ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2023, 28(1): 16–29 DOI: 10.53541/jams.vol28iss1pp16-29 Received 13 Feb 2022 Accepted 16 April 2022 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates Al-Shaaibi Sihama, Al-Alawi Ahmeda,*, and Wesonga Ronaldb Al-Alawi Ahmed( ) ahmed543@squ.edu.om, aDepartment of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat 123, Sultanate of Oman, bDepartment of Statistics, College of Science, Sultan Qaboos University, Muscat 123, Sultanate of Oman Introduction One of the major concerns on dates’ industry is the loss of freshly harvested dates during pick-ing, storage, and processing stages (Parn et al., 2015). Approximately, two million tons per year of dates are abandon as wastes (Mrabet et al., 2017). Low-quality dates are not consumed by humans but rather discard- ed or combined in animal feed (Parn et al., 2015) due to their hard texture, insect infestation or fungus con- tamination (Abbès et al., 2011; Mrabet et al., 2017). The discarded dates was found to have similar characteristics compared with high grade dates especially in sugars, di- etary fibers and safety (Besbes et al. 2009). Therefore, the low quality dates can be utilized into new products like biomass production or metabolites (Abbès et al. 2011). The gross economic loss in discarding low quality dates is believed to be much higher when the economic value of dates’ bioactive compounds are considered (Elleuch et al., 2008). Bioactive compounds in dates waste or low-quality dates products could be extracted and used as value-added constituents. Therefore, high value-add- ed products could be prepared from low-quality dates (Abbès et al., 2011). However, there are limited studies are reported on the dates’ product development (Ahmad & Abu Bakar, 2018). The main dates’ product used in the food industry is molasses (i.e. syrup) (Bedeir, 2014). Dates’ molasses can be consumed directly or as an ingredient in many food products (Abbès et al., 2011), such as jams, ice cream, bakery products, confectioneries and concentrated bev- erages (Abbès et al., 2011; Al-Mamary et al., 2014; Be- تقييم فعالية عدد من الراتنجات الكروماتوجرافية يف فصل سكر التمر و أتثري ذلك على املركبات األخرى املوجودة يف التمر سهام الشعييب، أمحد العلوي، ويسوجنا روانلد Abstract. Dates are one of the most important food commodities in the Middle East countries, including the Sultan- ate of Oman. The fruit is regarded as a highly nutritious and healthy food. However, there are two million tons per year of dates that are abandoned waste worldwide. The objective was to establish an optimized method to separate sugars from dates using chromatographic resins and know the effect of those resins on the other ingredients. Fard date and three strong-cation gel-type resins (DOW-Ca, PCR-Ca and PCR-Na) were used in this study. The free-sugar extracts were analyzed by HPLC-MS method to quantify sugars and polyphenols. Most of the sugars were adsorbed by all of the tested resins with a higher selectivity towards fructose compared to glucose and sucrose. DOW-Ca had the lowest sugar adsorption compared to the others. Minerals profile by ICP detected a sharp reduction in potassium content, which was the main mineral found in Fard dates. Polyphenols content showed drastic decrease after treatment. Overall, the project succeeded in defining a method to remove sugar from dates and extend the loss of minerals and polyphenols in the pro- cess. For future work, it is recommended to evaluate the efficiency of other chromatographic resins in sugar separation. Keywords: Resins, Sugar, dates, HPLC-MS, bioactive compounds. امللخص:تعتــر التمــور مــن أهــم الســلع الغذائيــة يف دول الشــرق األوســط مبــا يف ذلــك ســلطنة عمــان حيــث اهنــا تعتــر غــذاء صحــي ومغــذي للغايــة. ومــع ذلــك فــان هنــاك فاقــد كبــر، حيــث يتــم التخلــص مــن مليــوين طــن مــن التمــور ســنواي يف مجيــع أحنــاء العــامل. اهلــدف مــن هــذه الدراســة هــو إنشــاء طريقــة حمّســنة لفصــل الســكرايت عــن التمــر ابســتخدام الراتنجــات الكروماتوجرافيــة ومعرفــة أتثــر هــذه الراتنجــات علــى املكــوانت األخــرى. ومت اســتخدام متــر الفــرض كمثــال للتمــور يف هــذه الدراســة كمــا مت اســتخدام ثــالث راتنجــات مــن النــوع اهلالمــي قــوي الكاتيــون وهــي د.ا.و-كالســيوم و ب.ســي.ر- كالســيوم و ب.ســي.ر-صوديوم. مت حتليــل املســتخلصات الناجتــة مــن عمليــة فصــل الســكرايت جبهــاز الفصــل الكروماتوجــرايف الســائل عــايل األداء املــزود بكاشــف مطيــاف الكتلــة لقيــاس الســكرايت والبوليفينــول. أظهــرت النتائــج امتصــاص معظــم الســكرايت بواســطة مجيــع الراتنجــات املختــرة مــع انتقائيــة أعلــى جتــاه الفركتــوز مقارنــة ابجللوكــوز والســكروز. كان د.ا.و-كالســيوم أقــل امتصــاص للســكر مقارنــة ابلراتنجــات األخــرى. كمــا كشــف اختبــار حتليــل املعــادن بواســطة جهــاز البالزمــا عــن اخنفــاض حــاد يف حمتــوى البواتســيوم، وهــو املعــدن الرئيســي يف متــور الفــرض، كنتيجــة لعمليــة الفصــل. كمــا أظهــر حمتــوى البوليفينــول اخنفاًضــا حــاًدا بعــد فصــل الســكر. بشــكل عــام جنــح املشــروع يف حتديــد طريقــة إلزالــة الســكر مــن التمــور وحتديــد درجــة امتصــاص الراتنجــات الكروماتوجرافيــة للمعــادن والبــويل فينــول املوجــودة يف التمــر. وللعمــل املســتقبلي يوصــى بتقييــم كفــاءة راتنجــات كروماتوجرافيــة أخــرى يف فصــل الســكر. الكلمات املفتاحية: الراتنجات، السكر، التمور، جهاز الفصل الكروماتوجرايف السائل عايل األداء املزود بكاشف مطياف الكتلة، املركبات احليوية النشطة . 