Comparing Mitosis and Meiosis



Solution Preparation and Dilutions

Solutions are required for all functions in a laboratory setting. These reagents can be prepared based on four sets of calculations: mass/volume concentrations, % mass/volume concentrations, molarity concentrations, and dilutions of concentrated solutions.

In this laboratory you will:

a) learn proper metric conversions,

b) learn the calculations required for each of the solution types above,

c) determine the calculations for solutions required during the semester, and

d) prepare the solutions for use throughout the semester.

Materials

Lab handout

Calculator

Procedure

Part A. Making Metric Conversions

When preparing solutions, metric units must match for the proper calculations to occur. Use Table 1 to learn how metric units are properly converted.

Table 1. Commonly used metric units in the biotechnology field.

|Units |Equivalent Values |

|1 liter (L) |1000 (103) milliliters |1,000,000 (106) | | |

| |(mL) |microliters (μL) | | |

|1 meter (m) |0.001 (10-3) kilometer |100 (102) centimeters |1000 (103) millimeters |1,000,000 (106) micrometers |

| |(km) |(cm) |(mm) |(μm) |

|1 gram (g) |0.001 (10-3) kilogram |1000 (103) milligrams |1,000,000 (106) |1,000,000,000 (109) nanograms|

| |(kg) |(mg) |micrograms (μg) |(ng) |

Fill in the conversion table (Table 1) in the Practice Problems section for Part A. Making Metric Conversions at the end of this handout.

Part B. Making Solutions of Differing Mass/Volume Concentrations

Solutions are prepared with a certain mass of solute in a certain volume of solvent. 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/L nanograms per liter

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.

To determine how to prepare a certain volume of a solution at a certain mass/volume concentration, use the equation below. Convert units as necessary to make sure units that are used can be cancelled out.

Mass/Volume Concentration Equation

concentration desired x total volume desired = mass of solute in the total volume desired

(ex. g/mL) (ex. mL) (ex. g)

Ex. A technician needs 50 mL of 15 mg/mL pepsin solution for an experiment. Pepsin is a protein-digesting enzyme that is produced and functions in the stomach. Using the Mass/Volume Equation, the calculation would be as follows:

15 mg/mL x 50 mL = 750 mg = 0.75 g pepsin

The technician would add 0.75 g pepsin to a container and fill up to the 50 mL mark with solvent (usually deionized water). Most balances weigh in grams, so the conversion from mg to g was necessary.

Determine the calculations for the solutions in the Practice Problems section for Part B. Making Solutions of Differing Mass/Volume Concentrations at the end of this handout.

Part C. Making Solutions of Differing % Mass/Volume Concentrations

A lab technician must be able to make any solution at any concentration or volume. Most commonly, solutions are made with concentrations reported in one of these three measurements:

Measurement Example

mass/volume 4 mg/mL salmon sperm DNA solution

% (in mass/volume or volume/volume) 2% sucrose solution

molarity (moles/liter) 0.5 M TRIS solution

Technicians must be able to recognize which chemicals should be measured out, in what amounts, and what math must be done to calculate these amounts. It takes practice to consistently prepare solutions at the correct volume and concentration.

Preparation of % mass/volume solutions is presented here. Keep in mind that a 1% solution contains 1 g of solute in a total volume of 100 mL.

Use the % Mass/Volume Equation shown below to calculate the mass of each solute needed for a solution at some volume. Notice that as with preparing mass/volume solutions, the math is relatively simple. Multiply concentration desired (in decimals) with the volume needed (in mL). Make sure units that are used can be cancelled out and convert units as necessary.

% Mass/Volume Concentration Equation

Step 1. Convert the % to a decimal

% (percent value) = decimal value of the g/mL (grams needed divided by 100 mL)

Step 2. Determine the amount needed

decimal (g/mL) of the % concentration x total volume desired (mL) = grams of solute

Ex. A technician needs 5 mL of 10% NaOH solution for an experiment. Using the % Mass/Volume Equation, the calculation would be as follows:

Step 1. 10% = 10 g/100 mL = 0.1 g/mL

Step 2. 0.1 g/mL x 5 mL = 0.5 g NaOH

The technician would add 0.5 g NaOH to a container and fill up to the 5 mL mark with solvent.

Determine the calculations for the solutions in the Practice Problems section for Part C. Making % Solutions of Differing Mass/Volume Concentrations at the end of this handout.

Part D. Making Solutions of Differing Molarity Concentrations

The concentration of many solutions is reported as moles/liter (mol/L or M; the M is spoken “molar”) or some function 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 required only on 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 1023 molecules, but that is not really a very useful number. So, in biotechnology, 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 or by 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). If the concentration is very low, then the concentration could be reported in millimoles/liter (mmol/L or mM). 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, 1 M NaCl.

A liter of solution is a large volume for most research and development purposes. In research and development labs, mL or μL quantities are usually used. To determine how to mix up a smaller volume of a solution of some molarity, follow the example below.

