HPLC system contamination

The number of scientific articles published on meteorites has increased dramatically in the last few years few of these, however, concern themselves with small meteorites, the size of which lies between that of the normal meteorites (from centimetres to metres in size) and that of interplanetary dust particles. In the course of an Antarctic expedition, scientists (mainly from French institutions) collected micrometeorites from 100 tons of Antarctic blue ice (Maurette et al 1991). These micrometeorites were only 100 400 pm in size five samples, each consisting of 30-35 particles, were studied to determine the amount of the extraterrestrial amino acids a-aminoisobutyric acid (AIBS) and isovaline—both of which are extremely rare on Earth—which they contained. The analysis was carried out using a well-tested and extremely sensitive HPLC system at the Scripps Institute, La Jolla. Although the micrometeorites came from an extremely clean environment, the samples must have been contaminated, as they all showed traces of L-amino acids. Only one sample showed a significantly higher concentration of AIBS (about 280 ppm). The AIBS/isovaline ratio in the samples also lay considerably above that previously found in CM-chondrites. 

A representative example of this process is shown in Fig. 2. The spectra of the analyte peaks can be measured at the upslope, the top, and at the downslope, or the whole spectrum of the chromatography peak can be compared. In the latter case, the term totalpeak purity is used, and a purity curve of the peak can also be recorded. These operations can be performed by a HPLC system equipped with a DAD detector [13], or for TLC a densitometer that can measure the UV-Vis spectrum of the analyte spot should be used. If the value of the purity is 0.000 0.8900, it is not pure, and a purity of 0.9000-0.9500 means that the peak is contaminated (Shimadzu Class-VP, Chromatography Data System). 

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. 

The quality of the sample influences the quality of the data obtained by MALDI-TOFF. Contaminations inhibit ionization. As no in-line HPLC system is included in the system, contaminants cannot be removed from the sample. Fortunately, some new clean up tools (e.g., ZipTips) for removal of contaminants have been developed recently (Figure 5.5). 

The baseline noise as offered by many UV-Vis detectors is in the range 1 to 2 x 10 5 AU and much lower than the limit of detection and quantitation required for most applications. This value is achieved under optimum conditions, such as with a reasonably new lamp, an ultraclean flow cell, stable ambient temperature, HPLC-grade solvents, and no microleaks in the entire HPLC system. These conditions are always valid at the manufacturer s final test and probably at the time of installation in the user s laboratory. However, after some time, optical and mechanical parts deteriorate (e.g., the lamp loses intensity and the flow cell may become contaminated). If we repeat the test after 3, 6, or 12 months, the noise of 1 x 10 5 AU may no longer be obtained. The recommendation is to select acceptance criteria according to the intended use of the system. 

Using the HPLC system with a mass spectrometer as a detector forces the use of volatile buffers to avoid contamination of the analyzer. The buffers are still needed in many cases to control sample or column ionization to improve the chromatography, but must be removed in some way before they reach the detector flow cell. A table of volatile buffers and their pKa s is listed in Appendix C. 

Equipment in the laboratory that comes into direct contact with a potent compound requires thorough decontamination before it can be removed from the laboratory. Some equipment cannot be adequately cleaned and must be disposed of as hazardous waste. For example, it was found that cooling fans on pieces of equipment (e.g., Endeavor Hydrogenator, HPLC systems) had spread trace levels of contamination throughout the equipment. This equipment could not be cleaned to an acceptable level and therefore had to be disposed of as hazardous waste. The use of isolators or containment laboratories, which is discussed in the next section, is an alternative approach that addresses these issues. 

Also of importance to note with the PVDF method, we observed Triton to contaminate the HPLC system, most notably the flow cell, after multiple injections. In several instances, this was serious enough to produce many high UV-absorbing peaks and prevented collection of the in-progress map. Periodic, extensive washing of the system may help resolve this. 

The fourth major simplification of the procedure is to inject directly from the crimped vial to the HPLC system without a separation step. Fig. 12.3 shows the HPLC injection needle immersed in the aqueous phase sampling through the octanol phase without cross-contamination. 

Band tailing causes inferior resolution and reduced precision. Thus conditions resulting in tailing or asymmetric peaks should be avoided. Peak asymmetry or band tailing can arise from several sources partially plugged column frits, void(s) in the column, buildup of sample components and impurities on the column inlet following multiple sample injections, sample overload, solvent mismatch with reference to the sample, chemical or nonspecific interactions (e.g., silanol effects), contamination by heavy metals, and excess void volume in the HPLC system. 

Most of the SIA-HPLC systems have been applied for the separation and assay of radionuclides. The reason for selecting such a system is the potential radiation and contamination of an operator during the sampling process. By using SIA-HPLC systems, all steps are automated and the contact of the operator with... 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

Preliminary extraction of 5-HIAA may be used as an initial purification step before HPLC analysis. Organic solvents, anion-exchange resins, and other solid phase extraction procedures have aU been used. For many systems, direct injection of urine onto the analytical column is a common practice,and samples are often merely diluted with a buffer to protect the HPLC system from contamination. Methods that analyze 5-HIAA without prior sample cleanup rely on the selectivity of the HPLC separation combined with fluorescence or electrochemical detection to provide the requisite specificity. 

Corrosive eluents such as diluted hydrochloric acid and sodium hydroxide solutions are often used in ion chromatography. Therefore, all parts of the chromatographic system being exposed to these liquids should be made of metal-free inert materials. Conventional HPLC systems with stainless steel tubings and pump heads are only partially suited for ion chromatography, since even stainless steel is eventually corroded by aggressive eluents. Considerable contamination problems would result, since metal ions... 

Fraction II was identified as sulfanilamide by comparing its HPLC retention time and its H-NMR and mass spectra with those of the reference compound. This minor metabolite was detected in the urine of the intravenously dosed cow but not in the urine of the orally dosed cow. To our knowledge, this is the first report of an animal system cleaving a sulfonamide drug at the N -position to yield sulfanilamide. We considered the possibility that sulfanilamide was a contaminant in the dose given to the cow however, this seems very unlikely because the retention times of sulfamethazine d sulfanilamide differed by 14 minutes in the HPLC system used to purify C-sulfameth-azine. 

However, one has to be careful with tests for metal cootamisatioa of the silica surface. HPLC systems are usually made of steel, as is the column hardware, including the frits. All steel will slowly bleeo metals, especially iron, which accumulates on the surface of the packing. Thus the test for metal contamination is a test of the status of the contamination of a column rather than a test of a permanent property of the packing. The test is meaningful only, when carried out using an HPLC system with a metal-free fluid path and a nonmetallic column. 

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