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International Journal of FoodEngineering

Volume 4, Issue 1 2008 Article 13

Recovery of Polyphenolics from Apple JuiceUtilizing Adsorbent Polymer Technology

Zaid S. Saleh∗ Reginald Wibisono†

Katja Lober‡

∗HortResearch, New Zealand, [email protected]†HortResearch, New Zealand, [email protected]‡Hohenheim University, Germany, [email protected]

Copyright c©2008 The Berkeley Electronic Press. All rights reserved.

Recovery of Polyphenolics from Apple JuiceUtilizing Adsorbent Polymer Technology∗

Zaid S. Saleh, Reginald Wibisono, and Katja Lober

Abstract

A food grade divinylbenzene adsorbent resin was used to separate/recover plyphenolics fromdiluted apple juice concentrate. The work was carried out to investigate the effect of processingparameters on the adsorption process, to compare the efficiency of divinylbenzene adsorbent resinto that of ethylene glycol cross-linked polymethylmethacrylate adsorbent resin used in our previ-ous study and to determine the effect of oxygen on the adsorption of polyphenolics process.

A laboratory scale adsorption was determined by mixing weighed amounts of the polymer with adiluted apple juice concentrate at 10–60 oC and at pH values ranging from 2.0 to 3.9. At regularintervals, samples were withdrawn from the aqueous phase. Total phenolics, absorbance valuesat 280 and 420 nm, and the amounts of individual phenolics remaining in the liquid phase weredetermined. The analytical data were fitted to the Langmuir and Freundlich isotherms.

Findings from this study are in agreement with Kammerer et al. (2006) but, in contrast to pre-vious studies, the pH value significantly affected the adsorption onto the resin. HPLC analysesrevealed different affinities of individual compounds, which enabled selective enrichment of cer-tain phenolics in the liquid phase or on the sorbent surface. HPLC profiling showed that thepolyphenolics recovered did not differ significantly from those in the original apple extracts. Thishas confirmed that there is minimal damage during processing. The major losses in the processwere due to polyphenols not binding to, or not being recovered from, the adsorbent. Optimizationof the process would enable the losses to be minimized and up to 80% of the polyphenolics couldbe recovered in the extracts.

Desorption studies were performed using a resin with known amounts of adsorbed phenolic com-pounds. Elution was carried out by an automated pressurized liquid extraction system, studyingthe effects of temperature (40–180 oC) and solvents (water, ethanol, methanol). Again, individ-ual compounds behaved differently, depending on their hydrophobicity. Therefore, a systematicchange of parameters for the adsorption and desorption process can be helpful for the recovery ofpurified plant extracts enriched in certain target compounds.

KEYWORDS: polyphenolics, recovery, adsorption, desorption, resin

∗The authors are grateful to Dr. Janine Cooney for performing MS analyses. This work wassupported by the New Zealand Foundation for Research, Science and Technology, contractC06X0405.

1. Introduction

Increased consumption of fruit and vegetables is associated with a decreased risk of cancer, cardiovascular and other chronic diseases (Manach et al. 2005; Williamson & Manach 2005; Lotito & Frei 2006). These health effects are mainly attributed to polyphenolics present in foods such as fruits and vegetables which are known to have antioxidant activity. This means that they inhibit oxidative damage of cells by their abilities to scavenge free radicals and active oxygen species (Yao et al. 2004) which are thought to underpin pathologies associated with for the mentioned diseases.

Adsorbent resins have been used to separate and concentrate phenolic compounds from products and byproducts of food processing plants, for example debittering of citrus juices and removal of browning reaction products (Manlan et al. 1990; Couture & Rouseff 1992; Tomasbarberan et al. 1992; Carabasa et al. 1998; Grohmann et al. 1999; Di Mauro et al. 2000). The resins are nonpolar or slightly hydrophilic (acrylic) polymers with high adsorption capacity and possible recovery of the adsorbed molecules. They are relatively low in cost and easy to regenerate (Scordino et al. 2004).

Apple juice contains a wide range of different phenolic compounds as hydroxzbenzoic and hydoxycinnamic acids, flavanols, flavonols and dihydrochalcones which also can be partially glycolyzed (Sanchez-Rabaneda et al. 2004; Kahle et al. 2005). Depending on their structure, phenolic compounds are known to vary in their bioactivity and availability (Bravo 1998). Thus behaviour of individual compounds during the sorption process is also of interest.

