Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2019, 67, 5978−5988
Optimizing Nucleophilic Depolymerization of Proanthocyanidins in Grape Seeds to Dimeric Proanthocyanidin B1 or B2 Kui-Shan Wen,†,‡ Xiao Ruan,†,‡ Jing Wang,§ Li Yang,†,‡ Feng Wei,†,‡ Ying-Xian Zhao,†,‡ and Qiang Wang*,†,‡ †
Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, People’s Republic of China Ningbo Research Institute, Zhejiang University, Ningbo 315100, People’s Republic of China § Ningbo Osaki Biotech Co., Ltd, Ningbo 315800, People’s Republic of China Downloaded via BUFFALO STATE on July 30, 2019 at 09:47:22 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
ABSTRACT: Depolymerization of polymeric proanthocyanidins (PPCs) in grape seeds into oligomeric proanthocyanidins (OPCs), especially the dimers, has important academic significance and practical value. Reaction conditions including nucleophilic reagent/PPC mass ratio, HCl concentration, reaction time, and temperature were systematically optimized by central composite design to maximize the yield of the dimeric product B2 or B1. The yield of B2 reached 3.35 mg mL−1 under the conditions of (−)-epicatechin/PPC mass ratio 2.8, HCl concentration 0.06 mol, reaction time 16 min and temperature 36 °C, and that of B1 reached 3.64 mg mL−1 under the conditions of (+)-catechin/PPC mass ratio 2.8, HCl concentration 0.07 mol, reaction time 17 min, and temperature 34 °C. Overall, this study has provided theoretical guidance and a practical approach to improvethe reaction process and economic value of proanthocyanidins in grape seed proanthocyanidin extract. KEYWORDS: grape seed proanthocyanidin extracts (GSPEs), polymeric proanthocyanidins (PPCs), depolymerization, mechanism, optimization
■
INTRODUCTION As a group of very important polyphenolic compounds with vital functions of resisting microbial pathogens,1 insect pests,2 and larger herbivores,3 proanthocyanidins are widely present in natural plants of grape, cranberry, cocoa, etc., particularly in the skin and seed of these plants.4 Up to now, a great deal of research interest has been generated for proanthocyanidins due to their important roles in stabilizing color and enhancing mouth feel of red wines.5,6 As one of the most abundant sources of proanthocyanidins in nature, grape seed contains much richer polyphenolic compounds in comparison to its skin and stem. In fact, most products of proanthocyanidin in the market originally come from grape seed.7,8 Grape seed proanthocyanidin extracts (GSPEs) are composed of oligomers (OPCs) and polymers (PPCs) with flavan-3-ol monomeric units,9 such as (+)-catechin, (−)-epicatechin, and (−)-epicatechin-3-O-gallate (CATs), linked mainly through C4→C8 or C4→C6 (B type).10,11 The difference in constitutional units, bonding positions, and configurations gives structural diversity of the polymerized proanthocyanidins, and the number of isomers increases markedly with an increase in the degree of polymerization (DP).12 Substantial investigations have demonstrated that proanthocyanidins could broadly provide pharmacological and therapeutic functions against oxidative stress and degenerative diseases such as acute and chronic stress, gastrointestinal distress, neurological disorders, pancreatitis, cardiovascular dysfunctions, various stages of neoplastic processes, and carcinogenesis including detoxification of carcinogenic metabolites13,14 and curative efficacy. However, these functions largely depend on their structures and DP contents. Jiang et al.15 evaluated the effect of GSPE fractions with different DPs © 2019 American Chemical Society
