CENTRAL LUZON DOCTORS’ HOSPITAL – EDUCATIONAL INSTITUTION, INC

DEPARTMENT OF GENERAL EDUCATION

BIOCHEMISTRY LABORATORY

1ST SEMESTER 2021 - 2022

PRELIMINARY PERIOD

MODULE 2

Topic: Using Biochemical Reagents and Solutions

Overview: Biochemical reagents and solutions includes organic and inorganic chemicals that
have life science applications. They have biological effects or are required for biological assays.

Learning Objectives:
At the end of the laboratory discussion, the students should be able to:
1. Explain applications of water usage
2. Know the importance of cleaning laboratory glassware
3. Prepare and store solutions

Discussion
Water Purity
Water is the most common and widely used substance in the biochemistry laboratory.
Applications of water usage include: (1) solvent for preparing most buffer and reagent solutions;
(2) column chromatography; (3) high-performance liquid chromatography; (4) tissue culture; and
(5) washing glassware. Both the quality and quantity of water required must be considered for
each lab application. Ordinary tap water is relatively abundant, but its quality is very low. It
contains a variety of impurities including particulate matter (sand, silt, etc.); dissolved organics,
inorganics, and gases; and microorganisms (bacteria, viruses, protozoa, and algae). In addition,
the natural degradation of microorganisms leads to the presence of byproducts called pyrogens.
Tap water should never be used for the preparation of reagent solutions or for any sensitive
procedures. For most laboratory procedures, it is recommended that some form of purified water
be used.
  CENTRAL LUZON DOCTORS’ HOSPITAL – EDUCATIONAL INSTITUTION, INC
DEPARTMENT OF GENERAL EDUCATION

For most procedures carried out in the biochemistry lab, water purified by ion exchange,
reverse osmosis, or distillation is usually acceptable. Of these three processes, distillation is the
slowest, least energy-efficient, least pure (best is), and most high-maintenance—especially
in areas with hard water (needs regular de-scaling). Distilled water must also be stored to
prevent contamination by microbes.

Cleaning Laboratory Glassware

The results of your experimental work will depend, largely, on the cleanliness of your
equipment, especially glassware used for preparing and transferring solutions. There are at least
two important reasons for this: (1) many of the chemicals and biochemicals will be used in
milligram, microgram, or even nanogram amounts. Any contamination, whether on the inner
walls of a beaker, in a pipet, or in a glass cuvette, could be a significant percentage of the total
experimental sample; (2) many biochemicals and biochemical processes are sensitive to one or
more of the following common contaminants: metal ions, detergents, and organic residues. In
fact, the objective of many (any lab) experiment is to investigate the effect of a metal ion,
organic molecule, or other chemical agent on a biochemical process. Contaminated glassware
will ensure failure in these lab activities.
The preferred method for cleaning glassware is to begin with hot tap water. Rinse the glassware
at least 10 times with this; then rinse 4–6 times with distilled or de-ionized water. Occasionally it
is necessary to use a detergent for cleaning. Use a dilute detergent solution (0.5% in water)
followed by 5–10 water rinses with distilled or de-ionized water. Dry equipment is required for
most processes carried out in the biochemistry laboratory. When you needed dry glassware in the
organic laboratory, you probably rinsed the glassware with acetone, which rapidly evaporated,
leaving dry surface. Unfortunately, this technique coats the surface with an organic residue
consisting of nonvolatile contaminants found in the acetone. Because this residue could interfere
with your experiments, it is best to refrain from acetone washing. Glassware and plastic ware
should be rinsed well with purified water and dried in an oven designated for glassware, not one
used for drying chemicals. Never clean cuvettes or any optically polished glassware with
ethanolic KOH or other strong base, as this will cause etching. All glass cuvettes should be
cleaned carefully with hot tap water or 0.5% detergent solution, in a sonicator bath or in a
cuvette washer, followed by thorough rinsing with purified water.
  CENTRAL LUZON DOCTORS’ HOSPITAL – EDUCATIONAL INSTITUTION, INC
DEPARTMENT OF GENERAL EDUCATION

Solutions: Concentrations and Calculations

The concentrations for solutions used in the biochemistry laboratory may be expressed in
several different units. The most common units are:
 Molarity (M): concentration based on the number of moles of solute per liter of solution.
 Percent by weight (% wt/wt): concentration based on the number of grams of solute per
100 g of solution.
 Percent by volume (% wt/vol): concentration based on the number of grams of solute
per 100 mL of solution.
 Weight per volume (wt/vol): concentration based on the number of grams, milligrams,
or micrograms of solute per unit volume; for example, mg/mL, g/L, mg/100 mL, etc.

Preparing and Storing Solutions

In general, solid solutes should be weighed on weighing paper or plastic weighing boats, with the
use of an electronic analytical or top-loading balance. Liquids are more conveniently dispensed
by volumetric techniques; however, this assume that the density is known. If a small amount of a
liquid is to be weighed, it should be added to a tared flask by means of a disposable Pasteur pipet
with a latex bulb. The hazardous properties of all materials should be known before use (read
MSDS) and the proper safety precautions obeyed. The storage conditions of reagents and
solutions in the biochemistry lab are especially critical. Although some will remain stable
indefinitely at room temperature, it is good practice to store all solutions in a closed container.
Often it is necessary to store some solutions in a refrigerator at this inhibits bacterial growth and
slows decomposition of the reagents.

Some solutions may require storage below if these are aqueous solutions or others that will
freeze, be sure there is room for expansion inside the container. Stored solutions must always
have a label containing the name and concentration of the solution, the date prepared, and the
name of the preparer. All stored containers, whether at room temperature, or below freezing must
be properly sealed. This reduces contamination by bacteria and vapors in the laboratory air
(carbon dioxide, ammonia, HCl, etc.). Volumetric flasks, of course, have glass stoppers, but test
tubes, Erlenmeyer flasks, bottles, and other containers should be sealed with screw caps, corks,
  CENTRAL LUZON DOCTORS’ HOSPITAL – EDUCATIONAL INSTITUTION, INC
DEPARTMENT OF GENERAL EDUCATION

or hydrocarbon foil (Parafilm). Remember that hydrocarbon foil, a wax, is dissolved by

solutions containing nonpolar organic solvents like chloroform, diethyl ether, and acetone.

Bottles of pure chemicals and reagents should also be properly stored. Many manufacturers now
include the best storage conditions for a reagent on the label. The common conditions are: store
at room temperature; store at store below or store in a desiccator at room temperature, or below.
Many biochemical reagents form hydrates by taking up moisture from the air. If the water
content of a reagent increases, the molecular weight and purity of the reagent change.

Post Laboratory Activity

Questions:

1. Give at least five (5) purposes of water purity.

2. List at least five (5) applications of water purity aside from what you have learned.
3. What are the possible causes of not cleaning the laboratory glasswares? Give at least
three (3).
4. Explain the importance of concentrations and calculations in the biochemistry laboratory.
5. What are the possible causes of not storing solutions properly? Give at least three (3).