may need to convert units
within the metric system if necessary. Often, you are given a volume that is not in L (might be in mL or µL) or a
molarity that is in millimolar (mM) or micromolar (µM). If this is the case, you will need to convert the volume to
L and the molarity to mol/L, and then plug these numbers into the molarity concentration equation above.
Procedure:
You will do the calculations for and prepare the solutions in Table 8 below. The molecular weight (MW)
for CuSO4 (actually copper sulfate pentahydrate, or CuSO4•H2O) is 250 g/mol. The first calculation has been done
for you as an example.
1. Once you have done the calculations, label 3 tubes 1 through 3, and weigh out the needed amounts of
CuSO4 to make your solutions. Feel free to check your group’s calculations with the instructor before you
begin.
2. Prepare the solutions in labeled 15 mL test tubes, using deionized water as the solvent. Add the solute first,
then add some water to dissolve, using the vortex to mix. Then add more water to bring the final volume
up to 5 mL.
3. Are the differences in concentration of your tubes obvious? Compare the colors and volumes of your
samples to the “key” tubes made by the instructor. If any of the volumes or colors are obviously wrong, try
to identify where you may have made an error. Then dump them out in the proper disposal and remake
them.

Table 8. CuSO4 Molarity Solution Preparation

Tube CuSO4 Total Mass of CuSO4

Calculation of mass needed (g)
# molarity (M) Volume to use (g)
1 𝑚𝑜𝑙 250 𝑔
1 1.0 M 5 mL 1.25 g (0.005 𝐿) ( )( ) = 1.25 𝑔
𝐿 𝑚𝑜𝑙

2 0.5 M 5 mL

3 0.1 M 5 mL

Exercise 9 – Making Dilutions of Concentrated Solutions

In this activity you will learn how to make diluted solutions from a more concentrated stock solution. You will learn
how to use the dilutions equation and prepare several different dilutions from a concentrated CuSO4 stock solution.
Very often in biotech labs, you will dilute a concentrated “stock solution” to use as a “working solution.” This is
done to save time, as well as space in the lab. Also, it is often more accurate to prepare a concentrated stock solution
because it involves weighing larger masses of chemicals (therefore less error in measurements). The working
solution concentration is represented as 1X, and the concentrated stock solution is some multiple of this (5X, 10X,
etc.). For example, a buffer solution may be used at a concentration of 0.01 M Tris. This is the working
concentration of the Tris solution (1X). But because of shipping costs, a small amount of the buffer is shipped as a
10X solution with a concentration of 0.1 M Tris. When the technician is ready to use the buffer, it is diluted with
deionized water down to 1X (0.01 M Tris). To figure out how to dilute something from a concentrated solution, we
use a simple ratio equation shown here:
The C1V1 = C2V2 equation may be used with any concentration units (i.e. mass/volume, %, or molar) as long as the
units are the same on each side of the equation, for canceling purposes. Thus, you can’t have mL for V1 and L for
V2—you must change one to match the other. Once you have the equation set up and the numbers plugged in, then
solve for the variable you want to find. Here is an example. Let’s say you want to make 200 mL of 0.15 M NaCl
from a stock that is 3 M.
C1 = 3 M
V1 = the amount of stock to use (this is what you’re solving for)
C2 = 0.15 M
V2 = 200 mL
(0.15 𝑀) (200 𝑚𝐿)
(3 𝑀 ) (𝑉1) = (0.15 𝑀) (200 𝑚𝐿) 𝑉1 = = 10 𝑚𝐿
3𝑀

Therefore, to make the solution, you would measure out 10 mL of the 3 M NaCl stock solution and add 190 mL of
deionized water to dilute to the final concentration of 0.15 M.
When several dilutions must be made, and each is proportionally the same dilution as the one before, it is called a
serial dilution (see figure below). Doing a serial dilution makes sense for many experiments when many samples of
varying concentrations are needed. A serial dilution is also useful for preparing very dilute solutions that are hard to
make from scratch, because the solute masses can be too small to measure on a balance.
Procedure
1. Complete the following dilution calculations of a 300X stock CuSO4 solution in the tables below. The first
calculation has been done for you as an example.
Table 9.1. Dilutions of the 300X stock of CuSO4
Concen- Volume of Volume of
Volume Calculation of volume of stock needed
tration stock to use H2O to add
(300𝑋)(𝑉1) = (150𝑋)(5𝑚𝐿)
150 X 5 mL 150𝑋)(5𝑚𝐿 2.5 mL 2.5 mL
𝑉1 = ( ) = 2.5 𝑚𝐿
300𝑋

30 X 7 mL

15 X 4 mL

Table 9.2. Serial dilution (1:10) of the 300X stock CuSO4

Concen- Volume of V1 Volume of
Volume Calculation of volume of stock needed
tration to use H2O to add

30
3 mL
mg/mL

3
3 mL
mg/mL

0.3
3 mL
mg/mL