17Research Paper Al-Shaaibi, Al-Alawi, Wesonga deir, 2014). The syrup is rich in essential nutrients and contains high sugar content which provides rapid ener- gy to the human body (Abbès et al., 2011). The syrup consists mainly of moisture, fructose, glucose, and small amounts of sucrose, pectin, protein and calcium (Mo- stafazadeh et al., 2011). Dates’ type (Mostafazadeh et al., 2011) and processing methods (Abbès et al., 2013) affect the date syrup components. Besides that, dates’ syrup contains high amount of antioxidants that could reduce progression of many diseases, oxidative stress and inhib- it macromolecules oxidation. Antioxidants such as phe- nolic acid, carotenoids, flavonoids, and ascorbic acid has been identified in dates (Abbès et al., 2013). The func- tional ingredients in dates that are biologically benefi- cial to human health (Bedeir, 2014) are greatly enhance dates’ uses in nutraceutical businesses (Abu-Reidah et al., 2017). Additionally, phytochemicals substances en- hance date fruit’s organoleptic and nutritional proper- ties (Baliga et al., 2011). Epidemiological studies demon- strated that routine intake of fruits and vegetables could decrease the risk of several chronic diseases due to their dietary antioxidants content (Abu-Reidah et al., 2017). Therefore, dates’ syrup was recommended to be used as a functional food due to its biological components, e.g. antioxidants activity (Abbès et al., 2011). However, there is a concern of regular consumption of high amount of dates with high level of sugar, especially for non-com- municable diseases (NCD) patient. Fortunately, with modern food technology, bioactive compounds in dates could be extracted individually or collectively. On most dates, glucose and fructose are found in equal quantity whereas sucrose is at is lower level (Mo- stafazadeh et al., 2011). Due to the structural similarity of glucose and fructose, their isolation is difficult. There are several methods suggested to extract and purify sugars from sugar cane and beets including enzymatic break- down, sugar-binding protein; lectins, fractional freez- ing techniques, and carbonation (Buchele, 2010). Each method has disadvantages such as high cost, undesirable breakdown, toxicity and strong bound complexes. Oth- er studies had used toxic solvent to separate sugar from the media (e.g. juice), such as methanol, chloroform and acetonitrile. Also, there are chromatographic granular applied in the sugar refining process, which was found to be safe. Chromatographic carbohydrate separation mechanism is based on hydrophilic or hydrophobic interactions, ion exchange, size exclusion and ligand exchange (Gramblička & Polakovič, 2007; Nobre et al., 2009). The resins of sulfonated polystyrene-co-divin- ylbenzene (PS-DVB) are largely used in sugar industry due to their higher selectivity and capacity and they are chemically inactive (Luz et al., 2008). Generally, resins are classified into two major groups; macroporous and gel-type. Resins are functionalized with cations to form complexes with sugar hydroxyl group and according to the orientation of this group a selective adsorption occur. Therefore, cation and sugar conformation limits the cation-sugar affinity and distri- bution coefficient. In addition, number of OH groups have a major effect on cation-sugar complex stabilities (Tiihonen et al., 2002). Moreover, the formed adsorption forces between resins and sugars are weak forces and thus are easily broken by hot water (Buchele, 2010). Calcium gel-type resins are the mostly used resins in food industry for fructose-glucose separation (Luz et al., 2008). Never- theless, sodium and potassium cation resins are recom- mended for these sugar separation (Nobre et al., 2009). Several patents were registered that documented the use of chromatographic resins in producing sug- ar-free juice (Blase & Thomas, 2008; Buchele, 2010; Pease and Pu, 2016). Baikenov et al. (2020) published a work describing the optimal parameters for the sep- aration of glucose, fructose, and oligosaccharides from glucose-fructose syrup using chromatographic resins. In the case of dates, Mostafazadeh et al. (2011) implement- ed the same approach to separate sugars from date syrup. However, all the aforementioned methods aimed to optimize and model sugar separation process with- out studying the effect on the other important com- pounds such as polyphenols and minerals. Therefore, the objective of this study was to study the efficien- cy of different chromatographic resins (calcium and sodium resins) in removing dates’ sugars and their impact on other important compounds that exist in dates. The final aim was to produce date products. Materials and Methods Materials and Chemicals Date sample of Fard variety in fully ripe stage (Tam- ar stage) was purchased from the local market. All reagents and standards were of analytical grade