clean technologies

Article Study of Influential Parameters of the Extraction from Spent Grounds: From Brewing Coffee Method to the Waste Treatment Conditions

Alexandre Vandeponseele, Micheline Draye , Christine Piot and Gregory Chatel *

EDYTEM, University Savoie Mont Blanc, CNRS, F-73000 Chambéry, France; [email protected] (A.V.); [email protected] (M.D.); [email protected] (C.P.) * Correspondence: [email protected]

Abstract: This article aims to study the interest of spent coffee grounds (SCG) valorization through caffeine recovery. In an original way, this study takes into account all the parameters such as (i) the brewing coffee methods (household, coffee shops, etc.); (ii) the storage conditions, in particular the drying step; (iii) the solid/liquid extraction parameters such as the nature of solvent, the temperature, the extraction time and the solid/liquid ratio; and (iv) the liquid/liquid purification parameters such as the nature, the volume and the pH of extraction medium. Results have shown that spent coffee grounds from coffee-shops obtained by percolation contain a higher amount of caffeine than spent coffee grounds from households obtained from spent pods or filters. A drying treatment is   not required when extraction is performed under one week after the spent coffee grounds collection −1 with 96.4% of not degraded caffeine. Solid/liquid extraction performed with 25 mL.g SCG of Citation: Vandeponseele, A.; Draye, hydroalcoholic solvent (water/EtOH, v/v 60/40) at 60 ◦C during 15 min have given a caffeine yield M.; Piot, C.; Chatel, G. Study of −1 up to 4.67 mg.g SCG. When using ethyl acetate, 93.4% of the caffeine has been selectively recovered Influential Parameters of the Caffeine by liquid/liquid extraction. Finally, the extraction of caffeine for the valorization of spent coffee Extraction from Spent Coffee grounds is a promising and easy way, which fits with an already important and well established Grounds: From Brewing Coffee market. Method to the Waste Treatment Conditions. Clean Technol. 2021, 3, 335–350. https://doi.org/10.3390/ Keywords: spent coffee grounds; biomass valorization; caffeine; storage conditions; extraction cleantechnol3020019 parameters; purification

Academic Editor: Patrick Cognet

Received: 31 January 2021 1. Introduction Accepted: 3 March 2021 Coffee is one of the most being traded commodity with an annual world production Published: 2 April 2021 over 10 million tons in 2019 [1]. The path to produce a coffee beverage is long and leads to the generation of several by-products such as coffee husk, pulp, silverskin and spent Publisher’s Note: MDPI stays neutral coffee grounds (SCG). Coffee grounds represent the most valuable and available coffee with regard to jurisdictional claims in by-product produced by soluble industries, domestic houses, restaurants and coffee published maps and institutional affil- shops. Besides, it has been calculated that 650 kg of spent coffee grounds are generated iations. from one ton of green coffee beans turned into coffee beverage [2]. Spent coffee grounds has been studied for high value applications such as production of biodiesel [3,4], bioethanol [5,6], biopolymer such as polyhydroxyalkanoate (PHA) [7,8], adsor- bent for air depollution [9] or water depollution [10], and extracts of bioactive molecules Copyright: © 2021 by the authors. such as polyphenols and caffeine [11–21]. Indeed, 1,3,7-trimethyl-1H-purine-2,6(3H,7H)- Licensee MDPI, Basel, Switzerland. dione, also called 1,3,7–trimethylxanthine or caffeine for coffee, theine for tea or guara- This article is an open access article nine for guarana, is the most widely used psychotropic substance all over the world distributed under the terms and (Figure1)[22]. conditions of the Creative Commons Caffeine can be incorporated in daily life products such as sodas and energy drinks [23], Attribution (CC BY) license (https:// painkillers [24] or slimming creams [25]. Nowadays, coffee is the most im- creativecommons.org/licenses/by/ portant known process to recover caffeine [26,27]. Extracted caffeine is the same as synthetic 4.0/).

Clean Technol. 2021, 3, 335–350. https://doi.org/10.3390/cleantechnol3020019 https://www.mdpi.com/journal/cleantechnol Clean Technol. 2021, 3 336

Clean Technol. 2021, 3, FOR PEER REVIEWcaffeine, expect that its economic value is higher due to its naturalness. However, caffeine2

from spent coffee grounds is rarely purified from raw extracts of bioactive molecules [18].

FigureFigure 1.1. TheThe chemicalchemical structurestructure ofof 1,3,7–trimethylxanthine1,3,7–trimethylxanthine (caffeine).(caffeine).

Hence,Caffeine the can aim be of incorporated this work is toin proposedaily life a products full investigation such as ofsodas parameters and energy of caffeine drinks production[23], painkillers from [24] spent or slimming coffee grounds creams such [25]. as: Nowadays, (i) the brewing coffee decaffeination method in relation is the withmost theimportant origin of known spent process coffee grounds to recover (household, caffeine [26, restaurants/coffee27]. Extracted caffeine shops), is the (ii) same the storage as syn- conditionsthetic caffeine, with expect the influence that its of economic a drying value step, (iii)is higher the first due step to ofits solid/liquidnaturalness. extractionHowever, tocaffeine recover from caffeine spent from coffee spent grounds coffee is groundsrarely pu (naturerified from of the raw solvent, extracts extraction of bioactive tempera- mole- turecules and [18]. time, solid/liquid ratio) and (iv) the second step of liquid/liquid extraction to selectivelyHence, recover the aim caffeine of this work (nature is to and propose volume a offull the investigation solvent, pH). of parameters of caffeine production from spent coffee grounds such as: (i) the brewing method in relation with the 2.origin Materials of spent and coffee Methods grounds (household, restaurants/coffee shops), (ii) the storage con- 2.1.ditions Chemicals with the and influence Reagents of a drying step, (iii) the first step of solid/liquid extraction to recoverPure caffeine standard from of spent caffeine, coffee ethanol grounds (96% (nat purity,ure of notthe denatured),solvent, extraction acetic temperature acid (99.5% purity)and time, and solid/liquid dichloromethane ratio) wereand (iv) obtained the second from ACROSstep of liquid/liquid ORGANICS. extraction Acetonitrile, to ethylselec- acetate,tively recovern-heptane caffeine and sodium(nature hydroxideand volume were of the supplied solvent, by pH). Fisher Chemical. Chlorhydric acid (37% w) was obtained from Roth. All solvents and reagents were of analytical grade and2. Materials used as received.and Methods 2.1. Chemicals and Reagents 2.2. Plant Material Pure standard of caffeine, ethanol (96% purity, not denatured), acetic acid (99.5% pu- Spent coffee grounds and roasted coffee beans used in this study have been obtained fromrity) aand local dichloromethane bakery (R1) and were from obtained two restaurants from ACROS (R2, R3). ORGANICS. The bakery Acetonitrile, and restaurants ethyl prepareacetate, coffeen-heptane beverage and sodium using the hydroxide brewing were method supplied called by percolation Fisher Chemical. using professional Chlorhydric high-pressureacid (37% w) was coffeemakers. obtained fromThe roastedRoth. All coffee solvents beans and are reagents from the were same of provider analytical for grade each restaurantand used as and received. are composed of a blend of 80% of Arabica and 20% of Robusta coffee. Indi- viduals obtain spent coffee grounds through different ways: by percolation with household capsule2.2. Plant coffeemakers Material (C1, C2), by percolation with mocha coffeemaker (M1) and by filtration with filterSpent coffeemakers coffee grounds (F1). andThe roasted amount coffee of water beans required used in to this brew study the coffeehave been was differentobtained forfrom the a householdlocal bakery capsule (R1) and coffeemakers from two restau C1, C2rants (13 mL/g(R2, R3).coffee The), household bakery and filtration restaurants cof- feemakerprepare coffee F1 (18 beverage mL/gcoffee using), mocha the brewing coffeemaker method M1 called (unknown percolation ratio) and using restaurants professional R1, R2,high-pressure R3 (unknown coffeemakers. ratio). The The residence roasted time coffee of waterbeans are inside from coffee the same were provider unknown for in each all cases.restaurant and are composed of a blend of 80% of Arabica and 20% of Robusta coffee. Individuals obtain spent coffee grounds through different ways: by percolation with 2.3.household Storage ofcapsule Spent Coffeecoffeemakers Grounds (C1, (SCG) C2), by percolation with mocha coffeemaker (M1) and Theby filtration spent coffee with grounds filter coffeemakers from the bakery (F1). (R1)The wereamount used of for water the studyrequired ofstorage to brew and the solid/liquidcoffee was different extraction. for the A full household week was capsule required coffeemakers for the bakery C1, C2 to (13 fill mL/g up a 25coffee L), plastichouse- buckethold filtration with spent coffeemaker coffee grounds F1 (18 thatmL/g wascoffee), then mocha closed. coffeemaker The naturally M1 (unknown wet (59.25% ratio) water and w/wrestaurants) spent coffeeR1, R2, grounds R3 (unknown were then ratio). stored The atresidence standard time pressure of water and inside temperature coffee were in a ◦ closeunknown plastic in bucket.all cases. Dry spent coffee grounds were obtained after 24 h in a 50 C (12.50% water w/w). 2.3. Storage of Spent Coffee Grounds (SCG) 2.4. Solid/Liquid Extraction—General Protocol The spent coffee grounds from the bakery (R1) were used for the study of storage and Extractionssolid/liquid withextraction. dichloromethane, A full week was ethyl requ acetateired for or the a solution bakery to at fill 0, 20,up 40,a 25 60, L plastic 80 or 100%bucket of with EtOH spent in water coffee (v/v grounds) were that performed. was then A closed. total of The 1.43, natura 2, 3.33lly or wet 5 g (59.25% of dry spentwater coffeew/w) spent grounds coffee were grounds mixed upwere with then 50 mLstored of the at standard studied solution pressure in and a round temperature bottom flask in a magnetically stirred maintained at 20, 40, 60 or 80 ◦C in a water bath for 5, 10 or 15 min. close plastic bucket. Dry spent coffee grounds were obtained after 24 h in a 50 °C oven (12.50% water w/w).

