HPLC

Description

This simulation of a separation using reversed-phase high-performance liquid chromatography (RP-HPLC) follows the model described by Harris and Lucy1 using data from Shalliker, Kayillo, and Dennis.2 The approach is based on the empirical linear-solvent-strength model for reversed-phase separations which assumes a logarithmic relationship between a solute's retention factor, k, and the fraction of the organic component in the mobile phase, Ф:

log k ≈ log kw − SФ

In this empirical equation, kw is the value of k for the solute in pure water, and S is an estimate of the strength of the organic solvent. Both kw and S are determined experimentally by measuring k for a given solute at various values of Ф and plotting log k vs Ф. An equation for the linear region of this plot gives values for both S (slope) and log kw (y-intercept) for the solute.

In the simulation, the user selects a solvent dead time (tm), the number of theoretical plates (N), and the percentage of organic solvent in the mobile phase (Ф). (Note that Ф is limited to values between 55% and 80% as this is the range reported by Shalliker, Kayillo, and Dennis.2) A number of compounds can be chosen with measured values of log kw and S, or the user can add new compounds. The program then calculates the retention factor ( k = 10 (log kw – SΦ) ), the retention time ( tR = tM(k + 1) ), and the standard deviation of the peak ( σ = tR / N1/2 ) for each compound. The detector signal (y) for each compound is calculated with the following equation at each time (t) on the chromatogram:

y = [ relative area / (σ · (2π)1/2) ] · exp [ −(t − tR)2 / 2σ2 ]

The total detector signal is calculated by summing the y values for each selected compound at each time, t.

References:

1. Harris, D. C.; Lucy, C. A. High-Performance Liquid Chromatography. In Quantitative Chemical Analysis; W. H. Freeman, 2019; pp 739–742.

2. Shalliker, R. A.; Kayillo, S.; Dennis, G. R. Optimizing Chromatographic Separation: An experiment using an HPLC simulator. Journal of Chemical Education 2008, 85 (9), 1265. https://doi.org/10.1021/ed085p1265. 

Exercises and questions

1. Method Development: How does changing the percentage of organic solvent (methanol) affect the retention times of different compounds? What patterns do you observe between compound polarity and these changes, and how might this inform your approach to method development in real HPLC experiments?

2. Resolution and Separation: Select two structurally similar compounds (like phenol and p-cresol). What combination of parameters (theoretical plates, organic percentage) provides the best separation? How would you quantitatively define "best separation" in chromatography terms?

3. Structure-Retention Relationships: Compare the log kw and S values of the library compounds. What structural features seem to correlate with higher hydrophobicity (log kw)? How does the slope (S) value relate to molecular structure?

4. Peak Shape Analysis: How does the number of theoretical plates affect the chromatogram appearance? Explain the relationship between plate number, peak width, and resolution from a theoretical perspective.

5. Custom Compound Prediction: Based on patterns observed with the library compounds, predict appropriate log kw and S values for a new compound of your choice (e.g., benzyl alcohol, naphthalene). Add this compound to the simulation and evaluate how accurate your prediction was. What does this tell you about structure-retention relationships in reversed-phase HPLC?