LAB 1: INTRODUCTION TO BIOMOLECULAR SCIENCE LAB AND LAB SAFETY

A. GENERAL OBJECTIVES OF THE COURSE

i. Develop the basic laboratory techniques of a biological (biochemistry, microbiology,

molecular biology etc.) lab
ii. Supplement the lecture portion of the course, which deals predominantly with biological
techniques
iii. Develop critical thinking skills in students
iv. Encourage teamwork and accountability among students
v. Practice accuracy in calculations and in writing scientifically
vi. Develop multitasking skills
vii. Encourage students to take charge of their learning
viii. Learn the responsibilities associated with working environment

B. SPECIFIC OBJECTIVES OF THIS UNIT:

This is to help new students:

i. to familiarize with biomolecular laboratories and their environment

ii. to comprehend with lab regulations and lab safety

C. DOCUMENTATION:

Documentation in a lab reports is an essential skill for any biologists. The Food and Drug Administration's
(FDA) handbook states, "if it isn't written down, it wasn't done." Documentation details vary from lab to
lab but it is always done for one or all of the following reasons:

i. to record what an individual has done and observed

ii. to establish ownership for patent purposes and other legal uses
iii. to establish criteria used to evaluate a finished product or the process to make it
iv. to trace the manufacture of a product
v. to create a contract between a company and consumers and/or between a company and
regulatory agencies
vi. to prove that a procedure was done correctly
vii. to adhere to, evaluate, and develop standard operating procedures (SOP)

Even good lab work is worthless without documentation, and careful documentation can turn an
erroneous result or a failed procedure into a valuable learning experience by providing essential details
needed for trouble-shooting.

Instruction of lab report write up is in page iv.

D. LABELING
Labeling is very important in any lab. It is critical that you label every tube, bottle, flask, cuvette or other
container you use in the lab, whatever its contents. This is especially important for any hazardous
chemicals or pathogens, but be just as thorough with something as harmless as salt water. You must label
all containers with:

i. the identity of the contents and its concentration

ii. your initials or name of your group
iii. the date (and time, if applicable)
iv. your class (for example, BMS481)

If the container is destined to be kept on hand for more than a day, never use a number or letter
abbreviation; this will inevitably be found by someone else to whom your symbols mean nothing. Only
use the abbreviated labels if you will be disposing of the contents the same day. For example, if you are
doing column chromatography, you need only label the collection tubes with numbers in the order that
they come off the column. However, if your instructor wants to keep one of your fractions as a control for
the next semester‘s class, it is imperative that you label the tube with all the information above.

E. PROPER HANDLING & STORAGE OF CHEMICALS AND REAGENTS

There is no single simple formula for working safely in the laboratory, since each lab facility and each
experiment presents unique challenges. We will be addressing safety issues with each experiment that
we do in this course and give you some specific guidelines for safety throughout the semester.

1. MSDS (Material Safety Data Sheets)

While each chemical that you use will have its own unique properties, there are some common practices
that will aid you in treating them all with the level of respect that they are due. For example, labeling each
chemical is required under the law and should be thorough enough so that even a person who does not
work in the lab can identify any chemical. Also, every chemical in the laboratory should have a Material
Safety Data Sheet (MSDS) on file and readily available. The MSDS is a legally required technical document,
provided by chemical suppliers, that describes the specific properties of a chemical. Besides the MSDS on
file in the lab, several web sites offer MSDS databases. They are all broken down to the same 8 secti ons:

i. Chemical identity. The manufacturer‘s contact information is here, along with contacts for emergency
situations.

ii. Hazard ingredients/identity. Some reagents have multiple components, and many single-component
chemicals have alternative names. These are all listed here. Concentration limits for airborne exposure to
a chemical are listed here. Although these indices of toxicity are mainly of concern for production workers
in factories, they are also useful for evaluation of short-term exposures. The TLV (threshold limit value) is
the maximum airborne concentration of a substance to which workers can be repeatedly exposed without
adverse effects. The units used are usually parts per million (ppm) or mg/m3.

iii. Physical chemical characteristics. This list of physical properties tells you whether the chemical is
solid or liquid and how volatile it is.
iv. Fire and explosion hazard data. This is of particular interest in cases where fire-fighting methods must
be selected.

v. Reactivity data. This information is essential in determining the proper handling and storage of
chemicals. By knowing the reactivity patterns of a chemical, you know what substances or conditions from
which you must isolate the chemical. For example, acids and bases react with each other rapidly, giving
off large amounts of heat, so should not be stored next to each other. Others react with water and should
be stored in sealed containers with desiccants.

vi. Health hazards. The best source of specific toxicology data is given here, such as symptoms of acute
damage from exposure and some recommended emergency procedures. If a chemical has been tested
for carcinogenicity (cancer-causing potential) that information is listed here. In addition, levels at which a
chemical has been found to be lethal (called the LD50 for lethal dose for 50% of test animals) is listed
here. Since the LD50 is dependent on which type of animal it was tested on, as well as how the animal
was exposed to the chemical, this information always requires these specifics. For example, the lethal
dose for chemicals is much lower if injected than it is if ingested. The most common index reported is the
LD50 for a rat in mg of chemical per kg of animal, administered orally (ingestion). For volatile chemicals,
the toxicity of breathing it is measured as the LC50 (lethal concentration in air for half of the test animals),
measured in ppm; in all cases, the lower the number for the LD50, the more toxic the chemical.

vii. Precautions for safe handling and use. This describes how to deal with spills.

viii. Control measures. Specific recommendations for personal protective equipment (PPE) are given here.
2. NFPA Ratings (National Fire Protection Association)

