Concentration Measurement

Measurement of concentration of a known substance is the simplest kind of spectroscopic measurement. If you know molar extinction coefficient at any wavelength you can find concentration of sample according to Beer-Lambert law. Typically, extinction coefficients are listed for wavelength of maximal absorption.

When determining concentration it is important to subtract background absorption or to ensure that other substances do not absorb at particular wavelength.

Practical steps:

• Tryptophan:

  Abbreviation: Trp or Y
  Molecular mass: 204.2 Dalton

  1.0 M solution is 204.2 g/l or 204.2 mg/ml
  10.0 mM = 0.01 M = 2.042 mg/ml

  Measure Trp solid between 2 and 6 mg; dissolve in 2 ml of water.
  If necessary add 4-10 ml of concentrated (5 M) NaOH to increase solubility of Trp.

  Prepare 2 ml of approximately  10 mM stock solution of tryptophan (Trp) in water. Ensure that sample is fully dissolved.

• Familiarize yourself with operation of HPSpec. Turn on the spectrometer and both deuterium and tungsten lamps, allow to warm up for 20 min.

• Use quartz 1cm square cuvette. Be careful – cuvettes are fragile.  Using pipette place 2 ml of water in the cuvette. Check HP settings and measure blank as described in HPSpec section.

• Add 50 ml of Trp stock solution, mix. Mixing can be done using magnetic stirring bar, by repetitive withdrawing and dispensing 1 ml of solution using pipette, or by covering cuvette with parafilm and inverting it several time while holding parafilm down with your thumb. Before trying inversion method practice first using plastic cuvette and water.

• Coefficient of molar extinction for Trp at 279 nm is 5.57 mM^-1cm^-1;
  Actual reading is 1.47 A.U.
  Concentration in the cuvette is:
  C = 1.47 A.U. ÷ 5.57 mM^-1cm^-1 ÷ 1 cm = 0.264 mM

  0.05 ml of stock solution was diluted with 2.0 ml to a total volume of 2.05 ml or by a factor of 2.05 ÷ 0.05 = 41;Concentration of Trp in the stock before dilution is 0.264 mM × 41 = 10.8 mM.

  Replace cuvette in spectrophotometer and measure spectrum.

• After measurement is complete click on the spectrum trace and move arrow cursor to point at maximal absorption (279 nm). In the status bar read absorption value (O.D., optical density) at this position.

• Calculate actual concentration of the sample using molar extinction coefficient of 5.57 mM^-1cm^-1 at 279 nm.   

• Current concentration is 10.8 mM;
  Desired concentration is 10.0 mM;
  Required dilution is 10.8/10.0 = 1.08 times.

  2.0 ml should be diluted to final volume of 2.0 ml × 1.08 = 2.16 ml
  0.16 ml should be added to 2.0 ml to dilute stock to 10.0 mM

  Dilute stock solution to 10.0 mM concentration with deionized water.  If concentration was found to be below 10 mM, dilute to 5.0 mM.

• Repeat measurement of stock solution with new concentration; confirm that measured concentration is as expected.

• Cover /close stock solution container and label with  composition, date and your initials.