Multiply the volume desired (L) by the concentration (molarity) desired (mol/L), as you did in the mass volume calculations. Then, multiply the result by the compound’s molecular weight (g/mol) to account for measuring in moles, as in the following equation:

Molarity Concentration Equation

volume x molarity x molecular weight = grams of solute to be dissolved in

wanted desired of the solute solvent to the final desired volume

(L) (mol/L) (g/mol)

Convert smaller or larger units to these as necessary. The “L” units cancel out and the “mol” units cancel out, leaving the mass in grams of the solutes needed to make the solution.

Ex. A technician needs 50 mL of 0.5 M NaCl solution for an experiment. Using the Molarity Concentration Equation, the calculation would be as follows:

0.05 L x 0.5 mol/L x 58.5 g/mol = 1.46 g NaCl

The technician would add 1.46 g NaCl to a container and fill up to the 50 mL mark with solvent.

Determine the calculations for the solutions in the Practice Problems section for Part D. Making Solutions of Differing Molarity Concentrations at the end of this handout.

Part E. Making Dilutions of Concentrated Solutions

Making dilutions of concentrated solutions is a common practice in a biotechnology lab. A concentrated solution is generally called a “stock solution,” and the diluted solution is called the “working solution.” Preparing a concentrated stock solution saves a lot of time and is easier to store than large volumes of diluted working solutions. Making a working solution simply required diluting some volume of stock solution to the concentration needed.

The working concentration of a solution is represented as 1X. A concentrated solution could be represented as 10X if it has 10 times the amount of solute per unit volume compared with the working solution. A 50X stock has 50 times the concentration of solute as a working solution. For example, an enzyme storage buffer 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 enzyme 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).

When a number of dilutions must be made, and each is proportionally the same dilution as the one before, it is called a serial dilution. 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.

Each succeeding sample is made with the

same ratio of sample and diluent as the one before

1 M NaCl 0.5 M NaCl 0.25 M NaCl 0.125 M NaCl

Each of the above dilutions is one part previous sample and one part solvent. This is called a 1:2 dilution, or one part sample to two total parts.

If the technician needs 150 mL of the 0.1 M TRIS, then a dilution of the concentrated 1 M TRIS can be calculated. To figure out how to dilute something from a concentrated solution, we use a simple ratio equation as shown in the following equation:

Diluting Concentrated Solutions Equation

C1V1 = C2V2

C1 = the concentration of the concentrated stock solution (the starting solution)

V1 = the volume to use of the stock solution in the diluted sample

C2 = the desired concentration of the diluted sample

V2 = the desired volume of the diluted sample

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). Using the equation with the scenario above in which the technician needs 150 mL of the 0.1 M TRIS,

C1 = 0.1 M TRIS (the starting solution)

V1 = amount of the concentrate to use for the dilution

C2 = 0.01 M TRIS (the working solution)

V2 = 150 mL

(0.1 M)(V1) = (0.01 M)(150 mL)

V1 = (0.01 M)(150 mL)/(0.1 M)

V1 = 15 mL

Therefore, to make 150 mL of 0.01 M TRIS from the concentrated stock, measure out 15 mL of the concentrated 0.1 M TRIS stock and add 135 mL of deionized water to is and mix well.

Determine the calculations for the solutions in the Practice Problems section for Part E. Making Dilutions of Concentrated Solutions at the end of this handout.

Practice Problems

Part A. Making Metric Conversions

Table 1. Conversion Table

|Measurement |Conversion |

| 75.34 mg | g |

| 4.3 mL | μL |

| 440.3 mL | L |

| 3.33 g | μg |

| 0.34 g | mg |

| 34.0 g | kg |

| 0.004 L | mL |

| 80.34 μL | mL |

| 34 mg | μg |

| 0.25 g | ng |

Part B. Making Solutions of Differing Mass/Volume Concentrations

1. Describe how you would prepare 25 mL of a NaCl solution at a concentration of 2.5 g/mL.

2. Describe how you would prepare 2 L of a 0.5 g/mL dextrose solution.

Part C. Making Solutions of Differing % Mass/Volume Concentrations

1. Describe how you would prepare 200 mL of an 8% NaCl solution.

2. Describe how you would prepare 0.75 L of a 5% dextrose solution.

Part D. Making Solutions of Differing Molarity Concentrations

1. Describe how you would prepare 125 mL of a 10 M NaOH solution.

2. Describe how you would prepare 75 mL of a 0.1 M NaCl solution.

Part E. Making Dilutions of Concentrated Solutions

1. Describe how you would prepare 950 mL of a 1X CuSO4•5H2O solution from a 25X CuSO4•5H2O stock.

2. Describe how you would prepare 50 mL of a 5 mM NaCl solution from a 1 M NaCl stock.

-----------------------

500 mL

1M NaCl

500 mL

0.25M NaCl

500 mL

0.5M NaCl

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