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General Fluorometer FAQ
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What volume do your instruments measure?

One of the most common questions we receive is: What volume do your instruments measure?  With some of our instruments, such as the Trilogy, 10AU and AquaFluor, the volume is determined/limited by the cuvette or test tube size.  In the case of very high concentrations you will get an “OVER” error message on these instruments, meaning you have saturated the sensor and are outside the detectable range.   In this case you need to dilute your samples and do the appropriate calculations to correct the concentration of your sample.  With our Cyclops sensors, including the C3 and C6, the answer is a little more difficult to define.  The graphic below is a simplified version of how concentration dictates the volume of water sampled.  In this example, both Cyclops are in identical beakers with equal volumes of water.  The shaded portion represents the sampled volume, which is determined by the concentration of the fluorophore present.  In example A, as a result of the low concentration, the excitation and emission light travel further through the water, resulting in a larger volume being sampled.  In example B, as a result of the high concentrations, the excitation and emission light are attenuated, resulting in a smaller volume being sampled.

Volume

The Impact of the T Factor

The Turner Designs Technical Support Department strives to keep customers as informed as possible by constantly researching, updating, reviewing, and making resources available.  A good understanding of how temperature affects fluorescence is required for fluorometric analyses and we have references in our FAQ’s and application notes stating details and formulas regarding temperature corrections.  However, this is so critical to fluorescence measurements we thought we’d review it here.  In general, as temperature increases fluorescence decreases.

For some of our environmental customers, the temperature variance and subsequent effect on fluorescence is seasonal.   For some of our industrial customers that are using our instruments in boilers and cooling towers, the temperature variance is much greater.  In both cases, the difference between the temperature of the instrument during calibration and the temperature of the sample needs to be well-documented for correcting fluorescence data thereby maximizing accuracy of concentration estimates.  

For example, a two degree change in temperature for Rhodamine WT correlates to a 5.2% change in fluorescence due to temperature (2 x 0.026, expressed as a percentage).

Other examples for some common dyes:

Fluorescein
Temp Change Reading Drop
2 °C 0.72%
20 °C 7.20%
30 °C 10.80%
50 °C 18.00%
75 °C 27.00%
PTSA
Temp Change Reading Drop
2 °C 0.25%
20 °C 2.52%
30 °C 3.78%
50 °C 6.30%
75 °C 9.45%


Here are links to a few articles with more information about temperature’s affect on fluorescence:

A Temperature Compensation Method for CDOM Fluorescence Sensors in Freshwater

FAQ's: Fluorescence is temperature sensitive - coefficients and formula

Mysteries of the Solid Secondary Standard Revealed

A Solid Secondary Standard is available for most Turner Designs fluorometers. Solid Secondary Standards provide a quick and easy way to validate instrument performance. Questions related to the Solid Secondary Standards are very common.  I don’t think a day has gone by when I did not receive a phone call or email asking about our Solid Secondary Standards.  So I hope to dispel some of the mystery and confusion surrounding them.

What are they made of?  The outer black casing of the Solid Secondary Standard is made of delrin plastic and is very durable.  The core of the Solid Secondary Standard is one of several stable fluorescent plastic materials.  It holds up to the rigors of photo-decay and as long as it is stored under ambient conditions free from dust and humidity, it will provide you with stable consistent readings for many years.

solid-secondary-standard

What is the best way to use it?  I usually recommend that you use the Solid Secondary Standard as a stable reference check of the instrument and not as a calibration tool.  It is stable and unless adjusted, it should give stable readings.  If you notice a drift in readings greater than +/-2.5% from the initial value recorded, then we suggest recalibrating or contacting technical support.

But can I use it to calibrate my instrument?  Given the nature of the Solid Secondary Standard and the fact that it is a plastic material and not the fluorophore of interest, we do not advise using it as the primary standard for calibrating your instrument.  For applications such as dyes and turbidity there are known and tested primary calibration standards that can be used for calibrating the instrument.  However, for in vivo applications - there is no calibration standard.  These samples are raw water samples and are qualitative (relative) measurements.  It is important though to make sure that the instrument’s sensitivity is appropriately set for reading samples and the Solid Secondary Standard can be used as a tool to accomplish this.  We have a great video demonstrating this for the AquaFluor, see below, and you can contact Technical Support if you have questions about our other instruments.


What are the advantages of fluorescence measurement?

Sensitivity:  Limits of detection depend to a large extent on the properties of the sample being measured, but, on the whole, fluorescence achieves a high level of sensitivity.  Detectability to parts per billion or even parts per trillion is common for most analytes.  Fluorometers achieve 1,000 to 500,000 times better limits of detection as compared to spectrophotometers.  

Specificity: Spectrophotometers merely absorb light. Spectrophotometric techniques are prone to interference problems because many materials absorb light, making it difficult to isolate the targeted analyte in a complex matrix.  Fluorometers are highly specific and less susceptible to interferences because fewer materials absorb and also emit light (fluoresce).  And, if non-target compounds do absorb and emit light, it is rare that they will emit the same wavelength of light as target compounds.