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The obtained extracts were filtered on a Buchner funnel equipped with a cellulose filter and rinsed with 10 mL of solvent. The resulting liquid extracts were then filtrated with a syringe filter (0.22 µm, polyethersulfone (PES) membrane) before HPLC analysis.

2.5. Liquid/Liquid Extraction–General Protocol The previous aqueous spent coffee grounds extracts were acidified with 0.5 mL of a 10% HCl until pH = 2, basified by adding 1 mL of a 2 N NaOH until pH = 14 or remained neutral at pH = 7. Liquid/liquid extractions were performed under magnetic stirring to treat the extract obtained in solid/liquid extraction with 60 mL (6 × 10 mL) or 180 mL (6 × 30 mL) of dichloromethane, ethyl acetate, ethyl ether or n-heptane. Then, the mixture was decanted for 15 min in a separating funnel. The organic phases were evaporated and the obtained dried extracts were dissolved in 50 mL of water for HPLC analysis.

2.6. HPLC-UV Analysis HPLC analysis was achieved with a Perkin Elmer (Series 200) system equipped with an automated sampler. A reverse phase column (Surf C18 TriF 100A 3 µm 33 × 4.6 mm ImChem) was used at 25 ◦C. The sample injection was 5 µL. The chromatographic separation was performed using an isocratic elution with a mixture of 0.1% (w) of acetic acid in water (solvent A) and acetonitrile (solvent B). A constant flow of solvent of 0.4 mL.min−1 with A/B ratio of 90/10 (v/v) during 30 min was applied. Detection was accomplished with a UV/Visible diode at a wavelength of 273 nm [28].

2.7. Statistical Analysis Analyses were performed in duplicate. Experiments described in Section 3.3 were performed in duplicate. Linear regression was tested on the results in Section 3.2 to observe caffeine degradation during storage of wet spent coffee grounds (Figure 4). Student tests (t-test) were performed with the results in Section 3.3 to evaluate the significant difference on caffeine extraction with various %EtOH in hydroalcoholic solvent (Figure 6), temperature (Figure 7), time (Figure 8) and solid/liquid ratio (Figure 9). Linear regressions were tested on the results in Section 3.4 to observe the liquid/liquid extraction of caffeine with different organic solvent (Figure 10) and volume (Figure 11). Student tests (t-test) were performed in Section 3.4 to evaluate the significant difference on selectivity (Figure 13) and yield (Figure 14) with various pH when liquid/liquid extraction of caffeine. Kruskal- Wallis tests were carried out on the results of Section 3.2 to evaluate the significance of the influence of %EtOH in hydroalcoholic solvent, temperature, time and solid/liquid ratio when solid/liquid extraction of caffeine (Table 1). Statistical analyses were performed with the software R (4.0.3 version).

3. Results and Discussion 3.1. Influence of Brewing Methods The influence of the brewing methods on the caffeine content has been studied by comparing the caffeine content of roasted coffee beans (RCB, initial state) and of spent coffee grounds (after brewing) of different origin of roasted coffee beans and location of preparation (Figure2). Restaurants (R1, R2, R3) have the same RCB provider and prepare coffee through percolation using professional high-pressure coffeemakers. Two different household capsule coffeemakers (C1, C2) and a mocha coffeemaker (M1) lead to by percolation at lower pressures. Last, filter coffeemakers (F1) only involved filtration method. −1 Spent coffee grounds from restaurant are richer in caffeine with 2.8–4.0 mg.g SCG; 21.5–30.8% remaining caffeine compared to the one of domestic house filtration with −1 0.7 mg.g SCG; 5.6% remaining caffeine. Spent coffee grounds obtained by domestic percolation through capsule (C1, C2) have also revealed low concentrations of −1 caffeine 0.5–1.1 mg.g SCG; 2.7–9.3% of remaining caffeine. Spent coffee grounds from Clean Technol. 2021, 3, FOR PEER REVIEW 4

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Clean Technol. 2021, 3, FOR PEER REVIEW 4 −1 mocha apparatus (M1) exhibited a concentration of caffeine of 2.1 mg.g SCG; 16.4% remaining caffeine.

Figure 2. Caffeine content (left) in roasted coffee beans (RCB) and spent coffee grounds (SCG) and remaining caffeine SCG/RCB (% w/w) (right) as a function of the origin. A 4 h Soxhlet extraction was performed with water to totally remove the caffeine.

Spent coffee grounds from restaurant are richer in caffeine with 2.8–4.0 mg.g−1 SCG; 21.5–30.8% remaining caffeine compared to the one of domestic house filtration with 0.7 mg.g−1 SCG; 5.6% remaining caffeine. Spent coffee grounds obtained by domestic percola- tion through capsule espresso (C1, C2) have also revealed low concentrations of caffeine

