SOLUTION

PREPARATION
SOLUTIONS & REAGENTS
The vast majority of analytical procedures and
biochemical reactions performed within a medical
laboratory necessitate that reactants be present in a
dissolved state or that specific environmental
conditions, such as ionic strength or pH, be precisely
controlled.

Consequently, the in-house preparation of aqueous

solutions is a common practice.
SOLUTIONS & REAGENTS
For instance, physiological saline (0.9% w/v NaCl)
must often be prepared to provide an isotonic
environment suitable for suspending cells or diluting
biological samples without causing osmotic damage.
SOLUTIONS & REAGENTS
Similarly, buffer solutions, such as phosphate-buffered
saline (PBS) or Tris buffers, are precisely prepared to
specific pH values and buffering capacities to maintain
a stable hydrogen ion concentration, which is critical
for the optimal activity of enzymes and the integrity of
many biological molecules during assays.
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These examples
underscore why
understanding solution
preparation is
fundamental.
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A solution is basically a homogeneous mixture

composed of two or more substances.

Typically, it consists of a solute (the substance being

dissolved, often a solid or concentrated liquid) and
a solvent (the substance doing the dissolving, most
commonly purified water in biological applications).

The solute disperses uniformly throughout the

solvent at a molecular or ionic level.
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A reagent, more broadly, refers to any substance or

mixture used in a chemical or biochemical reaction to
detect, measure, examine, or produce other
substances.
Therefore, many reagents used in the lab are, in fact,
solutions prepared to specific concentrations, but the
term reagent can also encompass pure substances,
catalysts, enzymes, antibodies, or complex
formulated mixtures designed for specific analytical
procedures like staining, immunoassays, or molecular
diagnostics.
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The careful preparation of these reagents

according to established protocols (such
as Standard Operating Procedures, SOPs)
is necessary for ensuring the precision,
accuracy, and reproducibility of
laboratory results.
PURITY AND GRADES OF REAGENTS

The reliability of any prepared solution or reagent begins with the

quality of the starting materials. Chemicals are available in various
grades of purity, and selecting the appropriate grade is crucial for the
successful application, balancing analytical requirements with cost
considerations.
Using a reagent of insufficient Conversely, using an unnecessarily
purity can introduce high purity grade can significantly
interfering substances, leading increase costs without providing any
to inaccurate results or extra benefits for a specific
reaction failures. application.
PURITY AND GRADES OF REAGENTS
These reagents meet or exceed purity standards set by the American
Chemical Society (ACS). They are of very high purity and are suitable for
ACS / Analytical most quantitative and qualitative analyses performed in clinical and
research laboratories.
This designation indicates high purity suitable for general laboratory use
but may not have undergone the rigorous ACS testing. It is generally
Reagent acceptable for many routine procedures unless specific assays demand
higher purity.
These grades meet standards set by the United States Pharmacopeia (USP)
or the National Formulary (NF). They are primarily used for pharmaceutical
USP & NF and food applications, ensuring specific limits on impurities detrimental to
health, but may not always meet the stringent analytical purity required for
some laboratory reagents.
A grade of relatively good quality, suitable for qualitative work or
Laboratory / educational purposes, but usually with unknown levels of impurities.
Generally not suitable for preparing standard solutions or critical analytical
Chemical reagents.
Intended primarily for industrial use, this grade possesses minimal purity
Technical / and is generally unsuitable for laboratory applications where chemical
composition is critical.
Commercial
WATER THE PRIMARY SOLVENT 11

Crucially, the water utilized as the primary

solvent in reagent preparation must itself meet
appropriate purity standards, as impurities
present in tap water (such as dissolved ions,
organic compounds, particulates, and
microorganisms) can interfere with chemical
reactions or degrade reagents.

Various purification methods are employed in

laboratories to produce water suitable for
different applications.
 Filtered water has typically passed
through physical filters to remove
suspended particulate matter, but it
retains dissolved solutes.

