Fluorometric Assay of Quinine

Purpose: Instruments capable of measuring fluorescence are known as fluorometers or fluorescence spectrometers. They all have similar construction. The basic components are: (1) a radiant energy source (usually Xe or Hg arc lamp), (2) a device to isolate the Excitation Wavelength(s) of light, (3) a sample holder, (4) a device to isolate the Emission Wavelength(s) of light, and (5) a photodetector to measure the fluorescence. Consult your textbook for general instrument schematics and further information.

"True" fluorescence spectra are obtained when a correction for the lamp output vs. wavelength is made. The spectrofluorometer used here provides this correction automatically. At very low concentrations of fluorophore, the power of the emitted light is directly proportional to the amount or concentration of fluorophore. However, a variety of conditions adversely affect this linear relationship;

As always, clean glassware and spectral purity of reagents are requirements for accurate fluorometry.

The alkaloid base quinine (or quinidine) can be extracted from an alkaline solution of a biological matrix using the dichloromethane-isopropanol solvent system. A second extraction using this solvent system with dilute acid (0.1 N H2SO4) transfers virtually all the alkaloid into the acid layer. The emission fluorescence spectrum of this acid solution can then be used to identify and quantitate the extracted compound.

References: R. F. Chen, Analytical Biochemistry 19, 374-387 (1967), I. Sunshine, Ed., Methodology for Analytical Toxicology, Chemical Rubber Co. (1976), Application Data Sheet, "Clinical Analysis for Quinidine in Blood," M8090, Beckman Instruments, Fullerton, CA.

Equipment: Varian model SF-330 spectrofluorometer, centrifuge, plastic centrifuge tubes

Samples: 10 mL quinine water, 10 mL urine


Reagents


Procedure

  1. Label a series of 16 x 125 mm screw-cap test tubes as: Blank, STD-5, STD-10, STD-15, Urine, Control.

  2. Add 2.0 mL of appropriate sample to each tube, using distilled water for the BLANK. Then add 1.0 mL ammonia buffer to each of the six tubes and mix.

  3. Add 4.0 mL of extraction solvent to all tubes, caps, and mix by inversion for 5-10 minutes.

  4. Centrifuge the tubes for 2 minutes at 500 RPM using a clinical centrifuge. Use a setting of 8 on "Speed Control."

  5. Aspirate the (UPPER) aqueous layers and then decant each (LOWER) organic layer into a clean, correspondingly labeled 16 x 125 mm screw-cap tubes.

  6. Add 4.0 mL of 0.1 N H2SO4 to each tube. Cap tubes, and mix by inversion for 5-10 minutes.

  7. Centrifuge tubes for 2 minutes and 1500 RPM.

  8. Transfer each (UPPER) aqueous layer to a labeled test tube by means of a Pasteur pipet.

  9. Start the spectrofluorometer, following the instructions as listed in the instruction manual. Consult the instruction manual for guidelines on setting slit widths and scan speeds.

  10. Measure the spectra as listed below;

Calculations

Solution Solution
Fluorescence
Total
Fluorescence
Corrected Quinine
Conc. (mg/L)
Blank      
STD-5      
STD-10      
STD-15      
Control      
Unknown      

Comments

The silica cuvettes used in this experiment are expensive. Please treat them with utmost care.

To determine the correct excitation wavelength is not a trivial process. However, a judicious selection initially may save one large amounts of time. One starting point is that of the ultraviolet absorption spectrum for the compound in question. The excitation wavelength values are logical selections. Later, these wavelengths can be verified in the excitation wavelength scan.

The selection of both excitation and emission wavelengths involves determining the sensitivity, onset of quenching, interference, etc. It is appropriate in methodology set-up to scan the solution thoroughly before deciding on fixed wavelengths for measurement.


Report

Absorption, emission, fluorescence, phosphorescence, luminescence, photoluminescence, chemiluminescence, thermal relaxation, intersystem crossing, and internal conversion are commonly used terms in spectroscopy. Define each term. How are the terms related? Explain the chemistry involved in each process. What are the time scales involved with each process?

A discussion of the kinetics of fluorescence should be included in all reports. In your conclusions, comment on the utility and sensitivity of fluorescence. Those of you who wish to do your formal report on fluorescence spectroscopy should consider the following ideas.

  1. Compare the absorbance spectrum with the fluorescence spectrum. What are the similarities and differences? Account for these phenomena.

  2. A discussion on fluorescence quenching could be given.

  3. Why do chemicals fluoresce? Do all chemical species fluoresce?

  4. What is a Jablonski diagram?