Figure 2. Caffeine content (left0.5–1.1) in roasted mg.g− coffee1 SCG; 2.7–9.3% beans (RCB) of andremaining spent coffee caffeine. grounds Spent (SCG) coffee and remaining grounds caffeine from mocha appa- Figure 2. Caffeine content (left) in roasted coffee beans (RCB) and spent coffee grounds (SCG) and remaining caffeine SCG/RCB (% w/w)(right) asratus a function (M1) of exhibited the origin. Aa 4concentr h Soxhletation extraction of caffeine was performed of 2.1 withmg.g water−1 SCG to; 16.4% totally removeremaining caffeine. SCG/RCB (% w/w) (right) as a function of the origin. A 4 h Soxhlet extraction was performed with water to totally remove the caffeine. the caffeine. These preliminary results are in agreement with those of the literature, reporting that coffee beverage obtained from filtration is richer in caffeine than the coffee beverage ob- These preliminary results are in agreement with those of the literature, reporting tainedSpent from coffee percolation, grounds from due restaurantto longer timeare richer and biggerin caffeine volume with of2.8–4.0 extraction. mg.g−1 InSCG this; work that coffee beverage obtained from filtration is richer in caffeine than the coffee beverage 21.5–30.8% remaining caffeine compared to the one of domestic house filtration with 0.7 obtainedseveral minutes from percolation, and 120 duemL for to longer filtration time ar ande compared bigger volume to 30 ofs and extraction. 30 mL Infor this percolation mg.g−1 SCG; 5.6% remaining caffeine. Spent coffee grounds obtained by domestic percola- work[29,30]. several Thus, minutes a selective and collection 120 mL for of filtration spent coffee are compared grounds in to restaurants 30 s and 30 is mL the for most suit- tion through capsule espresso (C1, C2) have also revealed low concentrations of caffeine percolationable for caffeine [29,30]. Thus,recovery a selective [31]. This collection collection of spent strategy coffee grounds was implemented in restaurants in is the2020 by the −1 0.5–1.1moststart-up suitable mg.g “KaffeeSCG for; caffeine2.7–9.3% Bueno” recovery of in remaining Denmark [31]. Thiscaffeine. that collection co Spentllects strategycoffee spent grounds coffee was implemented grounds from mocha for in appa-free 2020 from res- ratus (M1) exhibited a concentration of caffeine of 2.1 mg.g−1 SCG; 16.4% remaining caffeine. bytaurants, the start-up hotels “Kaffee and Bueno”offices consuming in Denmark thatover collects 300 kg spent of coffee coffee per grounds month for [32]. free fromVakalis et al. These preliminary results are in agreement with those of the literature, reporting that restaurants,have reported hotels that and household offices consuming capsules over of sp 300ent kg coffee of coffee grounds per month are [the32]. most Vakalis difficult to coffee beverage obtained from filtration is richer in caffeine than the coffee beverage ob- etvalorize al. have due reported to their that householdhigh moisture capsules level of spentand the coffee additional grounds areissues the mostgenerated difficult by plastic tainedto valorize from due percolation, to their high due moistureto longer leveltime andand thebigger additional volume issuesof extraction. generated In this by plastic work from the capsule.[33] severalfrom the minutes capsule and [33 ].120 mL for filtration are compared to 30 s and 30 mL for percolation [29,30]. Thus, a selective collection of spent coffee grounds in restaurants is the most suit- able3.2.3.2. Influence forInfluence caffeine of of Spent recovery Spent Coffee Coffee [31]. Grounds GroundsThis Storagecollection Storage strategy was implemented in 2020 by the start-upTheThe “Kaffee influence influence Bueno” of of spent spentin Denmark coffee coffee grounds that grounds collects storage storage spent has coffee has been been grounds investigated investigated for free using from using visual res- visual ob- taurants,observationsservations hotels and and and measurement offices consuming of of caffeine caffeine over content 300 cont kg inent of spent coffeein spent coffee per coffeemonth stored stored during[32]. Vakalis fourduring weeks et al.four weeks have reported that household capsules of spent coffee grounds are the most difficult to (Figures(Figures3 and3 and4). 4). valorize due to their high moisture level and the additional issues generated by plastic from the capsule.[33]

3.2. Influence of Spent Coffee Grounds Storage The influence of spent coffee grounds storage has been investigated using visual ob- servations and measurement of caffeine content in spent coffee stored during four weeks (Figures 3 and 4).

FigureFigure 3. 3.Spent Spent coffee coffee grounds grounds after after collect collect (left) and(left four) and weeks four storageweeks (storageright) in ( aright closed) in bucket a closed at bucket roomat room temperature. temperature.

Figure 3. Spent coffee grounds after collect (left) and four weeks storage (right) in a closed bucket at room temperature.

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100 100

90 90

80 80 Wet Dry Wet Dry

Remain caffeine (% w/w) (% caffeine Remain 70

Remain caffeine (% w/w) (% caffeine Remain 70 y = −1.1361x + 102.17 y = 1.1361x + 102.17 − R² = 0.9792 60 R² = 0.9792 60 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Storage duration (days) Storage duration (days)

Figure 4. Evolution of the remain caffeine in wet (▲) and dry () spent coffee grounds after four FigureFigure 4. Evolution 4. Evolution of the of theremain remain caffeine caffeine in wet in wet(▲) (andN) and dry dry() spent () spent coffee coffee grounds grounds after afterfour four weeks storage in a closed flask at room temperature. A 4 h Soxhlet extraction has been performed weeksweeks storage storage in a in closed a closed flask flask at room at room temperature. temperature. A 4A h 4Soxhle h Soxhlett extraction extraction has hasbeen been performed performed with water to remove totally the caffeine. withwith water water to toremove remove totally totally the the caffeine. caffeine. Visual observations showed white moss that appeared at the surface of the sample VisualVisual observations observations showed showed white white moss moss that that appeared appeared at atthe the surface surface of ofthe the sample sample revealing the growth of fungi. In the inside of raw material, wet spent coffee grounds have revealingrevealing the the growth growth of offungi. fungi. In Inthe the inside inside of ofraw raw material, material, wet wet spent spent coffee coffee grounds grounds have have started to agglomerate into small balls filled with green fungus. startedstarted to toagglomerate agglomerate into into small small balls balls filled filled with with green green fungus. fungus. Experimental measurements have shown that more than 30% of caffeine in wet spent ExperimentalExperimental measurements measurements have have shown shown that that more more than than 30% 30% of ofcaffeine caffeine in inwet wet spent spent coffee grounds are degraded after 28 days when no degradation has been observed for coffeecoffee grounds grounds are are degraded degraded after after 28 28 days days when no degradationdegradation hashas been been observed observed for for dry dry spent coffee grounds. For wet spent coffee grounds, this slight decrease already ap- dryspent spent coffee coffee grounds. grounds. For For wet wet spent spent coffee coffee grounds, grounds, this this slight slight decrease decrease already already appears ap- pears after a week with 96.4% of not degraded caffeine. However, most significant degra- pearsafter after a week a week with with 96.4% 96.4% of not of not degraded degraded caffeine. caffeine. However, However, most most significant significant degradation degra- dation was observed after four weeks with 69.3% of remaining caffeine, in addition to a dationwas observedwas observed after after four four weeks weeks with with 69.3% 69 of.3% remaining of remaining caffeine, caffeine, in addition in addition to a strongto a whitestrong moss white development. moss development. Finally, Finally, the linear the regressionlinear regression model model suggests suggests that degradation that degra- strong white moss development. Finally, the linear regression model suggests that degra- woulddation keepwould going keep over going 28 over days 28 for days wet for spent wet coffee spent grounds. coffee grounds. dation would keep going over 28 days for wet spent coffee grounds. Batista et et al. al. have have identified identified AspergillusAspergillus genusas genusas as asone one of filamentous of filamentous fungi fungi that that de- Batista et al. have identified Aspergillus genusas as one of filamentous fungi that de- developvelop naturally naturally on on coffee coffee beans beans [34]. [34 ].The The mech mechanismanism of of the the degradation degradation of of caffeine by velop naturally on coffee beans [34]. The mechanism of the degradation of caffeine by Aspergillus strains has beenbeen describeddescribed byby GummadiGummadi etet al.al. asas successivesuccessive demethylationdemethylation ofof Aspergillus strains has been described by Gummadi et al. as successive demethylation of caffeine intointo 1,3-dimethylxanthine, 1,3-dimethylxanthine, theophylline, theophylline, into into 3-methylxanthine, 3-methylxanthine, then, then, into into xanthine xan- caffeine into 1,3-dimethylxanthine, theophylline, into 3-methylxanthine, then, into xan- (Figurethine (Figure5)[ 35]. 5) Hence, [35]. Hence, main differences main differen explainingces explaining those results those between results drybetween and wet dry spent and thine (Figure 5) [35]. Hence, main differences explaining those results between dry and coffeewet spent grounds coffee can grounds be related can be to related the presence to the presence of water of in water the raw in the material, raw material, which is which thus wet spent coffee grounds can be related to the presence of water in the raw material, which responsibleis thus responsible for fungal for development.fungal development. is thus responsible for fungal development.

