## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods
The cannabinoid acids were successfully separated using _n_-heptane/diethyl ether/formic acid (75:25:0.3, _v/v/v_) on C\({}_{18}\) F\({}_{254}\) plate and EtOH/H\({}_{2}\)O with 0.1% glacial acetic acid (75:25, _v/v_) on RP-C\({}_{18}\) F\({}_{254}\) plate [127]. Another study showed that, when using alkanes as eluents (isooctane, heptane, hexane and pentane/diethylether (90:10, _v/v_), the capability to separate \(\Delta^{9}\)-THC, CBD and CBN decreased as the length of the carbon-bearing chain increases [125]. Visualization of (HP)TLC plates is usually performed using 0.1% aqueous solution of Fast Blue B salt reagent [110; 125; 126; 127; 130; 147], alone, under white light (254 nm and 366 nm) [127], under UV (254 nm) [131], as solution in 0.1M NaOH [5] or under UV (206 nm) [110] or as 0.5% aqueous solution, followed by 0.1M NaOH [23; 125; 173]. Prior to Fast Blue B, diethylamine can be applied (50 mg:L\({}^{-1}\) H\({}_{2}\)O + 20 mL MeOH) [5]. RP-TLC plates are visualized using Fast Blue B in 0.1M NaOH or in 50 g:L\({}^{-1}\)H\({}_{2}\)O/acetone (9:1, _v/v_) [129]. Fast Blue RR was better for visualization of \(\Delta^{9}\)-THC, CBD, CBG and CBN than Fast Blue B salt [172]. As qualitative evaluation for the presence of cannabinoids during (HP)TLC analysis is based on color determination, it is often subject of analysts' erroneous determination. Recent studies made the pioneering efforts in developing a method for standardizing and naming colors using the Sci-Chromus(r) software, that significantly reduced the subjectivity of the color names in identifying \(\Delta^{9}\)-THC, CBD, CBN and CBG in cannabis extracts [172]. Apart from TLC and HPTLC, other planar chromatography methods are seldomly used, such as optimum performance laminar chromatography (OPLC) and automatedmultiple development (AMD) for phytocannabinoid profiling, despite their greater reproducibility due to complete automation. Moreover, OPLC offers extension as semipreparative technique for sample purification, while AMD offers best resolution. The only reported employment of OPLC in AMD in cannabinoid profiling is in hexane extracts of cannabis resins (dried and reconstituted in toluene) and in hexane extracts of _cannabis_ resin. OPLC was performed for determination of \(\Delta^{9}\)-THC, CBD and CBN on HTSorb BSLA 011 and HT Sorb BSLA 003 columns using isooctane/diethylether (90:10, _v/v_) as eluent. Semi-preparative OPLC was performed for isolation of CBD from _cannabis_ resin using hexane/diethylether (80:20, _v/v_). Using AMD, separation was performed on HPTLC with the elution gradient 1C acetone (100, _v/v_), disopropylether (100, _v/v_), hexane (100, _v/v_), hexane (100, _v/v_), hexane (100, _v/v_) and hexane (100, _v/v_) during 20 steps. For both OPLC and AMD, visualisation is performed with Fast Blue B salt reagent [125]. * HPLC Methods The HPLC technique is gaining popularity as the main choice for fingerprinting study for the quality control of herbal drugs [174], thus enabling chemical characterization of herbal medicines [175]. HPLC methods offer larger linear ranges and more consistent calibration curves for all phytocannabinoids in regards to GC-based methods [163]. In terms of reliability, reproducibility and sensitivity, it was shown that high-resolution GC/FID and HPLC-UV methods for quantification of \(\Delta^{9}\)-THC, CBD and CBN are comparable [98]. * HPLC Mobile Phases Most of the mobile phases used in HPLC/DAD analysis of phytocannabinoids consisted of buffered aqueous solutions of ammonium acetate [176], ammonium formate [83; 142], formic acid [11; 40; 50; 60; 61; 62; 63; 78; 173; 177; 178; 179; 180; 171; 17; 172; 173; 174; 175; 176; 177; 178; 179; 181; 17; 182], acetic acid [19; 183; 184], _o_-phosphoric acid [165; 185] or 5% MeCN/80% MeCN with 0.1% _o_-phosphoric acid [69]. Acid conditions are preferred for cannabinoid acids (\(\Delta^{9}\)-THC, CBD, CBA). Pharmacopoeial methods (DAB, Ph.Helv., Ph.Eur.)