The food processing industry also increasingly tries to utilize their waste materials for further products by isolating defined compounds (Peschel et al. 2006). These compounds could be used for a new sector of production of functional or nutraceutical foods. Only a few byproduct-derived antioxidants have been developed successfully from plant residues, from grape seeds and olive waste extracts (Alonso et al. 2002; Amro et al. 2002), but there is also a big interest in other crops like apple, tomato and artichoke (Lavelli et al. 2000; DuPont et al. 2002; Jimenez-Escrig et al. 2003). It has not been until recently that more detailed studies have been performed taking into account the effects of various process parameters on the adsorption and desorption efficiency(Kammerer et al. 2006). However, elucidating the effects of pH, temperature, solute/adsorbent ratio and resin parameters such as particle size, surface area and porosity and the chemical structure on the resin behaviour are required to achieve high recovery rates and economical production.

Apple juice was chosen as a model system with a complex phenolic profile for batch adsorption experiments in the present study. Kinetic and

1

Saleh et al.: Recovery of Polyphenolics Utilizing Adsorbent Polymer

Published by The Berkeley Electronic Press, 2008

equilibrium data of total phenolics were compared at various temperatures, pH values and solute/resin ratios in laboratory-scale adsorption experiments. The effect of oxygen on adsorption of polyphenols was also investigated. Furthermore, the elution of the molecules adsorbed by the resin was studied using an accelerated solvent extraction system to investigate the effects of different temperatures and solvent compositions on the recovery of polyphenols.

2. Materials

Deionized water was used throughout the experiment. Sodium carbonate was obtained from Merck (Darmstadt, Germany) and Folin Ciocalteu reagent was purchased from Sigma (St. Louis, MO, USA). All other solvents and reagents used were purchased from Ajax Finechem (Auckland, New Zealand) and they were all of HPLC or analytical grade. The divinylbenzene adsorbent resin (Alimentech P470) was provided by Bucher-Alimentech Ltd. (Auckland, New Zealand) and had the following characteristics: particle size 0.25–1.0 mm, surface area 800 m2/g, pore radius 200–300 Å, porosity 1.51 mL/g, density 1.01 g/L, moisture content 55–65%, temperature range 0–110 �C, pH range 0–14. The apple juice concentrate used in this study (72.2% total soluble solids (TSS)) was supplied by ENZAFOODS New Zealand Ltd. (Hastings, New Zealand).

3. Methods

3.1 Polyphenol Adsorption Studies

Resin was activated by overnight soaking in 96% ethanol (5 mL/g) prior to use in the experiment. Before performing the adsorption trials, the resin was rinsed with 2x volume of deionized water using a paper filter in a funnel to remove the alcohol.

Batch adsorption experiments were performed in 250 mL screw cap glass flasks. The apple juice concentrate was diluted using deionized water to give samples with 10, 15 and 25% TSS. The pH of the samples was changed with to pH 2 and pH 3 using concentrated HCl. The diluted concentrate (200 mL) was mixed with a known amount of pretreated resin (5, 10, 20 g) and stirred at 300 rpm in a temperature-controlled water bath at 10, 20, 40 and 60 �C,respectively. During the period of mixing, each time after 0, 10, 20, 30, 45, 60, 90, 120, 180 and 240 min, a 1-ml sample was taken for total phenolic analysis. To verify the influence of oxygen, an experiment was carried out using nitrogen to avoid any potential oxidation.

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International Journal of Food Engineering, Vol. 4 [2008], Iss. 1, Art. 13

http://www.bepress.com/ijfe/vol4/iss1/art13DOI: 10.2202/1556-3758.1383

3.2 Polyphenol Desorption Studies

In the desorption trials, 120 g of the resin was preconditioned and washed as described previously in the adsorption studies. For adsorptive loading of the resin, the sample was stirred at 300 rpm with 2400 mL of diluted apple juice concentrate (25% TSS) for 4 h at 20 �C. After this adsorption, the resin was separated, washed with 2000 mL of deionized water and dried using a CentriVap concentrator equipped with an Ultra-low cold trap (Labconco Corp., Kansas City, MO, USA). Mixtures of 1.74 g of the dried resin material and 6 g of diatomaceous earth (Celite®, Johns-Manville Corp., NY, USA) were filled into 34 mL stainless steel extraction cells for elution experiments by pressurized liquid extraction with an accelerated solvent extractor (ASE® 300, Dionex, Sunnyvale, CA, USA). Solventsused for eluting the phenolic compounds were deionized water, ethanol (v/v) and methanol (v/v) at temperatures of 40 and 180 �C, respectively. Parameters for all elution trials were as follows: three cycles, 60% flush volume and 2 min static time. The eluates were made up to a standard volume and used for total phenolic analysis. All adsorption and desorption experiments were performed in duplicate.

