## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.1 GC-Based Methods
\begin{table}
\begin{tabular}{l c c c c c} \hline \multicolumn{2}{c}{**Analytical Techniques**} & \multicolumn{1}{c}{**Advantages**} & \multicolumn{1}{c}{**Disadvantages**} & \multicolumn{1}{c}{**Note**} \\ \hline \multirow{2}{*}{SFC} & - & Green technique suitable for thermally & - & Availability of SPC equipment & / \\  & - & label compounds & & & \\ \hline NMR & - & Not sensitive to ballast compounds (chievophyls & - & High cost of this analyser & / \\  & and lipids) reference standards are not required & - & High cost of this analyser & / \\ \hline \multirow{2}{*}{RAMAN} & - & Rapid, versatile and non-invasive qualitative and quantitative profiling growth staging of cannabis & / & / \\  & plant and extracts & & & \\ \hline \multirow{6}{*}{FTIR, NIR, MIR} & - & Chemically fingerprint substances & - & Analysis of heterogeneous substances like cannabis samples and to determine the potency of & - \\  & - & Should be combined with chemometrics & / \\ \cline{1-1}  & - & Rapid on-site we for monitoring growth and curing & - & Less accurate for potency analyses & / \\ \cline{1-1}  & & & & \\ \cline{1-1}  &  
versatile silylation agents are used, ordered by reactivity: hexamethyldisilazine (HMDS) [88], N-methyl-(trimethylsilyl) trifluoroacetamide (MSTFA) [153], N, O-bistrifluoroacetamide (BSTFA) [90], alone or accompanied by a catalyst, usually 1% trimethylchlorosilane (TMCS) [58; 99; 100; 123; 124; 133] for TMS derivatization and N-_tert_-butyldimethylsilyl-N-methyltrifluoroacetamide (MBTSTA) alone or with 1% tert-butyldimethylchlorosilane (t-BDMCS) as a catalyst for TBDMS derivatization. A study comparing the derivatization efficiency of different alkylsilylation agents, HMDS + trifluoroacetic acid (TFA), MSTFA, activated MSTFA, MSTFA + 1% TMCS, BSTFA, BSTFA + 1% TMCS, MTBSTFA and MTBSTFA + 1% t-BDMSC, using pyridine as additional catalyst for CBC, CBD, CBG, CBN, \(\Delta^{9}\)-THC and \(\Delta^{9}\)-THCA in standard solutions and plant matrix concluded that maximum responses are obtained with HMDS + TFA. Responses were not influenced by catalysts, as well as reaction solvent (pyridine, EtAc, MeCN) [88]. Other less frequently exploited derivatization is methylation of the hydroxyl groups of the phytocannabinoids using trimethylsulfonium hydroxide (TMSH) [97]. Formation of alkylboronate-TMS derivatives using alkylboronic acid and BSTFA + TMCS showed that methyl- and n-butylboronates yielded derivatives that were stable for several weeks at 4 \({}^{\circ}\)C had improved GC peaks when compared to TMS derivatives, and MS characteristics, with preserved fragmentation patterns of the underivatized compound [116]. * GC Columns  
Various GC-based methods became popular and widely used during the 40-year span of phytocannabinoids profiling (Table S1). E.C. recommends use of glass capillary column 25 m long and 0.22 mm wide, impregnated with 5% non-polar phenyl-methyl-siloxane phase, that would allow good separation of phytocannabinoids [70]. Older GC-based methods employ glass columns packed with 2% OV-17 on Chromosorb WHP (mesh 100-120) [119], 2% OV-17 on 100-200 mesh GasChrom Q [98; 123; 140], 2% OV-17 [58] and glass column packed with 3% SE-30 on 100/200 mesh Gas Chrom Q [116]. Later, they were replaced by capillary columns with cross-linked and bonded stationary phases with various polarity. Here, analytes are separated based on differences in polarity, molecular mass and boiling point. As most frequently, phytocannabinoid profiling studies are focused on the most prominent phytocannabinoids, that are \(\Delta^{8}\)-THC, \(\Delta^{9}\)-THC, CBC, CBD, CBDA, CBDV, CBG, CBGA, CBN, \(\Delta^{9}\)-THCA and THCV. They contain aromatic, alkyl and alcohol moieties; it is expected that the proportion of phenyl groups in mixed dimethylpolysiloxane-silphenylene or mixed dimethylpolysiloxane-dimethyl-diphenyl stationary phases to have an impact on their chromatographic separation.