346
microparticles with a diameter of 2–5 μm or porous monolithic materials that significantly lead to a drop in pressure in the column. HPLC uses the selected structural
property of the substances and does not change their chemical nature during the
analysis. Depending on the chemical structure, the ingredients of the mixture spend
different time in the column because they have different retention affinities on the
stationary phase.
Using this chromatography technique, it is possible to perform structural and
functional analysis and purification of many molecules within a short time. This
technique yields perfect results in the separation and identification of amino acids,
carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active
molecules. In HPLC, mobile phase passes through columns under 10–400 atmospheric pressure and with a high (0.1–5 cm/sec) flow rate. In this technique, the use
of small particles and application of high pressure on the rate of solvent flow increases
separation power of HPLC, and the analysis is completed within a short time.
In liquid chromatography, the choice of detection approach is critical in order to
guarantee that all the components are detected. The most widely applied detectors
are UV detector, which is capable of monitoring several wavelengths concurrently,
photodiode array (PDA) detector, refractive index detector (RID), and mass spectrometry detector (MS). In the case of PDA, a wavelength range can be programed
and all the compounds that absorb within this range can be identified in a single
analysis. The PDA detector can also analyze peak purity by matching spectra within
a peak and finds its application in the method development. The refractive index
detector is the detector of choice when one needs to detect analytes with restricted
or no UV absorption, such as alcohols, sugars, carbohydrates, fatty acids, and polymers. Decent trace detection performance is secured through a low noise. This
detector has the lowest sensitivity among all detectors but is suitable at high-analyte
concentrations. Mass spectrometers operate by converting the analyte molecules to
a charged (ionized) state, with subsequent analysis of the ions and any fragment
ions that are produced during the ionization process, on the basis of their mass to
charge ratio (m/z). Several different technologies are available for both ionization
and ion analysis, resulting in many different types of mass spectrometers with different combinations of these two processes. The two most widely used ionization
interface systems are atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Which one will be chosen depends on the physicochemical
properties of the analytes (i.e., polarity and acidity). Ionization takes place at atmospheric pressure, and both are considered to be a soft ionization method, i.e., the
mass spectrum provides mainly the molecular weight information, unless fragmentation techniques are used. Since compounds partly co-elute from the chromatographic system, the clear assignment of the individual fragments cannot be
accomplished using only LC-MS, where only molecular ion masses are available.
To overcome this problem, tandem mass spectrometry (MS/MS), which involves
multiple steps of mass selection or analysis, is nowadays mainly used (LC/MS/MS
or LC/MSn). These systems are able to determine residues in the lower ppt range.
Among the different methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly employed for the separation of complex mixV. I. Petropulos and B. Balabanova
microparticles with a diameter of 2–5 μm or porous monolithic materials that significantly lead to a drop in pressure in the column. HPLC uses the selected structural
property of the substances and does not change their chemical nature during the
analysis. Depending on the chemical structure, the ingredients of the mixture spend
different time in the column because they have different retention affinities on the
stationary phase.
Using this chromatography technique, it is possible to perform structural and
functional analysis and purification of many molecules within a short time. This
technique yields perfect results in the separation and identification of amino acids,
carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active
molecules. In HPLC, mobile phase passes through columns under 10–400 atmospheric pressure and with a high (0.1–5 cm/sec) flow rate. In this technique, the use
of small particles and application of high pressure on the rate of solvent flow increases
separation power of HPLC, and the analysis is completed within a short time.
In liquid chromatography, the choice of detection approach is critical in order to
guarantee that all the components are detected. The most widely applied detectors
are UV detector, which is capable of monitoring several wavelengths concurrently,
photodiode array (PDA) detector, refractive index detector (RID), and mass spectrometry detector (MS). In the case of PDA, a wavelength range can be programed
and all the compounds that absorb within this range can be identified in a single
analysis. The PDA detector can also analyze peak purity by matching spectra within
a peak and finds its application in the method development. The refractive index
detector is the detector of choice when one needs to detect analytes with restricted
or no UV absorption, such as alcohols, sugars, carbohydrates, fatty acids, and polymers. Decent trace detection performance is secured through a low noise. This
detector has the lowest sensitivity among all detectors but is suitable at high-analyte
concentrations. Mass spectrometers operate by converting the analyte molecules to
a charged (ionized) state, with subsequent analysis of the ions and any fragment
ions that are produced during the ionization process, on the basis of their mass to
charge ratio (m/z). Several different technologies are available for both ionization
and ion analysis, resulting in many different types of mass spectrometers with different combinations of these two processes. The two most widely used ionization
interface systems are atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Which one will be chosen depends on the physicochemical
properties of the analytes (i.e., polarity and acidity). Ionization takes place at atmospheric pressure, and both are considered to be a soft ionization method, i.e., the
mass spectrum provides mainly the molecular weight information, unless fragmentation techniques are used. Since compounds partly co-elute from the chromatographic system, the clear assignment of the individual fragments cannot be
accomplished using only LC-MS, where only molecular ion masses are available.
To overcome this problem, tandem mass spectrometry (MS/MS), which involves
multiple steps of mass selection or analysis, is nowadays mainly used (LC/MS/MS
or LC/MSn). These systems are able to determine residues in the lower ppt range.
Among the different methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly employed for the separation of complex mixV. I. Petropulos and B. Balabanova