FigureFigure 5.5. PossiblePossible mechanismmechanism ofof enzymaticenzymatic caffeinecaffeine degradationdegradation inin spentspent coffeecoffee groundsgrounds underunder wetwet conditions,conditions, adaptedadapted Figure 5. Possible mechanism of enzymatic caffeine degradation in spent coffee grounds under wet conditions, adapted fromfrom GummadiGummadi etet al.al. [[35]35].. from Gummadi et al. [35]. 3.3. Solid/Liquid Extraction Optimization 3.3. Solid/Liquid Extraction Optimization The solid-liquid extraction process includesincludes threethree stages:stages: (i) permeation ofof the solvent throughThe solid-liquid the matrix, extraction (ii) solubilization process includes of the solute three and stages: (iii) diffusion(i) permeation of the of solute the solvent through through the matrix, (ii) solubilization of the solute and iii) diffusion of the solute through throughthe solvent the matrix, [36]. (ii) Pinelo solubilization et al. have of putthe so forthlute theand hypothesis iii) diffusion that of the the solute limited through stage in the solvent [36]. Pinelo et al. have put forth the hypothesis that the limited stage in solid- thesolid-liquid solvent [36]. extraction Pinelo et isal. the have diffusion put forth of the the hypothesis dissolvedsolute that the (step limited iii). Thestage diffusion in solid- of liquid extraction is the diffusion of the dissolved solute (step iii). The diffusion of the so- liquidthe soluteextraction is governed is the diffusion by the of Fick the laws dissolved [37]. Fick’ssolute first(step law iii). considersThe diffusion that of the the flux so- of lute is governed by the Fick laws [37]. Fick’s first law considers that the flux of the gradient lutethe is gradientgoverned of by solute the Fick concentration laws [37]. Fick’s goes first from law regions considers of high that concentrationthe flux of the gradient to regions of solute concentration goes from regions of high concentration to regions of low concen- of ofsolute low concentration goes with from a magnitude regions of that high is proportional concentration to to the regions concentration of low concen- gradient tration with a magnitude that is proportional to the concentration gradient (Equation (1)). tration(Equation with a (1)). magnitude that is proportional to the concentration gradient (Equation (1)). Equation (1): Fick first law Equation (1): Fick first law

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= − (1) where J is the diffusion flux expressed as amount of substance per unit area per time, D is the diffusion coefficient or diffusivity expressed as area per unit time, φ is the concentra- tion expressed as amount of substance per unit volume and x is the position expressed as the dimension of length. Hence, several parameters of solid-liquid extraction such as the nature of the solvent, temperature, time of extraction and solid/liquid ratio (w/v) or particle size can significantly affect the different stages of caffeine extraction and have been investigated. Only particle size has not been studied since spent coffee grounds is already a powder that does not need to be ground.

Clean Technol. 2021, 3 3.3.1. Influence of the Nature of the Solvent 340 Hydroalcoholic solutions, pure ethanol, pure water, dichloromethane and ethyl ace- tate were tested. The influence of hydroalcoholic solvent with different ratio EtOH/water (0/100 toEquation 100/0 v/v (1):) is Fick reported first law in Figure 6. Optimized conditions were observed with hy- droalcoholic solution at 40% ethanol (4.32 mg.g−1 SCGdϕ) compared to 100% water (3.63 mg.g−1 J = −D . (1) SCG) or 100% ethanol (0.26 mg.g−1 SCG). dx whereAs aJ isfurther the diffusion comparison, flux expressed Santana asand amount Macedo of substancehave determined per unit areaextraction per time, withD is H2theO/EtOH diffusion of 50/50 coefficient (w/w) oras diffusivityoptimum conditions expressed asfor area caffeine per unit recovery time, ϕ fromis the guarana concentration [38]. Thisexpressed is in accordance as amount with of substancethe fundamental per unit study volume of the and caffeinex is the solubility position expressed in hydroalco- as the holicdimension solutions of by length. Bustamante et al. reporting a higher caffeine solubility in H2O/EtOH with a Hence,40/60 ratio several (v/v) parameters between 5 ofand solid-liquid 40 °C [39].extraction such as the nature of the solvent, temperature,Zosel has timepatented of extraction a caffeine and extraction solid/liquid system ratio with (w/v )supercritical or particle size CO can2 at significantly tempera- turesaffect between the different 40 and stages 80 °C, of and caffeine pressures extraction between and 120 have and been 180 investigated. atm; 5 to 30 Only h were particle re- quiredsize hasto quantitatively not been studied extract since the spentcaffeine coffee [40]. groundsTodd and is Baroutian already a have powder reported that doesa com- not parativeneed to techno-economic be ground. analysis of the extraction of bioactive grape marc using super- critical CO2 vs. organic solvent extraction [41]. The costs of manufacture (COM) have been 3.3.1. Influence of the Nature of the Solvent reported as 87.0 NZ$/ton for organic solvents and 123.40 NZ$/ton for supercritical CO2; these resultsHydroalcoholic show that solutions,supercritical pure CO ethanol,2 is a more pure expensive water, dichloromethane process [41]. The and decaffein- ethyl ac- ationetate using were supercritical tested. The influence CO2 is efficient, of hydroalcoholic highly selective solvent and with eco-responsible different ratio EtOH/water for (0/100 without to 100/0 removingv/v) is reportedflavor. Unfortunately, in Figure6. Optimized the additional conditions cost can were seem observed questionable with hy- −1 −1 whendroalcoholic it comes solutionto treating at 40% waste. ethanol Indeed, (4.32 the mg.g residueSCG after) compared extraction to 100% is presently water (3.63 without mg.g −1 marketSCG) or value. 100% ethanol (0.26 mg.g SCG).

5 b b a ) a

SCG 4 c 3

2

1

Caffeine content (mg/g d

0 0 20406080100 EtOH (% v/v)

Figure 6. Influence of the EtOH/water ratio for the solid/liquid extraction of caffeine into spent Figurecoffee 6. groundsInfluence (Experimental of the EtOH/water conditions: ratio for 2 gthe of so SCGlid/liquid for 50 mLextraction of hydroalcoholic of caffeine into solvent spent at 20 ◦C coffee grounds (Experimental conditions: 2 g of SCG for 50 mL of hydroalcoholic solvent at 20 °C during 15 min, magnetically stirred). Values marked by the same letter are not significantly different during 15 min, magnetically stirred). Values marked by the same letter are not significantly differ- (p < 0.05) according to the Student test (t-test). ent (p < 0.05) according to the Student test (t-test). As a further comparison, Santana and Macedo have determined extraction with H2O/EtOH of 50/50 (w/w) as optimum conditions for caffeine recovery from guarana [38]. This is in accordance with the fundamental study of the caffeine solubility in hydroalcoholic

solutions by Bustamante et al. reporting a higher caffeine solubility in H2O/EtOH with a 40/60 ratio (v/v) between 5 and 40 ◦C[39]. Zosel has patented a caffeine extraction system with supercritical CO2 at temperatures between 40 and 80 ◦C, and pressures between 120 and 180 atm; 5 to 30 h were required to quantitatively extract the caffeine [40]. Todd and Baroutian have reported a comparative techno-economic analysis of the extraction of bioactive grape marc using supercritical CO2 vs. organic solvent extraction [41]. The costs of manufacture (COM) have been reported as 87.0 NZ$/ton for organic solvents and 123.40 NZ$/ton for supercritical CO2; these results show that supercritical CO2 is a more expensive process [41]. The decaffeination using supercritical CO2 is efficient, highly selective and eco-responsible for coffee bean without removing flavor. Unfortunately, the additional cost can seem questionable when it comes to treating waste. Indeed, the residue after extraction is presently without market value. Clean Technol. 2021, 3, FOR PEER REVIEW 7 Clean Technol. 2021, 3 341