3.3 Analysis of Total Phenols

The quantification of the total phenolic content was based on the method of Singleton et al. 1999. A 20 μL sample which had been diluted accordingly was taken from the adsorption and desorption experiments and placed in microplate cuvettes. A portion of 100 μL of Folin-Ciocalteu reagent was added into the sample. After 5 min, 80 μL of sodium carbonate solution (75 g/L) were added and incubated at 20 �C in the dark for 90 min. The absorbance of the sample was then read at 760 nm using a SpectraMax Plus384 microplate spectrophotometer equipped with SOFTmax® software v. 3.1.2 (Molecular Devices Corp., Sunnyvale, CA, USA).

3.4 Absorbance Readings

Aliquots of 200 μL of the samples taken during adsorption experiments were transferred into microplate cuvettes, and an absorbance reading was taken at 420 nm. Twenty-fold diluted samples were used for absorbance readings at 280 nm.

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3.5 HPLC and LC-MS Systems

Analysis of polyphenolic compounds in the apple juice was performed using a Shimadzu HPLC system (Koyoto, Japan) with a Phenomenex (Torrance, CA, USA) C18 Synergi Hydro-RP (250 x 4.6 mm i.d., 4 μm particle size) column and a C18 ODS (4.0 x 3.0 mm i.d.) guard column at 35 �C. The mobile phase consisted of 0.1% formic acid in acetonitrile (v/v, eluent A) and 0.1% formic acid in water and acetonitrile (95:5, v/v, eluent B) using a gradient programme as follows: 0% A to 8.7% A (5 min), 8.7% A isocratic (10 mni), 8.7% A to 17% A (10 min), 17% A to 20% A (5 min), 20% A to 30% A (9 min), 30% A to 50% A (4 min), 50% A to 95% A (5 min), 95% A isocratic (5 min), 95% A to 0% (2 min). Total run time was 65 min at a flow rate of 1.0 mL/min.

Peak assignment was performed by comparing the retention times and UV spectra with those of reference compounds. This was also confirmed by LC-MS analysis. For LC-MS, samples were purified by solid phase extraction (SPE). The SPE cartridges (StrataTM C18, endcapped, 2000 mg; Phenomenex, Torrance, CA, USA) were activated with 10 mL of methanol and rinsed with 20 mL of acidifiedwater (HCl; pH 2.0). Aliquots of 6 mL of the apple juice (25% TSS; pH 2.0) were applied to the cartridges and then washed with 40 mL of water. Phenolic compounds were eluted with 30 mL of methanol. The eluate was concentrated in a centrivap under vacuum, and the residue obtained was dissolved in 50% methanol (v/v).

Mass spectrometric analyses were carried out with an LCQ Deca ion trap mass spectrometer fitted with an ESI interface (ThermoQuest, Finnigan, San Jose, CA, USA) which was coupled to a SurveyorTM HPLC. Polyphenol separation was carried out with a Prodigy 5 �m ODS(3) column (150×2.0 mm; Phenomenex, Torrance, CA, USA) using a 0.2-�m in-line filter (Alltech, Deerfield, Illinois, USA) operated at 35 �C. The mobile phase consisted of acetonitrile+0.1% formic acid (eluent A) and of water+0.1% formic acid (eluent B) using a gradient programme as follows: 5% A (isocratic, 5 min), 5% A to 10% A(5 min), 10% A to 17% A (15 min), 17% A to 23% A (5 min), 23% A to 30% A (10 min), 30% A to 97% A (8 min), 97% A isocratic (5 min). The flow rate was 200 �L/min. ESI voltage, capillary temperature, sheath gas pressure and auxiliary gas pressure were set at ��C, 50 psi, and 10 psi, respectively.

Individual phenolics were quantified using a calibration curve of the respective reference compound or of a structurally related compound including amolecular weight correction factor if standards were not available.

