Multiple injection chromatography

A further extension of the DFG S19 method was achieved when polar analytes and those unsuitable for GC were determined by LC/MS or more preferably by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Triple-quadrupole MS/MS and ion trap MS" have become more affordable and acceptable in the recent past. These techniques provide multiple analyte methods by employing modes such as time segments, scan events or multiple injections. By improving the selectivity and sensitivity of detection after HPLC separation, the DFG S19 extraction and cleanup scheme can be applied to polar or high molecular weight analytes, and cleanup steps such as Si02 fractionation or even GPC become unnecessary. 

An extension of CC is Simultaneous Correlation Chromatography (SCC) (Smit et al. Fig. 13 shows an experimental set up. Three samples with naphthalene, anthracene and l,2-ben2anthracene are simultaneously injected, however, each controlled by a sequence uncorrelated with the others. The result is shown in Fig. 14. The peaks of naphthalene are used to construct a calibration line. The advantages are twofold The random fluctuations are reduced by multiple injection and averaging property, and both an unknown sample and calibration samples are measured simultaneously under exactly the same conditions, drift and uncertainty are reduced to a high extent. 

Another method, cross-correlation chromatography, was also reported for detection sensitivity enhancement (see Figure 7.12). Multiple injections were performed in a continuous but random sequence. A single point detection output was recorded. This output was correlated with the injection profile. This correlation enhanced the detection sensitivity due to the multiplex advantage [705]. 

Automation allows batch chromatography to be run as a continuous process. Multiple injections using a separate pump and fraction collection provide an opportunity for continuous unattended operation. In iso-cratic separations, sample injection is often made before previously injected product elutes from the column, thus reducing cycle time and solvent consumption. Continuous and automated processes are always used with smaller columns and lower amounts of expensive enantioselective stationary phases. One of the future goals for modern PHPLC optimization would be the creation of software that would allow computer simulation modeling of nonlinear effects in preparative chromatography. 

PDA analysis was also applied to the study of ribonuclease A in reversed-phase chromatography [33]. Ribonuclease A conformation has been the focus of numerous studies, and its dcnaturation is an excellent example of a reversible unfolding process. Cohen et al. [31] first reported the influence of a hydrophobic stationary phase on ribonuclease dcnaturation. In this work, the change in the 254/280 nm absorbance ratio was followed using multiple injections of the same sample. The increase in the ratio correlated extremely well with a number of other physical measurements of structural changes such as circular dichroism and intrinsic viscosity. 

The method for chloroacetanilide soil metabolites in water determines concentrations of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of alachlor, acetochlor, and metolachlor in surface water and groundwater samples by direct aqueous injection LC/MS/MS. After injection, compounds are separated by reversed-phase HPLC and introduced into the mass spectrometer with a TurboIonSpray atmospheric pressure ionization (API) interface. Using direct aqueous injection without prior SPE and/or concentration minimizes losses and greatly simplifies the analytical procedure. Standard addition experiments can be used to check for matrix effects. With multiple-reaction monitoring in the negative electrospray ionization mode, LC/MS/MS provides superior specificity and sensitivity compared with conventional liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/ultraviolet detection (LC/UV), and the need for a confirmatory method is eliminated. In summary,... 

The principle of LC LC separation with a narrow zone of an adsorb injected before sample solution called liquid chromatography under limiting conditions of desorption (LC LCD) is evident from Figure 16.12. It is also possible to apply multiple barriers in order to separate three and more components, such as statistical copolymers of different compositions or parent homopolymers from diblock copolymers [232]. The basic features of the LC LC methods are the following ... 

Use is made of this relationship in chromatography in calculating the total zone spreading that results from multiple sources. To get the total zone width a, the variances of the individual sources of zone spreading are summed and the square root is taken. For example, if a chromatograph has appreciable volumes in the tubing used to connect the column to the detector and to the injection valve, these will add to the total width of the peak. The total peak width aT is... 

By specifically and selectively reconstructing the total ion peak observed after a loop flow injection to display only the m/z values of interest, the area count for that ion can be extracted from the composite total ion peak. The measured area count can then be used to estimate the quantity of specific alkaloid by comparison to a calibration curve. Thus, component compounds in a mixture are separated by mass, as opposed to chromatography, for quantitation. To normalize the variability of the API response, an internal reference standard is added to the sample prior to loop injection. Alkaloids for which standards are not available are reported as equivalents of a closely related and available standard used to generate the calibration curve, for example, deltaline and methyllycaconitine have been used as calibration standards to represent the non-MSAL and MSAL types of alkaloids, respectively, in the plant material. Calibration curves for these two compounds were linear (r > 0.990) and there appears to be no selective suppression of lower-level alkaloids (figure 13.21). Multiple analyses of Delphinium barbeyi samples returned a level ofprecision that was less than 10 % (relative standard deviation) for all components [56]. 

High-performance liquid chromatography-tandem mass spectrometry method for the determination of gemifloxacin in human plasma was based on the protein precipitation of plasma samples with acetonitrile containing [ C Ha] gemifloxacin as an internal standard. The supernatant was injected onto a PLRP-S column without any further clean-up. The mass spectrometer was operated in positive ion mode, and the ions were detected in multiple reaction-monitoring (MRM) mode. The assay requires 50 gl of plasma and is precise and accurate within the range 10-5000 ng/ml [15]. 

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