Fluorescence detector laser-induced

The inability to detect precludes the ability to develop a separation. The selected technique is defined by the required limit of detection. If low-pg/mL levels are needed, it is fruitless to use a UV/visible absorbance detector. Laser-induced fluorescence (LIF) is usually appropriate, provided derivatization reagents are available if the solute does not have significant native fluorescence [2], Limits of detection of 10 10 M are easily achieved using LIF, provided the solute absorbs at a laser emission wavelength and has a reasonable fluorescence quantum yield. 

Photometric detectors are the most popular in CE instruments including diode array detectors. Laser-induced fluorescence (LIE) detection and electric conductivity detectors are also popular. LIE is particularly sensitive and powerful for detecting low concentration analytes. However, most analytes are not natively fluorescent and some derivatizations are necessary. Conductivity detector is useful for the detection of non-ultraviolet (non-UV) absorbing analytes such as inorganic ions or fatty acids. Both LIE detection and conductivity detectors are commercially available and easy to interface with conventional CE instruments. Electrochemical detectors are also useful for selective high-sensitivity detection. Several techniques have been developed to circumvent the problem of strong effects of electrophoretic field on electrochemical detection, but despite this, commercial electrochemical detectors are not used extensively. 

A new cyanide dye for derivatizing thiols has been reported (65). This thiol label can be used with a visible diode laser and provide a detection limit of 8 X 10 M of the tested thiol. A highly sensitive laser-induced fluorescence detector for analysis of biogenic amines has been developed that employs a He—Cd laser (66). The amines are derivatized by naphthalenedicarboxaldehyde in the presence of cyanide ion to produce a cyanobenz[ isoindole which absorbs radiation at the output of He—Cd laser (441.6 nm). Optimization of the detection system yielded a detection limit of 2 x 10 M. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Capillary electrophoresis coupled with a laser-induced fluorescence (LIF) detector has also been applied for the analysis of copper chlorophyll in olive oils. Samples were... 

The synthesis of a new near-infrared cyanine dye was monitored by CE and fluorescence detection. The chemicals structure of the dye and its synthetic precursor are depicted in Fig. 3.165. The analysis of the dye was realized in fused-silica capillaries of 75 and 100 /an i.d. The total and effective lengths of capillaries were 75 and 60 cm, respectively. The separation voltage was 30 kV and separations were carried out at ambient temperature. The running buffer was 2.5 mM Na2B407 (pH = 9.2). A near-infrared laser-induced fluorescence detector was applied. Electropherograms illustrating the separation of the dye are shown in Fig. 3.166. 

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

The wavelength accuracy and detector linearity and detector noise have the same effect on laser-induced fluorescence, as those of a UV absorbance detector. 

Many applications for ion analysis use a UV detector with indirect detection, though other electrochemical, laser-induced fluorescence (LIE), or mass spectrometry detectors have been described. The main advantage of UV detection is its availability on commercial instruments and that both UV-absorbing and non-UV-absorbing analytes may be detected. Nowadays, electrochemical detectors are also available specific background electrolytes (BGEs) must be used and the detector has to be adapted to existing CE instruments. 

Eluorescence or laser-induced fluorescence (EIF) detectors can be used in CEC to obtain higher sensitivities compared with UV detection. However, these detection systems are only limited to analytes that are intrinsically fluorescent or can be derivatized to fluorescent analogues. 

Fig. 6 Free-solution CE separation of PNA/DNA hybrid from excess PNA probe. M13 mpl8 ssDNA 4.2 X 10-8 M, and PNA probe 1.3 X 10-7 M. Detection LIF 488/520 nm. Buffer TBE, 7 M urea (pH 8.0). CE conditions 50-mm-i.d. polyacrylamide-coated capillary (27 cm in length and 20 cm to detector), 10 s gravity injection, separation voltage — 10 kV. Laser-induced fluorescence detection with excitation at 488 nm and emission at 520 nm. The buffer contained Triszborate (pH 8.0) with 7 M urea buffer. (From Ref. 37.)...

MS a more informative detector than conventional techniques such as UV absorbance, electrochemical or laser-induced fluorescence detection... 

In addition to the commercially available systems, several authors have described laboratory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown, which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, M A) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub-microliter samples. A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples. ... 

