## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods
The \"classic\" TLC became further less utilized due to its inconvenience to document for peer review, poor resolution due to systematic errors rising from hand-spotting, temperature/humidity control and imprecise R\({}_{\text{F}}\) measurement [167; 169]. Recent advances in TLC are in the development of HPTLC methods. Such reliable methods could offer advantages over both HPLC and GC techniques for cannabis profiling, including its ability to analyse multiple samples simultaneously and the consequently lower running costs and analysis runtime. Further, automation of sample application in HPTLC methods eliminate systematic errors, provide better resolution and generate reports for more convenient documentation for peer review of casework [170]. In that spirit, normal-phase HPTLC with an automated spotter is shown to achieve better separationthan TLC for the main neutral phytocannabinoids. The method is comparable within a small degree of error (\(\pm\)0.5%) to a validated HPLC method [110]. Older methods use plates spread with a layer of a slurry of alumina/CaSO\({}_{4}\)/H\({}_{2}\)O (22.0 g:3.0 g:50 mL), activated at 110 \({}^{\circ}\)C for 30 min and stored under anhydrous CaCl\({}_{2}\)[131], silica gel G plates [129], precoated silica gel G plates [171] and silica gel G layers impregnated with dimethylformamide [120]. Reverse phase (RP)-TLC is performed using RP-18 HPTLC plates [129] and RP-C\({}_{18}\) bonded silica gel F plates [18]. The more recent TLC/HPTLC methods most commonly use HPTLC silica gel 60 F\({}_{254}\) plates for successful separation of 11 phytocannabinoids (\(\Delta^{9}\)-THC, CBD, CBN, CBC, THCV, \(\Delta^{8}\)-THC, CBDV, CBG, CBGA, CBDA, \(\Delta^{9}\)-THCA) [127] or of \(\Delta^{9}\)-THC, CBD, CBN and CBG [172], or for separation of \(\Delta^{9}\)-THC, CBD and CBN only [125], silica gel 60 [110], silica gel 60F [135] or silica gel plates [5; 126]. For some methods, for instance, the type of TLC plate was not clearly defined [130; 147]. Most suitable mobile phases include xylene/hexane/diethylamine (25:10:1, _v/v/v_) [127], CHCl\({}_{3}\), with plate prewashing with MeOH [110], hexane/diethyl ether (80:20, _v/v_), which allowed clear separation between \(\Delta^{8}\)-THC, \(\Delta^{9}\)-THC, CBD and CBN [125], cyclohexane/toluene/diethylether (75:15:10, _v/v/v_) [126], benzene/_n_-hexane/diethylamine (25:10:1, _v/v/v_) [130], benzene/chloroform (50:50, _v/v_) [131], diethylether/petroleum ether (1:4, _v/v_) [120], benzene/_n_-hexane/diethylamine (25:5:0.5, _v/v/v_) [147], n-hexane/CHCl\({}_{3}\)/dioxane (89:8.75:2.25, _v/v/v_) [135], benzene, benzene/_n_-hexane (6:4, _v/v_), benzene/n-hexane/diethylamine (70:25:5, _v/v/v_) [129], MeOH/dioxane/hexane (1:2:7, _v/v/v_), hexane/EtAc (4:1, _v/v_), hexane/diethylether (4:1, _v/v_) [171], hexane/ethyl ether (8:2, _v/v_) [172], petroleum ether/deithylether (8:2, _v/v_), cyclohexane/diisopropyl ether/deithylamine (52:40:8, _v/v_/v) or _n_-hexane/dioxane/MeOH (7:2:1, _v/v/v_) [5]. For 2D TLC, first _n_-heptane/dichloromethane/butan-2-one (83:5:12, _v/v_/v) was used for first and n-hexane/acetone (86:14, _v/v_) for second development after 90\({}^{\circ}\) rotation [129]. RP-TLC employs McN:H\({}_{2}\)O (9:1, _v/v_) [129] or MeOH/5% acetic acid (19:1, _v/v_) [18] as mobile phase. Evaluation of 10 mobile phases (hexane/diethylether (80:20, _v/v_), toluene, _n_-heptane/diethyl ether/formic acid (75:25:0.3, _v/v_/v), CHCl\({}_{3}\), hexane/acetone (87:13, _v/v_), benzene, xylene/hexane/diethylamine (25:10:1, _v/v_/v_), 4-8% diethylamine in toluene, MeOH/H\({}_{2}\)O with 0.1% glacial acetic acid (75:25, _v/v_) and hexane/acetone (75:25, _v/v_) on Silica gel 60 F\({}_{254}\) plate showed that xylene/hexane/diethylamine (25:10:1, _v/v/v_) allows most precise bands and best separation of \(\Delta^{9}\)-THC, CBD and CBN, but without migration of \(\Delta^{9}\)-THCA, CBDA and CBGA.