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3.6 Adsorption Isotherms

The equilibrium of adsorption for a resin at a specific temperature can be described via adsorption isotherms. The two most important ones are the isotherms after Langmuir and the Freundlich. The Langmuir model describes a monolayer adsorption with energetically identical sorption sites and without mutual interactions between the adsorbed molecules. The model can be expressed as:

SL

SL

SL

SLms Ca

CKCaCaQ

q�

��

�11

(1)

which is converted into a linear form,

mSLS QCKq111

�� (2)

with qS as the maximum concentration achieved by the resin (mg/g) and CS as the concentration in the liquid phase after adsorption (mg/L). aL (ratio between sorption and desorption rate constants) and Qm (maximum sorption capacity) are the Langmuir constants. KL and Qm can be obtained directly by extrapolation of the analytical data, aL can be calculated with the use of the received values.

The Freundlich model assumes adsorption to heterogenous surfaces which is characterized by sorption sites at different energies. The model can be expressed as:

FbSFS CKq � (3)

which is in a linear form

log qS = log KF = bF log CS (4)

with qS and CS the same as in the other model. KF is a measure of the capacity and bF of the intensity of the adsorption process. They can be obtained by plotting the analytical data on a logarithmic scale.

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4. Results and Discussion

4.1 Adsorption Studies

4.1.1 Adsorption ProcessThe typical characteristics of adsorption of polyphenols to the resin are shown in Figure 1. Five, 10 and 20 g samples of resin were used in this trial and the total phenolic contents of the juice was measured over a series of adsorption times up to 240 min

0

100

200

300

400

500

600

700

800

900

0 50 100 150 200 250 300

Time (min)

Phen

olic

Con

tent

(mg/

L)

no resin5 g resin10 g resin20 g resin

Figure 1. Total phenolic content in juice during adsorption over 240 min at 20 �C, natural pH, 15% TSS and different amounts of resin.

The phenolic content in the juice remained almost constant without the addition ofresin as expected. The phenolics were adsorbed faster and more effectively with increasing resin quantity. When more resin was used, fewer phenolics remained in solution after 240 min adsorption. It took 25 min to adsorb half of the phenolics with 5 g of resin, while it took about 17 and 8 min with 10 g and 20 g of resin, respectively. The P-470 resin adsorbed half the phenolics in the juice much faster than P-495 resin (ethylene glycol crosslinked polymethylmethacrylate), used by Kammerer et al. (2006) in a previous study.

The difference of adsorption between the various amounts was more apparent between 5 and 10 g than between 10 and 20 g. The latter processes of adsorption became very similar after 90 min.

6



The chromatograms in Figure 2 below show the phenolic profile of apple juice used in this study after mixing with P-470 resin for 0, 20, 45 and 240 min.

1

2

3

4

5

6 7

89

10

11

12

13

Figure 2. HPLC-chromatograms of apple juice (25% TSS, natural pH) during the adsorption adsorption of phenolic compounds with 10 g resin.

Peak number Retention time (min) Phenolic compound1 12.8 Catechin2 13.8 Chlorogenic acid3 14.2 Caffeic acid4 17.1 B2 epicatechin-epicatechin dimer5 18.8 Epicatechin6 20.6 4-p-coumaroylquinic acid7 29.3 Rutin8 30.2 Quercetin-3-galactoside9 31.1 Quercetin-3-O-glucoside10 32.0 Quercetin-3-O-xyloside11 33.3 Phloretin-2-O-xylo-glycoside12 34.0 Quercetin-3-O-rhamnoside13 36.5 Phloridzin

Table 1. Different phenolic compounds identified in the apple juice used in this study and their retention times in 10 g resin at 20 ��.

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Peak no.

Rt(min)

� max (nm)

Peak assignment % of initial content

2 13.8 323 Chlorogenic acid 15.86 20.6 308 4-p-coumaroylquinic acid 10.37 29.3 208 Rutin 0.48 30.2 209 Quercetin-3-galactoside 1.611 33.0 213 Phloretin-2-o-xylo-glcoside 1.513 36.5 210 Phloridzin 0.9

Table 2. Relative loss of polyphenolics from 200 mL of apple juice (25% TSS) during adsorption using 10 g resin at 20 �� relative to the initial contents (t=0 min)

Some of the major compounds identified in the apple juice are displayed in Table 1. They were identified in the juice using standard compounds and the levels arein good agreement with those found in previous studies of apple juice (Kammerer, 2006 and Sanchez-Rabaneda, 2004). From Table 1, we can see that apple juice contains a variety of quercetin sugar derivative compounds. These compounds are normally present mainly in the apple skin (Siegelman, 1954).

