Analytical eco-scale (AES), national environmental method index (NEMI), green analytical procedure index (GAPI), and software-driven analytical greenness index (AGREE) are used in the distinctive HPLC approach. Use any method to conduct a detailed examination to assess the product's greenness profiling to ascertain its level of environmental consciousness [31, 32].

Considering its basic layout and simplicity of utilize, the NEMI assessment tool provides an in-depth assessment concerning the ecological implications related to the assessed approach that's readily comprehended by simply staring over the pictorial. The conditions for the characteristic are defined by the following terms: potentially hazardous, corrosive, waste, persistent, bioaccumulative, and toxic. The degree to which the plan satisfies the criteria determines either each quadrant is vacant or green. While an area has been filled colored green, it signifies that the plan of action satisfies the requirements for deciding on of that specific quadrant. Consequently, upon looking at the general characteristics associated with the recommended HPLC technique, the two sections appear green stated in Table 11. Considering the trash that is created is no more than 50 g or ml for each specimen and no methanol and acetonitrile comes under the PBT listed class, the suggested procedure spans two greenness examined regions [33,34,35,36].

The researchers have introduced the Eco-Scale, an indirect method for assessing how sustainable analytical approaches are. This application assigns points to penalties upon considering a number of variables, such as energy consumption, the creation of waste, and the quantity and severity associated with chemical elements. The most favorable green assessment is presented after deducting the entire quantity of penalties from 100. A score that falls between 50 and 75 is regarded as an average, whereas a score above 75 is superb green. Trash amount, equipment, and chemicals were among the study's variables. The approach is mentioned in Table 11 and obtained a score of 84. The eco-scale application suggests that the newly developed procedure is more sustainable in light of these outcomes [33, 34, 37,38,39].

A commonly employed greenness matrices for evaluating how techniques of analysis affect our surroundings is called GAPI. It serves as an efficient, trustworthy, and user-friendly application that provides useful information regarding how environmentally conscious a procedure is. A fantastic partially quantitative tool for the educational setting and laboratory is GAPI. Every step of a method of analysis can have external factors evaluated and quantified using the GAPI graphic. The diagram highlights the whole green character of the scientific procedure, from collecting the samples through the final comprehension, by color-coding the data in red, yellow, or green, according to the approach employed. The subjective information facilitates the comparison of various ways of analyzing.

The conclusion of the GAPI figure is shown in Table 11 consists of five pentagrams, every single representing a distinct stage within the analyzing procedure: gathering and preparing the sample, using the apparatus, using chemicals and solvents, and determining the objective of the analytical technique. The procedure in the GAPI assessment yields six green and six yellow, notably demonstrated in Table 11. Sections in require care are indicated by two red pentagrams. The suggested ecological HPLC method highlights possible risks to the ecosystem. Relying upon the GAPI pentagram, the suggested green technique is generally considered to be harmless to the environment. It uses a harsh solution to create red pentagrams, although it uses a lesser amount of energy, generates fewer waste products, and needs the preparation of samples in fewer than three stages [33, 40].

Table 11 shows the environmental friendliness of the HPLC technology using the AGREE numerical value as a stand-in. A score that was nearer to one signified a more ecological method, but a lesser score highlighted areas where the potential for increased environmental impact. The technique's greenness characteristic was assessed using the AGREE software, thereby created using the concepts of green analytical chemistry. An approach or process that scores 0.61 or higher is deemed beneficial to the environment and compliant with the standards of green analytical chemistry. High conformance toward GAC principles—which involve lowering trash creation, lowering or removing potentially dangerous substances, conserving usage of energy, and improving human health and safety—is indicated by an overall score of higher than 0.5. This suggests an extreme dedication to conservation and ecological responsibility [14, 41, 42, 45].

An approach is considered green if its evaluation score is greater than 0.5, whereas the center zone's comparatively weaker green color reflects this idea. The bright pictogram's regions 3 and 12 have been discovered to represent the most inefficient divisions. Sectors 11 and 12 stress the significance of worker protection due to the chemical's extremely harmful and combustible tendency. When the researchers attempted to transition between the two substances, ethanol and methanol, the analysis they generated was subpar. Moreover, component 7's orange hue indicates that 15 ml of debris are produced for every cycle [14, 33, 34, 41, 42, 46].

The first of the specifications for assessing the whiteness, greenness, as well as organized procedure verification standards of the methodology might be directly related to the total ChlorTox value, and this encompasses all aspects of the procedure. The developed method calculated the amount of mass of each agents used for a single analysis (m sub), the ChlorTox values indicating the level that is anticipated chemical threat, and the risk ratio with regards to chloroform (CH sub/CH CHCl3) estimated via the WHN and CHEMS-1 algorithms. The ChlorTox Scale, as mentioned in Table 11, is designed to assess techniques according to a couple of variables: the substance's quantities and its potentially dangerous characteristics. The designed HPLC process is not harmful therefore more ecologically conscious, according to the WHN and CHEMS-1 models [43, 45, 46].

Similar in concept to the prominent twelve concepts of GAC, which are the twelve guiding principles of white analytical chemistry (WAC), could facilitate the systematic and logical expression of all basic requirements on desirable approach characteristics covering green as well as other outstanding criteria. Three complimentary areas are identified by the following three columns: green (ecological compatibility and safety), blue (practical and financial issues), and red (analytical productivity). As shown in Table 11, recommended HPLC procedure displays the average result of the evaluation across all 12 concepts, or whiteness (%), at its bottom along with the results of the mathematical average of the defined basic attributes, which are R (%), G (%), and B (%)[44,45,46].

The outputs produced by RGB 12 and AGREE, which belong to the WAC as well as GAC notions, accordingly, must be regarded as equally robust when merely the judgment for greenness is taken into account. The two approaches identified the best ecologically conscious course of action.