and were obtained from Sigma-Aldrich unless otherwise stated. Methanol LC-MS grade from Fisher Scientif- ic (UK), Acetonitrile LC-MS grade from BDH Chem- icals (Germany), nitric acid for trace metal analysis from BDH (Germany) and ammonium acetate HPLC grade from J. T. Baker (Germany). The resins used were Dowex™ MONOSPHERE™ 99Ca/320 (DOW-Ca) (DOW CHEMICAL, USA), PCR641Ca 3282Q/18/2 (PCR-Ca) and PCR642Na MR7-916 (PCR-Na) (Purolite®, USA). Specifications of the used resins are shown in Table1. Table 1. Resins Characteristics Resin Type Ion- ic form Volume diameter Func- tional group DOW-Ca Strong acid Cation, gel Ca++ 300 - 330 μm Sulfonate PCR-Na Strong acid Cation, gel Na+ 315 ± 20 μm Sulfonic acid PCR-Ca Strong acid Cation, gel Ca++ 310 ± 10 μm Sulfonic acid 18 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates Sugar Separation Sample Preparation: The chart in Figure 1 illustrates sample preparation steps for sugar extraction. Sugar ex- traction was done similarly to the method described by Myhara et al. (2000) with some modifications. Briefly, 100 g of pitted dates were homogenized with 70% or 75 or 80% (70 oC) ethanol in a ratio of 1:4 (w: v) by ultra- mixer homogenizer (Ultra-Turrax, T-25 basic) at a speed of 13,000 rpm till a homogeneous slurry was produced. Then, the solution was sonicated in a water bath at 70 oC for 15 minutes. After that, the solution was centrifuged at 10,000 rpm, at 30 oC for 10 min. The supernatant was col- lected and filtered using glass microfiber filter paper (A, Whatman) while the residue was extracted again by eth- anol solution following the same procedure used above. The extraction step was repeated thrice for com- plete removal of sugar. Only the first extract was used in the subsequent analysis. Then, the residue was dried in an oven (Carbolite, UK) at 85 oC for 6 h, milled and stored in a freezer (- 20oC) till further analysis. Sugar concentration of the filtered supernatant was measured by an automatic refractometer (HI 96802, HANNA In- struments Inc., Romania) then diluted by 70% or 75 or 80% ethanol to produce 10% and 15% ethanolic sugar solution. Summary of the sample preparation steps are shown in Figure 1. Resin Conditioning: Three different types of resins were used for sugar extraction; DOW-Ca, PCR-Ca and PCR-Na. The resins were conditioned before their use as per the manufacturer instructions with minor mod- ification. Briefly, the resin was soaked in deionized wa- ter in 1:4 (v/v) ratio for 5 min. Then, the water was de- canted and 70% or 75% or 80% ethanol was added to the conditioned resin in a ration 1:4 (v/v) and left for 5 min to ensure complete water removal. Then the ethanolic solution was decanted. Sugar Extraction: The diluted ethanolic sugar solu- tion; 10% and 15% were sonicated for 5 min to remove gasses that could interfere sugar adsorption by resin as suggested by the resin manufacturer. Then, 50 ml of the conditioned resin was mixed with the diluted ethanolic sugar solution in different ratio (1:1, 1:2 and 1:3, resin: solution) in a reagent bottle. Two temperatures (20 oC and 40 oC) were used in this experiment that was ad- justed using water bath. Sugar brix was measured by an automatic refractometer (HI 96802, HANNA Instru- ments Inc., Romania) at different time intervals; 1, 10, 20, 30 and 40 min. The mixture was caped and kept un- der continuous mixing using low speed in a magnetic stirrer plate throughout the experiment. After 40 min, the ethanolic sugar solution was decanted carefully from the resin and collected in a separate beaker. The resin was washed and conditioned before another cycle of extraction. An aliquot (5 ml) of the ethanolic sugar solution was taken for further analysis and the rest was subjected to another extraction cycle using the same resin after it is being washed and conditioned using the same procedure used in cycle 1. The various experiment parameters are summarized in Table 2. Resin Washing: To release the adsorbed sugar from the resin before moving to the second extraction cycle; res- ins were washed using hot water as per the instructions of the manufacturer. Prior to the washing step, the resins were soaked in 100 ml de-ionized water (i.e. at room tem- perature, RT) for 5 min to remove any residual ethanol and then the water was discarded. Then, hot deionized water (70 oC) in a ratio 1:2 was added. The mixture was kept under continuous stirring using a magnetic stirrer in a hot plate. Brix was measured continuously till no fur- ther change in the reading; then water was removed and a new hot deionized water was added. The washing step was repeated three times. After washing, the resin was re-conditioned again by ethanol (70% or 75 or 80% de- pending on the starting ethanol solution) to remove any remaining water before starting a new extraction cycle. Figure 1. Summary chart of sample preparation 19Research Paper Al-Shaaibi, Al-Alawi, Wesonga Sample Concentration: Samples collected from first and second extraction cycles as well as from dates’ stock solution were dried by vacuum concentrator (Concen- trator plus, Eppendorf, USA) at 30 oC. Dried samples were stored