HPLC-DAD analysis

What is HPLC-DAD used for?

HPLC-DAD is used to assess chemical purity and contamination, which makes it useful across research and industry. In the food industry, HPLC-DAD is used to test products for certain preservatives, additives, and contaminants, such as benzoic acid and aflatoxins, which is essential for ensuring consumer safety and optimal product quality. The technique is also common in the pharmaceutical and food supplement industries for quality control, as it enables precise quantification of active ingredients, vitamins, and impurities.

How does HPLC-DAD work?

HPLC works by passing the sample through a chromatography column. This causes individual components within the sample to separate based on their chemical and physical properties. Each component will then be eluted individually from the end of the column, where a detector like DAD can analyze it.

The diode-array detector scans the samples with a beam of light that varies across the entire ultraviolet and visible light spectrum. It measures the amount of each wavelength of light that is absorbed against the time each chemical species was eluted from the column. Together these spectral and retention data help identify the different components within the sample and the quantities they are in.

Sample requirements and preparation

HPLC is performed in the liquid phase, which means that solid samples need to be dissolved in an appropriate solvent before testing. The presence of even a small number of solid particles can cause blockages in the column. Solvents (including water) must be of high purity to minimize contamination and possible effects on results. Liquids can be tested, but serial dilutions may be required to produce clearer results.

DAD vs UV-VIS detector

DAD and UV-Vis detectors both work on the same principle of measuring the amount of light absorbed by a sample. The difference is that the UV-Vis detector is set up to measure specific parts of the spectrum at any one time, while the DAD can measure the entire spectrum at once. This makes the DAD method more efficient and convenient than conventional UV-Vis detection for most uses. The HPLC-UV method can, however, be useful when quantifying a specific substance, such as benzoic or sorbic acid.

HPLC-DAD vs LC-MS

(High performance) liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is a similar technique that begins with liquid chromatography. The difference is that the separated sample is then passed through a mass spectrometer, where it is ionized, further separated, and detected based on each ion’s mass-to-charge ratio. This makes LC-MS a more sensitive technique, able to detect smaller quantities of each component. 

HPLC-DAD generally works best when the user already has some understanding of the contents of the solution, while LC-MS can be used to characterize a totally unknown sample. However, LC-MS can require more training and can be more difficult to interpret, hence why HPLC-DAD is often chosen as a more straightforward detection method.

Furthermore, the data gathered from the DAD detector is different from that gathered through mass spectrometry and can help to split up compounds that would be masked in MS, such as those with very similar chemical masses. Therefore, both techniques offer different insights and can be used in conjunction for a more comprehensive analysis.