on blood glucose, lipids, and hepatic oxidative stress in diabetic rats and confirmed that GSPE gave a positive effect on diabetes in rats, and its oligomeric form might be more effective than other forms. Moreover, GSPE exhibited potent abilities to scavenge free radicals in both in vitro and in vivo models. It has been lately verified that PPCs possess the highest antioxidant activities followed by OPCs, whereas CATs gave a lower antioxidant activity, indicating that the vitro antioxidant activities of proanthocyanidins were positively related to their DPs.16 In fact, the dimers, trimers, and tetramers of proanthocyanidin could be preferably absorbed and presented in blood circulation, whereas its polymers with higher molecular weight could not.17 In addition, Tomás-Barberan et al.18 proved that an increase of monomers in proanthocyanidins could enhance bioavailability. Among various proanthocyanidins, dimeric proanthocyanidins B1 and B2 have attracted more research interest; their physiological mechanism has been generally recognized and application value been highly affirmed so far.19−21 Extraction and separation of proanthocyanidins from plants still presents a major challenge to scientists due to the structural diversity and complexity. There have been a large number of studies on the fractionation of proanthocyanidins during the last two decades. A variety of separation methods such as size-exclusion chromatography,22 solid-phase extraction,23 enzymatic extraction,24 and normal-phase chromatography25 were tentatively adopted for the fractionation of Received: Revised: Accepted: Published: 5978
February 22, 2019 April 25, 2019 May 9, 2019 May 9, 2019 DOI: 10.1021/acs.jafc.9b01188 J. Agric. Food Chem. 2019, 67, 5978−5988
Article
Journal of Agricultural and Food Chemistry
Figure 1. Reaction mechanism and pathways in PPC depolymerization: (I) acid-catalyzed cleavage of interflavan bonds; (II) formation of dimeric proanthocyanidin by bonding C4−C8 or C4−C6; (III) formation of trimeric proanthocyanidin; (IV) formation of polymeric proanthocyanidin; (V) formation of the positively charged chalcane ion; (VI) formation of gambiriins.
proanthocyanidins corresponding to DP. Zhang et al.26 conducted the preparative separation of GSPEs by highspeed counter-current chromatography (HSCCC), and under the optimal conditions of HSCCC, GSPEs could be separated into seven distinct fractions (F1−F7) with an increase in the mean degree of polymerization from 1.44 to 6.95. Several methods of chemical reaction have been also developed to depolymerize proanthocyanidin polymers into oligomers for the enhancement of bioavailability.27−30 Acidcatalyzed hydrolysis is used to cleave the interflavanic C−C bonds in proanthocyanidin molecules into the terminal units (CATs) and the upper intermediate units or fragments as carbocations which react with nucleophiles to form stable conjugates. Foo and Porter27 heated palm proanthocyanidin polymers with epicatechin in ethanol/acetic acid at 95 °C for 22 h and then identified some dimers. Moreover, polymeric proanthocyanidins from grape seeds or hazelnut skins were reacted with flavan-3-ols under acidic conditions for the semisynthesis of dimers.30 Among these reaction experiments, the HCl-catalyzed depolymerization with catechin or epicatechin was convenient to operate with a simple apparatus and the yield of oligomers was relatively high.28 According to our
understanding, however, the depolymerization process of PPC from GSPE will generate a complex mixture of various intermediate products (see Figure 1). With a proper design based on the reaction mechanism and by optimization of reaction conditions, the use of HCl and the addition of (+)-catechin and (−)-epicatechin as nucleophilic agents may be be profitable to enhance the proportion of dimers proanthocyanidins B1 or B2 in the final mixture of products. In this work, the nucleophilic catalytic depolymerization of polymeric proanthocyanidins was conducted and the effects of various reaction variables on the yields of dimeric proanthocyanidins B1 and B2 were comprehensively investigated by using response surface methodology (RSM) for experiments of a central composite design (CCD) with four factors (reaction time, temperature, pH, and ratio of reactant PPC/nucleophile catechin or epicatechin) and five levels, and the maximum yield of dimers B1 and B2 under optimal reaction conditions was evaluated.
■
MATERIALS AND METHODS
Materials and Reagents. The powder of freeze-dried grape seed proanthocyanidin extract (GSPE) was supplied by Tianding, Inc.
