Flow cell fluorescence detection

Key words Flow cytometry, Viable cell, Fluorescence detection, Monoclonal antibody, Epitope... 

Chen et al.[58] described a sensitive and selective fluorimetric method for the determination of fluoride by measuring the fluorescence of the ternary complex of zirconium, Calcein Blue and fluoride retained on DEAE-Sephadex anion-exchanger in a 1.1 mm i.d. flow-cell. The detection limit for fluoride was 1 /g 1 at a sampling frequency of 30 h and consuming only 0.5 ml of sample. An excellent precision of 1% r.s.d. was reported, and the tolerance to most interfering ions has been improved substantially, some by an order of magnitude, compared to a manual method based on the same reaction. 

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

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. 

The combination of the specificity of the antigen-antibody interaction with the exquisite sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology (1). Within the last decade, flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa (2). 

A major contributing factor to the increased sensitivity of the improved HPLC system over that originally described (5 ) is the detector. The original method utilized a fluorescence spectrophotometer adapted for HPLC detection by use of a fabricated 40 ul flow cell. The present system utilizes a highly sensitive HPLC fluorescence detector and this contributes greatly to the improved detection limits. 

Drug residues in foods that strongly fluoresce can be more efficiently detected by fluorescence detectors. Typically, fluorescence sensitivity is 10-1000 times higher than that offered by a UV detector for strong UV-absorbing materials (125). Using a fluorescence detector, it has been possible to detect the presence of even a single analyte molecule in an LC flow cell. This type of detection is very versatile because of its ability to measure the intensity of the fluorescent radiation emitted from analytes excited by UV. 

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). 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

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