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100 FAQs on HPLC Troubleshooting 

“The best way to win is not to fight.”

 

This principle from Sun Tzu is also 100% true for HPLC. The best way to troubleshoot is to avoid problems altogether.

 

Unfortunately, existing HPLC troubleshooting guides focus on dealing with issues after they arise.


I would like to introduce a better strategy. Predict potential issues, anticipate the results of your manipulations with HPLC columns or equipment, and avoid HPLC troubles altogether.

 

This book will guide you. I wish you good luck and all the best.

 

Sincerely yours,
The Author

 

Buy E-book on Lulu.com: 23.21$
Buy Paperback on Lulu.com: 32.90$

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Table of Contents

I. Proper Operation of Reversed-Phase Columns

  

I.1. How to begin working with a new reversed-phase column?
I.2. How does washing differ from conditioning?
I.3. How to perform the initial washing of a reversed-phase column?
I.4. What volume of mobile phase is sufficient for conditioning a new reversed-phase column?
I.5. How much time is required to condition a new reversed-phase column?
I.6. What should reversed-phase columns be washed with?
I.7. Does it happen that the column is washed for no reason?
I.8. Is there a way to reduce the time spent on washes?
I.9. What precautions need to be taken during column washing?
I.10. What should reversed-phase columns be stored in?
I.11. What causes reversed-phase columns to fail?
I.12. Reasons for the natural wear and tear of reversed phases.
I.13. How can the service life of reversed-phase columns be extended?
I.14. Can reversed-phase columns be regenerated?
I.15. Can you "flip" the column (change its inlet and outlet)? 

 

II. Buffer Solutions in HPLC

 

II.16. What is a buffer solution? How does it work?
II.17. Why do we need different types of buffer solutions?
II.18. What is the purpose of using buffer solutions in HPLC?
II.19. What are the drawbacks of using buffers?
II.20. How to choose the buffer concentration?
II.21. Which buffer is better to use – phosphate or acetate (formate) one?
II.22. Why are mixtures of sodium and potassium salts sometimes used?
II.23. For preparing a phosphate buffer, a mixture of mono- and dibasic phosphates is often used. Why?
II.24. Is it worthwhile to calculate the buffer composition with a specific pH using a formula?
II.25. Why is sodium chloride sometimes added to a phosphate buffer? Can it be avoided?
II.26. What's the purpose of using perchlorates?
II.27. Can a solution of triethylamine or any ion-pairing additive be considered a buffer?

III. The Mobile Phases in RP HPLC. Ion-pair HPLC

  

III.28. Why do they sometimes use a mixture of acetonitrile and methanol for preparing the mobile phase?
III.29. Why do they sometimes use mixtures of acetonitrile and THF for preparing the mobile phase?
III.31. Does adding butanol to the mobile phase make sense?
III.32. Why is triethylamine sometimes added to the mobile phase? What are pros and cons?
III.33. How do ion-pair reagents work?
III.34. Which ion-pair reagents are the most popular?
III.35. In which cases ion-pair HPLC is used?
III.36. The main concern with ion-pair separations is that they are poorly transferable. Can anything be done to address this?
III.37. Are ion-pair separations compatible with gradient elution?
III.38. How can ion-pair separations be made more affordable?
III.39. How justified is the use of the ion-pair mode?
III.40. How is separation temperature determined in the reversed-phase mode?
III.41. How is the separation temperature selected in reverse phase mode? Is raising the temperature truly necessary?

 

IV. FAQs to an HPLC Method Transfer Manager

  

IV.42. Why are retention times unstable? How to deal with retention time instability?
IV.43. How to deal with phase collapse (dewetting)?
IV.44. What to do if the peak area is not reproducible?
IV.45. How should you properly inject a sample during manual injection?
IV.46. How to deal with ghost peaks on the chromatogram?
IV.47. What do injectors usually get contaminated with?
IV.48. What should you do if the expected peaks aren't present?
IV.49. What to do if the peak is split? What's the reason for peak splitting?
IV.50. How to fix peak broadening in reversed-phase chromatography?
IV.51. How can a column be overloaded?
IV.52. How does the difference in viscosity between the sample diluent and the mobile phase affect the peak shape? 
IV.53. How to address peak broadening and splitting caused by column overload?

