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Diagnostic: Volume vs. time
Many HPLC problems are caused by contamination or deterioration of the stationary phase. In some cases, the deterioration is abrupt, resulting from injection of a single dirty or corrosive sample. These cases are usually obvious. More commonly, the deterioration is gradual, with almost imperceptible changes from one sample to the next. The source of contamination or deterioration could be either internal (mobile phase or temperature related) or external (from injected sample). Generally, deterioration caused by sample contamination correlates with the number of injections, while deterioration caused by the mobile phase correlates with time or (assuming constant flow) with the volume of mobile phase pumped.

To diagnose, carry out a series of injections with different delay times between injections. The total number of injections or time must be large enough for a measurable deterioration to occur. Then plot the pertinent measure (e.g., retention time, resolution, tailing factor, peak area, etc.) both as a function of total elapsed time (or mobile phase volume pumped) and as a function of number of injections and compare the two plots.

  • If the trend correlates better with number of injections than with elapsed time (as in the lower plot on each of the two figures shown here), that means that the number of injections has a bigger effect on your results than does the amount of solvent pumped; you should suspect that some sample component is building up on the column and affecting the chromatography.
     
  • If the trend correlates better with time than with the number of injections (as in the upper plot on each of the two figures shown here), that means that the volume of solvent pumped has a bigger effect on your results than does the number of samples injected; you should suspect that some mobile phase component is building up on the column or that the stationary phase is deteriorating under your separation conditions. In this case, a further diagnosis can be made by
    - running a series of injections,
    - reducing the flow to a bare minimum for a substantial time,
    - restoring the flow to its original value, and 
    - running another series of injections. 
    Then plot the pertinent measure both as a function of time and as a function of the volume of mobile phase pumped and compare the two plots. If the trend better correlates with time than with volume pumped, you should suspect deterioration of the column packing under the mobile phase and temperature conditions used. If the trend correlates better with mobile phase volume, you should suspect contamination of the stationary phase with some component of the mobile phase.




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