The extracted caffeine for dichloromethane (1.7 mg.g−1 SCG) and ethyl acetate (0.5 −1 mg.g−1 SCGThe) are extracted worse than caffeine optimized for dichloromethane hydroalcoholic solution (1.7 mg.g (4.32 mg.gSCG)−1 andSCG). This ethyl is acetatesur- −1 −1 prising(0.5 mg.g since ShalmashiSCG) are worseet al. have than reported optimized that hydroalcoholiccaffeine solubility solution was higher (4.32 in mg.g dichloro-SCG). methaneThis is and surprising chloroform since compared Shalmashi to et water, al. have ethyl reported acetate, that ethanol, caffeine carbon solubility tetrachloride, was higher methanolin dichloromethane and acetone [42]. and chloroformHowever, according compared to toSondheimer water, ethyl et acetate,al., caffeine ethanol, occurs carbon in coffeetetrachloride, as chlorogenic methanol acid-caffeine and acetone complex [42]. However,where it is according hardly extractible to Sondheimer with chloroform et al., caffeine [43].occurs Hence, in coffeea polar as solvent chlorogenic such as acid-caffeine water-based complex solvent is where strongly it is recommended hardly extractible for ef- with fectivechloroform extraction [43 ].of Hence,caffeine a from polar coffee solvent matrices such as in water-based short time. In solvent addition, is strongly Bustamante recom- mended for effective extraction of caffeine from coffee matrices in short time. In addition, et al. have highlighted that caffeine display several functional groups with different hy- Bustamante et al. have highlighted that caffeine display several functional groups with drogen bonding abilities, explaining the affinity for water-based solvent [39]. Hence, we different hydrogen bonding abilities, explaining the affinity for water-based solvent [39]. can make the hypothesis that ethanol can decrease the polarity of water, while keeping Hence, we can make the hypothesis that ethanol can decrease the polarity of water, while strong hydrogen bonding interactions between water and caffeine. The Student test does keeping strong hydrogen bonding interactions between water and caffeine. The Student not show that an increase of the EtOH content in the hydroalcoholic solvent from 20% to test does not show that an increase of the EtOH content in the hydroalcoholic solvent from 40% increases caffeine extraction efficiency (p < 0.05). Therefore, extractions could be per- 20% to 40% increases caffeine extraction efficiency (p < 0.05). Therefore, extractions could formed with 20% EtOH to minimize the proportion of organic solvent. In our case, the be performed with 20% EtOH to minimize the proportion of organic solvent. In our case, following experiments were performed with hydroalcoholic solvent with 40% ethanol. the following experiments were performed with hydroalcoholic solvent with 40% ethanol.

3.3.2.3.3.2. Influence Influence of the of the Extraction Extraction Temperature Temperature TemperatureTemperature influence influence between between ambient ambient temperature temperature (20 (20 °C)◦ C)and and temperature temperature close close to ethanolto ethanol boiling boiling point point (80 °C) (80 was◦C) investigated was investigated on caffeine on caffeine extraction extraction (Figure (Figure7). A slight7). A ◦ −1 SCG −1 ◦ increaseslight (+11%) increase was (+11%) observed was observedbetween 20 between °C (4.23 20 mg.gC (4.23) mg.gand 60–80SCG )°C and (4.67–4.76 60–80 C −1 −1 mg.g(4.67–4.76SCG). mg.g SCG).

5 b cc a )

-1 4

3

2

1 Caffeine content (mg.g

0 20 40 60 80 Temperature (°C)

Figure 7. Influence of the temperature for the solid/liquid extraction of caffeine into spent coffee Figure 7: Influence of the temperature for the solid/liquid extraction of caffeine into spent coffee grounds (Experimental conditions: 2 g of SCG for 50. mL of H2O/EtOH (60/40 v/v), during 15 min, grounds (Experimental conditions: 2 g of SCG for 50. mL of H2O/EtOH (60/40 v/v), during 15 min, magnetically stirred). Values marked by the same letter are not significantly different (p < 0.05) magnetically stirred). Values marked by the same letter are not significantly different (p < 0.05) according to the Student test (t-test). according to the Student test (t-test). Linares et al. have found that a temperature between 40 and 70 ◦C did not affect the Linares et al. have found that a temperature between 40 and 70 °C did not affect the equilibrium concentration and extraction yield of yerba mate after 60 min of extraction [44]. equilibrium concentration and extraction yield of yerba mate after 60 min of extraction Hence, the temperature influenced the kinetic parameters since the pseudo first order [44]. Hence, the temperature influenced the kinetic parameters since the pseudo first order◦ kinetic constant (kobs) and effective diffusion coefficient (Dleaf) were higher at 70 C with kinetic constant (kobs3) and−1 effective diffusion 11coefficient2 −1 (Dleaf) were higher◦ at 70 °C with kobs = 3.155 × 10 s ,Dleaf = 9469 × 10 m .s compared to 40 C extraction with kobs = 3.155 × 103 s−1, 3Dleaf−1 = 9469 × 1011 m2.s−111 compared2 −1 to 40 °C extraction with kobs = 2.030 kobs = 2.030 × 10 s , Dleaf = 6092 × 10 m .s . The experiments that were performed 3 −1 11 2 −1 × 10(Figure s , D7leaf) showed= 6092 × similar10 m .s trends. The withexperiments an incomplete that were extraction performed at (Figure 20 ◦C compared7) showed to similar60–80 trends◦C. In with addition, an incomplete caffeine extraction is a very stableat 20 °C molecule compared even to 60–80 at high °C. temperature, In addition, as caffeinereported is a byvery Shalmashi stable molecule et al. [45 even]. The at Studenthigh temperature, test does not as showreported that by an Shalmashi increase of et the al. temperature[45]. The Student from 60test◦ Cdoes to 80 not◦C show increase that caffeine an increase extraction of the efficiency temperature (p < from 0.05). 60 Therefore, °C to 80 extractions°C increase were caffeine performed extraction at 60 efficiency◦C to reduce (p

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3.3.3.3.3.3. Influence InfluenceInfluence of the of the Extraction Extraction Time TimeTime TheThe time timetime of ofextractionof extractionextraction influence influenceinfluence was waswas investigated investigatedinvestigated on ononcaffeine caffeinecaffeine extraction, extraction,extraction, but butbut similar similarsimilar resultsresultsresults were werewere observed observedobserved from fromfrom 5 to 55 to15to 15min15 minmin of ofextractionof extractionextraction (Figure (Figure(Figure 8).8 ).8).

Figure 8. Influence of the time for the solid/liquid extraction of caffeine into spent coffee grounds FigureFigure 8. Influence 8. Influence of the of thetime time for forthe thesolid/liquid solid/liquid extraction extraction of caffeine of caffeine into intospent spent coffee coffee grounds grounds (Experimental conditions: 2 g of SCG for 50 mL of H2O/EtOH (60/40), at 60◦ °C, magnetically (Experimental(Experimental conditions: conditions: 2 g 2 of g ofSCG SCG for for 50 50 mL mL of of H H2O/EtOH2O/EtOH (60/40), (60/40), at 60 at 60°C, C,magnetically magnetically stirred). stirred). All values are not significantly different (p < 0.05) according to the Student test (t-test). stirred).All values All values are not are significantly not significantly different different (p < 0.05) (p < according0.05) according to the to Student the Student test (t- testtest). (t-test). ◦ LeeLee et al. et observedal. observed observed similar similar similar trends trends trends for for black for black black tea te extractiona tea extraction extraction with with water with water water at 25 at °C.25 at °C. 25The TheC. fast The fast caffeinefastcaffeine caffeine extraction extraction extraction might might mightbe bedue due be to due anto aneasy to aneasy accessibility easy accessibility accessibility of spentof ofspent spentcoffee coffee coffee grounds grounds grounds that that is that is is enhanced by the powder shape of spent coffee grounds due to the grinding step, like enhancedenhanced by bythe the powder powder shape shape of spentof spent coffee coffee grounds grounds due due to theto the grinding grinding step, step, like like the the the rolling step for black tea preparation [46]. The Student test did not show that times rollingrolling step step for for black black tea tea preparation preparation [46]. [46]. The The Student Student test test did did not not show show that that times times of of of extraction of 5, 10 or 15 min lead to significant extraction efficiency increase (p < 0.05). extractionextraction of 5,of 105, 10or or15 15min min lead lead to sitognificant significant extraction extraction efficiency efficiency increase increase (p <(p 0.05). < 0.05). Therefore, extraction was performed in 5 min to reduce energy consumption. However, Therefore,Therefore, extraction extraction was was performed performed in 5in mi 5 nmi ton reduceto reduce energy energy consumption. consumption. However, However, solid/liquid ratio experiments were performed in 15 min to more easily evaluate their solid/liquidsolid/liquid ratio ratio experiments experiments were were performed performed in 15in min15 min to moreto more easily easily evaluate evaluate their their in- in- influence. fluence.fluence.