at -60 oC until further analysis. Ash Determination Samples were ashed using a muffle furnace (CARBO- LITE, UK) at 550 oC for 18 h (AOAC 2000, Method p). Few drops of hydrogen peroxide were added to samples that had carbon. Samples that showed persistent black spots were suspended in a deionized water and filtered through an ashless filter paper (Whatman No. 541). The filtrate was dried in an oven and the filter paper with the black materials were ashed using the same conditions used initially. Minerals Profile by Induc tively Coupled Plasma (ICP) Sample Preparation: Some of the ashed samples were used to determine minerals profile by ICP, including date fruit, date residue, sugar extract and 15% (1:3) first cycle according to (EN act Oct1, 2017) method with slight modification. The ashed sample was digested by adding 2 ml concentrated nitric acid HNO3 and kept in a hot plate at 250 oC until it was dissolved. Then drops of deionized water were added to cool the solution, there- after, transferred to 10 ml volumetric flask and diluted with deionized water to the mark. After that, the solu- tion was filtered using ashless filter paper (Whatman #541) and then stored in a fridge at 2 oC until it was in- jected to ICP. ICP Conditions: Perkin Elmer-optima 8000 ICP-OES with spectral range 165-900 nm and resolution < 0.009 nm @ 200 nm was used. The detector was UV-sensi- tive, dual backside-illuminated Charge-Coupled Device (CCD) array detector with two photosensitive segments containing 176 by 128 pixels. The detector is cooled to -8 °C using a single-stage integrated Peltier cooler. The RF COIL power was 1500 W and the peristaltic pump flow rate was 1 ml/min. The plasma gas was argon with flow rate of 8 L/min and the shear gas was compressed air. The tested elements were arsenic, zinc, lead, cobalt, cadmium, nickel, iron, boron, mercury, manganese, chromium, copper, aluminum, sodium, potassium, mag- nesium and calcium. Sugar and Polyphenol Quantification by HPLC-MS Sugar and polyphenols were quantified using Nexera-X2 LC instrument (Shimadzu Corporation, Japan) com- posed of LC-30AD pump, SIL-30AC autosampler and mass-spectrometer LCMS-2020 following the manu- facture recommendation. The column used was Imtakt Unison UK-Amino column (250×3 mm, 3 µm) main- tained at 37 oC by the unit CTO-20AC. The ionization mode interface was DUIS (ESI and APCI) and DL tem- perature was 250 oC and heating block at 300 oC. Nitro- gen was used as nebulizing gas and drying gas at a flow rate of 1.5 L/min and 15 L/min, respectively. Four events were used, two for scan (positive and negative) and the other remaining two events were SIM positive and neg- ative according to m/z ± (H+) or + (CH3COOH-). In- terface voltage was 3.5 kV, DL voltage, quarry DC and RF voltage were sat at default. Scan speed was 10000 u/ sec from 100 m/z until 1000 m/z with event time 0.1 s. The mobile phase consisted of LC-MS grade acetoni- trile (mobile phase A) and 100 mM ammonium acetate (mobile phase B). The gradient for mobile phase B was programmed as follows, 5%-100% (0-15 min), then 100% for 5 min then 0% for 6 min. The concentrated samples were dissolved in 1 ml methanol and filtered into vials using 0.22 µm nylon syringe filter (ALWSCI Group, Chi- na). Sample injection volume was 1 µL and flow rate was 0.3 ml/min. Statistical Analysis The statistical analysis was carried out using Microsoft Excel 2021 (version 16.57,© 2021 Microsoft ) to calculate one-way analysis of variance (ANOVA) to test signifi- cance of the means of the samples. The significance of the difference between the analyzed group parameters was analyzed by Student’s t- test. Also, R program (ver- sion 4.1.1 (R Core Team, 2021)) used to test parameters correlations by Tukey test, Regression test and ANOVA interaction plots. P<0.05 was considered statistically sig- nificant. The collected data were expressed as mean ± standard deviation (SD). Results Optimization of Separation Condition Many tests were performed before finalizing sample preparation and extraction procedure. In reference to Table 2. Experimental parameters for sugar separation Parame- ters Conditions Sample sugar % 10% 15% Tempera- ture 20oC 40oC Extraction cycle 1 2 Resin DOW-Ca PCR-Ca PCR-Na Solvent Conc. 70% 75% 80% Ratio (resin: sample) 1:1 1:2 1:3 Time 1 min 10 min 20 min 30 min 40 min 20 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates the sample preparation, deionized water was compared with ethanol to check the best solvent for sugar solubi- lization that will facilitate sugar adsorption by the resin. It is noteworthy that Mostafazadeh et al. (2011) used de- ionized water to solubilize dates’ sugars. Also, mode of process was evaluated by comparing column (separatory funnel) to batch under vacuum (conical flask). The re- sults (not shown) found that resins adsorbed more sug- ar (higher capacity) when ethanolic solution was used. Moreover, ethanol offered many other advantages such as lower solubility of minerals and precipitation of wa- ter-soluble polysaccharides such as pectin. With regard to mode of separation, batch separation was better than column extraction