Detection, absorbance

To increase the absorbance detection sensitivity, both a collimating lens and a detection slit have been used in a PDMS chip to reduce collection of scattered light [707], An improved S/N ratio was also obtained by using tapered channel waveguides as the collimators as well as the elliptical lens for absorbance detection [708], 

To improve light collection for absorbance detection, a photosensor array was integrated on an acrylic chip with a smooth and transparent surface. This allows absorbance measurement (420-1000 nm) down to 0.0004 AU. Detection of 5 mg/ml blue dextran and 10 mg/mL each of lactic dehydrogenase (39 kDa) and P-galactosidase (116 kDa) has been carried out [240]. 

FIGURE 7.14 Micrograph of a U-shaped absorption cell with an optical path length of 1000 pm on a silica glass microchip. A 250-nM fluorescein solution was used for visualization [706], Reprinted with permission from Elsevier Science. 

A deeper channel (e.g., 100 pm) can be used to enhance absorbance detection because of the longer optical path length. However, this is feasible only when non-aqueous CE, which produces low electrical current, is used [622]. An increased path length (720 pm) needed for optical absorbance detection was also achieved by constructing a 3D fluid path [709]. 

In another approach, optical absorbance detection was achieved in an injection-molded PMMA flow cell consisting of 1800 pillars (25 pm high) that were used as diffraction elements. The LOD of Nile blue A perchlorate was determined to be 1.2 pM [713], 

Analogously to HPLC, photodiode array or multiwavelength fast scanning detectors can be used to increase the quantity and quality of information. These detectors allow the analyst to evaluate the on-line UV(-visible) spectra of the separated zones, and, by comparison with recorded reference spectra, to investigate peak purity and peak indentity. 

conductimetric detection has been used, by applying two electrodes into the capillary. In this detection mode, the separation of the high voltage part of the capillary (under dc) from the detection zone (under ac) can be accomplished also by electronic filtering. A capillary electropherograph, with a conductimetric detector, is commercially available. 

A variety of other detection techniques have been applied in CE, including laser-based thermooptical detection, refractive index detection, radioisotope detection, and, notably, mass spectrometric (MS) detection. 

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The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

N. M. Djordjevic and K. Ryan, An easy way to enhance absorbance detection on Waters Quanta-4000 capillary electi ophoresis system , ]. Liq. Chromatogr. 19 201-206 (1996). 

EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

Indirect UV absorbance detection in capillary zone electrophoresis has been used to analyze sodium alcohol sulfates. Excellent reproducibility was obtained when veronal buffer was used as UV-absorbing background electrolyte [302],... 

HPLC has also been used to determine the residue levels of dinifroaniline herbicides. Pendimethalin was quantified by HPLC under the following conditions apparatus, Spectroflow 400 solvent delivery system. Model 430 gradient former, and Kratos Model 783 with UV absorbance detection at 239 nm column, Cig reversed-phase (25cmx 3.0-mm i.d.) temperature, 40 °C mobile phase, acetonitrile-water (7 3, v/v) flow rate, 1 mL min . ... 

In the HPLC method for the simultaneous determination of dinitramine, ethalfluralin, trifluralin, pendimethalin, and isopropalin, a Spherisorb ODS-2 column (25 cm X 4.6-mm i.d.) was used the mobile phase was acetonitrile-water (11 9, v/v) at a flow rate of 1.0 mL min with UV absorbance detection at 220 nm. The average... 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

SCX column was used to separate oxalate and urate.140 In this separation, differential pulse and DC amperometric detection were compared. Differential pulse detection was found to allow better selectivity in detection. Anion exchange on Diaion CA08 was used to separate 20 carboxylic acids in the analysis of white wine, as shown in Figure 10.141 Because many carboxylic acids have a relatively weak absorbance, detection is difficult. The colorimetric detection scheme shown in the figure may be useful in some applications. 

Foret, F., Fanali, S., Nardi, A., and Bocek, P., Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection, Electrophoresis, 11, 780, 1990. 

