![[Readers Insight] Gradient vs. Isocratic Elution: Which to Choose?](http://www.welch-us.com/cdn/shop/articles/gradient-1080-105_750x.jpg?v=1767749867)
[Readers Insight] Gradient vs. Isocratic Elution: Which to Choose?
Author: Sepuxianyun
Previous article:
Hydrophilic Interaction Chromatography: A Nemesis to Polar Compounds
Introduction
The selection and configuration of elution conditions can be a significant source of challenge for those new to the field of chromatography. When should we choose gradient elution and when isocratic elution? This depends on the test we are conducting.
For example, if we are developing a new method for related substances, then we tend to utilize gradient elution due to the high number of potential impurity peaks. But if the analyte possesses only a single impurity with a polarity similar to the main component, isocratic elution may be more appropriate as it is sufficient to separate them and shortens analysis time. Though, in the pharmaceutical industry, the presence of only a single impurity is a rare occurrence.
Comparison of Isocratic and Gradient Elution
| Elution Mode | Advantages | Disadvantage |
|---|---|---|
| Isocratic Elution | Generally shorter total run times; does not require post-run re-equilibration. | Difficulty eluting components with widely varying polarities (low-polarity species may not elute); late-eluting peaks suffer from peak broadening. |
| Gradient Elution | Accommodates substances across a broad polarity range; maintains sharp peak shapes for both early and late eluters. | Requires post-run equilibration to reset the system, increasing the total cycle time per injection. |
What Scenarios Are Gradient and Isocratic Elution Suited for?
Isocratic Elution
- Assay Analysis: Since the target analyte is typically only the API (Active Pharmaceutical Ingredient), isocratic elution is preferred to minimize the run time per injection. The organic modifier ratio should be adjusted such that the retention time of the main peak falls within 1/3 to 2/3 of the total run time. When utilizing UPLC, run times can even be reduced to as little as one minute.
- Dissolution Testing: For drugs with low dosage strengths, HPLC is employed to determine dissolution profiles. The development strategy mirrors that of assay analysis; however, one must remain vigilant regarding solvent effects caused by the dissolution media.
- Chiral Isomer Analysis: Chiral stationary phases generally require longer equilibration times compared to C18 columns, particularly in normal-phase systems. Utilizing gradient elution with alkanes and alcohols can generate significant outgassing (bubbles), leading to baseline fluctuations. Therefore, manual premixing, ultrasonic degassing, and isocratic elution are standard. If reversed-phase gradient elution is necessary, the post-run equilibration time must be extended to ensure the column reaches equilibrium.
- Systems Utilizing Non-volatile Ion-Pair Reagents: Both cationic and anionic ion-pair reagents require extensive equilibration times. Consequently, isocratic elution is mandatory. Implementing a gradient in these systems often results in retention time instability and the frequent appearance of ghost peaks.
- Refractive Index Detection (RID): The detection principle of an RID involves comparing the refractive index between a sample cell and a reference cell. Because the reference cell contains a static mobile phase composition that cannot be adjusted in real-time, gradient elution is physically incompatible with this detector.
Gradient Elution
- Reversed-Phase Hydrophobic Interaction: This refers to standard RP-HPLC utilizing C18 columns, commonly used for related substances analysis. Since the primary driving force in a C18 system is hydrophobic interaction, the proportion of the organic phase (Methanol or Acetonitrile) is increased over time to elute weakly polar (hydrophobic) substances.
Technical Note: For a given compound, the gradient range typically spans from 100% aqueous (or 95% if the column is not 100% aqueous compatible) to 100% organic. The upper limit of the organic phase must be determined by the solubility of mobile phase additives. For example, 0.1% formic acid is highly miscible with organic solvents, allowing for 100% organic elution. However, when using phosphate buffers with acetonitrile, excessive organic proportions can cause in-line salt precipitation, leading to instrument blockages; the higher the phosphate concentration, the lower the permissible organic limit.
If modeling software is available, it can be used to predict the compound’s pH-logD profile. Generally, compounds with logD values between -2 and 5 are suitable for reversed-phase retention. A logD < -2 indicates excessive polarity (lack of retention), while a logD > 5 indicates extreme hydrophobicity (difficult elution). For multi-component mixtures without software assistance, a "scout gradient" (full gradient) should be performed first. Ideally, the first peak should elute at approximately 3×t₀ (dead time), and the final peak at approximately 2/3 of the total run time, followed by a high-organic wash to ensure total elution.
When designing gradients, the number of inflection points (steps) should be minimized to ensure method robustness. A high number of segments increases the risk of issues during method transfer, particularly due to differences in delay volume between different instrument brands. For peaks that are difficult to resolve, a shallow gradient plateau can be implemented.
- Temperature Gradient: Utilized in Gas Chromatography (GC) for solvent analysis, where the temperature is increased over time to elute substances based on their respective boiling points.
- Salt Concentration Gradient: Frequently used in Ion Exchange Chromatography (IEX). Generally, higher salt concentrations increase elution strength. The two mobile phases differ only in salt concentration, with the higher concentration acting as the strong eluent.
- pH Gradient: Also applied in IEX, where elution capacity depends on the charge state of the analyte and the type of stationary phase. This is most common in protein analysis.
Extra Words
In the process of method development, gradient optimization is often secondary to the selection of the appropriate stationary phase and mobile phase. It is a common occurrence that two peaks, which cannot be resolved regardless of gradient adjustments, separate immediately upon switching to a different column chemistry; such is the significance of selectivity (α).
Furthermore, some methods incorporate overly precise gradient steps, even as something like 11.5% acetonitrile. This is completely unnecessary and counterproductive. Aside from the inherent mixing errors of proportioning valves, if a 1% change in composition results in a loss of resolution, the method lacks the necessary robustness.
There is no "perfect" method, only the most appropriate one; the best method is the one that effectively meets the analytical objective.