due to longer contact time between resin and sugar molecules. Vacuum was not critical fac- tor on the experiment since the solution was degassed. Furthermore, use of refractometer to measure sugar content in ethanolic solutions was a challenge as ethanol itself gives a reading. This problem was solved by spiking the different ethanolic solutions (70%, 75%, 80%) with sugars to construct a calibration curve to normalize ef- fect of ethanol on brix readings. Standards were prepared by dissolving glucose (10% w/w) and fructose (10% w/w) in the different ethanolic solutions to produce 20% stock ethanolic sugar solution. Then, the solution was diluted into several concentrations. The curve equation for each ethanolic solution (70%, 75%, 80%) was used to deter- mine sugar concentration in all the solutions afterwards. Date Sugar In this study, glucose, fructose and sucrose were quan- tified in Fard dates using HPLC. The analysis was found to be the most important factor that affects resin chro- matographic separation (Baikenov et al., 2020). The results of sugar content and types are shown in Figure 2. This information was used to adjust dates’ sugar-eth- anolic extract to the desired concentration. As per the obtained data, in 100 g of dates, glucose and fructose level was ~ 35 g individually whereas sucrose was ~1.6 g. Level of ethanol (70-85%) was not significant in sugar extraction from Fard fruit. Parameters of the Separation Method Interactions of The Method Parameters: The aim of this work was to find an effective method to separate sugars from dates’ extract. Several factors and variables were tested to reach to the optimum separation conditions. These factors were adsorbent (resin) type, sugar con- centration, solvent concentration, resin to sample ratio, extraction temperature, experiment time and number of extraction cycle. Through R program, the variations between these factors with respect to sugar concentra- tion readings after each treatment were tested by Tukey multiple comparisons of means. Table 3 illustrates the results of this test for extraction cycle 1 and Table 4 for extraction cycle 2. Interaction of experimental time and resin types in extraction cycle 1 and extraction cycle two showed no significant differences as it is clear in Figures 3 and 4. However, there was a significant difference (P <0.05) between sample to resin ratio (1:1, 1:2, 1:3) within time in extraction cycle 1 (Figure 5) and extraction cycle 2 (Figure 6). Secondly, temperature (temp) did not show an effect (20°C vs. 40°C) in sugar separation in the ex- traction cycle 1 (Figure 7). However, in extraction cycle 2 the time interval from 20 min to 40 min was signifi- Figure 2. Effect of ethanol concentration on extraction of sugar from Fard dates. 21Research Paper Al-Shaaibi, Al-Alawi, Wesonga cantly different (P <0.05) in brix readings between 20°C and 40°C (Figure 8). Moreover, there was a significant difference (P <0.05) between the two tested concentra- tions (15% and 10%) according to brix readings with ex- periment time interval in extraction cycle 1 (Figure 9) and extraction cycle 2 (Figure 10). Resin-Sugar Holding Capacity: Due to the differences in the functional group and cation in the used resin, hold- ing capacity was different as illustrated in Figures 11-13. PCR-Ca was the highest sugar adsorbent (99%) followed by PCR-Na, however, the differences between the two resins was not significantly different (P<0.05). Dow-Ca resin showed the lowest adsorption capacity towards sugar; maximum adsorption was towards fructose (94%). As it’s clear from Figures 11-13, sugar holding capacity is proportional with resin:solution ratio. Also, 80% eth- anol concentration enhanced sugar absorptivity by resin compared to the other concentrations (70% and 75%). Table 3. Analysis of variance for cycle 1 Parameter Df Sum Sq Mean Sq F value Pr(>F) resin 2 4.4 2.2 1.990 0.138 Temp. 1 0.0 0.0 0.005 0.944 de.conc 1 1597.9 1597.9 1432.704 < 2e-16 *** ssratio 2 967.9 483.9 433.910 < 2e- 16 *** time.interval 4 1358.7 339.7 304.559 < 2e- 16 *** Residuals 529 590.0 1.1 de.conc: dates extract concentration (10 %& 15%), ssratio: solution: resin ratio, Signif. codes: ‘***’ = 0.0 Table 4. Analysis of Variance for cycle 2 Parameter Df Sum Sq Mean Sq F value Pr(>F) resin 2 5.5 2.8 2.618 0.07391 Temp. 1 13.0 13.0 12.305 0.00049 *** de.conc 1 435.4 435.4 411.591 < 2e-16 *** ssratio 2 2286.0 1143.0 1080.457 < 2e-16 *** time.interval 4 724.7 181.2 171.266 < 2e-16 *** Residuals 529 559.6 1.1 de.conc: dates extract concentration (10 %& 15%), ssratio: solution: resin ratio, Signif. codes: ‘***’ = 0.0 Figure 3. Correlation between resin types and time in cycle one 22 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates Resin-Sugar Affinity: All of the tested resins adsorbed most of the sugars in the sample, especially PCR-Na and PCR-Ca compared to DOW-Ca. However, fructose was adsorbed more than both glucose and sucrose. Resin Washing: As mentioned earlier, sugar separa- tion in this study was based on an adsorption cycle fol- lowed by a desorption cycle (Nobre et al., 2009). In the desorption cycle, sugars separated