Phenolic compounds were mostly removed from the juice and adsorbed to the resin between 0 and 45 min of adsorption as described above. HPLC analyses revealed significant differences in the adsorption of individual phenolic compounds using the percentage of initial content. The adsorption efficiency is mainly determined by the hydrophilicity of the molecules. This can be seen from Table 2 which illustrates the relative amounts of polyphenolics which remained in the aqueous phase at equilibrium when 5 g of resin where brought into contact with 200 mL of the 25% TSS apple solution at 20oC. The lower the percentage is,the better was the adsorption of this compound. Rutin was adsorbed very well,with only 0.4% remaining in solution after adsorption.

4.1.2 Adsorption at EquilibriumThe absorbance readings at 280 and 420 nm and total phenolic content wascompared in Table 3 at different temperatures, pH values and resin amounts.

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Temperature(oC)

pH Resin (g)

Total phenolics

A280 A420

20 natural 5 30.09 81.15 33.6920 natural 10 16.85 77.08 21.0120 natural 20 12.41 75.44 15.7520 3 5 33.81 80.10 29.6120 3 10 15.24 77.27 20.0420 3 20 11.76 75.14 16.8520 2 5 33.37 79.62 26.0620 2 10 15.63 74.69 16.3420 2 20 9.98 70.27 8.9810 natural 10 16.47 77.66 17.9140 natural 10 21.62 80.10 24.3960 natural 10 23.05 81.61 31.64

Table 3. Relative amounts of total phenolic content and absorbance values at 280 nm (A280) and 420 nm (A420) at equilibrium.

It is apparent that pH and temperature influenced the effectiveness of adsorption. Lower pH and temperature resulted in more polyphenols being adsorbed to the resin whereas the difference between 10 and 20 �C was minimal. The importance of lowering pH for adsorption was also found by (Kammerer et al. 2006).However, this is in contrast to another finding by Scordino et al. (2004) who used different styrene-divinylbenzene (SDVB) copolymers for adsorption of anthocyanins. Kammerer et al. (2006) concluded that this effect was caused by the changing ratio between protonated and unprotonated phenolic acid compounds which was dependent on the pH. The protonation hydrophobicity of these low-molecular compounds significantly changed, and thus their affinity to nonpolar adsorption resins also changed. The influence of decreasing adsorption with increasing temperature can be explained because adsorption in general is an exothermic process. This was also described previously (Juang & Shiau 1999; Kammerer et al. 2006).

The total phenolic content values obtained by the Folin-Ciocalteu method represent an average of low-molecular monomeric, oligomeric and polymeric compounds. Absorbance readings at 420 nm are often used as an index for polymerized phenolic contents, while readings at 280 nm show polyphenols in general. The values obtained with absorbance readings at 280 nm are obviouslyhigher than these, as other hydrophilic compounds like amino acids may also show absorbance at this wavelength, but no reaction with Folin-Ciocalteu. The relative reduction of total phenolics determined with Folin-Ciocalteu, absorption at 280 nm, and levels of polymerized phenolic substances were higher with higher

9



amounts of resin. The resin loading of all adsorption experiments carried out is displayed in Table 4.

Juice concentration (TSS %)

Resin amount (g) Temperature (�C) pH

Resin loading (mg/g resin) SD (n=2)

10 5 20 nc 14.09 0.6415 5 20 nc 25.80 0.3625 5 20 nc 36.67 1.3910 5 20 3 17.06 1.4015 5 20 3 22.18 0.1825 5 20 3 31.88 0.9610 5 20 2 15.96 0.4215 5 20 2 18.54 1.5625 5 20 2 32.56 0.80

10 10 20 nc 8.09 0.7215 10 20 nc 12.13 0.9525 10 20 nc 21.50 0.5110 10 20 3 10.98 0.0515 10 20 3 13.73 0.2225 10 20 3 21.75 0.5910 10 20 2 9.29 0.4315 10 20 2 13.73 0.3825 10 20 2 21.81 0.12

10 20 20 nc 4.50 0.1015 20 20 nc 7.08 0.0125 20 20 nc 10.93 0.0510 20 20 3 4.84 0.0715 20 20 3 6.27 0.2125 20 20 3 11.05 0.2010 20 20 2 4.26 0.0115 20 20 2 7.29 0.1625 20 20 2 12.16 0.14

25 10 10 nc 22.11 0.2625 10 40 nc 21.04 0.5425 10 60 nc 21.78 0.42

Table 4. Resin loading (mg total phenolics/g resin) for the adsorption experiments.