 

V. FAQs to a Technical Support Manager. Troubleshooting

  

V.54. What should be done if the pump system is not working properly? 
V.55. How to wash the pump's liquid system?
V.56. How to make sure that the pump works properly?
V.57. How to eliminate the check valve blockage?
V.58. How to replace the pump head? How to replace the inlet check valve?
V.59. What can be done with clogged inlet check valves?
V.60. Why do leaks in the liquid system happen, and how to deal with them? 
V.61. High back-pressure: is it dangerous, and how to deal with high back-pressure?
V.62. What to do if the capillary pops out of the column?
V.63. How to deal with high noise when using UV detection?
V.64. How does backpressure regulator work?
V.65. Why does noise increase when the mobile phase absorbs UV light?
V.66. How does the noise depend on wavelength?
V.67. What detector settings affect the noise, and how?
V.68. How to deal with high baseline drift when using UV detection?

VI. Proper Selection of HPLC Stationary Phases

  

VI.69. Is there a point in introducing a variety of stationary phases to the market?
VI.70. Why are stationary phases with different types of bonding chemistry produced for a single HPLC type?
VI.71. What does "complementary selectivity" mean?
VI.72. Why are more specific modes and stationary phases needed?
VI.73. Why do we need columns of different sizes, meaning different lengths and particle sizes?
VI.74. Are there "best" columns?

 

VII. HPLC Column Quality

  

VII.75. What is meant by the "quality" of a column? What makes a column considered high-quality?
VII.76. How are HPLC columns produced?
VII.77. What signs indicate issues in the column packing technology?
VII.78. What does "unstable packing" mean?
VII.79. Is it realistic to get a poorly packed column replaced by a column manufacturer?
VII.80. How can one protect themselves from buying a poorly manufactured column?
VII.81. What parameters characterize the quality of an adsorbent (a packing material)?
VII.82. Which quality parameters of the adsorbent are critical for the end-user?
VII.83. What does the chemical inertness of the adsorbent affect?

VIII. Stationary Phases for Reversed-Phase HPLC

  

VIII.84. What are the main types of reversed phases available on the market?
VIII.85. What types of bonding exist for reversed-phase silica gels?
VIII.86. What determines the retention on a given reversed phase?
VIII.87. Are there C18 phases with different retention?
VIII.88. What is the advantage of alkylamide (C16-Amide and similar) phases?
VIII.89. What is the advantage of aryl-bonded phases (PFP, PYE, and similar)?
VIII.90. What is the advantage of alkyl-bonded phases with ionic groups (C18/SAX, C18/SCX, C18/WCX)?
VIII.91. What is end-capping?
VIII.92. Is end-capping always done using TMS, or are there other reagents?
VIII.93. Are there advantages of polar end-capping?
VIII.94. How can one know the type of end-capping of a reversed-phase?
VIII.95. What do the various abbreviations in the names of reversed-phase columns signify?
VIII.96. What are the chemistry types of polymer-based reversed phases?
VIII.97. What is the point of using polymer stationary phases?
VIII.98. What are the advantages and disadvantages of using superficially porous reversed phases?

IX. Common Mistakes When Operating HPLC Equipment

  

IX.99. Poorly organized workspace.
IX.100. Not keeping a laboratory journal.
IX.101. Keeping a laboratory journal incorrectly.
IX.102. Not purging the instrument's liquid system at the end of the day. 
IX.103. Incorrect transition from the mobile phase with acidic (or alkaline) pH to the one with a neutral pH.
IX.104. Common use of dark glassware. 
IX.105. Not degassing the mobile phase manually.
IX.106. Improper preparation of the mobile phase.
IX.107. Not equalizing the temperature of the mobile phase with the one of the surrounding environment.
IX.108. Incorrect manual injection.

 

X. Useful Tips

   

X.109. Proper arrangement and placement of the HPLC instrument. 
X.110. Selecting the right capillaries and fittings. 
X.111. Biocompatible HPLC. 
X.112. A stand-alone HPLC pump for auxiliary operations.
X.113. Use of the mobile phase recycling system.

XI. Blunders and wasteful practices in HPLC

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