8. Detector
Detector allows for establishing both qualitatively nature of the individual compounds being eluted from the column, as well as their quantity.
There are many types of detectors that can be mounted to an HPLC system depending on the chemistry of the tested compounds. The most common detectors are below:
a) UV/VIS
b) Diode Array Detector
c) Mass Spectrometry
d) Fluorescence
e) Refractive Index
f) pH and Conductivity
g) Electrochemical
The vast majority of HPLC analyses are carried out using the first three types of detectors above. Historically, UV/VIS detectors were used due to the fact that most chemical compounds absorb energy in the UV/VIS spectrum (190 to 900 nm) and are visible to the detector in this range. Hence, the detector is able to see the compound and establish how much energy has been absorbed when the chemical compound is exposed to light at a specific wavelength (for instance 254 nm). The amount of absorbed energy at the given wavelength then corresponds to the quantity of the compound.
This elution of compounds that are visible to the detector gives rise to a chromatogram, which is a set of peaks (each corresponding to an individual compound isolated inside the column) corresponding to the time at which they eluted.
The photodiode array detector (PDA or DAD) is the same as UV/VIS detector, but is able to measure the absorbance across an array of wavelengths. In comparison, a simple UV/VIS detector works at a specified single wavelength. Most modern HPLCs use a diode array detector. The advantage is that compounds eluting from the column may be tested at various wavelengths at once. Some tested mixtures may contain compounds that absorb at a certain wavelength and other compounds that are visible at a different wavelength – hence can be tested at once.
The most modern liquid chromatography systems utilize mass spectrometers in combination with PDA or UV/VIS detector. Mass spec detectors are very expensive and very complex with many limitations, although they yield many advantages over above detectors, such as lower detection limits and the ability to identify tested compounds exactly. The basis for a mass spectrometer is that a sample eluted from the column is injected (usually sprayed) into a chamber, where the droplets are heated and ionized in a very high vacuum causing molecules to fragment. Then these fragments are carried into a high magnetic field, where they are separated based on the mass of the fragments followed by entering the mass detector. The pattern of mass fragmentation allows for exactly identifying the molecule as it forms a very specific pattern corresponding to the said molecule. In comparison, none of the other detectors listed above are capable of identifying compounds directly; instead, the compounds eluted from the column are matched to standards with known composition and identified based on the matching times they elute from the column.
