In chemistry, dilution is the process of reducing the concentration of a substance in a solution. Chain dilution is the repeated dilution of a solution to increase the dilution factor rapidly. This procedure is usually performed in experiments that require very dilute solutions with high accuracy. For example experiments involving concentration curves on a logarithmic scale or experiments to determine the density of bacteria. Chain dilutions are widely used in scientific experiments such as biochemistry, microbiology, pharmacology, and physics.
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Method 1 of 2: Performing Basic Dilution
Step 1. Determine the appropriate diluent solution
It is very important to determine the liquid to be used for dilution. Many solutions are soluble in distilled water, but this is not always the case. If you are diluting bacteria or other cells, do so on a culture medium. The liquid you choose will be used for all chain dilutions.
If in doubt about which diluent to use, seek help or look online. Look for examples from others who have done similar chain dilutions
Step 2. Prepare a number of test tubes containing 9 mL of diluent
Test tubes are used for empty dilutions. First, put the undiluted sample into the first tube, then do the dilutions in succession to the next tubes.
- Label all tubes used before starting the dilution so you don't get confused once the experiment is running.
- Each tube will be filled with 10 times the dilution, starting from the tube whose substance has not been diluted. The dilution in the first tube is 1:10, followed by 1:100 in the second tube, 1:1000 for the third, and so on. Determine the amount of dilution that must be carried out in advance, so you don't waste the diluent solution.
Step 3. Prepare a test tube containing at least 2 mL of undiluted solution
The minimum amount of solution to be diluted required to perform this chain dilution is 1 mL. If you only use 1 mL, there will be no undiluted solution remaining. Label BLM for undiluted solutions.
Mix the solution thoroughly before starting any dilution
Step 4. Perform the first dilution
Take 1 mL of the undiluted solution from the BLM test tube with a pipette and put it into a test tube labeled 1:10 containing 9 mL of diluent, then mix thoroughly. There is now 1 mL of the undiluted solution in 9 mL of the diluent. Thus, the solution has been diluted by a dilution factor of 10.
Step 5. Perform the second dilution
For the second dilution, take 1 mL of the solution from the 1:10 tube, then put it into the 1:100 tube which also contains 9 mL of the diluent. Make sure the solution in the 1:10 tube is completely mixed before adding it to the next tube. Again, make sure the dilution in the 1:100 tube is completely mixed. The solution from the 1:10 tube was diluted 10-fold in the 1:100 tube.
Step 6. Continue this procedure to perform longer chain dilutions
This process can be repeated as many times as necessary to obtain the desired solution concentration. In experiments using concentration curves, you can perform serial dilutions to produce a number of solutions with dilutions of 1, 1:10, 1:100, 1:1,000.
Method 2 of 2: Calculating the Dilution Factor and Final Concentration
Step 1. Calculate the ratio of the last dilution in the chain dilution
The total dilution ratio can be determined by multiplying the dilution factor from each step to the final step. The mathematical illustration is with the formula Dt = D1 x D2 x D3 x … x D , Dt is the total dilution factor and D is the diluent ratio.
- For example, let's say you do a 1:10 dilution 4 times. Plug the diluent factor into the formula: Dt = 10 x 10 x 10 x 10 = 10,000
- The diluent factor in the fourth tube in this chain dilution is 1:10,000. The concentration of the substance after the dilution is 10,000 times lower than before it was diluted.
Step 2. Determine the concentration of the solution after dilution
To determine the final concentration of a solution after a chain dilution, you must know its initial concentration. The formula is Cend = Cbeginning/D, Cend is the final concentration of the diluted solution, Cbeginning is the initial concentration of the original solution, and D is the predetermined dilution ratio.
- For example: If you start with a cell solution whose concentration is 1,000,000 cells per mL and the dilution ratio is 1,000, what is the final concentration of the diluted sample?
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By using the formula:
- Cend = Cbeginning/D
- Cend = 1.000.000/1.000
- Cend = 1000 cells per mL.
Step 3. Make sure that all units are the same
When performing any calculation, make sure that the units are always the same at the end of the calculation. If the initial unit is cells per mL, make sure that the units remain the same at the end of the calculation. If the initial concentration is parts per million (bpd), then the final concentration must also be in bpj units.