from resin by washing using hot water. According to the washing procedure, it involved three cycles of washing by hot deionized water. In each cycle, brix was measured to check effi- ciency of the desorption process. The sum of Brix read- ings for each washing cycle is illustrated in Figure 14. At washing cycle 1, most of the adsorbed sugars was released, > 60% of the total adsorbed sugar. DOW-Ca resins discharged the highest amount of sugars followed by PCR-Na then PCR-Ca. In cycle three, all of the tested samples recorded less than 1 g/ml of sugar. This makes the resin reusable for further sugar removal cycles. Sugar Content Glucose, fructose and sucrose were determined in the resin-treated ethanolic solution. The main findings are il- lustrated in Figure 15. HPLC-MS results were supporting Figure 4. Correlation between resin types and time in cycle two Figure 5. Dates solution ratios with time in cycle 1 23Research Paper Al-Shaaibi, Al-Alawi, Wesonga brix readings at the separation experiment. From Figure 15 we can conclude that Ca resins, DOW- and PCR-Ca, were more selective to fructose than glucose and su- crose. Similar observation was also reported by Baiken- ov1 et al. (2020). While Na-resin (PCR-Na) had no sugar selectivity towards any of the sugars (i.e. glucose and fructose). Thus, calcium resins made the solution rich in glucose than sucrose and fructose. However, Na-resin produced sucrose rich solution. Overall, sugar content in the resulting extract from exposure to the various types of resins was very low compared to the original sugar content in dates. This illustrates that the experiment suc- ceeded in removing sugars from dates’ ethanolic extract. Ash Content and Minerals Profile Ash contents: All three used resins in this study ad- sorbed most of the ash during the first extraction cycle (about 98%). Figure 16 illustrates the main minerals in the 75% ethanolic solution extract after treatment with the different resins (1:3 resin to sample ratio). Resins with the same cation have similar properties; DOW- and PCR-Ca. Calcium became the main mineral followed by sodium and potassium in solutions treated by DOW- and PCR-Ca resins. However, sodium content was not affected in the solution treated by PCR-Na resin. Figure 6. Dates solution ratios with time in cycle 2 Figure 7. Temperature effect in brix reading with time in cycle 1 24 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates Polyphenol contents: Dates fruits are rich in polyphe- nols content as many studies proved. In this study, the free polyphenols content was determined using LC-MS. Total of 18 compounds were detected in Fard date sam- ples as shown in Figure 17. Caffeic acid and the gallic acid were the dominant polyphenols. Similar to the observation with whole Fard dates fruits, 18 free polyphenols were identified in the treated extract but with different proportions. This is because the resins had adsorbed some of the polyphenols. Polyphenols’ ad- sorption varied with the resin type as shown in Figure18. Figure 18 displays the profile of polyphenols in the sug- ar ethanolic solutions resulted from treatment by the three different resins. It is very evident that PCR-Na and PCR-Ca resins adsorbed mainly sugars along with other compounds including free polyphenols. The percentage range of polyphenols hold by PCR-Na and PCR-Ca were 75% - 100% and 89% - 100%, respectively, depending on the type of the polyphenol. For example, m-coumaric acid was the lowest polyphenol adsorbed by PCR resins, whereas DOW-Ca resin adsorbed 95% of m-coumaric acid present initially in the solution. Figure 8. Temperature effect in brix reading with time in cycle 2 Figure 9. Sample concentration with time in cycle 1 25Research Paper Al-Shaaibi, Al-Alawi, Wesonga Discussion Carbohydrates are the major component in dates fruit; around 70%-80% (DW) (Bedeir, 2014). Glucose and fructose are the main simple reducing sugars in dates along with the disaccharide sucrose (Al-Harrasi et al., 2014). Al-Farsi et al. (2005) reported that Fard dates contain 28 g/100g (FW) glucose and the same for fruc- tose while the total sugar was 56.7 g/100g (FW). Howev- er, for the same variety, Myhara et al. (1999) found that 100 g (DW) contains 43.6 g glucose, 42 g fructose and 0.53 g sucrose. Al-Harrasi et al., (2014) determined the total carbohydrates in Fard dates which were 79 g/100 g (DW). The differences are attributed to moisture con- tent, sample preparation and quantification methods. In this study, Fard dates was found to contain ~82% sug- ars which is in line with what was reported earlier for the same variety (Myhara et al., 1999). Furthermore, as it is clear in the Figures 2 and 3, there was no signifi- cant differences in the extraction power of glucose or fructose or sucrose content (P <0.05) by different eth- anol concentration (70%, 75% and 80%). This means ei- ther solution can be used for this step (sugar extraction step) to prepare sugar ethanolic solution. Sugars exhibit high solubility in ethanolic solution and the percentage of solubilization decreases with the decrease in water percentage (Bockstanz et al., 