Frequently Asked Questions

Chlorophyll Reference Papers

Chlorophyll Standards

Using the Model 10 Analog, the 10-AU Digital, or the Trilogy Laboratory Fluorometer with EPA Method 445.0

"In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Phytoplankton by Fluorescence"1 by Elizabeth J. Arar & Gary B. Collins

The United States Environmental Protection Agency (USEPA) used the Turner Designs Model 10 Series Analog Fluorometer (Method 445.0, Section 6.1) (2) to evaluate a method for in vitro determination of chlorophyll and pheophytin a in marine and freshwater phytoplankton, Method 445.0 Rev.1.2. This method was published, post evaluation, in September 1997 and has since then been used in many laboratories, research institutions, water resource and water management facilities.

Turner Designs Model 10 Series Analog Fluorometer has been redesigned as a digital fluorometer. The newer Turner Designs Model 10-AU Digital Fluorometer performs all the functions and calculations of the previous model with added settings, a large digital display, and easy keypad functions. The Trilogy, Turner Designs' newest compact, solid state, touch screen fluorometer, is also capable of performing these and other calculations.

In addition to facilitating instrument use, there is a quick and accurate method, which was developed by Dr. Nicolas Welschmeyer (3), for measuring chlorophyll a in the presence of chlorophyll b and pheopigments. This method doesn't require the acidification step, which is described in more conventional fluorescence techniques. Conventional fluorescence methods for measuring chlorophyll a require samples to be measured twice: once before acidification and once after. And when using conventional acidification techniques, chlorophyll a can be underestimated by approximately 60% in situations where a 1:1 molar ratio of chl b/chl a is observed. The Welschmeyer method results in only a 10% overestimate of true chlorophyll a, even when working in aquatic systems that characteristically show high chl b/chl a ratios. It requires only a single fluorescence reading and is sensitive enough for estimates of euphotic zone chlorophyll a in all marine and freshwater ecosystems.

Configuring the Turner Designs Model 10-AU Digital or the Model 10 Analog Fluorometer for Method 445.0

Your fluorometer should be equipped with the following Turner Designs optical kit (or equivalent):

Optical Kit: PN: 10-037R
Lamp: PN: 10-045 Daylight White Lamp
Excitation filter: PN: 10-050R color specification 5-60
Emission filter: PN: 10-051R color specification 2-64
Reference filter: PN: 10-032 1 neutral density (1 ND), or the 10-035 2 neutral
density (2 ND), or the 10-052 color specification 3-66

Configuring the Turner Designs Trilogy Fluorometer for Method 445.0

For chlorophyll analysis using Method 445.0 the following module needs to be purchased with the Trilogy Fluorometer unit (PN: 7200-000):

Fluorescence Module: PN: 7200-040 (Chlorophyll a Acidification Module)

Using your Turner Designs Fluorometer with Method 445.0

Section 10.1 (Calibration and Standardization) of Method 445.0:

If you are using the 10-AU digital or Trilogy fluorometer you no longer need to calculate Fs. Follow the calibration instructions in the fluorometer's manual and the instrument will automatically calculate and save the Fs value. It will then use this value to calculate and display direct concentrations when measuring standards or samples. If you are using the Model 10 Analog Fluorometer, you must calculate the Fs value in this section.

Section 12.0 (Data Analysis and Calculations) of Method 445.0:

After proper calibration, the 10-AU Digital or Trilogy fluorometer will provide automatic sensitivity or range switching to give you the best resolution possible when reading standards or samples. All calculations or corrections for range or sensitivity switching will be performed by the fluorometer, so displayed values will reflect the direct concentrations of the solutions being read. If you are using the Model 10 Analog, you must manually switch sensitivity, if required, and perform the calculations necessary to correct for the sensitivity switch.

Configuring the Turner Designs Model 10-AU Digital or the Model 10 Analog Fluorometer for the NEW Chlorophyll a Method**

Optical Kit: PN: 10-040R
Excitation filter: PN: 034-0392 (436 nm)
Emission filter: PN: 034-0395 (680 nm)
Reference filter: PN: 10-032 1 neutral density (1 ND)
Lamp: PN: 10-089 Blue Mercury Vapor Lamp

Configuring the Turner Designs Trilogy Laboratory Fluorometer for the NEW Chlorophyll a Method**

For chlorophyll a analysis using the Welschmeyer Method, the following module needs to be purchased, along with the Trilogy Fluorometer unit (PN: 7200-000):

Fluorescence Module: PN: 7200-046 (Chlorophyll a Non-Acidification Module)

**The Welschmeyer chlorophyll a method requires that your fluorometer be equipped with a special optical filter kit or module, which will read chlorophyll a in the presence of chlorophyll b and pheopigments. Your 10-AU digital fluorometer should be equipped with the above Turner Designs optical filter kit (or equivalent). Your Trilogy Laboratory fluorometer should be equipped with the above-mentioned module.

Using Your Turner Designs Fluorometer with the NEW Chlorophyll a Method

For this procedure, follow the instructions in the Turner Designs procedure (P/N: 998-9000), "Measuring Extracted Chlorophyll a free from the errors associated with chlorophyll b and pheopigments." Procedures in Method 445.0 apply generally, EXCEPT you must NOT acidify your samples as described in Method 445.0 (Section 11.2.2). It is not necessary, as the special optical filter setup is designed to ONLY detect chlorophyll a and NOT chlorophyll b and/or pheopigments. It is not necessary to perform calculations described in Method 445.0 (Section 12.1). When properly calibrated with a known concentration of pure chlorophyll a, the sample reading without acidification represents the actual proportion of chlorophyll a relative to the standard.

References

1. To obtain copies of the complete EPA standard methods book, Methods for the Determination of Chemical Substances in Marine and Estuarine Environmental Samples, call the EPA in Cincinnati, Ohio at (513) 569-7562. The book contains Method 445.0 and several other useful procedures. Ask for item EPA/600/R-92/121.

2. A note about optics. For chlorophyll studies according to Method 445.0, the Turner Designs Model 10-AU digital fluorometer comes equipped with an excitation filter equivalent to the CS 5-60 excitation filter and an emission filter equivalent to the CS 2-64 emission filter (see Method 445.0, Section 6.1). We supply the F4T5D daylight white lamp.

3. The method was developed by Dr. Nicholas A. Welschmeyer of Moss Landing Marine Laboratories, Moss Landing, California. A paper by Dr. Welschmeyer, Fluorometric Analysis of Chlorophyll a in the presence of Chlorophyll b and Pheopigments, can be found in Limnology and Oceanography (1994) 39: 1985-1992.