5979
DOI: 10.1021/acs.jafc.9b01188 J. Agric. Food Chem. 2019, 67, 5978−5988
Article
Journal of Agricultural and Food Chemistry
(V/V) HCl in a dark water bath for 10 min at 20 °C, and then the absorbance was measured at 500 nm on a UV−vis microplate reader. In addition, the proanthocyanidin sample was dissolved in a minimum volume of methanol and diluted in glacial acetic acid. On the basis of the mass weight and molecular weight of proanthocyanidin, the mean degree of polymerization (mDP) could be calculated by the equation m mDP = M rn (1)
Ningbo, People’s Republic of China. Standard samples of (+)-catechin (≥98%), (−)-epicatechin (≥98%), gallic acid (≥98%), epicatechin-3-O-gallate (≥98%), proanthocyanidin B1 (≥96%), proanthocyanidin B2 (≥96%), proanthocyanidin B3 (≥96%), and proanthocyanidin B4 (≥96%) were provided by the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, People’s Republic of China. Vanillin, DPPH, acetonitrile, and methanol in spectral grade were purchased from Sigma Chemical, St. Louis, MO, USA, and all other chemicals and reagents in analytical grade were purchased from commercial sources. Isolation of Polymeric Proanthocyanidins. A 250 mL amount of an aqueous solution containing 2.5 g of GSPE powder was mixed with 750 mL of ethyl acetate, and then the mixture was refrigerated at 4 °C for 12 h. Next, the ethyl acetate supernatant and aqueous phase were separated, and the two fractions of three repeated operations were combined, respectively. Afterward, the solvents were removed from the ethyl acetate fraction and water fraction by using a rotary evaporator to give the OPC and PPC products, respectively. Of these, the PPC was used as a raw material for the depolymerization reaction. Depolymerization Reaction. The depolymerization reaction was conducted according to a procedure adopted in a previous study11,28 with minor modification. In a typical run, 100 mg of (−)-epicatechin (or (+)-catechin) and 100 mg of PPC were dissolved in 8.0 mL of methanol, resulting in a 1:1 mass ratio of epicatechin (or catechin) to PPC. Next, the solution was immediately mixed with 2.0 mL of methanolic HCl at 0.5 mol to give an H+ ion concentration of 0.1 mol and then incubated in a water bath at 40 °C for 40 min. The reaction was terminated by adding a certain amount of NaHCO3 solution to adjust the pH of reaction mixture to 5−6. At the end of the reaction, the products were filtered through a 0.22 μm nylon filter and sampled for UPLC analysis. UPLC Analysis. Chromatographic analyses of GSPE, extraction fractions, and deploymerization products were performed on an ultrahigh-performance liquid chromatography (UPLC) system (Agilent 1290, Santa Clara, CA, USA). This system contains a binary pump, an autosampler, a column thermostat, and a DAD detector. Chromatographic conditions were set as follows: injection volume 5 μL, flow rate 1 mL min−1, column temperature 30 °C, detected wavelength 280 nm, column with 1.8 μm, 4.6 mm × 100 mm i.d. (Agilent Technologies, Beijing, China), 0.2% aqueous formic acid (v/ v, solvent A) or acetonitrile (solvent B) as mobile phase. Gradient elution was performed linearly by 10−15% A at 0−2 min, 15−16% A at 2−5 min, 16−20% A at 5−8 min, 20−45% A at 8−9 min, and 45− 10% A at 9−10 min, followed by 5 min for column re-equilibration before the next injection. Gallic acid, catechin, epicatechin, dimeric proanthocyanidins B1−B4, and epicatechin-3-O-gallate as reference materials were assayed by UPLC. On the basis of the retention time eluted on the chromatogram, the contents of standard materials were calculated by referring