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Spectrophotometer Dilution Calculator

Work out how much to dilute a sample that reads too high on a spectrophotometer, with the sample and diluent volumes and the factor to correct the new reading.

Formula

DF=AmeasuredAtarget\mathrm{DF} = \dfrac{A_{\text{measured}}}{A_{\text{target}}}
Vsample=VfinalDFV_{\text{sample}} = \dfrac{V_{\text{final}}}{\mathrm{DF}}
Acorrected=Adilution×DFA_{\text{corrected}} = A_{\text{dilution}} \times \mathrm{DF}
target A\text{target A}
the absorbance you want to read, in the middle of the linear range (about 0.3 to 0.8)

How it works

Absorbance is proportional to concentration only in a limited range. A sample that reads 2.4 is outside it, and must be diluted to bring the reading back into range. Because absorbance is proportional to concentration, the dilution needed is the ratio of the reading you have to the reading you want.

The tool gives the sample and diluent volumes for a chosen final volume. After measuring the dilution, multiply the result by the dilution factor to get back to the original sample. Measure the diluted sample rather than trusting the calculated reading, since the original reading itself was outside the linear range.

Worked example

A culture reads A = 2.40 and you want a reading near 0.60, in 1 mL.

  1. Dilution factor = 2.40 / 0.60 = 4.
  2. Sample = 1 mL / 4 = 250 µL; diluent = 750 µL.

Mix 250 µL of sample with 750 µL of diluent. The diluted reading of about 0.60 is multiplied by 4 to report the original.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The absorbance is proportional to concentration across the range from the original reading down to the target.
  • The diluent is the same as the blank.

Common mistakes

  • Using the original reading's proportionality at A > 2, where the true absorbance is higher than measured.
  • Diluting in water when the blank is buffer, which changes the background.
  • Forgetting to multiply by the dilution factor.

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.