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;

• self-quenching
• external quenching
• decreased quantum efficiency
• contaminants.

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

• Quinine Sulfate, Stock Standard: 100 mg/L. Weight out 30.2 mg quinine sulfate ^. 2H₂O and dissolve in 250 mL 0.1 N H₂SO₄.
• Quinine Standards, Working: 5, 10 and 15 mg/L. Using 100 mL volumetric flask, dilute appropriate volumes of the quinine stock standard using 0.1 N H₂SO₄ as diluent. Mix thoroughly by inversion.
• Sulfuric Acid: 0.1 N. Add 9.0 mL dilute H₂SO₄ (6 N) to 500 mL volumetric flask and dilute to the mark with distilled water. Mix.
• Extraction Solvent: Mix 3 volumes of dichloromethane with 1 volume of isopropanol. Mix thoroughly. Do not make any more than you need. (4 mL solvent/test.)
• pH 8.5, 0.1 M Ammonia Buffer: Dissolve 5.4 g NH₄Cl in 90 mL (use a 100 mL vol. flask) distilled water, then add 2.5 mL 6 N NH₄OH. Adjust to pH 8.5 using 6 N NH₄OH or 6 N HCl. Dilute to mark with distilled water and mix.
• Quinine Control: bottled quinine water (Canada Dry).
• Urine Unknown: Further explanation of this sample will be provided at the beginning of the laboratory exercise.

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;

• Emission spectra of quinine in 0.1 N H₂SO₄: Use 10 mg/L standard as originally prepared. Set the excitation wavelength to 350 nm and scan from 200 nm to 750 nm using the emission monochromator.
  
  
• Excitation Spectra of quinine in 0.1 N H₂SO₄: Using the same solution, and an emission wavelength near the maximum found from the above step, scan the excitation wavelength from 200-340 nm.
  
  
• Set the excitation wavelength to 350 nm and perform an emission scan of blank solution from 300-700 nm.
  
  
• Set the excitation wavelength to 350 nm. Set emission wavelength to the optimum wavelength found from the emission scan. Place 15 mg/L extracted standard in the cuvette and adjust controls to just keep recorder pen on scale. Now run an emission scan from 300-600 nm.
  
  
• With controls adjusted, run emission scans on all remaining solutions including control and unknown.

Calculations

• Measure the fluorescence signal of all solutions and prepare table as shown below:

• Plot corrected fluorescence signal as a function of concentration (mg/L) and determine concentration of control and unknown in mg/L.

• Identify; a) Raleigh scattering peak, b) Raman spectra, and c) second order spectra, in the blank emission spectrum.

• Comment on spectra of control and urine relative to pure standard spectra.

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?