Few laser wavelengths are available…Isn't it a limitation to LIF applications ?

It is true that there are only a few lasers available at an affordable cost, but this does not really limit the range of applications for LIF detection because :

• Picometrics surveys the laser technology very closely to offer the broadest possible range of lasers compatible with the ZETALIF detectors. Up to now, Picometrics qualified 11 single and two dual wavelength lasers between 325 and 800 nm
• Using close UV lasers, such as the 325 nm , allows to detect a lot of various molecules which naturally express fluorescence when excited at this wavelength, e.g. PAH.
• Thanks to the broad range of laser wavelengths offered by Picometrics, a lot of fluorescent dyes are available with excitation between 325 and 800 nm. Many publications also describe the labeling methods. Finally Picometrics' application lab provides the customer support necessary to select the most appropriate labeling method for each application.
• Thanks to the large number of lasers available between 325 and 800 nm and the expertise provided by Picometrics scientists in selecting the best labeling method, LIF detection is becoming a widespread technology.
  

L.I.F. detection seems to be a very sensitive method…but some people say that this detection method is limited by the difficulty to label molecules at very low concentrations… what is Picometrics scientists' opinion ?

We often heard this argument but we do not support it. Our observation based on our long experience of derivatization reactions, led us to believe that the chemistry of labeling is not different that the one of any other chemical reaction. It depends on the compounds' reactivity.
Some authors have shown that reactive dyes can label molecules at 10^-10 M. Others have shown that for less reactive dyes, an excess ( in a range of thousands or even millions folds ) allows to label molecules at a concentration as low as 10^-12 M.
However, when a large excess of dye is used, impurities potentially present in the dye, can lead to separation problems and, consequently influence the detection process.

Fluorescein based dyes are commonly used in Biology but not so often in CE or HPLC, why ?

It is right! These reagents are widespread because they react very well with Argon-ion lasers at 488 nm. They offer two main benefits:

• A lot of reactive groups can be combined with this Fluorescein nucleus, allowing derivatization of nearly all chemical functions of the different analytes.
• They have a high molecular extinction coefficient and very good quantum fluorescence yield.

Unfortunately, they have five major drawbacks :

• They are highly sensitive to pH, and can only be used at basic pH, which strongly limits their use in HPLC
• They show poor stability, mainly due to photo degradation effects
• They have to be used in large excess quantities when it comes to derivatization of molecules at low concentrations
• They are not always available at highly pure grades, and impurities generate extra peaks in chromatograms
• Not only do they fluoresce when they are in their original state, but also when they are damaged (eg. Hydrolysis), or bound to molecules, which does not make the interpretation of electropherograms or chromatograms an easy task.
  

Capillaries of various Internal Diameters can be used as detection cells in HPLC. Depending on flow rates, how does one choose the right capillary ?

At a given mobile phase flow rate, the capillary I.D. will influence two parameters:

• the so called "dead volume" which directly influences the width of chromatographic peaks
• the linear speed of the fluorophore to be detected in front of the laser beam, that directly affects sensitivity.

Consequently choosing a capillary size, is a compromise between peak height and width.
As an example, for a large number of fluorophores, one will use,

• 50 µm I.D. capillary for flow rates lower than 10 µl/mn
• 150 / 200 µm I.D. capillary for flow rates of 100 µl/mn
• 320 µm I.D. capillary for flow rates of 500 µl/mn
  

 

In HPLC, does fluorescence depend on the flow rate of the mobile phase ? Why ?

Yes it does. Photodegradation can be important because of the large amounts of exciting photons provided by a laser. If the flow rate is too low, the molecules to be detected can stay too long a time in front of the excitation beam. This can result in a permanent damage particularly with energetic lasers at near UV or UV wavelengths. In that case fluorescence limitation is a direct consequence of the low flow rate.

To optimize the detection in HPLC, a compromise between flow rate and fluorescence collection, or between light intensity and fluorescence must be found.