On-line HPLC system

The Gilson Aspec automatic sample preparation system is a fully automated system for solid-phase extraction on disposable columns and online HPLC analysis. The Aspec system offers total automation and total control of the entire sample preparation process including clean-up and concentration. In addition, Aspec can automatically inject prepared samples into on-line HPLC systems. 

Gilson. The Gilson 215 Liquid Handler is a versatile, large-capacity, septum-piercing liquid handler for safe and efficient transfer. It helps transferring samples to other analytieal systems or inject samples directly to an on-line HPLC system. 

HPLC systems were helpful in monitoring cephalosporin production [174] and p-cresol degradation [392]. Other HPLC systems have been reported to serve for the control of penicillin production, namely via the precursor feed [62, 288],3-chlorobenzoate conversion [375] or naphthalenesulfonic acid reduction [279], as well as amino acids [444,445]. HPLC is also found to be useful when linked to biochemical assays [316]. Mailinger et al. [262] have discussed many important aspects of on-line HPLC systems. 

Figure 14-9. Schematics of an on-line HPLC system. (Reprinted from reference 37, with permission.)...

Figure 21-23. Flow scheme of an on-line HPLC system developed in-house (1, mobile phase 2, HPLC pump 3, heat exchanger 4, injector with 2-p.L loop 5, HPLC-column 6, heating oven 7, UV cell 8, LFV lamp 9, UV detector 10, reference sample 11, restrictor 12, valves).

HPLC and CE may also be coupled to give an on-line multidimensional set-up [960] CZE and HPLC are highly orthogonal systems. On-line HPLC-GC has been reviewed repeatedly [49,906,961-963], as well as MDHPLC [964] various textbooks are available [965,966]. 

CE instrumentation is quite simple (see Chapter 3). A core instrument utilizes a high-voltage power supply (capable of voltages in excess of 30,000 V), capillaries (approximately 25—lOOpm I.D.), buffers to complete the circuit (e.g., citrate, phosphate, or acetate), and a detector (e.g., UV-visible). CE provides simplicity of method development, reliability, speed, and versatility. It is a valuable technique because it can separate compounds that have traditionally been difficult to handle by HPLC. Furthermore, it can be automated for quantitative analysis. CE can play an important role in process analytical technology (PAT). For example, an on-line CE system can completely automate the sampling, sample preparation, and analysis of proteins or other species that can be separated by CE. 

As explained in Sections 16.4 and 16.5, the comprehensive characterization of complex polymer systems is hardly possible by the SEC alone. SEC employs only one retention mechanism which simnltaneonsly responds to all molecular characteristics of sample. Similarly, also the coupling of the different retention mechanisms within one single column only exceptionally allows fulfilling this task. Evidently several retention mechanisms should be applied in a tandem approach that is within at least two different on-line chromatographic systems. This is the basic idea of the two- and multidimensional polymer HPLC. In the present section, the principles of two-dimensional polymer HPLC, 2D polymer HPLC or (2D-LC) will be briefly elucidated. There are several reviews available [23-31,249,250] dealing with the 2D polymers. It is anticipated that also the three- and multidimensional polymer HPLC will be developed in future. 

V. Hakkinen, M. Virolaninen, et al., New on-line HPLC-GC coupling system using a 10 port valve interface, HRC CC J. High Res. Chromatogr. Chromatogr. Commun., 77 214-216(1988). 

Data from a CD detector can be used to successfully confirm the absolute stereochemistries of the eluted enantiomers and the elution order. This important application has been introduced by Salvadori and co-workers [23], More recently it was demonstrated that an on-line detection system can be made which measures the CD spectrum of the eluate by trapping and holding it in the HPLC cell. By comparing the spectrum with data in a CD spectral library, absolute configurations can be confirmed. Where spectra are unavailable, absolute configurations might be obtained by means of either empirical and semiempirical approach or by using nonempirical methods such as the exciton model or the DeVoe approach [22, 23]. 

One can no longer imagine performing GC/MS or HPLC/MS without an on-line data system. Such a system includes an acquisition processor, a magnetic recording device and a computer with its accessories. 

For structural identification of the fractions, the XH-NMR spectrometer was directly coupled via capillary tubing to the chromatograph. The injection of the sample into the HPLC system was automatically initiated by the NMR console via a trigger pulse when starting the acquisition of NMR data. Using an appropriate pulse sequence, both solvent resonances (ACN at 2.4 ppm and water at 4.4 ppm) could be suppressed simultaneously. As a result of the on-line HPLC-NMR experiment, a contour plot XH chemical shift vs. retention time was generated (see Fig. 39). Due to the efficient solvent suppression, the obtainable structural information relates to the entire chemical shift region. From the contour... 

Mobile phases should be degassed if an on-line degasser is not available on the HPLC system. 

This suitability of this technique has also been demonstrated for bioprocess monitoring, especially when it is coupled to biosensors through FIA systems, or when an on-line HPLC separation step is interposed. 

A Zymate II Pye system for automated analysis by the Zymark Corporation (USA) with on-line HPLC has been set up and used for content uniformity tests and assays, including process validation, for a variety of drug products in our QC laboratory since January 1992. The robotic system automatically performs the following operations preparation of sample and reference standard solutions, injection into HPLC, HPLC analyses, and generation of reports. 

The PROSPEKT is a fully automated SPE system with on-line HPLC injection that can process up to 96 samples automatically (Fig. 10.5, Table 10.5). Samples are automatically loaded onto disposable cartridges, purged with one... 

Our on-line GPC/NMR system consists of a JASCO TRI ROTAR-V chromatograph and a JEOL JNM-GX500 spectrometer (a 16-bit A/D converter was installed). A 2- or 3-mm (i.d.) glass tubing with tapered structure at both ends is employed as an NMR observation flow cell (Fig. 30) the detection volumes are 0.060 and 0.140 ml, respectively. A similar type of flow cell was used in the on-line HPLC/NMR experiments.340,341 The tapered structures at both ends are needed to prevent turbulent flow in the inlet path of the cell, which would cause broadening of the elution band. When a cell without tapering was used, some broadening of the band was observed.334... 

Unlike trapped ion spectrometers or sectors, oa-ToFs also have the advantage that they scan rapidly. This makes them ideal for carrying out accurate mass measurement with on-line separation systems such as GCs or HPLC [28]. A number of commercial instmments have been launched to do this. Accurate mass measurement requires good calibration and the stability of ToFs is such that an accurate mass can be obtained without adding calibrant. However, an electrospray system has been developed which allows stream switching between the LC effluent and a suitable calibration solution [29]. This has the advantage that the calibration sample need not be compatible with the analyte as it is being analysed independently. 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

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