The Ultimate Guide to Liquid Chromatography: Your Questions Answered

Liquid Chromatography Frequently Asked Questions

What is Mobile Phase Preparation?

Mobile phase preparation involves using aqueous and organic solvents, and mixtures of these solvents, in high-performance liquid chromatography (HPLC). The method of preparation can significantly impact analysis results, as demonstrated with phosphate buffer.

Why Choose Acetonitrile Over Methanol in Reverse Phase Chromatography?

The organic solvents acetonitrile and methanol are often used as the mobile phase in reversed-phase chromatography. Commercial prices of these solvents are relatively expensive, particularly Acetonitrile for HPLC analysis. Acetonitrile seems to be used more often than Methanol in literature and conditions specified by HPLC systems manufacturers.

How to Mix Solvents Correctly in Liquid Chromatography?

Usually, an HPLC solvent is mixed in terms of relative volumes (v/v) or relative weights (w/w). We have to remember that the volume of a solution will vary at different temperatures, therefore mixing solvents using relative weights ensures a high level of reproducibility of the mixture preparation. Due to the fact that this procedure is simply more awkward the mixing of solvents using relative volume seems to be more common. Consequently, if the mixing method is not specified in test method/application, it is reasonable to assume that mixing is performed by relative volume.

Why are Buffer Solutions Important in Liquid Chromatography?

The pH value of the mobile phase (eluent) is adjusted to improve peaks separation and extend the lifetime of your column. If possible, this pH adjustment should be performed using a buffer solution (liquid), rather than simply adding a few drops of acid or a base (alkali) solution to the solvent bottle in an uncontrolled fashion. Most certainly, it is not possible to manage reproducibility of “buffered” Mobile phase preparation when there is no buffering capacity.

Is Water Grade Important in Liquid Chromatography?

Water is one of the most important solvents commonly used in liquid chromatography applications. At present, across different laboratories, various grades of water are used, either being commercially available and purchased or generated in-house using commercial water purification systems. This practice is in pursuit of the best possible solvent for a given analysis type. One must also consider the chemical properties of residual impurities within the water, including whether they are organic/inorganic, ionic/non-ionic, and UV absorbent or non-UV absorbent, when using UV detection.

How to Handle Gradient Elution with TFA?

Gradient elution using mobile phase(s) composed of water/acetonitrile containing trifluoroacetic acid (TFA) is often employed for the analysis within most of the laboratories across the country. In gradient chromatography, baseline drift and ghost peaks are most likely to appear, and there are cases where it is difficult (nearly impossible) to decide on an objective data processing approach

What are Ion Pairing Agents and How to Use Them?

Ion pair chromatography (IPC) is one of the techniques used to separate charged substances and hydrophilic species. It is widely used to selectively analyze acids and bases, particularly with reverse phase chromatography. However, it is commonly known that setting analytical conditions for IPC can be troublesome and often good reproducibility is difficult to obtain. Most of the problems occur due to insufficient consideration given to selecting the correct ion pairing agent for your mobile phases, ions with the opposite charge of target components.

How to Avoid Quantitative Errors in Sample Preparation?

Looking at the simple task of sample or standard solution preparation, we typically neglect the fact that there are multiple, complex steps included which may potentially impact the accuracy of our results. Beyond obvious such as weighing and dilution errors, the unforeseen like adsorption to surface, compound stability, if not considered, will influence results reporting, which we all want to avoid.

What Causes Ghost Peaks and How to Address Them?

Sometimes when checking chromatograms, we notice peaks of unknown origin, so-called ghost peaks. Unknown peaks can be particularly troublesome when testing pharmaceuticals for impurities, both known and unknown impurities. There are various causes of ghost peaks but one possible cause that is often neglected is dissolved air in the sample solvent.

How Do Sample Solvent and Injection Volume Affect Peak Shape?

The impact of sample solvent and injection volume on peak shape in liquid chromatography is crucial. Different solvents and volumes can significantly alter the sharpness and retention time of peaks, measured by theoretical plates. Understanding and optimizing these factors ensures accurate and reliable chromatography results.

How to Manage System Pressure Issues in Liquid Chromatography?

Managing system pressure is essential to avoid blockages and equipment damage. This involves setting the maximum pressure, identifying common causes of pressure increases, and maintaining optimal system performance.

How to Use Internal Standards in Liquid Chromatography

The internal standard method improves quantitation accuracy by compensating for injection and instrumental variability. This method involves adding a known, stable compound to both standard and sample solutions, allowing for precise and consistent results.