Resin loading was best with low amounts of resin and a high concentration of total soluble solids in the juice as predicted. When the amount of resin was doubled from 5 to 10 g, the loading was still better than half of the loading with 5 g, while doubling the resin amount from 10 to 20 g made the loading worse than

10



half of the loading with 10 g. Temperature had a small influence on the loading. The lower the temperature, the better the loading as described above.Figure 3 illustrates the comparison of resin loading and loss of polyphenols remaining in the juice after adsorption when the juice concentration varied from 10 to 25% TSS with resin amount and pH kept constant.

10

15

20

25

30

35

0 5 10 15 20 25 30

Juice concentration (% TSS)

mg

Poly

phen

ols/

mg

Res

in

10

15

20

25

30

35

Loss

ofpo

lyph

enol

s(%

)

resin loading

loss

Figure 3. Ratio of polyphenols and resin amount and loss of phenolic compounds depending on juice concentration at pH 2 and 20 �C.

Both resin loading and loss of phenolic compounds from the juice increased almost linearly with the juice concentration. Thus the loss is biggest with 33% at 25% total soluble solids in the juice. At 15% total soluble solids, loss of phenolic compounds was much higher compared with the resin loading than at 10 and 25% TSS. While at 25% TSS, the difference between loading and relative loss was lowest.

Compared with the resin P-495 (Kammerer et al. 2006), the loading of P-470 is much better. Loading with 33% was higher at juice concentration of 25% TSS than that for P-495 of only ~17%. That could be attributed to the much greater surface area for P-470 than for P-495 (~800 m2/g cf. 450 m2/g). Thus loss for P-470 was also lower than for P-495.

4.1.3 Adsorption ConstantsThe results of the adsorption studies at 20 �C were plotted using the models of Langmuir and Freundlich. The corresponding graphs are shown in Figures 4 and 5.

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y = 7.4051x + 0.0084R2 = 0.6475

0

0.05

0.1

0.15

0.2

0.25

0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02

1/(PPC juice in mg/L)

1/(P

PC

resi

nin

mg/

g

Figure 4. Results of adsorption experiments on apple juice (25% TSS, natural pH, 20 �C) using Langmuir model.

A linear correlation between the results is apparent. Using the Langmuir equation,Qm (maximum sorption capacity) and aL (ratio between sorption and desorption rate constants) could be then calculated. Qm was 119.05 mg/g for the analyzed resin while aL was 0.0011 L/mg.

In a previous study (Scordino et al. 2004), different resin types were compared. The XAD-1180 resin, which was used for adsorption of anthocyanins, has similar physical characteristics to that of P-470, with aL of 0.018 L/mg, but with a much lower Qm value of 33.45 mg/g. This resulted from high variability, as indicated by the low stability index (R2 = 0.6475).

12



y = 0.8527x - 0.6525R2 = 0.7826

0

0.4

0.8

1.2

1.6

2

1.5 1.7 1.9 2.1 2.3 2.5 2.7

log Cs (mg/L)

log

qs(m

g/g)

Figure 5. Results of adsorption experiments using Freundlich model (25% TSS, natural pH, 20 �C).

If the results of the analytical data obtained by HPLC are plotted in the Freundlich model, a linear correlation can be shown. The Freundlich parameters KF (a measure for capacity) and bF (intensity of adsorption process) for P-470 were 0.534 L/g and 0.839, respectively. The Langmuir and Freundlich constants for XAD-1180 were KF = 0.811 L/g and bF = 0.797. These values are also similar to those obtained using P-470.

4.1.4 Influence of Oxygen on the AdsorptionThe influence of oxygen was tested using nitrogen to remove oxygen from the juice during adsorption. Chromatograms of the juice with and without nitrogen are illustrated in Figures 6 and 7.

13



Figure 6. HPLC-chromatogram of apple juice (25% TSS, natural pH) with and without oxygen contact before adsorption.

Figure 7. HPLC-chromatogram of apple juice (25% TSS, natural pH) with and without oxygen after adsorption.

By comparing Figure 6 and 7, it can be seen that the phenolic profile of the apple juice sample used in this experiment with and without oxygen contact are very similar.