to the calibration curve plotted with six concentration points of each material. The relative standard deviation (RSD) was calculated to evaluate the precision of the assay by a standard solution with six replicates. For catechin as an example, the RSD was 0.71%, and the accuracy was determined by recovery experiments in half- and doubleconcentration levels of the same solution, giving the determined recoveries to be 99.4 and 101.2%, respectively. Determination of Polymerization Degree. The mass content of proanthocyanidin was determined by a vanillin−methanol method and a previous procedure31 with minor modification. In detail, 1.0 mL of the sample was mixed with 5.0 mL of methanolic vanillin reagent containing 0.75% (M/V) vanillin and 4% (V/V) HCl in a dark water bath for 15 min at 25 °C, and then the absorbance was measured at 500 nm on a UV−vis microplate reader. A calibration curve was drawn using catechin as the standard material. Butler et al.32 revealed that when the reaction was carried out in a solvent of glacial acetic acid, only the terminal flavan-3-ol unit could react with vanillin, and therefore the molar number of proanthocyanidin would be determined. When the vanillin−acetic acid method was used, analogously, 1.0 mL of the sample was mixed with 5.0 mL of glacial acetic acid−vanillin solution containing 1.5% (M/V) vanillin and 4%
where m is the mass content (μg mL−1), n is the molecular content (μmol mL−1), and Mr is the relative molecular mass of catechin with a value of 290. Quantitative Analysis of Broad Peak in Chromatogram. Proanthocyanidins with DP values over 3 were eluted as a broad peak in the chromatogram.33 In order to measure the mass content of the substance in this broad peak, the product obtained from the water fraction (Isolation of Polymeric Proanthocyanidins) was taken as the reference to establish the calibration curve using UPLC. The water fraction was dried by a rotary evaporator to get a brownish solid product. Next, 10 mg mL−1 of methanolic product solution was prepared and diluted into six sample solutions at concentrations of 0.25, 0.5, 1.0, 2.5, 5, and 10 mg mL−1, respectively. Each of the sample solutions was be measured using the UPLC method in UPLC Analysis, and a calibration curve of peak area versus mass concentration could be drawn. The regression equation of the calibration curve was given as Y = 2010.2X − 485.7 with a linear range of 0.25−10.0 mg mL−1, where Y is the peak area and X is the concentration (mg mL−1). Assay of DPPH Radical Scavenging. The assay of DPPH radical scavenging activity was performed with a Biotek Synergy 2 MultiDetection Microplate Reader (Biotek, Winooski, VT, USA) according to the procedure described by Ruan et al.34 with minor modification. A set of samples was prepared with various concentrations in methanol. Fresh DPPH in methanol (3.9 mL) was added to 0.1 mL of each sample solution. In all of the experiments, methanol and lascorbic acid were selected as negative and positive controls, respectively. The absorbance was measured at 516 nm wavelength after incubation in the dark for 30 min at room temperature. The radical scavenging activity of DPPH was calculated as ÄÅ ÉÑ ÅÅ A Ñ ÅÅ negative control − A sample ÑÑÑ radical scavenging (%) = ÅÅÅ ÑÑÑ × 100 ÅÅ ÑÑ A negative control (2) ÅÇ ÑÖ Experimental Design and Evaluation. A central composite design (CCD) with four variables at five levels, including epicatechin or catechin to PPC mass ratio (X1 = 1, 1.5, 2, 2.5, 3), HCl concentration (X2 = 0.02, 0.04, 0.06, 0.08, 0.1 mol), reaction time (X3 = 10, 15, 20, 25, 50 min), and temperature (X4 = 30, 35, 40, 45, 50 °C), was applied to examine the effect of variables on the yield of proanthocyanidin B2 or B1 using Design-Expert Software (Stat-Ease Inc., Minneapolis, MN, USA). To predict the targeted response, a total of 30 experimental trials including 16 factorial, 8 axial, and 6 central runs were conducted to determine the parameters in a secondorder polynomial regression equation as follows: 4
