High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify components in a mixture. At the heart of HPLC lies the mobile phase, a critical component that plays a pivotal role in the separation process. The mobile phase is a solvent or a mixture of solvents that carries the sample through the chromatographic system.
The mobile phase serves multiple functions in HPLC
- Solvent for Sample Dissolution: The mobile phase dissolves the sample components, allowing them to be transported through the chromatographic column.
- Carrier for Sample Separation: As the sample moves through the column, the mobile phase carries it along and facilitates the separation of components based on their interaction with the stationary phase.
- Eluent for Detector Response: After separation, the mobile phase delivers the individual sample components to the detector for analysis.
Components of HPLC Mobile Phase
The mobile phase typically consists of two components:
- Solvent A: Often referred to as the "weak" solvent, it serves as the primary carrier for the sample. Common solvents include water or aqueous buffers.
- Solvent B: Known as the "strong" solvent, it aids in eluting the sample components from the column. Common solvents include organic solvents like methanol, acetonitrile, or tetrahydrofuran.
Optimizing HPLC Mobile Phase
To achieve optimal separation and analytical results, several factors related to the mobile phase must be considered:
- Selection of Solvents: Choosing appropriate solvents based on the sample's characteristics and the desired separation mechanism is crucial. Compatibility with the detector and column materials should also be considered.
- Gradient Programming: Utilizing gradient elution techniques, where the composition of the mobile phase changes over time, can enhance separation efficiency and resolution.
- pH Adjustment: Modifying the pH of the mobile phase with acids or bases can influence analyte ionization and interaction with the stationary phase, thus affecting separation outcomes.
- Degassing: Removing dissolved gases from the mobile phase is essential to prevent bubble formation, which can disrupt flow and affect chromatographic performance.
Common Challenges and Solutions
Despite its critical role, the mobile phase in HPLC can pose challenges:
- Baseline Drift: Inadequate solvent degassing or impurities in solvents can lead to baseline drift. Regular degassing and filtration of solvents can mitigate this issue.
- Poor Peak Shape: Incorrect solvent composition or column degradation can result in poor peak shapes. Adjusting mobile phase composition or replacing the column may be necessary.
- Gradient Reproducibility: Inconsistent mobile phase preparation can lead to variations in gradient profiles. Standardizing solvent mixing procedures can improve reproducibility.
Common Mistakes to Avoid When Using HPLC Mobile Phases
1. Measuring the pH of the mobile phase after the organic has been added
pH meters are calibrated to give the correct pH readback in aqueous solution – the buffers you verify this with are aqueous. If you measure the pH with the organic added, the pH will be different to that of measuring before the organic addition. However, the most important point is to be consistent. If you do always measure pH after the organic is added, make sure you state this in the method so that everyone does it the same way. It won’t be 100% accurate, but at least it will be consistent. This is probably more important than having the exact pH.
2. Not using a buffer
Buffers are present to control pH and resist a change in pH. Many other parts of the method (e.g. sample matrix, CO2 in air, source of water used for your mobile phase) can change the pH of the mobile phase causing shifts in retention, peak shape, and peak response. Formic acid, TFA etc. are not buffers.
3. Not using the buffer in its correct pH range
Each buffer salt has a 2 pH unit wide range over which it provides the optimal pH stability. Outside this window, the salt is ineffective at resisting changes in pH. Either use your buffer within the correct range or pick a buffer whose range covers the pH you require.
4. Adding buffer to organic
Mixing aqueous buffer into the organic phase carries a high risk of the buffer being precipitated – in many cases so finely that it may not be obvious it has happened. ALWAYS add the organic to the aqueous phase, this greatly reduces the risk of buffer precipitation.
5. Using the pump to mix gradients from 0%
Modern pumps are very effective at mixing mobile phases and degassing online, however not everyone who ends up using your method has a high-quality pump. Premix your A and B starting mix to a single solution that runs at 100% on line A. e.g. Prepare the starting mixture by mixing 950ml Aqueous with 50ml organic, then filter and degas. This reduces variability between HPLCs and reduces the risk of bubbles and precipitation in the system. Note however that 95:5 mixed on the pump will not give the same retention time as 95:5 premixed in the bottle – you normally need to add a few more percent organic when premixing.
6. Not using the correct pH modifying acid or base for your buffer
Only use the acid or base that forms the buffer salt you are using. E.g. sodium phosphate buffers should be adjusted with only phosphoric acid or sodium hydroxide.
7. Not stating the full information of your buffer in the method e.g. weigh 5g of sodium phosphate into 1000ml of water
The type of buffer (mono, di or tribasic) determines its pH buffering range. The required molarity is what determines the buffer strength. 5g or anhydrous sodium phosphate and 5g of monohydrate sodium phosphate will have different buffer strengths and will affect retention.
8. Filling lines with organic without checking what was in there before
If the previous method used buffer in line B and your method uses organic in line B there’s a good chance you will precipitate buffer in your pump tubing/pump head. I did it in my early days and it caused a lot of damage. If in doubt – flush it out (80:20 water : organic).
9. Propping up bottles to get the last drop out
It’s 5 to 5 and you’ve barely got enough mobile phase to finish the run – it’ll be running on fumes by the last few samples. Apart from the risk or running your pump and column dry, mobile phases evaporate from the surface, so the mobile phase at the top of the bottle will have changed the composition from the bulk. This portion from the top is exactly what will be running through the column if you use the last dregs in the bottle.
10. Using sonication to degas mobile phase
It’s great for making sure all your buffer salts have dissolved, but it’s the least effective method of degassing AND it quickly heats up the mobile phase causing the organic portion to evaporate. Save yourself problems later – take 5 minutes to vacuum filter your mobile phase – it degasses and filters in a single step.
Conclusion
The mobile phase is a fundamental component of HPLC, influencing separation efficiency, resolution, and analytical sensitivity. By understanding its role and optimizing its composition, chromatographers can achieve reliable and accurate results in their analytical endeavors.
Tags
Chromatography