Apple juice, 25% TSS, natural pH, 0 min

280 nm

with oxygen

without oxygen


280 nm


280 nm

with oxygen

without oxygen


280 nm

with oxygen

without oxygen


280 nm

with oxygen

without oxygen

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4.2 Desorption Studies

For estimating the desorption of polyphenols from the resin, different parameters were used. The saturated resin material was extracted using water and various alcohol/water mixtures at different temperatures. The recovery of phenolic compounds using water as the solvent at different temperatures is shown in Figure 8.

y = 0.2606x - 8.9546R2 = 0.9947

0

5

10

15

20

25

30

35

40

45

40 80 120 160 200

Temperature (oC)

Rec

over

yof

tota

lphe

nolic

s(%

)

Figure 8. Recovery of total phenolics from apple juice (25% TSS, natural pH)using water at different desorption temperatures.

From Figure 8, we can see that there is a linear correlation between desorption temperature and recovery of total phenolics using water with P-470, while recovery in pure was rather poor. At 40 �C, the recovery was only 2.5% while at the highest temperature used at 180 �C, it was 38.3%. The use of very high temperatures could not be considered in an industrial scale process. The recovered phenolics may also have changed their conformation through hydrolysis reactions,resulting in breakdown products and release of aglycones, which could contribute to the loss of bioactivity. The recovery of phenolics from this resin using water was worse than from the P-495 resin used in a previous study which achieved 7% at 40 �C, and ~56% at 180 �C (Kammerer et al. 2006).

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The recovery with different alcohol/water mixtures at 40 �C is displayed in Figure 9.

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80

Vol-% of solvent

Rec

over

yof

tota

lphe

nolic

s(%

)

EtOHMeOH

Figure 9. Recovery from apple juice (25% TSS, natural pH) of total phenolics at 40 �C with different ethanol and methanol amounts in the solvent.

Recovery rates of desorption experiments with ethanol and methanol were significantly higher than that for aqueous elution using the same temperature. At 40 �C, recovery was increased with increasing percentage of alcohol in the solvent with maximum achieved at 50% for both ethanol and methanol. With 25% solvent, recovery of phenolics using ethanol was substantially better, with 46.2% recovered compared with 24.1% using methanol. Otherwise, both recovery characteristics are very similar.

Desorption characteristics were very similar to those of P-495, with maximum desorption using ethanol between 50 and 75%, and at 75% using methanol. In Figure 10, recovery is shown with different alcohol/water mixtures at 80 �C.

16



0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80

Vol-% of solvent

Rec

over

yof

tota

lphe

nolis

(%)

EtOHMeOH

Figure 10. Recovery from apple juice (25% TSS, natural pH) of total phenolics at 80 �C with different ethanol and methanol amounts in the solvent.

Recovery at 80 �C in water combined with 25% of either solvent was better than using only water or the 25% alcohol/75% water combinations at 40 �C. Recovery of phenolics using 50% solvent from either ethanol or methanol (Figure 10) was very similar to that obtained at 40 �C, with about 70% of total phenolics eluted from the resin. The difference between the alcohols used was still present at the 25% solvent concentration, with better recovery using ethanol. At 75% solvent,recovery of polyphenols using methanol was almost equal to that using 50%, while recovery decreased below 50% using ethanol and was even worse than that obtained when using 25% ethanol.

Increasing temperature would result in increasing desorption. This wouldonly happen when the amount of alcohol is up to a certain limit, in this case 50%.However, the effect of temperature decreased when more than 50% alcohol was used.

17



5. Conclusion

A study of separation of polyphenolics from apple juice using resin ion exchange adsorption chromatography led to the following conclusions:

� The adsorption of apple phenolics to a polymeric resin was affected by temperature, pH value and solute/resin ratio

� The present study may contribute to the production of purified plant extracts with various health-beneficial effects

� The data obtained in such studies can be used for prediction and with it the optimization of purification and separation processes, which are based on the recovery of phytochemicals using polymeric resins

� The presence of oxygen did not cause any polyphenols oxidation during the adsorption process

� No differences were observed in phenolic compound concentrationsbetween 40 and 80 �C using higher amounts of alcohol

� In comparison to P–495, P–470 resulted in faster adsorption, more phenolic adsorption/loading to the resin and slightly better elution tendency when using solvents

� ASE is proven to be a good method for extracting polyphenols from resin which could be used to simulate industrial extraction

6. References

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