Dasgupta PK, Genfa Z, Poruthoor SK, et al. 1998. High sensitivity gas sensors based on gas permeable liquid core waveguides and long-path absorbance detection. Submitted to Analytical Chemistry. 

The experiments below use reverse phase chromatography with bonded silica columns and uv absorbance detection. If more extensive experimental facilities are available, some additional experiments are suggested. These are concerned with the preparation and evaluation of columns, and with the use of other detectors and modes of hplc. It should be possible to complete each experiment within a three hour practical period. 

In uv absorbance detection, it is often useful to be able to detect different peaks in the chromatogram at different wavelengths. This may be because certain solutes have only a small absorptivity at the wavelength selected. At other times, a wavelength change can be used to edit out unwanted peaks. It is also useful to be able to record the spectrum of each component this can tell us the optimum detection... 

For which of the following analyses do you think that uv absorbance detection would not be suitable if uv absorbance is unsuitable suggest an alternative detector. 

Ethers contain additives to stabilise them against peroxide formation. For instance, tetrahydrofuran is commonly stabilised by the addition of small amounts of hydroquinone. This absorbs uv radiation strongly and so interferes with uv absorbance detection. It can be removed by distilling the solvent from KOH pellets. If you use inhibitor-free tetrahydrofuran, it should be stored in a dark bottle and flushed with nitrogen after each use. Any peroxides that form should be periodically removed by adsorption onto alumina. 

Detection UV absorbance detection is typically used for capillary electrophoresis. However, the short optical pathlength of the capillary results in poor detection limits... 

He, Y., Yeung, E.S., Chan, K.C., Issaq, HJ. (2002). Two-dimensional mapping of cancer cell extracts by liquid chromatography-capillary electrophoresis with ultraviolet absorbance detection. J. Chromatogr. A 979, 81-89. 

The MCLW sensor has been exploited by several groups for a wide range of experiments. However, both the design of the sensor and the detection scheme used in connection with the monitoring vary. This section reports on experiments using angular interrogation, absorbance detection and fluorescence detection. 

FIGURE 6.9 Individual flow cell employed for UV absorbance detection in /rPLC systems. The shaded channels represent fluid (sample + mobile phase) pathways the clear channels represent the light pathways. The flow cell counts with a U-shape design since liquid is transported in a U-shape within the flow cell. 

While separation takes place in the cartridge housed in the instrument, samples elute from each column through 24 individual exit ports as shown in Figure 6.5. The ports are connected to a bank of 24 flow cells employed for UV absorbance detection. Figure 6.9 depicts one of the individual cells employed. Figure 6.10 shows an overall view of the UV absorbance detection mechanism. 

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. 

FIGURE 6.17 Chromatogram overlay for 24 consecutive runs performed on a single column. (A) results of overlay for the chromatograms obtained with UV absorbance detection. Peaks are identified as (with increasing retention time) uracil (dead volume marker), methyl paraben, and propyl paraben. (B) results of overlay for chromatograms obtained from fluorescence detection (peak identified as rhodamine 110 chloride). 

Change in the absorption coefficient is found by arranging the measurements and voxel combinations in vector-matrix form as y = Ax where y is the change in absorbance detected at each source-detector pair, A is the so called system matrix derived from Lj and x is the optical parameters of interest, namely the absorption coefficients. 

J. Liu, J. Chao, G. Jiang, Y. Cai and J. Liu, Trace analysis of sulfonylurea herbicides in water by on-line continuous flow liquid membrane extraction-C18 precolumn liquid chromatography with ultraviolet absorbance detection. J. Chromatogr.A 995 (2003) 21-28. 

R. Loos, M.C. Alonso and D. Barcelo, Solid-phase extraction of polar hydrophilic aromatic sulfonates followed by capillary zone electrophoresis-UV absorbance detection and ion-pair liquid chromatography-diode array UV detection and electrospray mass spectrometry. J. Chromatogr.A 890 (2000) 225-237. 

C.W. Huck, M.R. Buchmeiser and G.K. Bonn, Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)coated silica and electrospray ionization tandem mass spectrometric detection. J. Chromatogr.A 943 (2002) 33-38. 

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