1989). Ethanol offers many advantages over other organic solvents, such as safety and re-usability while eliminates extraction of other in- gredients (such as pectin) that could complicate the sub- Figure 10. Sample concentration with time in cycle 2 Figure 11. DOW 99 Ca sugar holding capacity Figure 12. PCR642 Na sugar holding capacity 26 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates sequent treatments (Rayo-Mendez et al., 2019). During resin treatment, ethanol was unfavorable medium for sugars, thus sugar migrated into the polar resins. Other tested parameters were significantly (P >0.05) affected in sugar adsorption at extraction cycle 1, except type of resin and temperature. However, in extraction cycle 2, resin type and waiting time (20-40 min) did not affect the separation significantly. A previous study employed Na+ and K+ cation resins for similar separation at 25 °C and 40 °C concluded that in mono-component sugar mixture higher temperature decreased adsorption while sugar adsorption increased with increased temperature in multi-component sugar mixtures (Nobre et al., 2009). Overall, the tested parameters had different effect on sugar adsorption. Temperature and the used resin types were not significantly effective variables in sugar adsorp- tion. While sample concentration and resin:sample ratio were significantly effective (P >0.05) in sugar extraction within the different variables used. Figure 13. PCR641 Ca sugar holding capacity Figure 14. Sugar concentration in each washing cycle for each resin Figure 15. Fructose, glucose and sucrose ratio in sugar-free dates extract 27Research Paper Al-Shaaibi, Al-Alawi, Wesonga PCR-Ca was the highest in adsorbing sugar (99%) with no significant differences (P <0.05) within sugar type. However, DOW-Ca resin was the lowest in sugar ad- sorption. Cation type had effect in sugar adsorption and affinity. As proved by this study, the solution treated by calcium resins contained more glucose than sucrose and fructose. However, Na-resin-treated-solution contained higher sucrose. This result is supported by Mostafazadeh et al. (2011) findings where they used PCR-Ca to separate fructose from date syrup. Moreover, this finding was also observed elsewhere with other resins; potassium gel- type and sodium macroporous resins (Nobre et al., 2009). Additionally, sugar selectivity was found to be unaf- fected by temperature. Work done by Nobre et al. (2009) on Na resins reported similar results to our findings. This was attributed to the similarity in molecular weight of sugars. However, fructose/sucrose selectivity was higher anticipated to fructose-resins strong adsorption. Also, gel-type resins are higher selective than macroporous res- in towards fructose (Gramblička & Polakovič, 2007; No- bre et al., 2009). Moreover, Tiihonen et al. (2002) showed that higher ethanol content are better suited for sugar separation by Ca2+ column than Na+ and La3+ columns. Whereas water sugar mixture is best for La3+ columns. The main mineral in Fard dates was potassium which had declined after exposure to the three tested resins. Also, the main free polyphenol in Fard dates fruits were caffeic and gallic acids. This finding is somehow different than other studies that reported gallic acid the main free polyphenol in Omani dates (Ahmed et al., 2013; Al Harthi et al., 2015). However, after exposure to the resin, rutin Figure 16. Main minerals in 75% ethanolic solution extract after treatment with 1:3 resin:sample ration Figure 17. Polyphenols compounds extracted by different ethanol concentration from Fard dates 28 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of Several Chromatographic Resins on the Separation of Dates Sugar and their Impact on other Compounds in Dates and procyanidin B2 became the highest in the samples treated by DOW-Ca resin. For samples treated by PCR resins, m-coumaric acid and vanillic acid became the highest in PCR-Na and PCR-Ca treatments, respectively. Conclusion The method presented in this work showed that resins had different power and selectivity towards different sugars. PCR-Ca had the highest adsorbing power to- wards sugar (99%) followed by PCR-Na and DOW-Ca, respectively. Calcium based resins had more affinity to- wards glucose, whereas sodium based resin had more af- finity towards fructose. Furthermore, all resins adsorbed minerals and polyphenols along with sugars. Tempera- ture was found to be not critical. References Abbès F, Bouaziz MA, Blecker C, Masmoudi M, Attia H, Besbes S. (2011). Date syrup: Effect of hydrolytic enzymes (pectinase/cellulase) on physico-chemical characteristics, sensory and functional properties. LWT-Food Science and Technology 44(8): 1827–1834. Abbès F, Kchaou W, Blecker C, Ongena M, Lognay G, Attia H, Besbes S. (2013). Effect of processing condi- tions on phenolic compounds and antioxidant prop- erties of date syrup. Industrial Crops and Products 44: 634–642. Abu-Reidah IM, Gil-Izquierdo Á, Medina S, Ferreres F. (2017). Phenolic composition profiling of different edible parts and by-products of date palm (Phoenix dactylifera L.) by using HPLC-DAD-ESI/MSn. Food Research