Y = b0 +
4
3
4
∑ biXi + ∑ biiXi 2 + ∑ ∑ i=1
i=1
i=1 j=i+1
bi jXiXj
(3)
where Y is response, Xi and Xj are independent variables, b0 is the offset term, bi and bii are the linear effect and the quadratic effect of Xi, respectively, and bij is the interaction effect between Xi and Xj. The fitted polynomial equation was expressed as a 3D response surface. Parameters R2, adjust-R2, variance analysis, and residuals analysis were employed to evaluate the model. The software was used to fit the response surface to optimize the reaction conditions. The predicted optimal yield of dimeric proanthocyanidin was examined by experiments with the selective optimal values of variables. Statistical Analysis. Data were expressed as mean ± standard deviation. The standard curves were plotted using Origin Pro 8.1 (Origin Lab, Northampton, MA). The significant difference among 5980
DOI: 10.1021/acs.jafc.9b01188 J. Agric. Food Chem. 2019, 67, 5978−5988
Article
Journal of Agricultural and Food Chemistry
Figure 2. UPLC (280 nm) chromatograms of various samples: (A) standard materials with peak identification of (1) gallic acid (1.52 min), (2) proanthocyanidin B1 (2.46 min), (3) proanthocyanidin B3 (2.93 min), (4) catechin (3.11 min), (5) proanthocyanidin B4 (3.32 min), (6) proanthocyanidin B2 (3.49 min), (7) epicatechin (4.02 min), (8) epicatechin-3-O-gallate (ECG, 7.74 min); (B) GSPE; (C) ethyl acetate extract of GSPE; (D) water raffinate (PPC) of GSPE; (E) the product from PPC depolymerization in the epicatechin/HCl system under the conditions PPC:epicatechin ratio of 1:1, HCl concentration of 0.1 mol/L, 40 °C and 20 min; (F) the product from PPC depolymerization in the catechin/ HCl system under the conditions: PPC:epicatechin ratio of 1:1, HCl concentration of 0.1 mol/L, 40 °C and 20 min; (G) the product from PPC depolymerization catalyzed by 0.1 mol/L HCl at 40 °C for 20 min. parameters of the reaction were first examined by ANOVA (p < 0.05) and then analyzed for multiple comparisons among the different treatments by Fisher’s LSD test with using SPSS software (IBM, New York, USA).
established in UPLC Analysis. All of the calibration curves showed excellent linearity (R2 > 0.999) in a wide concentration range of 0.01−1.0 mg mL−1. The chromatogram of GSPE is demonstrated in Figure 2B. All the references mentioned above and some unidentified substances eluted in time range of 0−9 min gave insignificant contents of less than 50 mg g−1, as presented in Table 1. A notable single broad peak appeared at 9.63 min and accounted for 881.0 mg g−1 of GSPE, attributed to a mixture of proanthocyanidins with mDP over 3.28 After the GSPE was separated into two fractions by extraction of ethyl acetate/ water at 3/1 volume ratio, the supernatant fraction of ethyl
■
RESULTS AND DISCUSSION Characterization of GSPE and Extraction Fractions by UPLC. The peaks of standard monomeric and dimeric proanthocyanidins in the UPLC chromatogram were identified, as shown in Figure 2A. The retention time of each compound was measured, and the contents of various samples were calculated in turn from the related calibration curves 5981
DOI: 10.1021/acs.jafc.9b01188 J. Agric. Food Chem. 2019, 67, 5978−5988