3.3.4. InfluenceInfluence of the Solid/Liquid Ratio Ratio 3.3.4. Influence of the Solid/Liquid Ratio −1 The solid/liquid ratio ratio (5–35 (5–35 mL.g mL.g−1) of) caffeine of caffeine extraction extraction was was studied studied (Figure (Figure 9). The9). The solid/liquid ratio (5–35 mL.g−1) of caffeine extraction−1 was studied−1 (Figure 9). The −1 The major difference was observed between 5 mL.g−1 (2.35 mg.g−1 SCG) and 15 mL.g−1 major difference was observed between 5 mL.g (2.35 mg.g SCG) and 15 mL.g (3.23 major difference−1 was observed between 5 mL.g−1 (2.35 mg.g−1SCG) and 15 mL.g−1 (3.23 (3.23 mg.g SCG). The Student test does not show that a variation of the solid/liquid mg.g−1 SCG). The Student test does not show that a variation of the solid/liquid ratio from mg.gratio−1 SCG from). The 15 Student to 25 mL.g test− 1doessignificantly not show increase that a variation caffeine extractionof the solid/liquid (p < 0.05). ratio The from exper- 15 to 25 mL.g−1 significantly increase caffeine extraction (p < 0.05). The experiments could 15 imentsto 25 mL.g could−1 significantly be therefore increase performed caffeine with extraction a 15 mL.g (−p1

5 5 )

-1 c ) 4 bb -1 4 bbc

3 a 3 a 2 2

1 1 Caffeine content (mg.g Caffeine content (mg.g

0 0 5152535 5152535 Solid/liquid ratio (mL.g-1) Solid/liquid ratio (mL.g-1)

Figure 9. Influence of the solid/liquid ratio for the solid/liquid extraction of caffeine into spent ◦ coffee grounds. (Experimental conditions: 2 g of SCG for 50 mL of H2O/EtOH (60/40), at 60 C during 15 min, magnetically stirred). Values marked by the same letter are not significantly different (p < 0.05) according to the Student test (t-test).

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Ho Row et al. investigated the influence of solid/liquid ratio from 5 to 60 mL.g−1 in ◦ decaffeination of coffee bean waste with EtOH/H2O 50/50 (v/v) at 80 C during 60 min [47]. They reported an increase of the decaffeination with bigger volume of extraction. More precisely, up to 78%, 92% and 98% of caffeine were extracted with 5, 10 and 20 mL per gram, respectively. According to the authors, when the amount of solvent increases, the chance of bioactive components coming into contact with the solvent goes up, leading to higher leaching out rates. In addition, the bad diffusion of solutes cannot be compensated by strong agitation since Silva et al. have observed weak influence of stirring (0–400 rpm) on the yield of extraction [48]. Kruskal-Wallis comparative analysis of the influence of the different factors is summa- rized in Table1. The results show, with a probability superior to 99.9%, that solvent and temperature significantly influence caffeine extraction in the range of values that has been studied.

Table 1. Kruskal-Wallis test of influencing parameters during solid/liquid extraction of spent coffee grounds.

Kruskal-Wallis Degree of Freedom Probability Value Chi-Squared (fd) (p-Value) Solvent 22.044 5 0.0005136 Temperature 20.022 3 0.000168 Time 6.1869 2 0.04535 Solid/Liquid ratio 9.0544 3 0.02858

3.4. Purification of Caffeine by Liquid/Liquid Extraction The solid/liquid extract is rich in caffeine and impurities such as polyphenols, melanoidins and other polar molecules. To purify caffeine, a liquid/liquid extraction step is required. Instead of hydroalcoholic extracts, water extracts have been used for this study to simplify the liquid/liquid extraction. Hence, the following results can be extrapolated to hydroalcoholic extracts that can be evaporated and solubilized into water. This part will focus on the optimization of liquid/liquid extraction such as nature and volume of organic solvent and pH of aqueous phase.

3.4.1. Influence of Different Organic Solvents for Liquid/Liquid Extraction of Caffeine The choice of the organic solvent is the most important parameter in a liquid/liquid extraction [49]. It should be immiscible with the aqueous phase and the solutes need a higher affinity for the solvent of extraction to be recovered. It is measured as K, the coefficient distribution/partition, provided in Equation (2). Equation (2): Distribution/Partition coefficient in liquid/liquid extraction.

Solute concentration in organic phase (extraction solvent) K = (2) solvent Solute concentration in aqueous/hydroalcoholic phase (original solution)

Since the distribution coefficient is a ratio, unless K is very large, a single extraction is not enough to extract all the solute. A multiple extraction is recommended, that is, in different steps, at equilibrium, the solute has the same partition/distribution coefficient K. Then, to perform liquid/liquid extraction in appropriate conditions, the volume of extraction has been split into six small extractions of 10 mL instead of a unique extraction of 60 mL. Among the organic solvents with immiscibility for water, dichloromethane, ethyl acetate, diethyl ether and n-heptane have been tested (Figure 10). Diethyl ether and n- heptane are not reported in Figure 10 since no caffeine was recovered in these solvents. Interestingly, Table2 shows that dichloromethane or ethyl acetate have the dipolar moment Clean Technol. 2021, 3, FOR PEER REVIEW 10

Among the organic solvents with immiscibility for water, dichloromethane, ethyl ac- etate, diethyl ether and n-heptane have been tested (Figure 10). Diethyl ether and n-hep- tane are not reported in Figure 10 since no caffeine was recovered in these solvents. Inter- estingly, Table 2 shows that dichloromethane or ethyl acetate have the dipolar moment and the Hildebrand function the closest of those of caffeine, and are the solvents the most appropriate here for the transfer of caffeine.

Table 2. Dipolar moment μ and Hildebrand function δ of caffeine and organic solvents for liq- uid/liquid extraction [50–53].

Dipolar Moment Hildebrand Function

μ (Debye) δ (cal/cm3)1/2 Caffeine 3.46 13.8 Dichloromethane 1.600 9.93 Ethyl acetate 1.780 9.10 Diethyl ether 1.098 7.62 n-heptane 0 7.4

Clean Technol. 2021, 3 After six cycles, 80% of caffeine transferred (Equation (3)) in dichloromethane instead344 of 57% in ethyl acetate. Equation (3): Yield of liquid/liquid extraction. ℎ and the Hildebrand function the= closest of those of caffeine, and are the solvents the most(3) / appropriate here for the transfer of caffeine.

100 Ethyl Acetate (6x10 mL) y = 1.3462x + 8.3118 R² = 0.9539 80 Dichloromethane (6x10 mL)

60 d extraction (% w) (% extraction d

40 y = 0.9631x + 0.9768 R² = 0.9938 20

Yield of liquid/liqui 0 0 102030405060 Volume (mL)

Figure 10. Evolution of the yield of caffeine transferred (%w) from aqueous extract (60 mL) into Figure 10. Evolution of the yield of caffeine transferred (%w) from aqueous extract (60 mL) into 6 × 106 mL× 10 of mLdichloromethane of dichloromethane (▲) or (ethylN) or acetate ethyl acetate() during () liquid/liquid during liquid/liquid extraction extraction at room temper- at room ature.temperature.

TableMohammed 2. Dipolar momentet al. haveµ and performed Hildebrand liquid/liqui functiondδ extractionof caffeine of and caffeine organic with solvents dichloro- for liq- methane,uid/liquid since extraction it is the [50 –most53]. widely used solvent for decaffeination. Their experience al- lowed recovering 98 to 99% of caffeine [54]. The efficiency of dichloromethane compared Dipolar Moment Hildebrand Function to ethyl acetate is supported by the experimental measurements of mole fraction solubility µ (Debye) δ (cal/cm3)1/2 of caffeine in dichloromethane that is 10 times higher than the one in ethyl acetate [42]. However, dueCaffeine to green chemistry concerns 3.46it is more suitable to use an 13.8eco-compatible solvent suchDichloromethane as ethyl acetate. In addition, ethyl 1.600 acetate extraction curve follows 9.93 a linear regressionEthyl model. acetate This suggests that caffeine 1.780 extraction has not yet reach a 9.10 plateau for the maximumDiethyl ethyl acetate ether volume that was used 1.098 in the present study. 7.62

n-heptane 0 7.4

After six cycles, 80% of caffeine transferred (Equation (3)) in dichloromethane instead of 57% in ethyl acetate. Equation (3): Yield of liquid/liquid extraction.

