Precolumn sampling techniques

It is felt that the precolumn sampling techniques deserve much attention in future studies, as they can serve a double function in biochemical investigations (a) removal of solvents or derivatization agents and (b) protection of the analytical column from non-volatile impurities. Chemical nature of the precolumn packing can also be varied to suit a particular sample type. Further investigations aiming at the optimization and automation of the precolumn sampling techniques appear desirable. 

The most common precolumn chromatographic techniques discussed here are SFE (Section 3.4.2), SPE (Section 3.5.1) and SPME (Section 3.5.2). However, sampling methods such as thermal desorption, pyrolysis and headspace (Section 4.2.2) may also be classified in this category. 

Several precolumn derivatization techniques are available for those who wish to trade extra sample preparation time for the expense and maintenance of post column pumps and reactors. The more popular derivatives are dansyl-(32), OPA-(33), PTH-(34) and PITC-(35) amino acids. There are problems and limitations with some of these systems, however, analysis time is only 15-25 min. compared to 90-240 min. of the ion-exchange post column systems. 

Another method of reconcentrating the components at the head of the column is to keep the column temperature low enough to condense the solutes (cold trapping).A general guideline for the use of this precolumn concentration technique is that compounds with boiling points 100°C higher than the column temperature will be cold trapped. Therefore, splitless injection should be used when component concentrations are too low for detection by split injection (<0.1% sample) or when only a very limited amormt of sample is available. 

The sample introduction system must be capable of introducing a known and variable volume of sample solution reproducibly into the pressurized mobile phase as a sharp plug without adversely affecting the efficiency of the column. The superiority of valve injection has been adequately demonstrated for this purpose and is now universally used in virtually all modern instruments for both manual and automated sample introduction systems [1,2,7,31,32]. Earlier approaches using septum-equipped injectors have passed into disuse for a several reasons, such as limited pressure capability, poor resealability, contamination of the mobile phase, disruption of the column packing, etc., but mainly because they were awkward and inconvenient to use compared with valves. For dilute sample solutions volume overload restricts the maximum sample volume that can be introduced onto the column without a dramatic loss of performance. On-column or precolumn sample focusing mechanisms can be exploited as a trace enrichment technique to enhance sample detectability. Solid-phase extraction and in-column solid-phase microextraction provide a convenient mechanism for isolation, concentration and matrix simplification that are easily interfaced to a liquid chromatograph for fully or semi-automated analysis of complex samples (section 5.3.2). 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

When a first column of a very short length (and therefore a low selectivity) is used (this is especially suitable for multiresidue methods), we talk about an on-line precolumn (PC) switching technique coupled to LC (PC-LC or solid-phase extraction (SPE)-LC). This is particulary useful for the enrichment of analytes, and enables a higher sample volume to be injected into the analytical column and a higher sensitivity to be reached. The sample is passed through the precolumn and analytes are retained, while water is eliminated then, by switching the valve, the analytes retained in the precolumn are transferred to the analytical column by the mobile phase, and with not just a fraction, as in the previous cases. 

Using nano LC-MS at submicroliter per minute flow rates requires special attention to plumbing, system dead volume, valve switching, large volume sample injection, precolumn methodology, automation, online sample clean-up, and multichannel parallel operation of a single MS. The techniques discussed below are particularly useful for nano LC-MS-MS applications. 

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

Despite their distinct advantages, on-line SPE and column-switching proce-dures do not always represent ideal separation techniques. In many cases, only a small number of samples can be analyzed before contamination of the precolumn by proteins occurs. Alternative techniques that prevent the adsorption of macromolecules onto column packings and allow direct injection of sample extracts are those based on use of specific LC columns. Shielded hydrophobic phase (27), small pore reversed-phase (28), and internal surface reversed-phase (29, 30) columns can be used to elute proteins in the excluded volumes, allowing small... 

Increased use of liquid chromatography/mass spectrometry (lc/ms) for structural identification and trace analysis has become apparent. Thermo-spray lc/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions Liquid chromatography/thermospray mass spectrometric characterization of chemical adducts of DNA formed during in vitro reaction lias been proposed as an analytical technique to detect and identify those contaminants in aqueous environmental samples which have a propensity to be genotoxic, t.e.. to covalently bond to DNA. 

Sample preparation is similar to that of Basic Protocol 1. The precolumn can be viewed as the ideal technique for sample cleanup. No other sample cleanup is necessary. If the sample contains nonvolatile material, cleanup procedures 2 to 4 (see Basic Protocol 1) would help prolong the life of the precolumn, but it would not improve the quality of the separation on the analytical column. 

A multiresidue technique—matrix solid-phase dispersion (MSPD)—was used to purify the meat samples. The prewashed Cl8 bulk material was gently ground with the blended sample. The resultant C18/tissue matrix mixture was transferred to a 10-ml syringe barrel. The precolumn prepared in this way was washed with hexane, and FZD residues were eluted with dichloromethane, with 13 other veterinary drugs assayed. The recoveries varied from 44% to 87%, depending on the concentration level, and the LOD was established at 2.5 ng/kg (20). 

A schematic diagram of a heart-cut LC-LC system is depicted in Figure 5.4. The column switching technique was developed by employing two high-pressure four-way pneumatic valves inserted before and after the precolumn (39). The front-cut and the end-cut of the sample eluted from the first column were vented to waste. The valves were manipulated to transfer only the heart-cut of the analyte of interest to the analytical column. The detailed operational conditions for the four-step sequence of this system can be described as follows ... 

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