**2.4 Matrix assisted laser desorption ionization (MALDI)**

MALDI operates on principle of ionization of analytes dissolved in a matrix consisting of organic compound (sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid) and evaporated to dryness on a target plate [34]. Matrix crystallizes up on drying and the analyte dissolved with in it also gets co-crystallized. Firstly, laser beam hits the dried sample and ionize organic compound, which later ionize the analyte molecules. Laser beam causes both desorption and ionization of analytes. Nitrogen laser emitting at 337 nm and Nd: YAG laser emitting at 355 nm are the most widely used ones (**Figure 4**). MALDI is considered as a very

#### **Figure 3.**

*Mass Spectrometry - Future Perceptions and Applications*

mode primary ions such as N2+

be the peak response. One important requirement in APCI for optimal sensitivity is the sample has to be completely evaporated, before subjecting to ionization. First an aerosol of mobile phase is formed with the assistance of nebulizer gas. This aerosol is further subjected to heating at 200–550°C in a ceramic tube enabling complete evaporation. Even though higher temperatures are employed, the actual temperature felt by analyte molecules is way lesser due to a phenomenon called evaporative cooling effect/evaporation enthalpy. Next, analyte molecules in gas phases were bombarded with electrons formed from corona discharge needle [26, 27]. In positive

to ESI is, APCI is not suitable for thermolabile compounds as typical temperatures experienced by the analyte molecules are ~150°C. However, in case of ESI, molecules

APPI by design is similar to APCI, with only difference being the replacement of corona discharge needle with gas discharge krypton lamp (10.0 eV) that produces ultraviolet photons [28–30]. Evaporation of liquid phase happens in pneumatic nebulizer. While ionization potential for most of the analytes is less than 10 eV; mobile phase constituents such as water, acetonitrile and methanol has higher potential requirements. Presence of dopants such as toluene or acetone will help in enhancing the sensitivity of analyte ions. Dopant molecules absorb photon energy and eject an electron, resulting in the formation of radical cation. Ionization of analytes can happen by two processes: 1) Charge transfer between analyte and radical cations generated from dopant molecules. 2) Charge transfer between dopant molecules and mobile phase components and finally from mobile phase components to analytes (**Figure 3**). Similar to other atmospheric pressure ionization techniques, APPI is also suitable for negative mode of ionization. Sensitivity of APPI is flow rate dependent and better sensitivities have been reported at low flow rates. When compared to APCI, APPI offers lesser matrix effects and minimal source contamination. Success of APPI as an ionization technique was reported in

react with water in several steps by charge transfer to form H3O+

encounter temperatures ~40°C in the process of evaporation.

**2.3 Atmospheric pressure photo ionization (APPI)**

analytes occurs then by proton transfer from H3O+

either by: (i) resonance capture (AB to AB−

(iii) ion–molecule reaction (BH to B−

are formed by electron impact. These ions further

), (ii) dissociative capture (AB to B−

). One disadvantage with APCI when compared

. Ionization of the

) or

. In negative mode ions are formed

**42**

**Figure 2.**

*Mechanism of atmospheric pressure chemical ionization process.*

the analysis of steroids and quinones [31–33].

*Schematic representation of atmospheric pressure photo ionization process.*

**Figure 4.** *Mechanism of matrix assisted laser desorption ionization process.*

soft ionization technique that causes minimal fragmentation of the analyte ions and is also suitable for analysis of large molecules ranging from peptides to proteins, lipids and polymers [35–38]. It is also amenable to high throughput and target sample plates can be readily stored for future use. One major advantage of MALDI-MS is chromatographic separation of analytes is not required. However, due to lack of separation, matrix interferences impact the analytical results [39, 40]. Additionally, MALDI is not suitable for low molecular weight compounds. Also, MALDI needs TOF as an analyzer to cover high mass range in a linear mode, whereas ESI can be coupled with any mass analyzer. Recently, MALDI has been coupled to triple quadrupole and successfully used for the analysis of small molecules [41, 42].