Another quick assessment of a chemical‘s health hazards that is usually available on its container is a rating
by the National Fire Protection Association (NFPA). A colour-coded diamond shape lists numbers rating a
hazard as:

https://www.safetysign.com/help/h89/nfpa-hazard-rating

3. General Safety Precautions in Handling Hazardous Chemicals in the Lab

There are generally four routes to exposure to hazardous chemicals that you should keep in mind while
handling them:

i. Inhalation: avoid by the use of fume hoods and masks

ii. Skin & eye contact: avoid by the use of lab coats, gloves, and goggles

iii. Ingestion: avoid eating or drinking in the lab or leaving the lab without removing gloves
and washing hands

iv. Injection: dispose of broken glass and needles properly

Because chemicals pose so many different kinds of hazards, there are no simple rules of thumb for safe
handling of them all except for some common sense measures:

 Treat all chemicals as if they were hazardous until you learn otherwise
 Label all containers with contents, including concentrations and date that they were
transferred
 If a hazardous material is contained, label it with a warning
 Think through your experiment BEFORE doing it, making sure that you wil l not be
combining incompatible chemicals
 Clean your bench top before and after use
 Wash hands often and ALWAYS before leaving the lab
 Take off lab coats and gloves before leaving the lab
 Always remove gloves before touching phones, doorknobs, light switches, etc.
 Ensure proper waste disposal and labeling .

Here are some specific tips for handling the different types of hazardous chemicals:

Flammables: Do NOT heat these reagents unnecessarily, and never in the presence of a flame or source
of a spark. In general, only open containers in fume hoods. When storing more than 10 gallons of
flammable liquids, a special explosion proof storage cabinet is required.

Corrosives: Wear personal protective equipment (PPE) such as lab coats, goggles and gloves, and always
add strong acids or bases to water when making solutions. Neutralize slowly to avoid rapid generation of
heat and gases. Strong acids and bases should never be stored together.

Reactive chemicals: Wear PPE such as lab coats, goggles and gloves, and know the reactive properties of
the chemical. Always store oxidizing chemicals away from flammable materials.

Toxic chemicals: Wear personal protective equipment (PPE) such as lab coats, goggles and gloves, and
know the toxic properties of the chemical. When working with a dry powder, wear a mask to avoid
breathing the dust. Be aware of the waste disposal procedures for unused reagents and materials that
come in contact with the chemical

Some of the most common hazardous chemicals that you will encounter in the biology lab:

Carcinogens – formaldehyde Mutagens – ethidium bromide

Neurotoxins – acrylamide Teratogens – formamide
Nephrotoxins – acetonitrile Hepatotoxins – chloroform
Corrosives – phenol, strong acids & bases
F. BIOLOGICAL SAFETY

You will be working with live organisms in many biology/ microbiology labs, so it is important to be able
to assess any biological hazards that they may pose and to treat them accordingly. In general, a live
organism is considered a biological hazard if its release into the environment could have an effect on the
health of the environment in general or humans in particular. This includes known pathogens to humans,
plants, or animals, as well as benign organisms containing recombinant DNA that could render the
recombinant host dangerous. In fact, the recombinant DNA itself should be treated as a biohazard, since
it is usually inserted into a vector that could transform organisms in the environment if released. Similarly,
tissue cultures of human or animal cells should be treated as a biohazard: while they would not survive if
released into the environment, most immortalized tissue culture cells contain recombinant DNA.

The routes of exposure to infectious agents are the same as those of hazardous chemicals: inhalation,
contact with eyes and skin, ingestion, and injection. The same general precautions should be taken in
handling biological hazards as the guidelines above for handling chemical hazards, especially toxic ones.
Here are some additional general practices to maximize biological safety:
i. Limit access to the lab at the discretion of the lab director,

ii. Use PPE at all times, and keep all PPE inside the lab.

iii. Avoid touching your face with your hands or gloves.

iv. Keep personal items such as coats and book bags out of the lab or in a designated work area.

v. No mouth pipetting; use mechanical pipetting devices.

vi. Minimize splashes and aerosol production.

vii. Disinfect work surfaces to decontaminate after a spill and after each work session.

viii. Disinfect or decontaminate glassware before washing.

ix. Decontaminate all regulated waste before disposal by an approved method, usually by
autoclaving.

x. Use a laminar flow biological safety cabinet when available.

More than 70% of recorded laboratory-acquired infections are due to inhalation of infectious particles, so
special precautions should be taken to avoid producing aerosols when working with pathogens. While
performing activities that mechanically disturb a liquid or powder, students should make the following
adjustments.

 Shaking or mixing liquids: mix only in closed containers

 Pouring liquids: pour liquids slowly
 Pipetting liquids: use only cotton plugged pipettes
 Removing a cap from a tube: point tubes away when opening
 Breaking cells by sonication: in closed containers
 Removing a stopper or cotton plug: remove slowly
 Probing a culture with a hot loop: cool loop first
Disinfectants such as bleach and ethanol are used extensively to decontaminate glassware and work
areas, and it is important to realize that the effectiveness of disinfectants depends on the type of living
microorganisms. Biological agents (bacteria, viruses, fungi, prions) are classified into four Hazard Groups.
Classification is based on whether:

 the agent is pathogenic to humans

 the agent is a hazard to employees
 the agent is transmissible to the community
 there is effective prophylaxis or treatment available

The hazard groups are defined as:

Hazard Group 1 Hazard Group 2 Hazard Group 3 Hazard Group 4

Unlikely to cause Can cause human Can cause severe Causes severe human
disease disease human disease disease

May be a hazard to May be a serious Serious hazard to

employees hazard to employees employees

Unlikely to spread to May spread to the Likely to spread to the

the community community community

Usually effective Usually effective Usually no effective

prophylaxis or prophylaxis or prophylaxis or
treatment available treatment available treatment available

G. LABORATORY WASTE DISPOSAL

Sharps Yellow sharps containers

Contaminated sharps

Broken Glass
(chemically clean,
including domestic
glass)

Biohazardous Yellow bags or containers

labelled with black biohazard
symbol.
 Radioactive Red bags or container labelled
with radioactive symbol.

Cytotoxic Container labelled with

cytotoxic symbol.

Old or unlabeled chemicals in These bottles can be taken

supplier bottles directly to waste pick up,
ensuring that a waste
tracking log is completed.