Amount of caf feine in organic phase Yield = . (3) liquid/liquid Amount of caf feine in aqueous extract

Mohammed et al. have performed liquid/liquid extraction of caffeine with dichloro- methane, since it is the most widely used solvent for decaffeination. Their experience allowed recovering 98 to 99% of caffeine [54]. The efficiency of dichloromethane compared to ethyl acetate is supported by the experimental measurements of mole fraction solubility of caffeine in dichloromethane that is 10 times higher than the one in ethyl acetate [42]. However, due to green chemistry concerns it is more suitable to use an eco-compatible solvent such as ethyl acetate. In addition, ethyl acetate extraction curve follows a linear regression model. This suggests that caffeine extraction has not yet reach a plateau for the maximum ethyl acetate volume that was used in the present study.

3.4.2. Influence of the Volume of Extraction Solvent The influence of the volume of ethyl acetate with 6 × 30 mL compared to 6 × 10 mL to treat 60 mL of aqueous extract has been reported during liquid/liquid extraction (Figure 11). The curve of the yield has shown quite linear increases even up to 6 × 30 mL of ethyl acetate. Better results with 6 × 30 mL have reached 93.4% of extraction yield. By increasing Clean Technol. 2021, 3, FOR PEER REVIEW 11

3.4.2. Influence of the Volume of Extraction Solvent The influence of the volume of ethyl acetate with 6 × 30 mL compared to 6 × 10 mL to Clean Technol. 2021, 3 345 treat 60 mL of aqueous extract has been reported during liquid/liquid extraction (Figure 11). The curve of the yield has shown quite linear increases even up to 6 × 30 mL of ethyl acetate. Better results with 6 × 30 mL have reached 93.4% of extraction yield. By increasing thethe volume, volume, solvent solvent of of extraction extraction is is not not saturated saturated in in solute, solute, th thus,us, coefficient coefficient distribution distribution is is increasedincreased in in favor favor of of the the solvent solvent of of extraction extraction.. Results Results with with 6 6 ×× 3030 mL mL of of ethyl ethyl acetate acetate are are competitivecompetitive with with those those obtained obtained with with 6 6 ×× 1010 mL mL of of dichloromethane. dichloromethane.

100 Ethyl Acetate (6x10 mL) Ethyl Acetate (6x30 mL) 80

60 y = 0.9631x + 0.9768

d extraction (% w) (% extraction d R² = 0.9938 y = 0.5229x + 1.7279 R² = 0.9778 40

20

Yield of liquid/liqui 0 050100150 Volume (mL)

Figure 11. Evolution of the yield of caffeine transferred (%w) from aqueous extract (60 mL) into Figure 11. Evolution of the yield of caffeine transferred (%w) from aqueous extract (60 mL) into 6 × × × 106 mL10 ( mL) or ( 6 )× or 30 6 mL30 ethyl mL acetate ethyl acetate (♦) during () during liquid/liquid liquid/liquid extracti extractionon at room at temperature. room temperature. 3.4.3. Influence of Aqueous Extract pH 3.4.3. Influence of Aqueous Extract pH The influence of the pH of the solution is an important parameter that can affect both, yieldThe and influence selectivity of the of the pH liquid/liquid of the solution extraction is an important of caffeine. parameter The affinity that can of affect caffeine both, for yieldthe aqueous and selectivity or organic of the phase liquid/liquid is strongly extraction related to of the caffeine. different The pKa affinity of caffeine of caffeine and for pH theof theaqueous aqueous or organic phase. phase Charged is strongly molecules relate haved to better the different affinity topKa aqueous of caffeine phase and through pH of thehydrogen aqueous bonds. phase. Charged The selectivity molecules can have be observed better affinity indirectly to aqueous through phase the through area ratio hy- of drogencaffeine bonds. in chromatograms The selectivity (Equation can be observed (4)). indirectly through the area ratio of caffeine in chromatogramsEquation (4): (Equation Area ratio (4)). of caffeine. Equation (4): Area ratio of caffeine. Area of caf feine peak in the chromatogram extract at 273 nm Area ratio of caf feine = ℎ ℎ 273 (4) = Area of all the peaks in the chromatogram extract at 273 nm (4) ℎ ℎ ℎ 273 The influence of pH on the selectivity and yield has been illustrated through the analysisThe influence of chromatograms of pH on the (Figure selectivity 12), the and area yield ratio has of been caffeine illustrated (Figure through 13) and the yields anal- of ysisextractions of chromatograms (Figure 14). (Figure 12), the area ratio of caffeine (Figure 13) and yields of ex- tractionsThe (Figure observation 14). of chromatograms after liquid/liquid extraction was reported. Ethyl acetate was yellow colored after liquid/liquid extractions performed at pH = 2 and 7 and colorless at pH of 14. At pH = 2 and pH = 7, the chromatograms contain several peaks with moderate to high intensity in addition of caffeine (Figure 12). By contrast, at pH = 14, chromatogram contains only the peak of caffeine peak (Figure 12). This results in an area ratio of caffeine described in Equation (4) that is higher at pH = 14 than at pHs = 2 and 7, indirectly indicating a higher purity (Figure 13). In accordance with those observations, Student tests show significant differences between the values of the area ratio of caffeine (Equation (4)) at different pH (p < 0.05). On the other hand, Student tests do not show significant difference on the yield of caffeine transferred in ethyl acetate as a function of pH (p < 0.05). To conclude, statistical analyses of the pH influence unequivocally suggest that pH exclusively affects the selectivity of the liquid/liquid extraction of caffeine.

Clean Technol. 2021, 3, FOR PEER REVIEW 12 Clean Technol. 2021, 3 346 Clean Technol. 2021, 3, FOR PEER REVIEW 12

Figure 12. Chromatograms of caffeine and impurities transferred in ethyl acetate at pH = 2 (red, Figure 12. Chromatograms of caffeine and impurities transferred in ethyl acetate at pH = 2 (red, behind), pH = 7 (green, middle) and pH = 14 (blue, forward) during liquid/liquid extraction at room behind),Figure 12.pH Chromatograms = 7 (green, middle) of caffeine and pH and= 14 impurities (blue, forward) transferred during in liqui ethyld/liquid acetate extraction at pH = 2 at(red, roomtemperature.behind), temperature. pH = 7 (green, middle) and pH = 14 (blue, forward) during liquid/liquid extraction at room temperature.

Figure 13. Influence of the pH on the area ratio of caffeine of liquid/liquid extraction of aqueous Figure 13. Influence of the pH on the area ratio of caffeine of liquid/liquid extraction of aqueous extract (60 mL) treated with ethyl acetate (60 mL) at room temperature. Values marked by the symbol extractFigure (60 13. mL) Influence treated of with the pHethyl on acetate the area (60 ratio mL )of at ca roomffeine temperature. of liquid/liquid Values extraction marked of by aqueous the symbol*extract are significantly * (60 are mL) significantly treated different with different (p ethyl< 0.05) (acetatep < according 0.05) (60 according mL to) at the room to Student the temperature. Student test ( t-testtest). Values (t-test). Values marked Values marked bymarked the by the bysymbolsymbol the symbol ** * are significantlysignificantly** are significantly different different ((pp < 0.01)0.05) (p < according 0.01) according to the Studentto the Student test (t-t- test).test).test ( t-Valuestest). marked by the symbol ** are significantly different (p < 0.01) according to the Student test (t-test).

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Clean Technol. 2021, 3, FOR PEER REVIEW 34713

Figure 14. Influence of the pH on the yield of liquid/liquid extraction of aqueous extract (60 mL) treated with ethyl acetate (60 mL) at room temperature. All the values are not significantly differ- ent (p < 0.05) according to the Student test (t-test).