Simplicity and Speed: Fluorometry is a relatively simple analytical technique.  Fluorometry’s sensitivity and specificity reduce or eliminate the sample preparation procedures often required to concentrate analytes or remove interferences from samples prior to analysis.  This reduction in or elimination of sample preparation time not only simplifies, but also expedites the analysis.

I have heard that fluorescence is temperature sensitive. Can I correct my data to compensate for this?

Fluorescence is temperature sensitive.  As the temperature of the sample increases, the fluorescence decreases.  For greatest accuracy, record the sample temperature and correct the sensor output for changes in temperature.  
The following are Temperature Coefficients for common fluorescent applications:
gen-table-1
If you need to correct various samples to a standard temperature the equation is:

Fr = Fs [n (Ts-Tr)]

Where:
Fr = the calculated fluorescence reading at the reference temperature, Tr.
Fs = the observed fluorescence reading of the sample at the time of reading the sample temperature, Ts.
e = the base of natural log.
n = a temperature coefficient.
Ts = the sample temperature at time of reading Fs.
Tr = the reference temperature.

Note that usually when presented, n has a negative value, but here the equation is arranged in simpler form, with n being positive.  All temperatures are in degrees centigrade. For small temperature differences, the values may be used directly.  For a two-degree rise in temperature for rhodamine WT, the reading will drop 5.2% (2 X 0.026, expressed as a percentage).

I am conducting a stream study and I need a Dye Injection Pump. Does Turner Designs sell these?

We no longer carry dye injection pumps, however we recommend contacting the following manufacturer:

Fluid Metering, Inc.
5 Aerial Way, Suite 500 Syosset, NY 11791
Telephone: 1-516-922-6050
Toll-free: 1-800-223-3388
Fax: 1-516-624-8261
E-mail: pumps@fmipump.com
Web: fmipump.com

There are three basic types of constant-rate injectors: constant displacement pumps, constant-head (gravity-feed) devices, and regulated pressure systems.  Fluid Metering, Inc., should be able to advise you on which pump is best for your application.

What variables will effect the linearity of a sample?

Fluorescence intensity is typically directly proportional (linear) to concentration.  However, when a concentration is too high, light cannot pass through the sample to cause excitation; thus very high concentrations can have very low fluorescence.  This happens when the sample is “quenched”.  The fluorometer readings will decrease, even though the concentration is still increasing.  Diluting a sample 1:1 or some other convenient ratio will check linearity.  If it is linear, the reading will decrease in direct proportion to the dilution.

How does photochemical decay effect fluorescence?

Molecules with fluorescent properties can be bleached or destroyed by light (sunlight, UV, etc.).  As the molecules are destroyed fluorescence readings as a result decrease.

What factors influence in vivo chlorophyll fluorescence?

Three items that can influence the fluorescence sensitivity coefficient are:

1)    Species Composition.  The K value – calibration coefficient - is species dependent.  The ratio of chlorophyll to plant carbon can be anywhere from 25 to 100%.  Differences in cell packaging, chloroplast shape and cell morphology can also result in varying K values.

2)     Physical Environmental Parameters.  The in vivo  fluorescence efficiency of chlorophyll is dependent on light exposure and temperature.  At low light levels, algal cells can optimize the light uptake by pushing chloroplasts to the outer edge of the cell or by producing more chlorophyll per cell.  Both of these responses can result in fluorescence data that falsely represents the algal biomass.  Temperature is also a consideration.  Fluorescence is indirectly correlated to temperature.  As the temperature increases, the fluorescence decreases.  This can be corrected through temperature compensation.  The temperature coefficient for in vivo chlorophyll is 1.4%/ °C or .78%/ °F.

3)     Physiological state of the cell.  The presence of senescent cells (or dying cells) will also affect the K value.  There are three outcomes of light energy that are absorbed by chlorophyll contained in algal cells; 1) it is channeled towards photosynthesis, 2) it is given off as heat, or 3) it is re-emitted as fluorescence.  This is why healthier cells fluoresce less than senescent cells.
See our Application page for a complete description of in vivo  chlorophyll theory, measurement and calibration considerations.

What is the Max acid ratio (FM) and why is it important?

The EPA method 445.0 is available on the Application page.  This method explains in detail all the variables involved in determining Chlorophyll using the acidification method.  Pay close attention to section 10.0 for detailed explanations.  This is the method we used in our labs to calibrate and/or determine chlorophyll concentrations.

The FM value or max acid ratio value is the ratio of the response of Chlorophyll (Standard or Sample) before acidification to the response of the same (Standard or Sample) after acidification.  Typically, in natural waters you will see Fm values vary from 1 to 2.  Values near 1, indicate that you have a high concentration of pheopigments in your system.  Values near 2 indicate a lower concentration of pheopigments.  In a perfect world, the highest max acid ratio you will get is 2.1.

Why you would want to check the acid ratio of your Standard?

To determine if the Standard hasn't been degraded or converted to pheopigments (i.e. a double check to see if your calibration standard is good).