# Deep Eutectic Solvents as Extraction Media for
## 2 Factors Affecting the Extraction of Flavonoids Using DES Separation Techniques
### Separation Techniques  
The majority of the research with regards to the use of DES in flavonoid extractions still revolves around the optimization of conditions and evaluating the performance of the extraction at a level deemed satisfactory after having modified certain parameters. Most cited attempts employed HPLC (High Performance Liquid Chromatography), UV-Vis (Ultra-Violet - Visible Spectroscopy) [12], or other instrumental analysis, aiming to analyze the extract in order to evaluate the performance of the DES mixtures to capture the target compounds. Therefore, some reviews do not capitalize upon the separation of the flavonoids from each other or from a group of organic compounds extracted via the DES as a research point in itself, rather than as a means to examine the performance of the initial extraction. For example, research aiming at simply verifying the flavonoid content of the extract to confirm a successful NaDES extraction employed the Folin-Ciocalteu reagent in a properly prepared extract sample, which, after reaction and incubation, could be studied through UV-Vis or another method to verify the total phenolic content, and subsequently, the flavonoid content [52]. Whenever deemed necessary, often due to the extraction methodology, some researchers opted for filtration or centrifugation prior to the commonplace HPLC analysis that would confirm the extraction of the target flavonoids [53]. A popular and efficient solution for the enrichment and the separation of the flavonoids from the DES extract involves the use of column chromatography through a packed column with a macroporous resin (such as ME-2 [44], NKA-9, or AB-8 [54]), which is cleaned with deionized water, and then after exposure to the extract is eluted with aqueous ethanol. This method, however, requires further processing of the solution to isolate a particular flavonoid from a potential group of flavonoids extracted from a source. The packed column method may provide exceptionally high yields of up to 98.92% [55]. A back extraction using an antisolvent is a simpler method of isolating the flavonoids from the DES. After centrifugation of the sample, the supernatant is diluted with an organic antisolvent such as methanol [46], and subsequently centrifuged again to create a biphasic system. The newly occurring supernatant is the target system, leaving only the solvent to be evaporated (i.e., by vacuum centrifugal evaporation [8]). Finally, a novel method for extracting the flavonoids from the DES involves the mimicking of DNA denaturation in the DES, as described by [PERSON] et al. [56]. According to their research, the main goal in removing the flavonoids from the DES is the breakdown of the hydrogen mesh that holds the DES together, which is a process similar to denaturation. The group designed an effective method of extracting the flavonoids on a chrome metal organic framework (Materials of Institut Lavoisier: MIL-100 (Cr)), from which the isolation of the flavonoids becomes easier and more selective. Initially, the DES is diluted in water (10% DES Solution), then NaCl is added, causing the HB mesh to breakdown. The subsequent addition of the MIL allows for the readily collectible flavonoids to attach to it and be easily removed from the diluted DES. An overview of all the applications of NaDES on flavonoid extractions examined in this review is presented in Table 2, including the bioactivity of the target flavonoids as mentioned in each study.