Department of Biology

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  Making Solutions

You should become completely comfortable making up solutions. To do so, you need to to familiarize yourself with Molarity, Buffers and practice basic calculations.This provides you with a step-by-step guide. Eventually you should be able to dispense with this and do the calculations automatically.

Before you start:
List all of the ingredients that will be needed; note what the final concentration of each should be. Also note the total volume of the finished solution and what pH it should be.

  1. Measure out the appropriate amount for all the ingredients. (see the directions below).
  2. Dissolve all of the ingredients in about 75% of the required final volume.
  3. If any of the ingredients are very bulky, or one is a liquid that will make up a large portion of the liquid, make sure that the total volume is no more than 75% of the final needed volume. For example, if you are making 500 mls of 40% ethanol, you will initially mix 200 mls of ethanol with about 175 mls of water (not 375mls).
  4. Check the pH and correct if needed.
  5. Which acid or base to use to correct the pH will usually be given in the instructions? In some cases you will have to do this while dissolving the solids. For example, high concentrations of Tris will not fully dissolve unless you reduce the pH by adding acid. If no acid or base is mentioned, use HCl to lower the pH and NaOH to raise it.
  6. Bring to the correct volume.
  7. This should be done in a volumetric flask unless the desired volume is not one for which a flask is available, in which case use a graduated cylinder. Pour your solution into the flask and then rinse the beaker with distilled water and add that to the flask. This way you get any material that was not poured into the flask initially. Finally, bring the volume up to the desired level with water.
  8. Label the container and store as needed.
  9. Pour the solution into a storage bottle. Label with the name of the solution, the date it was made and your initials. If it needs to be kept cold, place it in a fridge or cold room.

Beginning with the first ingredient, follow the flow chart below:

  1. Are there any more ingredients to be added?
  2. Is this a directed amount? For example, do the directions say something like "add 50 grams of sucrose"?
  3. Is the needed concentration a molarity?
  4. Weigh out the appropriate amount and set aside.
  5. Is the needed concentration a percentage?
  6. Measure out the appropriate amount and set aside.
  7. Is this a dilution of a concentrated stock? Typical concentrates are running buffers or destaining solutions. They will be labeled 'Running buffer 10x' or 'Destaining 4x'. Measure out the amount needed.

Note: if none of the above choices apply to your situation, check with the instructor.

Molarity: calculating the amount needed.
First: are you working with the pure chemical (solid or liquid) i.e., you have a bottle of solid NaCl; or are you using a concentrated solution, such as a 1.5M stock solution of NaCl?
Pure chemical | Stock solution

Working from the pure chemical
You need to know three things:
The Molecular (or Formula) Weight of the compound (MW). This is usually printed on the label of the bottle.
The final Molarity needed (M). Remember milliMolar mM is 1/1000 Molar, so a 50 mM solution is 50/1000M, = 0.05M.
The final Volume needed in liters (V). Remember milliliter ml is 1/1000 Liter, so a 200 ml is 200/1000M, = 0.20 Liter.


The weight you need in grams is MW x M x V.


Note: this is true whether the material is a solid or a liquid, you need to weigh out the correct amount.

Examples:

  1. You need to make 400 mls of a 2.2 M solution of Tris (solid) Molecular weight of Tris is 121.
    Therefore the weight you need is 121 x 2.2 x 0.4 = 106.48 grams.
  2. You need to make 1.5 liters of a 75 mM solution of NaCl (solid) Molecular weight of NaCl is 58.5.
    Therefore the weight you need is 58.5 x 0.075 x 1.5 = 6.58 grams.
  3. You need to make 750 mls of 100 mM glycerol (liquid) Molecular weight of glycerol is 92.
    Therefore the weight you need is 92 x 0.1 x 0.75 = 6.9 grams.

Note: do not measure out 6.9 mls, this will not weigh the correct amount.
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Dilution to a known molarity from a concentrated stock
In some cases the stock solution is made up at a specific molarity and will be labeled as such. i.e., 2.5M NaCl. You can make dilutions of these as needed.

The three things you need to know are: the starting concentration (S), the final concentration needed (F) and the final volume (V). The amount of concentrate that you will need is V x F/S.

Example: you need to make 500 mls of 50 mM NaCl and you have on hand a 2.5M stock.

Volume needed is 500 x 0.05/2.5 = 10 mls of stock solution.

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Creating a percentage solution
Nearly all percentage solutions will be either weight:volume (w:v) or volume:volume (v:v). For a w:v solution you dissolve the appropriate weight of material into the desired final volume of solution. So a 10% w:v NaCl solution would have 10 grams of NaCl dissolve in every 100 mls of final solution. By contrast, a v:v solution contains the appropriate volume of material in the desired final volume of solution. For example a 25% v:v solution of methanol will have 25 mls of methanol in every 100 mls of final solution.

  1. Making a percentage solution from a solid or pure liquid, i.e. it is 100%.

    First, check the desired final volume (V) and the final percentage (P) of your finished solution. The amount of material you will need is V x P.

    Example: You need 500 mls of 30% ethanol (v:v).
    The amount of ethanol needed is 500 x 30% = 150 mls. Measure this out in a graduated cylinder.

    Example: If the same recipe had called for 500 mls 30% ethanol (w:v).
    Then the amount of ethanol needed is 500 x 30% = 150 grams. Weigh this in a beaker.

  2. Making a percentage solution from a pre-existing stock solution. This will be labeled as such: i.e. 60% sucrose (w:v) or 90% ethanol (v:v). You can make dilutions of these as needed.

    Note: if the stock solution is not labeled w:v or v:v generally assume that solids are dissolve as w:v and liquids as v:v.

    The three things you need to know are: the starting concentration (S), the final concentration needed (F) and the final volume (V). The amount of concentrate that you will need is V x F/S.

    Example: you need to make 500 mls of 50 mM NaCl and you have on hand a 2.5M stock.
    Volume needed is 500 x 0.05/2.5 = 10 mls of NaCl stock.

    Example: you need 1.5 liters of 10% sucrose and you have a 60% sucrose stock.
    Volume needed is 1500 x 10/60 = 250 mls mls sucrose stock.

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Concentrated stock solutions
Some stock solutions are made up at a high concentration and can simply be diluted as needed. In such cases you do not need to know the content, or the molarity of the ingredients, that has already been taken care of. For example, running buffer for electrophoresis might be made up 25 times stronger than needed. To use it, you will need to dilute it 25 times. To determine the volume of the concentrate that you need, you need to know the final volume needed (V) and the concentration of the stock (C). The volume of the stock you will need is V/C.

Example: You have a 5x stock solution of transfer buffer, and want to make up 2 liters of the final buffer. You will need 2000/5 = 400 mls of the concentrated stock solution.

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