STUDY GUIDE FOR EXAM IV
HPLC Methodologies

Please start studying from the slide, which has a chart on different types of HPLC methodologies.
What is the basic principle for separation in normal phase, reversed phase, ion-exchange and gel permeation chromatography?
Normal and Reversed Phase Chromatography
What is the relationship between polarity of the mobile phase vs. elution time and resolution in normal phase and reversed phase chromatography? Study slide 6 very carefully that illustrates that the use of stronger mobile phase in normal and reversed phase chromatography decrease run time but also decrease resolution and vice versa.
You should be able to calculate the strength of the mobile phase by using the equations for isolelutropic mobile phase to determine which binary mobile phase is stronger. Several examples were given in the homework to solve this. Once you calculate that strength you can easily two sets of mobile phase to determine how making a mobile phase stronger or weaker influence retention and resolution between two closely eluting peaks.
What is adsorption chromatography? What are the possible stationary phase and their order of polarity strength in adsorption chromatography?
What is partition chromatography? What are the possible stationary phases and their order of polarity strength?
What is the difference between competition and solvent interaction model in normal phase chromatography? Which model is the most accepted one?
What is the difference between partitioning and solvophobic interaction model in reversed phase chromatography? Which model is the most accepted one?
What are the three important disadvantages for normal phase chromatography?
Study how the effect of solvent strength influence resolution and run time in normal and reversed phase chromatography. Several slides were shown to illustrate this phenomenon
What is the relationship between capacity factor and polarity index (P’) in normal and reversed phase chromatography? How these equations can be used to predict the mobile phase composition. You must practice the problems solved for both normal and reversed phase chromatography? Please remember the log/antilog significant rule when solving this problem.
What is meant by the term bonded phases? Study the chemical reaction which results in the production of bonded phase for normal phase and reversed phase chromatography?
The two major type of back bones used in chromatography are silica vs. polymeric? What are the advantages and disadvantages of silica versus polymeric backbone in any type of HPLC?
What is meant by end-capping? How end-capping is done? What is the main advantage of endcapping?
Study the selectivity changes and the elution time when the stationary phase is changed in normal and reversed phase chromatography.
Identify the type of compounds which are highly selective for normal phase and reversed phase chromatography. Several examples were provided to illustrate this difference.
Use of Gradient in HPLC
What is meant by isocratic and gradient elution? What is the difference between linear convex and concave gradient? Which gradient is usually best for separation of homologues of a test mixture?
What is the major difference between high pressure and low pressure gradient in HPLC? Which one is more cost-effective (i.e., less expensive system)
Size or Gel Exclusion Chromatography
Review the basic principle of Gel or Size Exclusion Chromatography. What is importance of K value and how it is calculated for several compounds? What is the value tells us about the size of the molecule? What is meant by exclusion limit and permeation limit? What is the difference between gel filtration and gel permeation chromatography?
Ion Exchange Chromatography
--What is the difference between anion exchange and cation