Deionized (DI) water, also known as

Distilled water is
demineralized water, is produced by passing
produced by boiling
water through ion-exchange resins that
water and condensing
remove dissolved
the resulting steam; this
inorganic ions (cations
process effectively
and anions), resulting in
removes non-volatile
water with high electrical
solutes like mineral salts but may not
resistivity but potentially
remove volatile organic impurities or
containing non-ionic
dissolved gases.
organic contaminants or
microorganisms.
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• Often, these methods are used in combination, or

followed by additional steps like activated carbon
filtration, ultraviolet (UV) sterilization, or ultrafiltration,
to achieve the very high purity levels necessary for
sensitive procedures such as molecular diagnostics or
trace element analysis.

• Therefore, regardless of the specific purification system

employed, standard laboratory purified water must be
assessed or known to possess the requisite quality—in
terms of ionic content, organic contaminants, and
biological purity—to ensure it is appropriate for the
specific reagent being prepared and will not compromise
the integrity of the subsequent laboratory procedure.
FUNDAMENTAL CONCEPTS IN 14

SOLUTION PREPARATION
Accurate solution preparation relies on
understanding fundamental chemical principles
and units of concentration.
Density

%
Molarity
Solution

Normalit
Molality
y
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DENSITY (Ρ)
Density (ρ) is the mass of a substance per unit volume (typically g/mL or g/cm³ for liquids and
solids). It is an intrinsic property of a substance at a given temperature and pressure.

Density is particularly important when preparing solutions from

liquid solutes, as it allows conversion between mass (which is
accurately measured on a balance) and volume (which might be
more convenient to measure with a pipette or cylinder, though
potentially less accurate than weighing).
For example, if a protocol requires a specific mass of a
concentrated acid, but measuring its volume is more practical,
the density (provided on the reagent bottle or MSDS) must be
used for the conversion:

Mass (g) = Volume (mL) * Density (g/mL)

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CONCENTRATION
This term describes the amount of solute present in a given
amount of solvent or solution. Essentially, concentration
quantifies the proportion of solute dispersed within a
given quantity of solvent or the total volume of the
solution, effectively defining the solution's strength or
composition.
This precise relationship is critical, as the physical and
chemical properties of the solution—such as its reactivity,
osmotic pressure, or absorbance—are directly dependent
on this ratio. Various units are used to express
concentration, each suitable for different contexts and
facilitating consistent communication and calculation in
laboratory work.
MOLARITY (M)
Perhaps the most common unit in chemistry and biology, molarity is defined as
the number of moles of solute per liter of solution (M = moles / L).
A mole represents Avogadro's number (6.022 x 10²³) of particles (atoms,
molecules, ions) and has a mass equivalent to the substance's molecular weight
(MW) or formula weight (FW) in grams.
To prepare a molar solution, one calculates the required mass of solute using the
formula:

Mass (g) = Desired Molarity (mol/L) * Desired Volume (L) * Molecular Weight (g/mol)
The calculated mass of solute is dissolved in a portion of the solvent, and then sufficient
solvent is added to reach the final desired total volume of the solution, typically using a
volumetric flask for high accuracy.

Molarity is temperature-dependent because the volume of the solution

changes slightly with temperature.
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MOLALITY (m)

Molality is defined as the number of moles of solute per kilogram of solvent (m

= moles / kg solvent).

Unlike molarity, molality is independent of temperature because mass does not

change with temperature.

While less commonly used in routine medical laboratory solution preparation

than molarity, it is important in physical chemistry and when studying
colligative properties.
NORMALITY (N)
Normality is defined as the number of gram equivalents of solute per liter of solution
(N = equivalents / L).
An equivalent represents the mass of a substance that can react with or
supply one mole of hydrogen ions (H⁺) in an acid-base reaction, or one
mole of electrons in a redox reaction. Normality depends on the specific
reaction the solute participates in.