H. LAB TOUR

Students will be brought around to other labs and be introduced to some common equipment used in
biomolecular labs.
EXERCISES

Student’s Name:

Date:

1. Before each practical lab begins, students should:

i.

ii.

iii.

2. You have accidentally broken a test tube and spilled a chemical on a table. Explain what should
you do to clear it up.

3. Long hair, hanging jewelry, and loose clothing can be dangerous in a lab. If you possess one or
more of these, what should you do to ensure your safety in the lab?

4. List three important points to remember while you are using heat in a lab.

i.

ii.

iii.
LAB 2: HOW TO USE A MICROPIPETTE

Objective:

To learn how to measure a small volume of liquid samples correctly using micropipettes.

The micropipette is used to transfer small amounts (< 1 ml) of liquids. The scales on micropipe ttes are in
microliters (1000 µl = 1 ml). They are varies in size and by the number on the round button on the plunger.
The value is the maximum volume in microliters that can be transferred with that size pipette. They are
used in conjunction with disposable sterile plastic tips. The following is an illustration of a micropipette:

Figure 1: The micropipette

Using a micropipette:

A. NEVER exceed the upper or lower limits of these pipettes.

The limits are:

P10: 1.0 - 10.0 µL
P20: 2.0 - 20.0 µL
P200: 20 - 200 µL
P1000: 200 - 1000 µL

Warning: to exceed these values will put the

pipet out of calibration
The examples below show how to read the volume on the micropipette:

B. How to choose a right size of micropipette.

RULE OF THUMB: Always select the SMALLEST size pipette that will handle the volume you wish to move
to achieve the greatest accuracy. Accuracy decreases as you use unnecessarily large pipette for small
volumes.

C. Micropipette tips. Use blue tips for P1000 pipettes and clear tips for P200 and all smaller sizes.
Note:
i. Use filter tips when performing PCR or working with RNA.
ii. Close the tip box to maintain sterility.
iii. Do not allow the pipette tip to touch any objects (including your gloves, clothes, hair, skin, bench).

D. Load the samples.

i. The plunger will stop at two different positions when it is pressed. Push the plunger down slowly to the
point of first resistance: this is the load volume.
ii. While holding the plunger at the load volume set point, put the tip into the solution so that it is
immersed just enough to cover the end (3-4 mm), not as deep as possible.

iii. Slowly release the plunger to draw up the liquid making sure to keep the tip immersed.
NOTE: If the solution you are pipetting is viscous, allow the pipet tip to fill to final volume before removing
it from solution to avoid the presence of bubbles in the tip, which will result in an inaccurate volume.
iv. Visually inspect the load to make sure it is correct - there should be no air space in the distal end tip.
E. Deliver the samples.
i. Place the tip into the receiving vessel.
ii. Press the plunger to the first stop point. Wait a second
iii. Continue to press the plunger all the way to the bottom (second stop point)- this expels all the liquid.
Note: This second stopping point is used for the complete discharging of solutions from the tip. You should
not reach this second stop when drawing liquid into the pipette, only when expelling the last drop.
iv. THEN, WITHOUT RELEASING THE PLUNGER, withdraw the tip.

F. Discharge the tip.

While holding the tip over an appropriate waste container, press the discharge slider on the back of the
grip.

G. Small Volumes Technique

With small volumes, especially the 1-10µl range, you must keep track of the droplets you pipette.
i. Carefully expel the liquid droplet on the side wall of the tube so that you can see it, drawing the tip
away/out carefully BEFORE releasing the plunger.
ii. If adding to a larger volume, flush the tip with the solvent liquid after expelling the droplet to make sure
you get all the delivery liquid. With small volumes you'll usually need to centrifuge and then vortex the
tube to get a good mixing of the reagents

H. Reverse Pipetting Technique

In the reverse technique, a larger volume than the desired volume of solution is drawed into the tip. An
exact (set) amount is delivered, but liquid remains in the tip. This is infrequently used, but may be
preferred when pipetting very viscous or foamy liquids. Pre-rinsing is NOT necessary.

Note:
a. Never point a pipette up. This may cause liquid to run down into the pipette destroying it.
b. When withdrawing liquids with the pipette, always release the plunger slowly. This prevents liquid
from rushing into the end of the pipette and clogging it up. This is especially important with large
volume pipettes (200-1000µl).
c. Always use a new tip for each different liquid.
d. Use the correct pipette for the volume that is to be dispensed. Never use the 200-1000µl pipette to
dispense volumes below 200µl. Going below or above the range of the micropipette may damage the
instrument.
Exercise:
1. Micropipette Calibration
Ref: Seidman, L. S. & C. J. Moore. Basic Laboratory Methods for Biotechnology. Prentice Hall. (1999)

Calibration of micropipettes is to determine the difference between the dispensed volume and the
selected volume. The correct functioning of a micropipette is determined by checking the accuracy and
precision of the volumes it delivers. Adjustment means altering the pipette so that the dispensed volume
is within certain specifications.

The weight of water samples delivered by the instrument is converted to a volume based on th e density
of water. Check the calibration of your micropipettes by using the fact that 1 ml of deionized (or distilled)
water has a mass of 1 g. Calibrations should be checked at two volumes: one at the maximum setting and
another at half the maximum setting—so this means two sets of measurements for each micropipette.

Micropipette Calibration Procedures:

Micropipettes
Distilled water
Analytical balance
Weighing boats

i. Work in pairs and select an adjustable micropipette. Each member of the group will perform the
pipetting for one of the two settings described above (max and ½ max volume.)
ii. The analytical balance used to check the micropipette must be well -maintained and properly
calibrated. It must also be in a draft-free, vibration-free environment.
iii. Document all relevant information including date/time, micropipette serial number, pipette volume
range, temperature, and name of the person performing the evaluation. **Note: If the water and
micropipette are not stored at the same temperature, allow at least 2 hours for temperature
equilibration.
iv. Set the micropipette volume (maximum volume for first setting, then 50%).
v. Tare (zero) the balance to a weighing boat or beaker.
vi. Measure and deliver a volume of water into the weighing boat.
vii. Immediately record the weight measurement.
viii. Tare (zero) the balance between each measurement.
ix. Working quickly, repeat steps v-vii for a total of 10 weight measurements.
Note: Avoid prolonging this process, as you want to avoid evaporative losses.
x. Repeat the 10 measurements at the second volume setting of your micropipette.
xi. For each volume setting, calculate the mean delivered weight ( x̅ ) of the water for the 10
measurements.
xii. Calculate the Mean Volume Delivered (Vt) at the mean water temperature of about 25.0 OC using the
following formula. Please note, “Z” is the conversion factor (in µL/mg) for the mean water
temperature. Let say “Z” for this lab is 1.0041.

