Molecular Fluorescence: Quinine Assay

Reference: D. A. Skoog, D. M. West, F. J. Holler, and S. R. Crouch, Analytical Chemistry: An Introduction, 7th ed. Chapter 23, pp. 594-631.



Quinine, C20H24N2O2, molar mass 324.43, is an alkaloid extracted from the bark of the cinchona tree. It has been used for many years as an antimalarial agent; although it does not cure malaria, it is effective in alleviating the symptoms of malarial attacks. The usual medicinal form is quinine dihydrochloride or quinine sulfate, (C20H24N2O2)2 H2SO4 2H2O, molar mass 782.97. Quinine is a strongly fluorescing compound, especially in dilute acid solution, and thus can be detected in very trace amounts.

Quinine in 0.05 M H2SO4 is said to have two analytically useful excitation wavelengths, 250 and 350 nm. Regardless of which excitation wavelength is used, the wavelength of maximum fluorescence intensity is 450 nm. The instrument used in this experiment uses glass filters (rather than some type of expensive grating or prism monochromator) to isolate the appropriate excitation and fluorescence wavelengths.


Turner Quentech Digital Filter Fluorometer.

Source: A quartz-halogen lamp, which emits intense broadband radiation from 340 nm to 750 nm.

Excitation Wavelength Selection: A narrow-band 360-nm filter with a bandpass of 40 nm.

Emission Wavelength Selection: A sharp cut-in, long-wavelength-pass filter which transmits essentially all light with wavelength > 415 nm.

Turn the instrument on at least 15 minutes before using. (The ON-OFF switch is in the back near the power cord). The instrument runs a countdown timer during which it undergoes self tests.

Preparation of Stock and Standard Solutions

1. Prepare a 1000 ppm stock solution of quinine by carefully weighing exactly 0.1207 g of quinine sulfate onto a glassine weighing paper, transferring into a 100-mL volumetric flask, and pipetting in 5.00 mL of 1 M H2SO4. (NOTE 1: Carefully dissolve all the quinine; a few squirts from a wash bottle may help to wash the solid material from the neck. Dissolve completely before diluting.) Dilute to volume with distilled water and mix thoroughly.

2. Prepare an intermediate 10.0 ppm stock solution by pipetting 5.00 mL of the 1000-ppm solution into a 500-mL volumetric flask, adding 25.0 mL of 1 M H2SO4, and diluting to volume with distilled water.

3. Prepare 0.1, 0.3, 0.5, 0.75, and 1.0 ppm solutions by pipetting 1.00, 3.00, 5.00, 7.50, and 10.00 mL of the 10 ppm solution into 100-mL volumetric flasks and diluting to volume with 0.05 M H2SO4.

4. Your unknown solution is obtained from the teaching assistants in a 500-mL volumetric flask. Add 25.0 mL of 1 M H2SO4 and dilute to volume with distilled water. Mix thoroughly.

Measurement of Emission Intensities

1. Into separate cuvettes place the blank (0.05 M H2SO4), each of the five standards and each unknown sample.

2. At the main menu, press ENTER.

3. Use the right cursor (®) to select "Quinine"; press REUTRN.

4. The instrument asks if filters are correct; press RETURN.

5. The instrument asks for the number of points for the calibration curve; use the up arrow to increase the number to 5. Press RETURN.

6. Beginning with the highest concentration standard; enter the concentration using the up-arrow to change the values. Use (¬) to move the cursor to the appropriate position. Use the up-arrow to set the appropriate units (ppm).

7. The instrument asks to insert the sample followed by RETURN.

8. Repeat for other standards.

9. Instrument asks the operator to insert the BLANK; press RETURN; the instrument displays READ and gives the value.

10. Insert the unknown and PRESS RETURN.

11. Readout shows READ and provides the value.

To compare values calculated by the instrument based on the calibration curve with values obtained based on the calibration curve that you establish.

1. At main menu, select RAW FLUORESCENCE as the units of measure.

2. The instrument requests value for the standard; press RETURN.

3. Instrument instructs that you insert the standard (USE the 1.0 ppm STANDARD), press return and the instrument adjusts internal gains.

4. The instrument asks "insert blank sample?"

5. Select YES, followed by RETURN.

6. Insert the blank and the instrument will take a reading.

7. Now insert standard solutions and unknown and record emission in fluorescence units .

8. Make at least 3 measures for each sample.

9. Construct a calibration curve and calculate the concentration of the unknown sample.


Analysis of Data

Prepare appropriate calibration curve(s) from your replicate sets of data. Depending on the nature of the data and any drift in the instrument, it may be best to average all the net emission intensities for a particular solution and to prepare one calibration curve; or it may be best to prepare several calibration curves from the separate sets of data, obtain several values for the concentration of quinine in your unknown, then average these values.

Report the concentration of quinine in your unknown in ppm.


1. Parts-per-million (ppm) is a mass-ratio unit. In dilute aqueous solutions, 1 ppm is equivalent to 1 mg/mL or 1 mg/L because the density of the solution is 1.00. A 1000 ppm solution of quinine sulfate (1.000 g of quinine sulfate per liter of solution) would be an 828.7 ppm solution of quinine. If you cannot weigh out exactly 0.1207 g of quinine sulfate, try to get it close, and simply calculate the correct values of the concentrations of the standards.

2. 1 M H2SO4 Solution. Slowly and carefully add 56 mL conc. H2SO4 to about 500 mL deionized water with stirring.

3. 0.05 M H2SO4 Solution. Add 100 mL 1 M H2SO4 to about 500 mL deionized water, mix, dilute to 2.0 L and mix thoroughly. The 0.05 M H2SO4 should have been prepared for your use. If you use all (or most of what is there), prepare a new batch.

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This page was last updated October 21, 2002