In chromatographic testing, the precision of measurement of a few system suitability parameters such as resolution, and plate counts is crucial. This article offers an in-depth analysis of why resolution and plate count must be measured by using peak width at half height rather than just the peak width at the base.
Peak Width at the Base vs. Peak Width at Half Height:
Traditionally, peak width at the base has been a standard measure. However, recent advancements and methodologies suggest that peak width at half height might offer a more accurate and convenient approach (USP General Chapter, Chromatography <621>).
Calculation of Plate number (N) as per USP GC 621:
Moreover, as per USP40, it is mentioned that where electronic integrators are used, it may be convenient to determine the number of theoretical plates, by the same equation as mentioned in USP41. This indicates that even older versions of USP also mentioned calculating plate numbers by using peak width at half height.
Note: USP41 has removed the calculation formula for the measurement of plate numbers by using peak width at the base.
Calculation of Resolution (R) as per USP GC 621:
Moreover, as per USP40, it is mentioned that where electronic integrators are used, it may be convenient to determine the resolution, by the same equation as mentioned in USP41. This indicates that even older versions of USP also mentioned calculating resolution by using peak width at half height.
Note: USP41 has removed the calculation formula for the measurement of resolution by using peak width at the base.
Why peak width at the half-height?
For peak width at the base, the process involves drawing tangents at the inflection points of the peak and measuring the width at the baseline. In contrast, peak width at half height is measured as the width of the peak at 50% of its maximum height.
Measuring peak width at half height is typically simpler and more straightforward. It doesn't require the drawing of tangents to the peak as needed for the peak width at the base. This simplicity becomes particularly advantageous when using modern chromatography data systems, which can automatically calculate peak width at half height with high precision.
When using peak width at the base, any irregularities at the baseline or low-intensity parts of the peak can significantly affect the measurement. By focusing on the half-height, where the peak is more defined and intense, there's less variability due to baseline noise or minor peak shape distortions.
Many modern chromatographic systems use electronic integrators (software) for peak analysis. These integrators are often better suited for automatically determining peak width at half height. They can accurately and consistently measure this parameter across various peak shapes and sizes.
Regulatory guidelines and standard methods often recommend using peak width at half height. For example, the United States Pharmacopeia (USP41) and other regulatory bodies have shifted their preference to peak width at half height in their guidelines, making it a standard practice in many pharmaceutical and analytical laboratories
The plate number or efficiency of a chromatographic column is a measure of its ability to separate compounds. Measuring peak width at half height provides a more accurate representation of this efficiency, especially in cases where peak asymmetry might otherwise skew the results.
In summary, using peak width at half height for measuring resolution or plate number in chromatography offers greater accuracy, ease of use, compatibility with modern software, adherence to regulatory standards, reduced variability, and a more accurate representation of column efficiency. These advantages make it a preferred method in many chromatographic analyses