Vt= (x̅ ) * Z
 Table: Values for Z factor as a function of temperature and pressure for distilled water

xiii. Determine the % inaccuracy or the “inaccuracy” by calculating the percent error of the micropipette
at each volume setting as follows:

% error = Vt - expected volume x 100

Expected volume

xiv. Log your result on the report form. Confirm that the pipette setting and the mean volume
measurements are in the same units. If not correct, using 1000 µL/ 1mL.

Note: % error can be lower (a negative % error) or higher (a positive % error) than the intended volume
setting.

xv. Determine the precision (repeatability) of the micropipette at each volume setting by first calculating
the standard deviation (SD) as follows, using the original 10 observations in grams (g):

Xi = each weight measurement

x̅ =mean delivered weight measurement
n is the number of measurements (10)

Alternatively, you can calculate SD using Excel and the original 10 observations in grams (g). Log your
result on the report form.

xvi. Determine the imprecision by calculating the coefficient of variation (CV%). Log your result on the
report form. (You may use scientific calculator functions to determine SD and CV)
CV = SD x 100%

xvii.Check the manufacturer’s specifications, below, for the micropipette to see if your inaccuracy (%error)
and imprecision (CV) measurements are within the manufacturer’s inaccuracy and precision limits.
Document if “out” or within manufacturer’s limits. See full report form example below.
 EXAMPLE

Micropipette Calibration Report

Room Temperature:____21.7 °C___

Pipette Serial number:___X12345N_
Pipette Volume Range:__20-200μL___

Setting 1:

Name of person calibrating:_Rohana Mat Nor_

Calibration Date/Time:____24/1/2018 18:00 __

SETTING 1 volume:
100μL

RUN Weight Measurement Mean Weight Mean Volume

(in g) Delivered (𝑊) Delivered (Vt )
1 0.0985 RAW in g RAW in mg
2 0.0987
0.09841 g 98.41 mg
3 0.0985
4 0.0982
5 0.0984 Converted to mg using Converted to µL using the
(1000mg/1g) “Z” conversion factor
6 0.0984
7 0.0981 0.09841g (1000mg/1g)= 98.41mg (1.0033μL/mg)=
8 0.0983
9 0.0982 98.41 mg 98.73 μl
10 0.0988
Total 0.9841
SETTING 1 volume:
100μl
% Error Standard Coefficient of Manufacturer’s Manufacturer’s
(inaccuracy) Deviation Variation (CV%) Inaccuracy limits Imprecision limits
(SD) (within range/out of (within range/out of
range) range)
(98.73μl-100μl) = 0.000223*100 =
100μl 0.09841g Out of range Within range
0000223
-0.0127*100 = 0.22622% (-1.27% vs +/- 1%) (0.23% vs +/- 0.3%)

-1.27%
 Micropipette Calibration Report

Room Temperature:___________________
Pipette Serial number: _________________
Pipette Volume Range: ________________

Setting 1:

Name of person calibrating:_______________

Calibration Date/Time:_________________

SETTING 1 volume:

RUN Weight Measurement Mean Weight Mean Volume

(in g) Delivered (𝑋) Delivered (Vt )
1 RAW in g RAW in mg
2
3
4
5 Converted to mg using Converted to µL using the
(1000mg/1g) “Z” conversion factor
6
7
8
9
10
Total
SETTING 1 volume:

% Error Standard Coefficient of Manufacturer’s Manufacturer’s

(inaccuracy) Deviation Variation (CV%) Inaccuracy limits Imprecision limits
(SD) (within range/out of (within range/out of
range) range)
2. Reverse Pipetting Technique

1000µL pipette
Glycerol: 80%, 40%
Eppendorf tubes

Procedure:
i. Select 1.0ml volume on the 1000µl pipette.
ii. Measure 1.0ml of distilled water and dispense it in an Eppendorf tube. Mark the volume with a
permanent marker pen.
iii. Repeat the same as in ii for one more tube.
iv. Remove the water and dry the tubes using a clean tissue paper. Now you should have 2 marked
Eppendorf tubes.
v. With a fresh tip, measure 1.0ml of 40% glycerol by pressing the plunger to the second stop.
vi. Dip the tip 2-5 mm below the meniscus of the liquid.
vii. Remove the tip from the liquid.
viii. Wipe off excess liquid from the outside of the tip.
ix. Place the tip of the pipette against the wall of the transfer container (Eppendorf tube)
x. Press the plunger just to the first stop.
xi. Keep holding the plunger in this first stop position and remove the tip from the tube.
xii. Return the remaining liquid to the original vessel by pressing the plunger to the second stop or
dispose of the remaining liquid in a Biohazard/sharps container by pressing the ejection button.
xiii. Repeat steps v to xii using 80% glycerol.
xiv. Observe the volume of the measured glycerol and compare them with the marked volume of water
on the tube wall.
LAB 3: BUFFERS AND SOLUTION

OBJECTIVES:

The unit is for students to master in the concentration calculation/measurement of buffers and solution.

CONCENTRATION CONCEPTS

Concentration refers to the relative amount of a substance in a given volume of solution; but in some
cases alternative expressions are used. Different ways of expressing concentration are listed below,
which are mostly used in biochemical calculations.

