Peak-detection mode

Steady-state and peak-detecting methods may both be used in single- and multichannel systems. Advantages of the peak-detecting mode are increased analytical speed and considerably reduced sample volume. However, the advantages are offset by increased sensitivity to environmental changes (temperature, flow changes, etc.) and more complicated data evaluation. 

The automated methods presented here are CFA methods which may be operated in the steady-state or peak-detection mode. 

The required reaction time for the manual method is about 10 min, which suggests about 5 min for the steady state and 3 min for the peak-detection flow method. The slowest step of the reaction is the formation of the phosphomolybdate complex which starts after the addition of the first reagent (mixed reagent). Consequently mixing and system flow after the first reagent addition (about 1.5 min) already contribute to the reaction time leaving a necessary delay of about 3.5 min for steady state and 1.5 min for peak detection mode. 

In the peak-detecting mode, the heating procedme is more delicate than in the steady-state mode, because small changes in temperature may drastically modify the state of reaction achieved. 

Reduced flow-system lengths and, consequently, reduced flow-mixing generally cause steeper slopes of the peak fronts in the peak detection mode. 

In the steady-state mode the peak height is determined by averaging about 10 s of spectrophotometer signal well before the peak ends (synchronised by the sampler signal). It is important to exclude the very peak end, i.e., the last few seconds, because small density differences between subsequent samples or sample and wash solution often produce a small additional signal at the peak end and a negative one at the peak start (Fig. 10-26). In the peak-detection mode the real peak top has to be determined. 

In the peak-detecting mode the insertion of zero samples is not necessary because peaks are always followed by a zero (wash) reference. However, to detect possible contamination of the wash solution, a zero check is recommended at intervals (fe, every 3-4 calibrations). Standards should be inserted more frequently as compared with the steady-state mode, Le., about every lO or 20 sample. 

Using the same conditions as for the blank run, and following the same rigorously standardized schedule (see 11.1), inject an appropriate aliquot of the calibration mixture (see 7.4) into the chromatograph. Record the data in such a manner that retention times and areas for each component are obtained (peak detection mode). 

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

While our research was concerned with developing wet chemical methods, we confirmed our data with analyses from an available spark source mass spectrometer (SSMS). The SSMS operating parameters are given in Table I. The instrument used was an AEI MS-7 (I, 2) equipped with electrical detection. It was used in the peak switching mode only to provide more precise analyses. 

One limiting component of HPLC systems for the analysis of different food additives is the choice of the detector. In recent years, monitoring peak elution via the absorption of ultraviolet (UV) light has been the most common method, because the vast majority of compounds have some absorbance in the UV or the visible region. The popularity of this detection mode is primarily due to its sensitivity toward a large number of constituents in the range of 210-280 nm. 

Stop-Flow Mode The LC-ARC StopFlow controller performs two major functions. First, the controller maintains the back pressure during stop-flow data acquisition. This function is crucial for maintaining the shape and resolution of a peak. The second function is to deliver a liquid scintillation cocktail necessary for peak detection by the radiochemical detector. 

It is also worth briefly reiterating here the main operational modes for HPLC-NMR and HPLC-NMR-MS. There are currently five main options which can be employed for HPLC-NMR using either isocratic or gradient elution. These are continuous-flow, stop-flow, time-sliced stop-flow, peak collection into capillary loops for post-chromatographic analysis, and automatic peak detection with UV-detected triggered NMR acquisition. 

Figure 8.12 Chromatography of anthracene derivatives with different detection modes (A) laser-induced fluorescence (B) UV absorbance. Conditions capillary, 115.1 cm X 10 /un I.D. with 1.27-/im coating stationary phase, silicone acrylate/ethylhexyl acrylate Vy Vm = 0.65 mobile phase, acetonitrile detection, (A) laser-induced fluorescence (AeX 325 nm, Aem 380 nm), pressure 13.8 bar, (B) UV (258 nm), pressure 13.0 bar. Peaks 1, salicylate 2, anthracene-methanol 3, anthracenecarbonitrile 4, anthracene 5, fluoranthracene 6,1,2-benzanthracene 7, 9-phenylanthracene. (Reprinted from Ref. 11 with permission.)...

Unlike an optical microscope, a scanning electron microscope can reach very high depths of field (it is, for example, possible to obtain a sharp image of all the points of a cube or sphere occupying the entire image). The detection mode employed, allowing the observer to see into holes or behind peaks, enables this field depth to be exploited to a maximum. 

A variety of tools are available for the detection and characterization of the metabolites. Unless advanced data-acquisition strategies (Ch. 10.4.4) are applied, at least two LC-MS analyses are required. The first injection is performed in full-spectrum LC-MS mode, preferable using an MS system with improved full-spectrum sensitivity, such as (linear) ion trap and time-of-flight (TOP) instruments. The analysis of a blank extract next to real samples greatly facilitates peak detection and avoids further work on endogenous peaks. The data are processed to find the relevant /w/z-values of potential metabolites. These /w/z-values are used as precursor ion m/z in a subsequent (time-scheduled) product-ion LC-MS-MS analysis. Again, instruments with improved full-spectrum sensitivity, such as ion-traps, quadrapole-linear ion trap hybrids (Q-LIT), or quadrapole-TOF hybrids, are favourable. 

Fig. 3 Collection in Time Program plus Peak plus Time mode. This multimode protocol involves two collection time windows, peak detection (based on slope) within those time windows, and subfractionation of the peaks by time per tube.

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