Chapter 2:
Advantages of Fluorescence

Chapter 3:
Instrumentation

Chapter 4:
Variables of Fluorescence

Chapter 5:
Calibration and Standards

Filter fluorometers can be calibrated by a number of different techniques. The most common calibration of a filter fluorometer consists of compensating for blank (solution containing zero concentration of the substance to be read) and adjusting the instrument to reflect a known concentration of sample (the standard). Filter fluorometers can also be calibrated by correlating their raw fluorescence signal compared to a standard laboratory method like EPA method 413.1.

Fluorescence is a relative measurement and the optics and electronics of each instrument vary, certainly from manufacturer to manufacturer, but also among instruments from the same manufacturer. A fluorometer must be calibrated and recalibrated whenever the optics or filters are changed.

Fluorescence is subject to temperature and other environmental effects; it is important to calibrate the fluorometer in conditions as close as possible to the actual conditions for your study. Recall that fluorescence is an extremely sensitive measurement. Sample readings are only as accurate as the standard and blank used to calibrate the instrument. It is important to be rigorous in laboratory procedures, such as cleaning labware and carefully preparing standards.

Instrument stability is critical to accurate readings. If frequent calibration is impractical or for long-term studies or on-line monitoring, a fluorometer with stable reference circuitry (closed-loop) like the Turner Designs 10-AU or TD-4100 should be used. See section 3.53.

5.21 Standards. A blank should be obtained before measuring sample. The blank is water or matrix liquid for the sample, taken before any of the substance to be measured has been added. This liquid should be used as the make-up liquid for standards and to set the instrument’s reading to zero.

Normally, the standard will be a known concentration of the material to be quantitated. For single-point calibrations, and where a known concentration is needed, it is best to choose a standard with a concentration approximately 80% of the highest concentration to be read. A less concentrated standard may be used, but a higher concentration will provide greater accuracy.

If concentrations above the linear range are to be read, several standards should be used so a calibration curve may be prepared. For example, if the substance is linear to 250 ppm and measurable with a calibration curve to 1000 ppm, you might calibrate with a 200 ppm standard and take readings at 500, 750, and 1000 ppm for a calibration curve.

In some cases, calibration with a standard of known concentration is not necessary. In procedures such as in vivo chlorophyll or certain flow measurements, instrument sensitivity will be set with an unknown concentration. For in vivo chlorophyll measurements, for example, in most cases the "standard" will be an unknown sample from the body of water under investigation. During the sensitivity-setting procedure, while the unknown is running through the fluorometer, you should take a grab sample of the unknown immediately after it passes through the flow cell. This grab sample will be extracted later and the actual chlorophyll concentration determined. The researcher will then use a ratio calculation method to compare in vivo sample readings with extracted readings to determine the actual concentration of field samples.

Where the instrument has more than one range or "door" factor, many researchers will want to check for range-to-range correlation. Some fluorometers, such as the Turner Designs 10-AU are designed with excellent range-to-range correlation, making this kind of comparison unnecessary in most cases.

The stability of the standard is an important issue for accurate readings. Some substances, such as Rhodamine WT are stable for months. Others, such as chlorophyll will become inaccurate in as little as a few hours when exposed to light or temperature changes. Properly store standards to ensure stability or make new ones just prior to calibration. In some cases, purchasing premade standards is a practical and economical option. Contact Turner Designs for information about extracted chlorophyll and Rhodamine WT standards.

5.22 Secondary Standards. The stability and accuracy of standards is a critical factor in fluorescence measurements. In cases such as chlorophyll measurements, where standards are not stable over the long term, a stable secondary standard can considerably simplify procedures, is relatively inexpensive, and can provide confidence in the accuracy of readings.

A secondary standard is basically a substance that fluoresces at wavelengths similar to the substance being measured. Turner Designs, for example, offers secondary standards for many applications that are stable for years with no special storage requirements. Secondary standards are used in place of the primary standard after the instrument has been calibrated at least once with the primary standard.

For example, if measuring chlorophyll, the researcher would first obtain or prepare accurate chlorophyll standards and calibrate with these primary standards. (Premade, ready-to-use chlorophyll standards in 90% acetone are available from companies such as Turner Designs. Chlorophyll a in a dry powder ready for diluting in 90% acetone or methanol is available from chemical companies such as Sigma or Fluka.) The researcher would then read the secondary standard and record the concentration as compared to the primary standard. After this, the secondary standard could be used in place of the primary standard when calibrating.

Another advantage to a stable secondary standard is as a check on instrument stability. A secondary standard can be read periodically, and if the reading has not changed from the last calibration, the researcher can proceed with confidence that the instrument is providing accurate readings. If the reading has changed significantly, simply recalibrate using the secondary standard. A secondary standard is especially useful for instruments that lack reference circuitry. A secondary standard greatly minimizes the need to constantly prepare fresh standards, which is expensive and time-consuming, as well as potentially introducing error from contaminated or improperly prepared standards.

1. Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.

2. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York.

3. Id., p. 7.

4. Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.

5. G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence and Chemiluminescence: Instruments and Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca Raton, FL, 1985), pp. 45-47.

6. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.

7. Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.

8. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 67-69.

9. Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.

10. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 57-58.

11. Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, Laurin Publishing Co., Inc., Pittsfield, MA, p. 119.

12. Kodak Filters for Scientific and Technical Uses, Eastman Kodak Company, 3 ed. 1981.

13. Andover Corporation Optical Filter Guide, Andover Corporation.

14. Id.

15. Kodak Filters for Scientific and Technical Uses, Eastman Kodak Company, 3 ed. 1981.

16. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.

17. Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.

18. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 30.

19. Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.

20. Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998 communication from Molecular Probes, Eugene, Oregon).

21. Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.

22. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 172.

23. Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.

24. Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.

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