1. Percent solution

The concentration of a solution frequently can be denoted in terms of percentage, that is in parts per
hundred. Some of these percent solutions are “true” percent solutions, referring to so many parts out of
100 identical parts; others are “hybrid” percent solutions, referring to so many parts out of 100 differing
parts. The most common three methods for percentage determination are defined as follows:

i. weight percent (%w/w) = weight of solute x 100

weight of solution

ii. volume percent (%v/v) = volume of solute x 100

volume of solution

iii. weight-volume percent (% w/v) = weight of solute (g) x 100

volume of solution (mL)

A % (w/w) solution is a “true” percent solution in which a specific number of grams of solute contained
in 100 g of solution. Thus a 5% (w/w) NaCl solution is the one containing 5.0 g of NaCl in 100 g of
solution.

A % (v/v) solution is likewise a “true” percent solution in which a specified number of mL liquid are
contained in 100 mL of solution. Thus a 5% (v/v) solution of ethanol in water is the one containing 5.0 mL
of ethanol in 100 mL of solution.

On the other hand a 5% (w/v) NaCl solution is the one containing 5.0 g of NaCl in 100 mL of solution, which
may be indicated as a 5% NaCl solution. Strictly speaking, this is a “hybrid”, not a “true” percent solution
since one refers to 5.0 gm of NaCl in 100 mL of solution.

Weight/Volume percent is often used for routine laboratory solutions where exact concentration is not
too important.
2. Concentration

Conentration expression for very dilute solutions: When we deal with very dilute solutions, as is often the
case in biological sciences and medicine, we need other expressions for concentration. It is not very
convenient to say that drinking water should contain no more than 0.000195 percent by weight of sodium
fluoride (the additive used to prevent tooth decay).

Milligram percent (mg%) is the number of milligrams present in 100 mL of solution.

mg% = mg of solute
100 mL of solution

Parts per million (ppm) is frequently encountered in pollution-control work and refers to the number of
parts of solute per million parts of solution.

There are two ways of calculating and interpreting the ppm expression, depending on whether the solvent
is a liquid or gas. In liquids, ppm means the number of milligrams of solute per liter of solution. For
example 1.0 L of solution that contains 5 ppm of CdCl2 means it contains 5 mg of CdCl2.

In air pollution control, the term ppm is based on volume measurements rather than weight
measurements, and may be expressed as microliters (μL) of solute (1.0 μL = 0.001 mL or 1.0 x 10 -6) per
liter of air. For example, 5 ppm of SO2 in the air means 5 μL of SO2 per liter of air.

ppm (gases) = μL of solute

L of air

Example: Consider that 0.8 g of solid NaOH used for the preparation of 500 mL solution. Express the
concentration of this solution in terms of g/L, %w/v, mg %. (MW: NaOH= 40)

The solution contains 0.8 g/ 500 mL, or 1.6 g/L.

%(w/v) => 1.6 g/L = 0.16 g/100 mL = 0.16 %

Mg % => 0.16 g/100 mL = 160 mg/100 mL = 160 mg%

Example: How many grams of NaCl are necessary to prepare 500 mL of a 10% (w/v) solution?

%(w/v) = g of solute/100 mL of solution

10% (w/v) = 10 g NaCl/100 mL.

0.1 g NaCl/mL; so for 500 mL of a solution: 0.1 x 500 = 50 g NaCl.

3. Molarity

The molarity, M, of a solution defines the number of gram molecular weight (or moles) of a species in 1 L
of solution or the number of millimolecular weights in 1 mL of solution. A 0.2 M solution of BaCl2 contains
0.2 mole of the salt barium chloride in a liter of solution. Molarity is a very convenient unit for laboratory
work since aqueous solutions of known molarity can be prepared by weighing out small amounts of solute
and measuring the volume of solution in calibrated containers.

Molar concentrations are usually given in square brackets, for example, [H+] = molarity of H+ ion. To
calculate M, we need to know the weight of dissolved solute and its molecular weight (MW) such as:

The following are the fundamental relationships:

volume (L) x molarity (M) = number of moles

volume (mL) x molarity (M) = number of millimoles
volume (μL) x molarity (M) = number of micromoles
Dilute solutions are often expressed in terms of millimolarity, micromolarity and so on, where;

Example: How many grams of solid NaOH are required to prepare 500 mL of 0.04 M solution? Express
the concentration of this solution in terms of %(w/v). (MWNaOH= 40).

M = number of moles of NaOH required

volume (L)

Therefore;

liters x M = number of moles of NaOH required

0.5 x 0.04 = 0.02 mole.

Number of moles = Weight MW x mass in grams = 40 x 0.02 = 0.8g

Therefore, weigh out 0.8 g NaOH, dissolve in water and dilute to 500 mL. The solution contains 0.8 g /500
mL or 1.6 g /L NaOH.

%(w/v) = g per 100 mL. 1.6 g/L = 0.16 g/100 mL = 0.16%.

Example: How many moles of NaCl are present in 150 mL of a 1.5 M solution?

number of moles = M x V

where V = 150 ml = 0.15 L and M = 1.5 moles/L

1000 ml
number of moles = 1.5 moles/L x 0.150 L = 0.225 mole NaCl;

MW NaCl= 58.5 which is also equal to 0.225 x 58.5 = 13.1125 g NaCl.

4. Normality

Normality refers to the concentration of a solution expressed in terms of the number of equivalent-
weights (gram-equivalent weights, equivalents e.g.) of solute in 1 L of solution. This is same as the number
of milliequivalent weights (milligram-equivalent weights, milli equivalents “meq”) in one mL of solution.
Normality is indicated by the symbol N and is used, in calculations involving acid-base (neutralization) and
oxidation-reduction (redox) reactions.