For instance, sulfuric acid (H₂SO₄) has two acidic protons, so a 1 M

H₂SO₄ solution is 2 N for acid-base reactions. While historically
common, particularly in titration chemistry, the use of
normality is declining in favor of the less ambiguous
molarity.
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PERCENT SOLUTIONS
This method expresses concentration as parts of solute per 100 parts of the total solution. It is
crucial to specify the basis of the percentage:

Percent Weight by Volume (% w/v): Defined as the mass of

solute in grams per 100 milliliters of solution.

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

This is very common for preparing solutions from solid solutes in

liquids (e.g., 0.9% w/v NaCl).
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PERCENT SOLUTIONS

Percent Volume by Volume (% v/v): Defined as the volume of

liquid solute in milliliters per 100 milliliters of solution

% v/v = (mL of solute / 100 mL of solution) * 100

Used when mixing two liquids (e.g., 70% v/v ethanol).

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PERCENT SOLUTIONS

Parts Per Million (ppm) and Parts Per Billion (ppb): Used for
expressing very dilute concentrations, often for trace contaminants or
analytes.

For dilute aqueous solutions,

ppm is roughly equivalent to milligrams of solute per liter of solution
(mg/L)
ppb is roughly equivalent to micrograms of solute per liter of solution
(µg/L).
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DILUTIONS

Preparing a less concentrated solution from a more concentrated stock solution

is a frequent laboratory task.

The fundamental principle governing dilutions is that the amount of solute

remains constant before and after dilution.

This is expressed by the formula: C₁V₁ = C₂V₂

where: C₁ = Concentration of the stock solution C₂ = Desired concentration of the final solution

V₁ = Volume of the stock solution to be used V₂ = Desired final volume of the diluted solution

This formula allows calculation of the volume of stock solution (V₁) needed to
achieve the desired final concentration (C₂) and volume (V₂).
PRACTICAL CONSIDERATIONS IN 24

PREPARATION
Beyond the calculations, precise technique is
essential:
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WEIGHING (gravimetric measurement)
Weighing (Gravimetric Measurement): Use an analytical
balance appropriate for the required precision. Ensure the
balance is calibrated and level.
Use clean weigh boats or paper, tare the container
accurately, and handle the chemical carefully, avoiding
spills. Record the exact mass weighed.
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VOLUME MEASUREMENT (volumetric measurement)

Use clean, calibrated glassware appropriate for the required

accuracy. Volumetric flasks are designed to contain a precise
volume at a specific temperature and are used for preparing
solutions of accurate concentrations (molarity, %w/v).

Graduated cylinders are less accurate and suitable for

approximate volumes.

Pipettes (volumetric or graduated/serological) are used for

accurately transferring specific volumes, especially for
dilutions. Always read the bottom of the meniscus at eye
level for aqueous solutions.
MIXING

Ensure the solute dissolves completely. Use magnetic stirrers

or manual agitation as appropriate.

Be aware that some dissolution processes are exothermic

(release heat) or endothermic (absorb heat), which can affect
the final volume

If temperature changes are significant; allow solutions to

return to room temperature before final volume adjustment
in a volumetric flask.
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STORAGE AND LABELING

Store reagents in appropriate containers (glass or plastic, clear

or amber for light-sensitive substances) at the recommended
temperature (room temperature, refrigerated, or frozen).

Labels must be clear, durable, and contain essential information:

unambiguous identity of the reagent expiration date (if applicable)

concentration preparer's initials
date of preparation storage requirements

and any necessary hazard warnings (e.g., corrosive, flammable).

Always consult the Safety Data Sheet (SDS) before handling any
chemical.

Wear appropriate Personal Protective Equipment (PPE), including

gloves, eye protection, and lab coats. SAFETY
Perform operations involving volatile or hazardous chemicals in a
chemical fume hood. Be aware of chemical incompatibilities and
follow proper waste disposal procedures.