Fluorescence flow cells

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

Four different fluorescence flow cell designs were compared to see if they were significantly different. The following results represented relative fluorescence intensities for four replicate measurements. 

A similar analysis using a Farrand fluorometer fitted with a 10-pL flow cell gave limits of fluorescence detection of 1 pmol ... 

Radiation from a xenon or deuterium source is focussed on the flow cell. An interchangeable filter allows different excitation wavelengths to be used. The fluorescent radiation is emitted by the sample in all directions, but is usually measured at 90° to the incident beam. In some types, to increase sensitivity, the fluorescent radiation is reflected and focussed by a parabolic mirror. The second filter isolates a suitable wavelength from the fluorescence spectrum and prevents any scattered light from the source from reaching the photomultiplier detector. The 90° optics allow monitoring of the incident beam as well, so that dual uv absorption and fluorescence... 

In a second experiment, Cy5-labelled antiBSA antibodies were immobilised on a silanised glass slide precoated with metallic nanoislands using a polydimethylsiloxane (PDMS) flow-cell. The antibody solution was left for 1 hour to attach and then the cell was flushed with deionised water. The slide was then dried with N2. For this experiment, a portion of the slide was not coated with metallic nanoislands, in order to act as a reference. Figure 20 shows the image recorded using the fluorescence laser scanner mentioned previously. The enhancement in fluorescence emission between those areas with and without nanoislands (B and A, respectively) is again evident. For both chips, an enhancement factor of approximately 8 was recorded. There is considerable interest in the elucidation and exploitation of plasmonic effects for fluorescence-based biosensors and other applications. 

The chemical composition of particles can be just as varied as their shape. Commercial particles can consist of polymers or copolymers, inorganic constructs, metals and semiconductors, superparamagnetic composites, biodegradable constructs, and synthetic dendrimers and dendrons. Often, both the composition of a particle and its shape govern its suitability for a particular purpose. For instance, composite particles containing superparamagnetic iron oxide typically are used for small-scale affinity separations, especially for cell separations followed by flow cytometry analysis or fluorescence-activated cell sorting (FACS). Core-shell semiconductor particles, by... 

The MCLW chip with moulded grating and flow cell is also demonstrated for fluorescence detection. Figure 15.12 shows the MCLW for fluorescence detection using different concentrations of fluorescein solutions (10 13 10 7 M). The limit of detection was determined to be 10 13 M for fluorescein solution. 

Some, uPLC systems are equipped with UV absorbance detection, and other systems allow for both UV absorbance and fluorescence detection. Fluorescence detection increases the sensitivity and selectivity of certain applications and is the method of choice in many separation-based assays. The liquid (mobile phase + sample) leaving the individual flow cells designated for UV detection is transferred through capillaries to a bank of 24 flow cells designated for fluorescence detection. 

A PMMA-Phe film was seated in the flow cell and was continuously exposed to 290 nm radiation throughout the experiment (Figure 1). The Phe fluorescence was monitored at 365 nm maximum which did not shift appreciably with a change in MEK concentration in PMMA-Phe. Therefore, the decay in the Phe fluorescence intensity provides an accurate measure of MEK diffusion and its SCP. 

The solvent pump was turned on at t 0 sec. It takes ca. 20 sec for the solvent to reach the flow cell containing the PMMA-Phe sample. A significant reduction in fluorescence intensity signals the arrival of solvent at the PMMA-Phe surface. 

Figure 1. Flow Cell for Monitoring solvent Permeation and PMMA Film Dissolution Simultaneously. The cell is placed in the sample chamber of a fluorescence spectrometer. (Reproduced with permission from Ref. ll. Copyright 1988 Wiley Sons.)...

Luminescence instrument LS-3B luminescence instrument LS-5B Accessories low flow cell, cell holders, bioluminescence spectroscopy, fluorescence spectro scopy, recorder/printers, low-temperature luminescence, fluorescence plate reader, polarization accessory, microfilm fluorimeter LS-2B... 

Radiation from a Xenon-radiation or a Deuterium-source is focussed on the flow cell through a filter. The fluorescent radiation emitted by the sample is usually measured at 90° to the incident beam. The second filter picks up a suitable wavelength and avoids all scattered light to reach ultimately the photomultiplier detector. 

For a fluorescence detector, quinine sulfate is used as the standard compound. The flow cell is filled with a standard solution and the fluorescence intensity is measured. The value is compared with that measured by a fluorescence spectrophotometer. This standard solution is also used for fixing the wavelength and position of the flow cell. The Raman spectrum of water can also be used for this purpose. 

A fluorescence-activated cell sorter (FACS) is a flow cytometry instrument used to separate and identify cells in a heterogeneous population. Cell mixtures to be sorted are first bound to fluorescent dyes such as fluorescein or phycoerythrin. The labeled cells are then pumped through the instrument and are excited by a laser beam. Cells that fluoresce are detected, and an electrostatic charge is applied. The charged cells are separated using voltage deflection. 

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