One equivalent weight (EW) of an acid or base is the weight that contain 1g-atom (1 mole) of replaceable
hydrogen, or 1 g-ion (1 mole of replaceable hydroxyl). The EW of a compound involved in an oxidation-
reduction is the weight that provides or accepts 1 Faraday (1 mole) of electrons. In general;

As an example the equivalent weight of HCl is identical to its molecular (formula) weight and a 1.0 M HCl
solution is also a 1.0 N solution, but the equivalent weight of H2SO4 is equal to one half of its molecular
(formula) weight. Hence, a 1.0 M H2SO4 solution is a 2.0 N H2SO4 solution. Likewise, 1.0 M NaOH is also
1.0 N NaOH but 1.0 M Ba(OH)2 is 2.0 N Ba(OH)2. As a result, it can be stated that the molarity and normality
are related by N = nM

Example: What is the normality of 1.0 L of an aqueous solution of 98.0 g of H2SO4 (MW of H2SO4 = 98
g/mole)?
5. Ionic Strength

Ionic strength measures the concentration of charges in solution and formulated as:

Only the net charge on an ion is used in calculating ionic strength. Thus unionized compounds (e.g.
unionized acetic acid) or species carrying an equal number of positive and negative charges (e.g. a
neutral amino acid) do not contribute toward the ionic strength of a solution.

Example: Calculate the ionic strength of a 0.02 M solution of Fe2(SO4)3.

Changing concentrations: It is often necessary to change the concentrations of one solution to that of a
more dilute solution. This can be done by utilizing the fundamental relationship just discussed for molar
and normal solutions. It is clear that, in the process of dilution, the total amount of solute remains
unchanged, only its concentration is decreased. Hence, it follows that;

V1 x C1 = V2 x C2

where: V1 = volume of initial solution

C1 = concentration of initial solution
V2 = volume of desired (final, diluted) solution
C2 = concentration of desired (final, diluted) solution
This equation can be used regardless of the units in which concentration is expressed. If the entire volume
of the solution has to be diluted, the actual initial volume is used for V1. If only an aliquot (a part or fraction
of the whole) of the solution has to be diluted (and it is not clear how much will be needed), 1.0 mL is
used for V1 for purposes of calculations; then one decides how many multiples of 1.0 mL are to be used.

Example: Dilute a 1 mL of 12.0 M solution to a 3.0 M solution.

1 x 12.0 = V2 x 3.0

V2 = 4 mL.

For every 1.0 mL of initial solution; add 3 mL of diluent, or V2 = 4 times of V1.

Example: Prepare 50.0 mL of a 2.0 N solution from a 5.0 N stock solution.

V1 x 5.0 = 5.0 x 2.0

V1 = 20.0 mL

To 20.0 mL of stock solution add 30.0 mL of diluent.

Example: Dilute 7.0 mL of a 5.0% (w/v) solution to a 3.0% (w/v) solution.

7.0 x 5.0 = V2 x 3.0

V2 = 11.7 mL

To 7.00 mL of initial solution add 4.7 mL of diluent.

Example: Describe the preparation of 2L of a 0.4 M HCl starting with concentrated HCl solution: 28% (w/v)
HCl, ρ (specific gravity, s.g.) = 1.15 g/mL.

Liters x M = number of moles.

2 x 0.4 = 0.8 mole HCl is needed

Weight = number of moles x MW

= 0.80 x 36.5 = 29.2 g pure HCl is needed

The stock solution is not pure HCl but only 28% HCl by weight.

therefore, 29.2 = 104.3 g stock solution is needed

0.28
Instead of weighing out 104.3 g stock solution, we can calculate the volume required.

Therefore measure out 90.7 mL stock solution and dilute to 2L with water.
DEMO: Calibration of pH meter

A. The Operation and Calibration of a Glass Electrode

Calibrate the pH meter with the three standard buffers supplied, pH 4, 7 and 10 (or only two; The expected
pH has to be in the range of calibtared buffers)

1. Use 5 mL or 10 mL beakers and add enough “pH = 7.0” standard buffer to cover the end of the
electrode.

2. Two adjusting controls, one usually labelled “calibrate” or “buffer adjust” and a second labelled
“temperature”, are required to match the meter reading to the two buffers. Adjust the meter needle with
the “calibrate” control to the pH listed on the bottle while the electrode is immersed “pH = 7.0” buffer.

3. Rinse the electrode (the tip of a glass electrode is delicate and care must be taken to avoid breaking
this expensive piece of apparatus). The electrode must be rinsed with distilled water and touched (not
rubbed) with absorbent tissue between measurements of different solutions. Immerse the electrode into
“pH = 4.0” buffer and and adjust the needle to match the “pH = 4.0” buffer using the temperature control.

4. Repeat with ph 10.

5. Repeat the calibration with all the buffers until the pH meter reading matches both buffer without
further adjustments.

6. Discard standard buffers; do not return them to stock bottles.

7. At the end of an experiment the electrodes should be left in a slightly acidic (0.1 N HCl) aqueous
solution

Exercise:

Measure the pH of the following solutions with the pH meter with directly dipping the electrode into the
desired solutions; tap water, distilled water, 0.05 N HCl, 0.05 N KOH, 0.05 N NaOH; record the results in
the notebook.
QUESTIONS

1. What is the % (w/v) of 1.0 L solution that contains 5.0 g of NaCl?

2. Find the percent volume of ethanol in a solution prepared by diluting 30.0 mL of ethanol to 250 mL.

3. Carry out the following calculations:

a) What is the concentration in mg % of a solution that contains 5.0 mL of water?

b) How many ppm of NaCl is present in the solution?

4. a) How many moles of NaCl (MW = 58.5) are required to prepare 100 mL of a 1.6 M solution?

b) How many grams of NaCl are required?

5. How many moles of HCl are present in 50 mL of 3.0 M HCl solution?

6. What are the normalities of (a) 0.213 M HCl, and (b) 0.010 M Ca(OH)2?

7. How would you prepare 200 mL of a 1.0% (w/v) solution of glucose from a 6.0% (w/v) solution? How
would you prepare 2.5 L of a 2% (w/v) glucose solution from 5% (w/v) aqueous glucose?

8. A solution contains 15 g of CaCl2 in a total volume of 190 mL. Express the concentration of this solution
in terms of (a) g per L, (b) % (w/v), (c) mg%, (d) M, (e) What is the ionic strength of the solution? (MW of
CaCl2 = 110.986 g/mole)

9. What is the molarity of pure ethanol; that is, how many moles are present in 1 L of pure ethanol? The
density of ethanol is 0.789 g/mL. The MW of ethanol is 46.07g/mole.