The observation of chromatograms after liquid/liquid extraction was reported. Ethyl acetate was yellow colored after liquid/liquid extractions performed at pH = 2 and 7 and colorless at pH of 14. At pH = 2 and pH = 7, the chromatograms contain several peaks with moderate to high intensity in addition of caffeine (Figure 12). By contrast, at pH = 14, chromatogram contains only the peak of caffeine peak (Figure 12). This results in an area ratio of caffeine described in Equation (4) that is higher at pH = 14 than at pHs = 2 and 7, indirectly indicating a higher purity (Figure 13). In accordance with those observations, Student tests show significant differences between the values of the area ratio of caffeine FigureFigure(Equation 14. 14. InfluenceInfluence (4)) at differentof of the the pH pH onpH on the the(p yield < yield 0.05). of of liquid/ liquid/liquidOn theliquid other extraction extraction hand, ofStudent aqueous of aqueous tests extract extract do (60 not (60mL) show mL) treatedtreatedsignificant with with ethyl ethyldifference acetate acetate on (60 (60 the mL) mL) yield at at room room of caffeinetemper temperature.ature. transferred All the values in ethyl are notacetate significantlysignificantly as a function differentdiffer- of ent(pHp < (p 0.05)( p< 0.05)< 0.05). according according To conclude, to theto the Student Student statistical test test (t-test). (analysest-test). of the pH influence unequivocally suggest that pH exclusively affects the selectivity of the liquid/liquid extraction of caffeine. ThePolyphenolsPolyphenols observation likelike of chlorogenicchlorogenic chromatograms acidsacids after areare thethe liquid/liquid mainmain impuritiesimpurities extraction inin aqueousaqueous was reported. extract,extract, Ethyl thus,thus, acetatepotentiallypotentially was transferred yellowtransferred colored in in ethyl ethylafter acetate acetateliquid/liquid [55 [55,56].,56]. Gazzaniextractions Gazzani et al. etperformed haveal. have reported reportedat pH that = 2 chlorogenicthat and chloro- 7 and colorlessacidsgenic fromacids at pH greenfrom of 14. coffeegreen At pH beanscoffee = 2 and werebeans pH more were= 7, the efficientlymore chromatograms efficiently extracted extracted contain in ethyl several in acetate ethyl peaks acetate at acidic with at moderatepHacidic [57 ].pH This to [57]. high might This intensity due might to the indue lackaddition to ofthe affinity lackof ca of offfeine affinity protonated (Figure of protonated form12). ofBy chlorogenic contrast, form of at chlorogenic acids pH = since 14, chromatogramtheiracids pKa since are their pKa contains 1pKa= 3.50, are only pKapKa the21 = peak 8.423.50, andof pKa caffeine pKa2 = 8.423 = peak 11.00 and (Figure [pKa58].3 For= 12). 11.00 our This experimental[58]. results For ourin an results,experi- area ratioatmental pH of = caffeineresults, 2 and 7,describedat thepH –OH= 2 andin function Equation 7, the –OH of (4) chlorogenic thatfunction is higher of acids chlorogenic at stayedpH = 14 inacids than a totally stayedat pHs or in= partially 2a and totally 7, indirectlyprotonatedor partially indicating form, protonated which a higher resultedform, purity which in lower (Figure resulted affinity 13). in forIn lower accordance aqueous affinity phase with for and thoseaqueous higher observations, transferphase and in Studentethylhigher acetate. transfertests show At in pH significantethyl = 14, acetate. all thedifferences –OHAt pH functions = between14, all ofthe the chlorogenic –OH values functions of acidsthe area of were chlorogenic ratio deprotonated of caffeine acids − (Equationintowere –O deprotonated, which(4)) at favoreddifferent into –O hydrogen pH-, which (p < 0.05). bondsfavored On with hydrogenthe aqueous other bondshand phase,, withStudent resulting aqueous tests in nonedophase, not transfer result-show significantofing chlorogenic in none difference transfer acids inof on ethylchlorogenic the yield acetate. of acids caffeine in ethyl transferred acetate. in ethyl acetate as a function of pH (pCaffeineCaffeine < 0.05). To hashas conclude, lowlow interactioninteraction statistical withwith analyses aqueousaqueous of phasethephase pH at atinfluence pHpH == 2,2, 7unequivocally7 andand 1414 sincesince suggestcaffeinecaffeine thatpKapKa pH areare exclusively pKapKa11 == 0.6–0.70.6–0.7 affects andand the pKapKa selectivity22 = 14 (Figure of the 1515) liquid/liquid)[ [59–61].59–61]. ThisThis extraction resultsresults inin of aa caffeine. similarsimilar yieldyield ofof caffeinecaffeinePolyphenols transferredtransferred like atat chlorogenic allall thethe studiedstudied acids pHpH are (Figure(Figure the main 14 14).). impurities in aqueous extract, thus, potentially transferred in ethyl acetate [55,56]. Gazzani et al. have reported that chloro- genic acids from green coffee beans were more efficiently extracted in ethyl acetate at acidic pH [57]. This might due to the lack of affinity of protonated form of chlorogenic acids since their pKa are pKa1 = 3.50, pKa2 = 8.42 and pKa3 = 11.00 [58]. For our experi- mental results, at pH = 2 and 7, the –OH function of chlorogenic acids stayed in a totally or partially protonated form, which resulted in lower affinity for aqueous phase and higher transfer in ethyl acetate. At pH = 14, all the –OH functions of chlorogenic acids wereFigureFigure deprotonated 15.15. Acid/baseAcid/base intoequilibrium –O-, which of caffeine. favored hydrogen bonds with aqueous phase, result- ing in none transfer of chlorogenic acids in ethyl acetate. CaffeineNoNo experimentexperiment has low has interaction been been carried carried with outaqueous out with with pHphase pH < <0.7 at 0.7 andpH and =pH 2, pH ≥7 14and≥ to14 14confirm to since confirm caffeinethat that caf- pKacaffeinefeine are cation pKa cation 1or = or0.6–0.7anion anion has and has more pKa more 2affinity = affinity14 (Figure for for water. water.15) [59–61]. This results in a similar yield of caffeine transferred at all the studied pH (Figure 14). 4. Conclusions The present study promotes the valorization of the most popular food waste, spent coffee grounds, into caffeine molecule, presenting a well-established market for applications in agrifood, cosmetic, nutraceutic or pharmaceutic industries. This paper proposes a complete investigation and optimization for the production of caffeine from spent coffee grounds, including all key steps such as collection, storage, solid/liquid extraction and purification by liquid/liquid extraction. Investigation of collection has shown that spent coffee grounds from restaurants are Figure 15. Acid/base equilibrium of caffeine. richer in caffeine with 30% remaining caffeine. Studies on storage have reported that dryingNo is experiment an unnecessary has been step carried when spent out with coffee pH grounds < 0.7 and extractions pH ≥ 14are to confirm performed that below caf- one week of storage because degradation of caffeine is lower than 4%. Investigation of feine cation or anion has more affinity for water. solid/liquid extraction indicates that extraction of 2 g with 50 mL hydroalcoholic solvent

40% EtOH and 25 mL.g−1 solid/liquid ratio at 60 ◦C during 5 min are the optimized conditions. Investigation of caffeine purification by liquid/liquid extraction illustrates that

Clean Technol. 2021, 3 348

extraction of 60 mL of aqueous phase at pH = 12–14 with 6 × 30 mL of green solvent like ethyl acetate is efficient to recover 93.4% of purified caffeine. In accordance with circular economy and the need to 100% waste valorization, further studies will be carry out to perform (i) the valorization of decaffeinated extract that is rich in polyphenols [18,62] and (ii) the valorization of solid coffee residue after hydroalcoholic extractions that is a rich lignocellulose and lipid material [4,5].

Author Contributions: Experimental work at lab, A.V.; writing—original draft preparation, A.V.; writing—review and editing, A.V., M.D., C.P., G.C.; Student supervision, M.D., C.P., G.C.; project coordination, G.C. All authors have read and agreed to the published version of the manuscript. Funding: The authors gratefully acknowledge the Auvergne Rhône Alpes French Region for the awarding of the PhD scholarship to Alexandre Vandeponseele and for equipment funding through the Pack Ambition Recherche program. They also acknowledge the TRIALP company and the Université Savoie Mont Blanc Foundation for their financial supports through soMAR project. Acknowledgments: The authors warmly acknowledge David Gateuille and Lise Marchal for their help in performing statistical analyses. Conflicts of Interest: The authors declare no conflict of interest.

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