IV. ANALYTICAL TECHNIQUES

5. High Performance Liquid Chromatography (HPLC) including Liquid Chromatography/Mass Spectrometry (LC/MS)

  1. Define the following terms: chromatography, band broadening, void volume, UV cutoff, capacity factor (aka retention factor, k’), selectivity, isocratic elution, gradient elution, resolution.
  2. Who is primarily responsible for ensuring that laboratory instrumentation is properly maintained?
  3. According to your standard operating procedures, how often should the intensity check and wavelength calibration be performed on the HPLC’s diode array detector (DAD)?
  4. Why is it important to degas the HPLC mobile phase / solvents?
  5. Name and describe three techniques for degassing the HPLC mobile phase / solvents.
  6. When a sample is prepared for analysis using HPLC, should it be dissolved in a solvent which is weaker or stronger than the mobile phase?
  7. Arrange the following solvents in order of increasing elution strength in a reverse-phase HPLC system: methanol, isopropanol, H 2O, acetonitrile.
  8. Describe the following parts of an HPLC system: solvent/mobile phase reservoir, pumping system (aka solvent delivery system), sample injection system, column, and detector.
  9. List 5 of the most commonly used HPLC detectors.
  10. List three reasons why it is important to filter both the eluent and the sample.
  11. Which of the following techniques is the most effective method of controlling the capacity or retention of the sample molecule: decreasing the flow rate, increasing or decreasing the solvent strength, or decreasing the column oven temperature?
  12. In general , k’ values less than ____ are unreliable as your sample components may be eluting with other sample components.
  13. One advantage of using HPLC columns of small particle size is the increase efficiency. What is a disadvantage?
  14. Which of the following analytes will require the use of an RI detector? (Hint: look at values for λ max ) heroin, morphine, sucrose, LSD.
  15. What are some of the advantages that HPLC has over GC?
  16. If two solutes have a resolution of 0.7 and k’ values of less than 1, what would be the easiest approach to increase resolution?
  17. Under what circumstances would you add an amine to a mobile phase buffer?
  18. What is the purpose of a buffer in an HPLC mobile phase?
  19. What are 3 possible causes of fronting peaks and how might each be resolved?
  20. What are 3 possible causes of a retention time shift and how might each cause be resolved?
  21. What is the UV cutoff (in nm) for the following solvents? Acetonitrile, Water, Methanol, Tetrahydrofuran.
  22. What is the primary advantage that HPLC-MS has over GC-MS systems?
  23. What is the main problem encountered when combining HPLC and MS systems? How is this problem overcome?
  24. Define the three major steps involved in the production of gas-phase ions from electrolyte ions in solution by electrospray.
  25. One of the advantages of LC over GC is that certain properties of the mobile phase can be manipulated. List some of these properties.
  26. Which instrumental technique (LC or GC) will give better resolution? Explain.
  27. List some of the advantages of HPLC analysis over GC analysis when it comes to seized drugs.
  28. List some common drugs benefit from HPLC analysis.

Last Update June 2018

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