10. Consider that 0.8 g of solid NaOH used for the preparation of 500 mL solution. Express the
concentration of this solution in terms of g/L, %w/v, mg %. (MWNaOH= 40)
LAB 4: SPECTROPHOTOMETRY

OBJECTIVES:
Students will be introduced to the concepts of spectrophotometry as well as how it is used to
measure the concentration of compounds in solution.

INTRODUCTION:
Spectrophotometry is a procedure that is frequently utilized in biological laboratories. Probably the most
common application in biology of this technique is in the measurement of the concentration of a
compound in solution.

Spectrophotometry is the measurement of the interaction of light with matter. Many kinds of biological
substances absorb visible light (400 to 700 nm) selectively and, as a result, appear coloured. Substances
that do absorb visible light are called pigments. Other substances do not absorb light of visible
wavelengths and appear colourless. We can study colourless compounds in two different ways. One is
that they can often react with other substances to form coloured derivatives that we can see and measure.
Another is that colourless compounds usually absorb light in the region of the spectrum that is not visible
to the naked eye. Absorption of this light can be measured, even if we cannot observe it unaided.

A second application of spectrophotomerty is the determination of the absorption spectrum of a

compound. Both of these can be applied to colourless as well as colored solutions since a
spectrophotometer can measure absorbance of light that we cannot see.

Absorption spectra and color

Regardless of whether a solution is colourless or coloured, the wavelength(s) absorbed are distinctive. A
solution of a particular compound, such as hemoglobin, always absorbs light of specific wavelengths and
reflects light of other wavelengths. Furthermore, absorption of light is not absolute. A compound will
characteristically absorb a certain proportion, anywhere from 0% to 100%, of light of a specific
wavelength. For every compound, if you measure the proportion of light absorbed, for any wavelength,
you will always get the same answer. This is the basic idea of the absorption spectrum of a specific
compound, which is the proportion of light absorbed for each wavelength of the spectrum.

The wavelength(s) absorbed by a substance in the visible part of the spectrum is complementary to the
colour that we perceive. The "colour" is a function of human perception, but absorption of specific
wavelengths of light is a function of molecular interaction with light. If a substance absorbs blue and red
light, but not green light, we will see it as green since that is the only light that reaches us from that
substance. Therefore, when you look at the absorption spectrum of a green solution, it should show low
absorbance of wavelengths in the green part of the spectrum but high absorption of light in the red and
blue parts of the visible spectrum.

While the "colour" of a substance depends on the person observing it, absorption of specific wavelengths
depends on the molecular structure of the substance. This allows qualitative analysis (i.e., identification)
of some substances by determination of the absorption spectrum.
Measuring concentration

The basis of this application of spectrophotometry is that the proportion of light that is absorbed by a
solution of a particular compound is a function of the concentration of that compound. This allows for a
quantitative analysis of concentration of a substance from the Beer-Lambert relationship (below). A
spectrophotometer will direct light of a specific wavelength on your solution. The light that passes through
the solution is the transmitted light. The absorbance (A) of the solution is the log of the ratio of these two
measures:

A (Absorbance, or Optical Density) = log 10 (Intensity of incident light / Intensity of transmitted light)

The spectrophotometer will calculate and display the absorbance. Once we know the absorbance,
concentration of the solution follows from the Beer-Lambert equation:

A= E* C* L

in which:

E (Molar Absorption) = absorbance of a l M solution of the substance measured through

a l-cm light path. This is a constant for the substance at a given wavelength.

C = concentration, in moles/liter.

L = length of the light path through the solution, in cm. For the spectrophotometer we
will be using, L is equal to 1 cm.

Therefore, since L equals 1, C = A / E.

In order to apply the Beer-Lambert equation you must know the Molar Absorption Value (E) for the
substance (compound) and wavelength you are using. This is defined as the Absorbance of a 1 M solution
so it can be measured easily by the obvious – reading Absorbance of a 1 M solution. However, for
substances with strong absorbance, the Absorbance of a l M solution is too great to read in an analytical
instrument. In this case a standard curve is produced by measuring Absorbance of a number of dilute
solutions, each of known concentration. When Absorbance is plotted against concentration the slope of
the line is the relationship between concentration and Absorbance. This is the E value (E=A/C, if L=1) so
the slope of the standard curve gives you E. Once you have measured E, you can find the concentration
of any solution (of that compound) by measuring Absorbance at the same wavelength used to calculate
E.

A spectrophotometer measures the intensity of light transmitted through a solution. It consists of two
principal parts: a spectrometer and a photometer. Using a white light source and a monochromator (a
prism), the spectrometer of the instrument is designed to provide discrete wavelengths of light at a known
intensity.
 Diagram: Spectrophotometer - The photometer consists of a photoelectric tube
sensitive to the wavelengths of light provided by the spectrometer and a
galvanometer to quantitate the intensity of the light. The sample to be measured
is inserted between the spectrometer and the photometer. By comparing the
intensity emitted by the spectrometer to the intensity measured by the
photometer, absorbance by the solution can be calculated by the machine.

LAB EXERCISES: BIURET COLORIMETRIC ASSAY OF PROTEIN

Proteins react with copper ions in alkaline solution to form a violet-colored complex that absorbs light at
550 nm. This reaction is the basis of the biuret assay for protein. Biuret reagent is a solution of CuSO4 in
NaOH. When you add biuret reagent to a protein solution the reaction produces a solution of the protein-
biuret complex in a concentration that is the same as the original concentration of protein. Therefore, by
measuring the concentration of the complex, using A550 (Absorbance at 550 nm), you are also measuring
the concentration of protein.