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

Since the development of HPLC as a separation technique, considerable effort has been spent on the design and improvement of suitable detectors. The detector is perhaps the second-most important component of an HPLC system, after the column that performs the actual separation it would be pointless to perform any separation without some means of identifying the separated components. To this end, a number of analytical techniques have been employed to examine either samples taken from a fraction collector or the column effluent itself. Although many different physical principles have been examined for their potential as chromatography detectors, only four main types of detectors have obtained almost universal application, namely, ultraviolet (UV) absorbance, refractive index (RI), fluorescence, and conductivity detectors. Today, these detectors are used in about 80% of all separations. Newer varieties of detector such as the laser-induced fluorescence (LIE), electrochemical (EC), evaporative light scattering (ELS), and mass spectrometer (MS) detectors have been developed to meet the demands set by either specialized analyses or by miniaturization. 

FIGURE 7.6 Schematic of a laser-induced fluorescence detector. A lamp with focusing optics and an appropriate band-pass filter could be used in place of the laser excitation when tightly collimated light is not required. The emitted fluorescence is detected by a PMT that can be operated in current mode or photon counting mode. Inset shows the mutually perpendicular arrangement of excitation, capillary, and detection optics. 

Relatively recently, AIS Sommer GmbH of Germany delivered a laser-induced fluorescence (LIP) analyzer for quality control in minerals and mineral processing (Broicher 2000). The LIP analyzer includes two light detector systems with three photomultipliers each, which evaluate three spectral bands in two time windows each. It was done in the Kiruna phosphorous iron ore mine, Sweden. The limitation of LIP analysis is that its accuracy depends on the complexity of the composition of the ore and the concentration and fluorescence properties of the critical minerals in relation to all the other minerals present. The phosphorous iron ore in Kiruna is ideal for LIP analyzes, because its iron minerals are practically non-luminescent, while magmatic apatite is strongly fluorescent with intensive emissions of Ce and Eu ". ... 

Tunable laser spectroscopic techniques such as laser-induced fluorescence (LIF) or resonantly enhanced multi-photon ionization (REMPI) are well-established mature fields in gas-phase spectroscopy and dynamics, and their application to gas-surface dynamics parallels their use elsewhere. The advantage of these techniques is that they can provide exceedingly sensitive detection, perhaps more so than mass spectrometers. In addition, they are detectors of individual quantum states and hence can measure nascent internal state population distributions produced via the gas-surface dynamics. The disadvantage of these techniques is that they are not completely general. Only some interesting molecules have spectroscopy amenable to be detected sensitively in this fashion, e.g., H2, N2, NO, CO, etc. Other interesting molecules, e.g. 02, CH4, etc., do not have suitable spectroscopy. However, when applicable, the laser spectroscopic techniques are very powerful. 

Laser-induced fluorescence (LIF) has also been utilized as a highly sensitive detection principle for CE [48-51]. However, while the LIF detector is now able to achieve zeptomole (10 21) detection limits, conventional derivatization techniques are inefficient at these exceptional levels [52]. Also, CE has successfully been coupled with mass spectrometry (MS) [53], nuclear magnetic resonance (NMR) [54, 55], near-infrared fluorescence (NIRF) [56, 57], radiometric [58], flame photometric [59], absorption imaging [60], and electrochemical (conductivity, amperometric, and potentiometry) [61-63] detectors. A general overview of the main detection methods is shown is Table 1 [64]. 

Musenga et al. [55] described a capillary electrophoresis method for determination of vigabatrin in human plasma after precolumn derivatization with 6-carbox yfluorescein-N-s ucc i n i m i d i d yl ester. Optimal separation and detection were obtained with 50 mM borate buffer (pH 9.0) containing 100 mM N-methylglucamine with laser-induced fluorescence detector (Aexc = 488 nm). The assay was rectilinear over the concentration... 

Abbreviations AOD, Acousto-optical deflection BCB, bisbenzyocyclobutadiene CCD, indirect contact conductivity detection CL, chemiluminescence ECD, electron capture detector FCS, fluorescence correlation spectroscopy FRET, fluorescence resonance energy transfer ICCD, integrated contact conductivity detection GMR, giant magnetoresistive LED-CFD, light emitting diode confocal fluorescence detector LIF, laser-induced fluorescence LOD, limit of detection MALDI, matrix-assisted laser desorption ionization PDMS, poly(dimethylsiloxane) PMMA, poly(methylmetha-crylate) SPR, surface plasmon resonance SVD, sinusoidal voltammetric detection TLS, thermal lens spectroscopy. 

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