Flow cell contamination

Flow cell contamination. Clean detector cell. 

The characteristics of an equipment may change over time, e.g., LfV detector lamps lose intensity, or pump piston seals abrade, or short-term noise affecting the LOD is increased because of flow cell contamination. These changes will have a direct impact on the performance of... 

The characteristics of an equipment may change over time, e.g., UV detector lamps lose intensity, or pump piston seals abrade, or short-term noise affecting the LOD is increased because of flow cell contamination. These changes will have a direct impact on the performance of the HPLC instrument. The frequency of performance tests will be determined by experience and is based on need, type, and history of equipment performance. Intervals between the checks should be shorter than the time the instrument drifts outside acceptable limits. New instruments need to be checked more frequently, and, if the instrument meets the performance specifications, the time interval can be increased. 

Wastage was caused by classic long-term underdeposit corrosion. The combined effects of oxygen concentration cells, low flow, and contamination of system water with high chloride- and sulfate-concentration makeup waters caused corrosion. 

Do not connect the column to the detector. This precaution will prevent any contaminants precipitating in the flow cell that could damage it. 

UV detector—replace deuterium source and clean/rebuild detector flow cell if contaminated. 

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. 

The porosity of the flow cell was found to be 34.5%, and the absolute permeability was 16 darcies. These values are typical of unconsolidated media. All experiments were performed using a completely water-wet glass system. A new flow cell was constructed for each fluid pair studied to eliminate the possibility of contamination. For each individual system the cell was flushed with several pore volumes of alternating water and acetone between experiments, assuring reproducible results. 

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. 

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. 

A noisy baseline of an RI detector can be caused by inadequate mobile phase degassing or temperature thermostatting. However, low light energy can also be caused by a contaminated detector flow cell or high UV absorbance of the mobile phase (Figure 10.7d). 

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