Sample-injection systems, HPLC

A flow scheme for the basic form of ion chromatography is shown in Fig. 7.3, which illustrates the requirements for simple anion analysis. The instrumentation used in IC does not differ significantly from that used in HPLC and the reader is referred to Chapter 8 for details of the types of pump and sample injection system employed. A brief account is given here, however, of the nature of the separator and suppressor columns and of the detectors used in ion chromatography. 

Sample Injection System There are in all three different modes of sample injection system that are used in HPLC, namely ... 

High Performance Liquid Chromatography (HPLC) (Chapter 30) gives an elaborate discussion of theoretical aspects. Instrumentation encompasses the various important components e.g., solvent reservoir and degassing system pressure, flow and temperature pumps and sample injection system ... 

HPLC apparatus consists of a mobile-phase reservoir, a sample injection system, a column, a detector, and a recorder. The operation of most of these components is self-evident, and a schematic diagram of an HPLC apparatus is shown in Fig. 3. 

Automated sample injection systems and multiport injection valves (HPLC) have good reproducibility so that a series of injections can be made with a... 

High-performance liquid chromatographic determination Inject an aliquot (Vi) of the soil and crop samples into the HPLC system. 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

Figure 7.5 Schematic representation of a coupled SFE-HPLC system employing a recirculating extraction manifold interfaced to HPLC via a sample injection valve. After Lynch [54]. Reprinted from T. Lynch, in Chromatography in the Petroleum Industry (E.R. Adlard, ed.), J. Chromatography Library, 56, 269-303, Copyright (1995), with permission from Elsevier...

Typical protein precipitation procedures use one volume of plasma plus three to six volumes of acetonitrile or methanol (or a mixture) with the internal standard at an appropriate concentration for the assay. Poison et al.102 reported that protein precipitation using acetonitrile eliminates at least 95% of the proteins after filtration or centrifugation, the supernatant can often be directly injected into the HPLC/MS/MS system. Usually this step is performed using 96-well plates that are ideal for semi-automation of sample preparation. Briem et al.103 reported on a robotic sample preparation system for plasma based on a protein precipitation step and a robotic liquid handling system that increased throughput by a factor of four compared to a manual system. 

In some cases, so called direct plasma injection techniques may be used23 83 104 108 instead of protein precipitation for loading plasma samples onto an HPLC/MS/MS system. Some direct plasma injection systems use a column switching technique in which the plasma is loaded onto an extraction column that retains the small molecules. The other plasma components are sent to waste and the flow is switched so that the small molecules are eluted onto an analytical column that connects to the MS/MS.23 83 108 One variation of the column switching method is turbulent flow chromatography commercialized by Cohesive Technologies (now part of Thermo, San Jose, CA).23... 

Figure 7.5 Schematic diagram of a high performance liquid chromatography (HPLC) system. The solvent(s) are pumped through the system, and the sample injected just before the column where separation occurs. Detection is often by UV/visible spectrophotometry at a fixed wavelength.

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. 

Compare HPLC with GC in terms of (a) the force that moves the mobile phase through the stationary phase, (b) the nature of the mobile phase, (c) how the stationary phase is held in place, (d) what types of chromatography are applicable, (e) application of vapor pressure concepts, (f) sample injection, (g) mechanisms of separation, (h) detection systems, (i) recording systems, and (j) data obtained. 

A guard column is a short, less-expensive liquid chromatography column that is placed ahead of the analytical column in an HPLC system. The purpose of a guard column is to adsorb and retain mixture components that would contaminate the more expensive analytical column. In-line filters are relatively coarse filters (compared to prefilters) placed in the mobile phase line to filter out particulates that maybe introduced on-line, such as from sample injection. 

Figure 6. scheme for on-line orthogonal HILIC-RP HPLC system and one detector step (A) sample injection into HILIC column (for polar analytes) and RP column (for non-polar analytes), step (B) analysis of HILIC retained analytes, step (C) analysis of RP phase retained analytes. 

HPLC system consisting of a model LC-20 AT pump unit SPD-20A uv-vis detector, 7,725 20 iL sample injection, a computer and a 10 gm alphabond Cj (300 x 3.9 mm) RP column was used. Elution was obtained by using the following gradient steps of acetonitrile and 0.03 M pH 3 phosphate buffer. Acetonitrile Buffer (15 85)... 

The majority of commercial developments which relate to the automation of GC and HPLC pay little attention to sample preparation. There are few examples where pretreatment is not required. A fully automated system was developed by Stockwell and Sawyer [23] for the determination of the ethanol content of tinctures and essences to estimate the tax payable on them. An instrument was designed and patented which coupled the sample pre-treatment modules, based on conventional AutoAnalyzer modules, to a GC incorporating data-processing facihties. A unique sample-injection interface is used to transfer samples from the manifold onto the GC column. The pretreated samples are directed to the interface vessel hy a simple hi directional valve. An ahquot (of the order of 1 ml) can then he injected on to the GC column through the capillary tube using a time-over pressure system. 

Set the flow rate of the HPLC system to 1 ml/min across a 5-pm x 250-mm x 4.6-mm Prodigy ODS-3 column (or equivalent) at ambient temperature. Set the detector at 520 nm. Inject 50 pi sample into the HPLC system and start a gradient similar to that... 

Injection volume will vary according to sample. For typical HPLC systems, injection volumes range between 1.0 and 100 pi. It is important that the analytical column not be overloaded. Therefore, when exploring conditions for a new sample, always start with lower injection volumes and adjust to high volumes as needed. As a rule of thumb for most analyses, injection volumes between 25 and 50 pi work best. 

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