Dilution is a fundamental technique used across various scientific disciplines, representing the process of reducing the concentration of a substance in a solution. This is accomplished by adding more of the solvent, which is the liquid that dissolves the substance, without changing the total amount of the substance itself. Mastering this technique is important for preparing reagents and samples for analysis, ensuring experimental results are accurate and reproducible.
Defining Key Terms
A solution is composed of a solute, which is the substance being dissolved, and a solvent, which is the medium that does the dissolving. The liquid added during a dilution is specifically called the diluent, which is often the same substance as the original solvent, such as water or a buffer solution.
The starting material for a dilution is referred to as the stock solution, which has a known, higher concentration. Common ways to express concentration include molarity (moles of solute per liter of solution) and percent concentration (parts of solute per 100 parts of solution by mass or volume).
Calculating the Dilution
The mathematics of dilution relies on the principle that the total amount of solute remains unchanged during the process. This relationship is described by the universal dilution formula: $C_1V_1 = C_2V_2$. Here, $C_1$ and $V_1$ represent the concentration and volume of the initial stock solution, and $C_2$ and $V_2$ represent the concentration and volume of the final, diluted solution.
For example, if a researcher wants to prepare 500 milliliters ($V_2$) of a 0.5 M solution ($C_2$) from a 2.0 M stock solution ($C_1$), the formula is rearranged to solve for the volume of stock needed ($V_1$). The calculation becomes $V_1 = (C_2 \times V_2) / C_1$, which is $(0.5 \text{ M} \times 500 \text{ mL}) / 2.0 \text{ M}$. Solving this equation yields a required stock volume ($V_1$) of 125 milliliters. The concentration units used for $C_1$ and $C_2$ must match, and similarly, the volume units for $V_1$ and $V_2$ must be the same.
Performing a Simple Dilution
This technique, known as a simple or single-step dilution, requires accurate measuring equipment, typically a pipette for measuring the stock volume and a volumetric flask for the final solution. Safety considerations are important, and appropriate personal protective equipment should be used, especially when handling concentrated stock solutions.
The measured volume of the stock solution is carefully transferred into the volumetric flask or container intended to hold the final volume. A portion of the diluent is added to the container first, and then the stock solution is introduced and gently mixed to start the dissolution process. The container is then filled with the remaining diluent until the liquid’s meniscus aligns precisely with the volume mark etched on the neck of the glassware, a technique known as “diluting to volume.” Mixing the solution thoroughly is the final step, usually by capping the container and inverting it several times.
When to Use Serial Dilutions
A serial dilution is a specialized technique involving a sequence of repeated simple dilutions, performed in a step-wise manner. This method is employed when the final concentration required is extremely low, making a single-step dilution impractical or inaccurate due to the need to measure an impossibly small volume of stock solution. This technique is frequently used in fields like microbiology to estimate the number of microorganisms in a sample, such as a bacterial culture.
By creating a logarithmic progression of concentrations, the sample is diluted until the number of colonies that grow on a plate is countable, typically between 30 and 300. The total dilution factor for a serial dilution is cumulative and determined by multiplying the dilution factor of each individual step. For example, performing three consecutive 1:10 dilutions results in an overall dilution factor of 1:1000 ($1/10 \times 1/10 \times 1/10$). A serial dilution involves transferring a fixed volume of the mixed solution from one tube to the next, which already contains a known volume of diluent.