Credit: ACD/Labs
by Baljit Bains, Marketing Communications Specialist, ACD/Labs
In chromatography, nothing is more coveted than a sharp symmetrical shape on a flat baseline—the Gaussian peak. Ideal peak shape is essential to achieve enhanced resolution (Rs) and improved accuracy in quantitative analysis. The allure of the Gaussian peak is heightened by the common occurrence of peak abnormalities (fronting, tailing, and splitting). Peak abnormalities can occur for one, a few, or all peaks and are indicative of whether the problem is arising prior to or after separation. Close attention must be paid to the samples and columns used in chromatography experiments to minimize peak abnormalities.
What is peak fronting?
Peak fronting occurs when an asymmetric peak is broader in the first half and narrower in the second half. Several factors may cause peak fronting:
- Poor Sample Solubility: If the sample has poor solubility, it cannot be evenly dissolved into the mobile phase. This can be resolved by either reducing the injected sample’s volume or the solute’s concentration.
- Column Collapse: This is a sudden physical change in the column which can be due to inappropriate conditions such as the temperature or pH of the column. Column collapse can be avoided by modifying the method so that the column is used within recommended limits, replacing the column with a more robust column, or routinely replacing the column (i.e., every 500 injections).
- Saturation/Overload of the Column: This is where the maximum sample capacity is exceeded, causing saturation of the mobile phase. If this happens, the additional molecules cannot partition between the stationary and mobile phases and will elute faster. This can be prevented by reducing the amount of sample loaded on the column.
What is Peak Tailing?
Peak tailing is an asymmetrical peak, with a second half that is broader than the front half. It is difficult to eliminate all peak tailing. Quantifying tailing allows for an acceptable peak tailing limit to be established. There are two main methods for defining peak tailing: The Tailing Factor and the Asymmetry Factor.
The Tailing Factor (Tf) measures the peak width at 5% of the peak height and is widely used in the pharmaceutical industry— Tf = (a+b)/2a, where a is the width of the front half of the peak and b is the width of the back half of the peak. Whereas, the Peak Asymmetry Factor (As) measures the peak width at 10% of the peak height, and is used in most industries outside of pharma— As = b/a.
Either Tf or AS can be used to measure peak tailing, but not interchangeably. Good peak shape can be defined by a tailing factor of 1.0, high efficiency, and narrow peak width. When Tf or AS is less than 1, net fronting can occur. Conversely, when Tf or AS is greater than 1, net tailing can occur.
Peak tailing can occur for one, a few, or all peaks in a chromatogram, depending on the causing factor:
Secondary Interactions – Strong interactions between acidic silanol groups on column packing and basic functional groups of the analyte create secondary analyte interactions. Resultantly, not all molecules will travel through the column at the same speed, and this causes peak tailing. Secondary interactions can be minimized by:
- Operating at a lower pH—Performing chromatographic separation at lower pH can ensure silanol groups are protonated.
- Using a Highly Deactivated Column—Utilize an “end-capped” column to reduce surface activity. End-capping involves converting residual silanol groups to less polar surface functional groups.
- Adding Buffers to the Mobile Phase of the Chromatography System—Using buffers in mobile phases can control pH and mask residual silanol interactions.
Packing Bed Deformation—This can occur as the result of the creation of a void at the inlet of the column, the presence of channels in the packing bed, or a collection of particles at the inlet frit. To resolve this issue, determine whether there is a column void or blocked inlet frit. This can be done by substituting the column. If there is a suspected void, then reverse the column and wash with a strong solvent to remove any blocking contamination. Regularly replacing solvent filters and using in-line filters and guard columns can help avoid blockage of column frits.
Column Overload—If all peaks tail, then consider the possibility that the column has been mass overloaded. Column overload can be assessed by diluting the sample and re-assessing the resulting peak shapes. It can be prevented by using a higher capacity stationary phase either with increased % carbon or pore size, using a column with a larger diameter, or decreasing the amount of sample introduced to the column.
Excessive Column Dead Volume—This usually affects the peak shape of early eluting peaks. To avoid excessive dead volume in the column, exercise extra caution when pre-packing the column.
Impurities—Contaminants in the material can enhance secondary interactions and cause peak tailing. Using the purest packing material possible will help to reduce the presence of contaminants.
Why is Peak Tailing a Problem?
Peaks are Harder to Integrate—The transition from the baseline to peak or vice versa is much more gradual, making the peak harder to integrate. Peak tailing also causes sloping baselines which makes it difficult to determine peak limits.
Shorter Peaks—Peaks with larger Avalues tend to have shorter peak heights. Peak height is a limiting factor in determining detection limits, so method limits can be affected.
Larger Time Window to be Eluted—Peaks with tails can take longer to return to baseline resolution between peaks and therefore the sample will require a longer run time. Inaccurate assignment of the end of the peak may cause the peak area to be miscalculated.
What is Peak Splitting?
Peak splitting is when a shoulder or ‘twin’ appears on a Gaussian peak and can indicate deficiencies in method development. Split peaks can be caused by column overload, a mismatch between the strength of the mobile phase and injection solvent, a void/channel in the column, or a plugged frit.
How Many Peaks Are Affected and How to Fix Peak Splitting— If only a single peak has split, the problem is likely due to the separation itself. It may be the case that two components are eluting close together. One way to check this is by injecting a smaller sample volume and observing whether this results in two distinct peaks. It may also be that the mobile phase and sample solvent are incompatible. Here, the solvent needs adjusting, and samples need to be injected into the mobile phase. To improve separation resolution, parameters like mobile phase, temperature, flow rate, or column type need to be reconsidered.
If there is peak splitting for all the peaks, a problem occurs before separation and affects every peak similarly. The two common causes of this are a blocked frit and a void in the packing at the head of the column.
Blocked Frit—This is where part of the sample is delayed in entering the column and causes the sample delivery to the column to be spread out thus affecting sample separation and causing all the peaks to split. It can be resolved by using in-line filters, reverse flushing the column, or replacing the frit.
Void in the Packing Head—A void in the packing material can appear as a settled packing bed or a wormhole in the packing, causing some of the sample material to travel faster in the column. The sample is therefore spread out before it enters the column, and all the peaks in the chromatogram are similarly affected and split. The solution for this is to use a guard column, use a less aggressive mobile phase, use a more stable column, do better sample clean-up, or a combination of all these.
Addressing Peak Shape Abnormalities with Software
Commonly encountered peak abnormalities can significantly impact the accuracy and reliability of analytical results. It is crucial to identify, understand, and correct the causes.
Column selection plays a large role in the quality of separations. Factors that must be considered when selecting columns include the type of column (normal phase, reverse phase, etc.,) and the dimensions of the column (diameter and length). There are tools available in various software programs, for example, Method Selection Suite, that allow the selection of the most appropriate column. The Column Selector tool screens a full set of columns to find the most effective retention mechanism.
The mobile phase and stationary phase are other factors to consider. The mobile phase is responsible for transporting analytes through the column. The stationary phase determines the degree of interaction between the analytes and the column. The physicochemical properties of the analyte determine the type and extent of the molecular interactions between the analyte and the mobile and stationary phases. Tools in Method Selection Suite predict physical properties like pKa and logD from chemical structures. This helps to predict analyte retention, guiding the selection of the most appropriate pH and buffer and helps determine the composition of the mobile phase.
Selection of the most appropriate column, mobile, and stationary phase is crucial to successful peak separation and method development. Using software to determine the most appropriate parameters, and saving successful methods to databases for later use, gives a better starting point and saves time. By minimizing peak shape abnormalities, chromatogram accuracy is improved and leads to robust and reproducible methods.