Article
Journal of Agricultural and Food Chemistry Table 1. Basic Data of GSPE, Extract and Raffinate from Ethyl Acetate Extraction of GSPE, and Products of PPC Depolymerization X/GS PE GSPE extract of GSPE raffinate of GSPE (PPC) PPC depolymerization with epicatechin and HClc PPC depolymerization with catechin and HCld PPC depolymerization with HCl onlye
1 0.19 0.81
mDP
gallic acid (mg/g)
C (mg/g)
EC (mg/g)
3.56 2.21 5.65 1.74
2 9 0 0.5
35 178.1 0 9
25 143 0 351.5
1.68
0.6
341.5
11.5
4.46
0.8
24.2
10.5
a
b
B1 B2 (mg/g) (mg/g) 4.8 25.4 0 0.9 109 4.1
B3 B4 (mg/g) (mg/g)
ECG (mg/g)
broad peak (mg/g)
DPPH IC50 mg/mL
881 330 991 251.5
51.2 53.3 50.6 53.6
10 45.3 0 98.2
1.8 11 0 1.9
2.3 13.4 0 11.5
1.4 6.7 0 18
3.5
12.6
0.8
3.5
254
51.7
10.1
1.3
1.5
1.07
878.4
52.3
Mean degree of polymerization. bStandard material same as those in Figure 2. c10 mg mL−1 epicatechin reacted with 10 mg mL−1 PPC in 0.1 mol HCl methanolic solution. d10 mg mL−1 catechin reacted with 10 mg mL−1 PPC in 0.1 mol HCl methanolic solution. e10 mg mL−1 PPC reacted with 0.1 mol HCl methanol solution. a
As showed in Figure 1, those newly formed dimers as nucleophiles would continuously interact with carbocations of epicatechin or catechin to produce trimeric proanthocyanidin (path III), so that a broad peak with an elution time near 10 min was still found in the chromatogram (Figure 2). Theoretically, such a similar reaction would continue to synthesize higher polymers of proanthocyanidin (path IV) and form a closed circle of reaction pathway in the end. On the basis of the suggested reaction mechanism and pathways of PPC depolymerization, it can be concluded that various variables such as flavan-3-ol to PPC ratio, HCl concentration, reaction temperature, and time would influence the formation yields of the intermediate products in different ways and degrees. On the other side, the positive hydrogen ion H+ could also attack and protonate the oxygen on the ring of flavan-3-ol, causing the ring to open and form the positively charged ion of chalcane during the reaction process. These chalcane carbocations would compete as nucleophiles with the flavan3-ol carbocation of the cleaved PPC to generate chalcan-falvan3-ol derivatives, so-called gambiriins.28 The formation pathways of byproducts are shown as V and VI in Figure 1. The structures of B-type dimeric proanthocyanidins are shown in Figure 1, and their conformations and yields from the PPC depolymerization in the HCl/nucleophile system depended on the carbocation configuration, nucleophile type, and interflavan bonding patterns.36 When the interflavan bonded through C4→C8, dimeric proanthocyanidins B1−B4 were formed, and if it was bonded through C4→C6, the formed dimers were B5−B8. By comparison, the dimeric proanthocyanidins B1−B4 occur more widely in nature due to the preferable conformation of C4→C8 bonding. The extensional units of grape seed proanthocyanidins were predominantly epicatechin, and thus the cleaved carbocations were epicatechin positively charged ions as well.37 When epicatechin was added, the yields of proanthocyanidin B2 and B4 were 98.2 and 11.5 mg g−1 respectively, giving a ratio of about 8.5:1. When catechin was added, the yields of proanthocyanidins B1 and B3 were 109.0 and 12.6 mg g−1, respectively, giving a ratio of about 8.6:1. In other words, the dimeric products were abundant in B2 (or B1) and proportionally accompanied by less B4 (or B3). Therefore, we have further targeted the maximum yield of proanthocyanidin B2 or B1 as the objective to optimize the reaction conditions of PCC depolymerization.