Materials: Bovine serum albumin (BSA) (10mg/ml)

Biuret reagent
NaCl (0.9%)
Spectrophotometer
Protein X (unknown concentration)

Procedures:

i. To prepare the standard curve from known concentration of protein (BSA), prepare solutions as follow:

Tube NaCl (ml) BSA

1 1.0 0
2 0.9 0.1
3 0.8 0.2
4 0.6 0.4
5 0.4 0.6
6 0.2 0.8
7 0 1.0

ii. Mix the solution well.

iii. Add 4.0ml of biuret reagent to each of the tubes and mix well.

iv. Allow the solutions at room temperature (bench top) for about 30 mins.

v. Measure the absorbance of each reaction mixture at 550nm wavelength by using tube 1 mixture as the
blank.

vi. Simultaneously, add 4.0 ml of the biuret reagent into 1.0ml of Protein X tube. Mix well, incubate and
measure the absorbance.

vii. On a page of graph paper, plot the absorbance readings of the BSA samples (on the Y axis) against BSA
concentration in each reaction mixture (on the X-axis).

viii. Using any two points on the straight line, calculate the slope of the standard curve. This is E from the
Beer-Lambert equation, the relationship between concentration and absorbance.

ix. Determine the concentration of protein X sample using the E value.

LAB 5: PAPER CHROMATOGRAPHY

OBJECTIVES:

Students will be able to:

• Gain understanding of the purpose of chromatography.
• Measure and graph pigment separation.

INTRODUCTION

Chromatography is a term used to describe several laboratory techniques that separate mixtures on the
basis of their differing affinities for a stationary phase. As a result of these differences in mobilities, sample
components will be separated from each other as they travel through the stationery phase. It is one of
the most common chemical and biochemical separation techniques. In a variety of chromatographic
methods, chemists use liquid solvents or mobile phase to move compounds over a stationary phase (a
solid, gel, or polymer).

Figure: Illustration of chromatography

There are several types of chromatography, such as, thin layer chromatography (TLC), high performance
liquid chromatography (HPLC), paper chromatography (PC), and gas chromatography (GC). They are
designed to separate specific types of mixtures. Table 1 includes the most common chromatography
techniques and their phases.
 Table 1: Types of chromatography

Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying
compounds, determining their purity and following the progress of a reaction. It also permits the
optimization of the solvent system for a given separation problem. In comparison with column
chromatography, it only requires small quantities of the compound (~ng) and is much faster as well.

Stationary Phase: A special finely ground matrix (silica gel, alumina, or similar material) is coated on a glass
plate, a metal, a paper or a plastic film as a thin layer (~0.25 mm). In addition a binder like gypsum is mixed
into the stationary phase to make it stick better to the slide. In many cases, a fluorescent powder is mixed
into the stationary phase to simplify the visualization later on (e.g. bright green when you expose it to 254
nm UV light).

Preparation of chromatography (TLC) paper:

Do not touch the paper on the side with the white surface. In order to obtain
an imaginary start line, make two notches on each side of the paper. You can
also draw a thin line with pencil. Do not use pen. The start line should be 0.5-1
cm from the bottom of the paper.
Spotting the TLC paper:
The thin end of the spotter is placed in the dilute solution; the solution will
rise up in the capillary (capillary forces). Touch the plate briefly at the start
line. Allow the solvent to evaporate and spot at the same place again. This
way you will get a concentrated and small spot. Try to avoid spotting too much
material, because this will deteriorate the quality of the separation
considerably (‘tailing’). The spots should be far enough away from the edges
and from each other as well. If possible, you should spot the compound or
mixture together with the starting materials and possible intermediates on
the plate. They will serve as internal reference since every TLC paper is slightly
different.

Developing a TLC:
A TLC paper can be developed in a beaker or closed chamber. Place a
small amount of solvent (= mobile phase) in the container. The solvent
level has to be below the starting line of the TLC, otherwise the spots will
dissolve away. The lower edge of the paper is then dipped in a solvent.
The solvent (eluent) travels up the matrix by capillarity, moving the
components of the samples at various rates because of their different
degrees of interaction with the matrix (=stationary phase) and solubility
in the developing solvent. Non-polar solvents will force non-polar
compounds to the top of the plate, because the compounds dissolve well
and do not interact with the polar stationary phase.

Analysis
Example:
The components, visible as separated spots, are identified by
comparing the distances they have traveled with those of known
reference materials. Measure the distance of the start line to the
 solvent front (=d). Then measure the distance of center of the spot to
the start line (=a). Divide the distance the solvent moved by the
distance the individual spot moved. The resulting ratio is called Rf-
value. The value should be between 0.0 (spot did not moved from
starting line) and 1.0 (spot moved with solvent front) and is unitless.

The Rf (=retardation factor) depends on the following parameters:

- solvent system
- absorbent (grain size, water content, thickness)
- amount of material spotted temperature

LAB EXERCISE
Procedures:

A. Sample preparation

Material: Leave samples, pestle and mortar, 80% ethanol,

i. Using a clean pestle and mortar, grind 5g of leave sample in 3 ml of 80% ethanol.

ii. Separate the liquid and debris using a piece of cotton gauze muslin cloth.

iii. Allow the liquid in laminar flow at room temperature for about 5 minutes.

iv. Use the paste as your sample.

B. Paper chromatography

Material: Chromatography paper, hexane: etyl acetate (1:1) solvent, capillary tubes, a glass chamber

i. Cut the TLC paper to fit the size of the mobile phase chamber.

ii. Draw a line at 1 inch above the bottom edge. Use a pencil to draw and label the spot. Be careful not
to touch the paper.

iii. Use a capillary tubes to spot the sample onto the TLC paper. Allow the spot to dry before adding
more sample.

iv. Place the paper in the mobile phase chamber and dip the paper in the solvent.

v. Allow the solvent to travel up the plate until ~1 cm from the top.
vi. Take the plate out and mark the solvent front immediately. Do not allow the solvent to run over the
edge of the plate.

vii. Next, let the solvent evaporate completely.

viii. The spots on the TLC paper should be circled (marked) to have a permanent record how far the
compound traveled on the paper.

ix. A sketch of the developed plate should be placed in your lab report.

x. Calculate the Rf value for each of the samples.