Liquid chromatography is a traditional separation technique used to separate individual analytes from a mixture. HPLC is an advanced version of liquid chromatography that can be employed over a broad range of applications including, food analysis, drug testing, and pharmaceuticals. HPLC assays are particularly beneficial for small non-volatile organic compounds that are difficult to process with gas chromatography.
HPLC stands for high-pressure liquid chromatography or also known as high-performance liquid chromatography. Therefore, as the name suggests, a pump drives the solvent in HPLC under high pressure, whereas the solvent in liquid chromatography travels via gravity.
The difference between HPLC-UV and LC-MS/MS analysis
The fundamental difference between both these techniques is the detector unit. LC-MS method is HPLC with MS detector units, whereas HPLC-UV has UV detectors. Both techniques have similar chromatographic conditions, but instead of a phosphate buffer in HPLC-UV, researchers use formic acid in LC-MS. Hence, choosing the right buffer becomes crucial during LC-MS method development and HPLC method validation.
Using a chromatogram (a graph), individual components are separated based on their retention time. The x-axis represents retention time, while the y-axis measures the absorbance intensity. Hence, depending on the method used, the detector could be mass spectrometry (MS) or a UV detector.
MS detectors are highly sensitive and provide higher resolution than UV detectors. They separate components according to their mass to charge ratio by ionizing the atoms or molecules in the mixture. Comparatively, MS detectors are about 1000X more sensitive than UV and are widely used for biological targets that are typically in the picogram range. Hence, combining HPLC with two units of MS (LC MS Analysis) is one of the most powerful analytical methods that can be used to reduce errors and improve assay sensitivity.
An HPLC-UV unit shows when compounds elute from the column based on their rate of retention. However, MS detection adds a new perspective of mass to charge ratio to the analytical signal. Hence, it provides not only the retention time of the analytes but also the sequence in which the individual components elute from the column. Such added dimension to the output signal increases the clarity and certainty about the results, saving time and resources.
UV detector measures in the ultraviolet or visible spectra. However, the generated graph can be complex to decode. For example, two analytes can have similar retention times and can be difficult to distinguish from the UV spectra. On the other hand, MS detectors can provide real-time peak identification based on their molecular weight. So even when two analytes have similar UV or visible spectra, they are unlikely to have identical masses.
UV detectors specialize in light-absorbing compounds. So when they provide better sensitivity for these compounds, it is still questionable whether all components are individually identified. But with the addition of MS, researchers can detect individual analytes with poor chromophores and even co-eluted compounds.
The road ahead
Historically, HPLC-UV has been a major driving force in analytical and bioanalytical innovations. However, with enhanced selectivity, sensitivity, and identification, LC-MS/MS in practice can improve productivity while saving time and money.
Published by Abhitha Ahilya