International 100: 494–500. Ahmed J, Almusallam A, Al-Hooti SN. (2013). Isolation and characterization of insoluble date (Phoenix dac- tylifera L.) fibers. LWT - Food Science and Technol- ogy 50(2): 414–419. Ahmad SZ, Abu Bakar AR. (2018). Emirates Dates: a case of growth strategy dilemma. Emerald Emerging Markets Case Studies. Al-Harrasi A, Rehman NU, Hussain J, Khan AL, Al-Rawahi A, Gilani SA, Al- Al Harthi SS, Mavazhe A, Al Mahroqi H, Khan SA. (2015). Quantification of phenolic compounds, evaluation of physicochem- ical properties and antioxidant activity of four date (Phoenix dactylifera L.) varieties of Oman. Journal of Taibah University Medical Sciences 10(3): 346–352. Al-Mamary M, Al-Habori M, Al-Zubairi AS. (2014). The in vitro antioxidant activity of different types of palm dates (Phoenix dactylifera) syrups. Arabian Journal of Chemistry 7(6): 964–971. AOAC (2000). Official Methods of Analysis. 17th Edi- tion, The Association of Official Analytical Chemists, Gaithersburg, MD, USA. Method 940.26. Baikenov AO, Muslimov N, Baigenzhinov KA, Yessimo- va ZA, Kim YV. (2020). Mathematical model of de- pendence of factors for chromatographic separation of fructose from glucose-fructose syrup. IOP Confer- ence Series: Materials Science and Engineering 994(1). Baliga MS, Baliga BRV, Kandathil SM, Bhat HP, Vayalil PK. (2011). A review of the chemistry and pharma- cology of the date fruits (Phoenix dactylifera L.). Food Research International 44(7): 1812–1822. Bedeir (2014). Evaluation of Pan Bread and Pies Made By Partial Substitution With Dates Syrup (Dibs). Egyptian Journal of Agricultural Research 92(3): 1025–1044. Besbes S, Drira L, Blecker C, Deroanne C, Attia H. (2009). Adding value to hard date (Phoenix dactylifera L.): Compositional, functional and sensory character- istics of date jam. Food Chemistry 112(2): 406–411. Blase DJ, Thomas CB. (2008). Juice Processing. Unit- ed States Patent Application, Patent No. US 20080044531 A1. Figure 18. The main polyphenols (%) in resin treated sugar-ethanolic solution 29Research Paper Al-Shaaibi, Al-Alawi, Wesonga Bockstanz GL, Buffa M, Lira CT. (1989). Solubilities of alpha anhydrous glucose in ethanol-water mixtures. Journal of Chemical and Engineering Data 34(4): 426–429. Broumi M, Ali L. (2014). Nutritional assessment and an- tioxidant analysis of 22 date palm (Phoenix dactylif- era) varieties growing in Sultanate of Oman. Asian Pa- cific Journal of Tropical Medicine 7(S1): S591–S598. Buchele WN (2010). Sugar extraction process. Unit- ed States Patent Application, Patent No. US 20100068373 A1. Elleuch M, Besbes S, Roiseux O, Blecker C, Deroanne C, Drira NE, Attia H. (2008). Date flesh: Chemical com- position and characteristics of the dietary fibre. Food Chemistry 111(3): 676–682. Gramblička M, Polakovič M. (2007). Adsorption equi- libria of glucose, fructose, sucrose, and fructooligo- saccharides on cation exchange resins. Journal of Chemical and Engineering Data 52(2): 345–350. Luz DA, Rodrigues AKO, Silva FRC, Torres AEB, Caval- cante CL, Brito ES, Azevedo DCS. (2008). Adsorptive separation of fructose and glucose from an agroin- dustrial waste of cashew industry. Bioresource Tech- nology 99(7): 2455–2465. Mostafazadeh AK, Sarshar M, Javadian S, Zarefard MR, Haghighi ZA. (2011). Separation of fructose and glucose from date syrup using resin chromatographic meth- od: Experimental data and mathematical modeling. Separation and Purification Technology 79(1): 72–78. Mrabet A, Rodríguez-Gutiérrez G, Rubio-Senent F, Hamza H, Rodríguez-Arcos R, Guillén-Bejarano R, Sindic M, Jiménez-Araujo A. (2017). Enzymatic con- version of date fruit fiber concentrates into a new product enriched in antioxidant soluble fiber. LWT 75: 727–734. Myhara RM, Al-Alawi A, Karkalas J, Taylor MS. (2000). Sensory and textural changes in maturing Omani dates. Journal of the Science of Food and Agriculture 80(15): 2181–2185. Myhara RM, Karkalas J, Taylor MS. (1999). The compo- sition of maturing Omani dates. Journal of the Sci- ence of Food and Agriculture 79(11): 1345–1350. Nobre C, Santos MJ, Dominguez A, Torres D, Rocha O, Peres AM, Rocha I, Ferreira EC, Teixeira JA, Ro- drigues LR. (2009). Comparison of adsorption equi- librium of fructose, glucose and sucrose on potassi- um gel-type and macroporous sodium ion-exchange resins. Analytica Chimica Acta 654(1): 71–76. Parn OJ, Bhat R, Yeoh TK, Al-Hassan AA. (2015). De- velopment of novel fruit bars by utilizing date paste. Food Bioscience 9: 20–27. Pease S, Pu G. (2016). Chromatographic separation of sugars using blend of cation exchange resins. United States Patent Application, Patent No. US 9441280 B2. R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Rayo-Mendez LM, Gómez AV, Tadini CC. (2019). Ex- traction of soluble sugars from banana puree to ob- tain a matrix rich in non-starch polysaccharides. Food Chemistry 294: 539–546. Tiihonen J, Markkanen I, Paatero E. (2002). Complex stability of sugars and sugar alcohols with Na+, Ca2+, and La3+ in chromatographic separations using poly(- styrene-co-divinylbenzene) resins and aqueous or- ganic eluents. Chemical Engineering Communica- tions 189(7): 995–1008.