**3. Mass spectrometry coupled with liquid chromatography**

High-performance liquid chromatography (HPLC) is an innovative type of LC used in various fields, including food analysis and pharmaceuticals. It is primarily beneficial for low or non-volatile organic compounds that are not suitable for GC. The main difference between HPLC and LC is the solvent's mobility. In the case of LC, the solvent moves by force of gravity, while in HPLC, it moves under high pressure obtained through pumps. The use of the pumps ensures the overcome of the pressure drop in the column and reducing the separation time. The combined technique between MS and HPLC is generally identified as LC–MS (**Figure 7**).

LC coupling with MS is more complicated than with GC because of the need to generate gas-phase ions for the MS. Furthermore, the necessity to eliminate the elution solvent is another downside of LC–MS. In the case of water, if the column used has a small diameter permitting a maximum flow rate of 0.1 ml min−1, which is equal to 0.1 g min−1 of water, generating a flow rate of 135 cm3 min−1 of gas at atmospheric pressure. This flow is too high to be injected under a vacuum into a source. In order to overcome this downside, numerous methods are used [11–13].

**Figure 7.** *Schematic representation of LC–MS.*

*Mass Spectrometry Coupled with Chromatography toward Separation and Identification… DOI: http://dx.doi.org/10.5772/intechopen.100517*

#### **3.1 Ionization and ions source**

The coupling of HPLC and MS became possible with the installation of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in the commercial apparatus [14, 15].

#### *3.1.1 Electrospray ionization*

Electrospray ionization (ESI) consists of pressing the analytes present in the solution through a capillary. The charged droplets form when a high voltage is applied (between 1.5 and 5 kV) [16]. The charge density improves with the elimination of solvents from the droplets via continuous evaporation. In addition, the surface area increases due to splitting droplets into smaller droplets at a specific charge density (Coulomb explosion). At the end of this process, the remaining microdroplets emit single ions, or the droplets only contain single solvated ions that will be entirely desolvated upon further drying [17–19]. The transfer of the ions into the high vacuum of the MS is carried out via a capillary or small hole in the front plate through electric fields (**Figure 8**).

ESI is applicable for various compounds such as proteins and peptides, oligosaccharides, bio-organic molecules, polymers, and non-covalent complexes.

#### *3.1.2 Atmospheric pressure chemical ionization*

APCI has attracted considerable attention due to its ability to produce ions from solution and analyzing rather nonpolar compounds. Like electrospray, the liquid analyte is directly injected into the ionization chamber via an APCI probe (**Figure 9**). The analyte solution is submitted to a nebulization to produce fine droplets of aerosol spray, which will undergo rapid heating in the nitrogen stream and then emerge at the end of the probe as a stream of a vaporized analyte. In the area of the corona discharge needle, the reagent ions are formed. The analyte molecules react with these ions and form protonated or deprotonated analyte ions that are singly charged [20, 21].

Generally, the transfer of proton happens in the positive mode to generate [A+ H]+ ions. However, the negative mode may also occur, and the M<sup>−</sup> and [A− H]− are formed from electron transfer or proton loss, respectively. During ionization, the solvent clusters and high gas pressure influence the reagent ions resulting in reduced fragmentation and intact quasi-molecular ions. The process is considered more energetic than ESI, which results in the absence of multiple charging [22].

#### *3.1.3 Matrix-assisted laser desorption ionization*

The matrix-assisted laser desorption ionization (MALDI) is another ionization technique, which permits high molecular weight molecules injection into the

**Figure 8.** *Schematic representation of ESI.*

#### *Biodegradation Technology of Organic and Inorganic Pollutants*

**Figure 9***. Schematic representation of APCI.*

gas phase as intact ions. MALDI technique gives desorbed analyte with a relative mass of 300KDa. In MALDI, the analytes are crystallized using an excess matrix compound (DBA, Sinapic acid, etc). Then the crystalşized analyte is carried into the high vacuum of the MS and irradiated via laser. Finally, the analyte molecules are carried into the gas phase after the matrix evaporated the absorbed laser energy (**Figure 10**). The transfer of protons between the matrix and analyte molecules is responsible for ionization [23–25]. The downside of this technique is the connection to the chromatography, which needs to be indirect either manually or through robotics. Currently, MALDI is limited to scanning applications where a matrix sprayed sample is scanned in two-dimension via a laser beam to get a mass distribution to produce false-color images [26, 27].

No fragmentations due to ionization are obtained when ESI, APACI, and MALDI are used, hence the "soft" reference. Furthermore, because of their covered polarity and molecular weight array, ESI and MALDI are perfect for bio-molecules analysis.

**Figure 10***. Schematic representation of MALDI.*

*Mass Spectrometry Coupled with Chromatography toward Separation and Identification… DOI: http://dx.doi.org/10.5772/intechopen.100517*

ESI and MALDI, in particular, are ideal for bioanalytics (proteins, peptides, etc.) due to their covered polarity and molecular weight range.

#### **3.2 LC: MS domaine of applications**

The LC–MS found application in numerous fields. In this section, the most crucial area will be discussed.


### **4. Outcomes recording and treatment**

Regardless of GC–MS or LC–MS, an online data system is present, containing an acquisition processor, a magnetic recorder, and a computer.

#### **4.1 Outcomes recording**

As a function of time, the spectrometer offers two series of outputs: the number of ions detected and, at the same time, the mass of these ions is given. The mass of each ion emerges with a particular distribution over some time, as displayed in **Figure 11**. Thus, the number of ions detected can be computed from the area under the curve, whereas the centroid of the peak displays the ion's mass. The mass determination is effectuated via the acquisition processor, where the signal related to the number of ions accumulates quickly.

For instance, in 1 s, a spectrometer covers 500 mass, which means in 2 ms 1 mass. For this period, eight measurements of the number of ions ought to be conducted, meaning 0.25 ms assigned for each sample. In other words, 4000 samples ought to be measured per second, and the frequency of the sampling is 4 kHz. The ions detector's current goes through a resistance 4000 times a second, and at the end of the resistance, the acquisition processor is responsible for reading the potential difference relative to the number of ions detected and then digitalize it. The obtained output value corresponds to the y axis of the mass spectrum. The x-axis value corresponds to the reading of the mass indicator. The bar graph is the result of an algorithm that permits the processor to define the limits of the peak and centroid. The number of ions corresponds to the sum of the values read within these limits, whereas the ion's mass corresponds to the interruption of the indicator value at the centroid. A representative obtained bar graph is given in **Figure 12**.

In the case of a broader mass range scanning or high-resolution usage, increasing the sampling speed is needed, increasing the data points per unit time.

**Figure 11.** *Schema of a chromatogram.*

One of the essential characteristics of the data acquisition process is the dynamic range, connected in part to the signal digitization possibilities. For instance, an ion detector may identify one to a million ions that reach the detector at once.

Its dynamic range, the largest to the lowest measurable signal ratio, is equivalent to 106 . In an ADC with 16 bits, the numerical obtained values are between 1 and 216. Therefore the dynamic range is considerably lower than that of the detector. However, this problem can be overcome by reading different value ranges consecutively.
