Key Takeaways
HPLC separates components based on how they interact with a stationary phase and a mobile phase.
A chromatogram plots detector response against time and can reveal separated peaks.
Retention time can support identification when used with appropriate controls, but retention time alone is not necessarily a specific identity test.
Peak areas can be used for quantitative calculations when the method has been appropriately developed and validated.
What is HPLC?
High-performance liquid chromatography, commonly abbreviated HPLC, is a separation technique used to resolve components in a liquid sample. A pump moves a mobile phase through a chromatographic column containing a stationary phase. Compounds interact differently with the stationary phase and therefore travel through the column at different rates.
How separation happens
A sample is introduced into the mobile phase and carried into the column. Some molecules spend more time interacting with the stationary phase while others remain preferentially in the mobile phase. The difference in interaction produces separation. The detector then records the compounds as they leave the column.
Reading a chromatogram
The typical HPLC output is a chromatogram with time on the horizontal axis and detector response on the vertical axis. Individual peaks may correspond to separated components. Important features include retention time, peak height, peak area, baseline quality, resolution, and peak shape.
Retention time
Retention time is the elapsed time between injection and detection of a peak. It can be useful for comparing a sample with a reference under the same method conditions. However, a single retention time is not automatically a definitive identity test. FDA guidance on specifications notes that identification should be specific and that combinations such as HPLC/MS can provide stronger identification than retention time alone.
Purity and peak area
In an appropriate method, chromatographic peak areas can be used to estimate the relative amount of components detected by the method. The reported result depends on the detector, method conditions, integration approach, reference standard, and assumptions behind the calculation. ‘HPLC purity’ should therefore always be interpreted in the context of the actual method.
Method development and validation
Analytical procedures need to be suitable for their intended purpose. ICH Q14 addresses science- and risk-based analytical procedure development, while Q2(R2) addresses validation principles.
Frequently Asked Questions
What does HPLC measure?
HPLC primarily separates components and produces a detector response for compounds as they elute from the column. The exact measurement depends on the detector and analytical method.
What is a chromatogram?
A chromatogram is the graphical output of a chromatographic separation, commonly showing detector response versus retention time.
Can HPLC identify a compound?
It can provide useful identification evidence, especially when compared with an appropriate reference and method. For greater specificity, laboratories may combine chromatography with mass spectrometry or another orthogonal technique.
NorthPep Research Note
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