acetate predominantly contained oligomeric proanthocyanidin (OPC) with a yield of 19% GSPE (g/g) and an mDP of 2.21; the polymeric proanthocyanidins (PPCs) with a yield of 81% GSPE (g/g) remained in the water phase and gave an mDP of 5.65. In the extracts of ethyl acetate, the contents of monomers and dimers were remarkably enhanced, such that the contents of catechin and epicatechin significantly increased from 35 and 25 mg g−1 to 178 and 143 mg g−1, respectively (Figure 2C and Table 1). On the other hand, the residues in the water phase only contained PPC at an estimated content of 991.0 mg g−1, and almost no signals of monomers or hardly visible signals of dimers appeared in the chromatogram (Figure 2D). Depolymerization of Polymeric Proanthocyanidins (PPC). In the depolymerization of PPC catalyzed by an acidic catalyst such as HCl, positive hydrogen ions H+ initiated the cleavage of interflavan bonds in polymeric proanthocyanidins, so that the terminal unit was released as a catechin (or epicatechin) while the rest successively turned into several carbocations with positive charged on C4 (pathway I in Figure 1).27 In the absence of additional nucleophilic agents, these liberated carbocations might react backward with the released catechin (or epicatechin) and eventually reach a dynamic equilibrium with the forward reaction, thus giving a low apparent conversion of the original reactant (PPC) and a small reduction of mDP, as indicated by the data on the bottom line of Table 1 or by the chromatogram in Figure 2G. Once nucleophiles such as flavan-3-ol (epicatechin or catechin) were added to the reaction system, however, these carbocations would be immediately captured by the nucleophile as bottom units to form dimeric proanthocyanidins (path II in Figure 1).35 As seen in Figure 2D and Table 1, the original PPC material contained no monomers or dimers. After depolymerization with addition of epicatechin or catechin as nucleophilic promoter, however, the dimeric products increased from zero (mg/g) to a visible level (Table 1 and Figure 2). Moreover, in the case of epicatechin as a reaction promoter, the predominant dimeric product was B2 with a yield of 98.2 mg g−1 (Table 1), while the released terminal unit of catechin and other dimeric products B4, B1, B3, and ECG as well as some unidentified compounds were also detected (Figure 2E). Similarly, in the case of catechin as promoter, the primary dimeric product was B1 with a yield of 109 mg g−1 (Table 1), and several other products were simultaneously found in the chromatogram (Figure 2F). 5982
DOI: 10.1021/acs.jafc.9b01188 J. Agric. Food Chem. 2019, 67, 5978−5988
5983
variable
0.08 0.06 0.08 0.08 0.06 0.06 0.04 0.06 0.04 0.08 0.08 0.08 0.06 0.08 0.04 0.04 0.06 0.08 0.02 0.04 0.04 0.06 0.06 0.06 0.1 0.06 0.06 0.04 0.04 0.06
X2 (mol/mL)b 15 20 25 25 20 20 15 20 25 25 15 15 20 25 15 15 20 15 20 25 15 20 10 20 20 20 20 25 25 30
X3 (min)c 35 40 45 35 40 40 35 40 45 35 35 45 40 45 45 35 30 45 40 45 45 40 40 50 40 40 40 35 35 40
X4 (°C)d 2.46 3.03 2.56 3 3.12 2.94 3.22 2.16 2.51 2.51 3.26 3.15 3.09 2.9 3.09 2.51 2.85 2.51 2.65 2.84 2.46 3.16 2.91 2.9 2.8 2.98 3.04 2.99 2.44 2.78
B2 2.72 3.32 2.83 3.19 3.35 3.19 3.54 2.34 2.77 2.79 3.58 3.42 3.22 3.15 3.22 2.67 3.07 2.77 2.86 3.09 2.7 3.41 3.11 3.14 3 3.31 3.25 3.24 2.73 3.12
B1
yield (mg/mL) 2.365 1.803 0.013 0.052 0.003 0.001 0.077 0.024 0 0.002 0.002 0.245 0.168 0.062 0.047 0.048 0.025 0.022 2.413 0.981 0.962 0.927 28.31
pure error cor-total R2 Adj-R2 cPred-R2 adeq precision
sum of squares
model X1 X2 X3 X4 X1, X2 X1, X3 X1, X4 X2, X3 X2, X4 X3, X4 X12 X22 X32 X42 residual lack of fit
source
B2
0.005
0.169 1.803 0.013 0.052 0.003 6 × 10−4 0.077 0.024 4 × 10−4 0.002 0.002 0.245 0.168 0.062 0.047 0.003 0.003
mean square
X1 = EC (or C) to PCC mass ratio. bX2 = HCl concentration. cX3 = reaction time. dX4 = reaction temperature.
1.5 2 1.5 2.5 2 2 2.5 1 1.5 1.5 2.5 2.5 2 2.5 2.5 1.5 2 1.5 2 2.5 1.5 3 2 2 2 2 2 2.5 1.5 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
a
X1 (mol/mL)a
trial no.
