Resins for ion-exchange chromatography

Anion exchange column chromatography [6]
This is the most commonly applied method in both polysaccharide purification and column chromatography at present. In particular, anion exchange column chromatography is usually used at first for bulky polysaccharide solution. Polysaccharide solution can be concentrated and preliminarily purified through this method, even some polysaccharides can be purified homogeneous fractions. The widely used anion exchanger so far are DEAE-cellulose, DEAE-Sephadex and DEAE-Sepharose, among which DEAE-cellulose is usually the first choice. DEAE-cellulose possesses an open framework and polysaccharide molecules can freely enter this carrier and diffuse rapidly. DEAE-cellulose has a big surface area. Although its ion-exchange capacity is only 0.70⿿0.75 mmol/g, the absorption quantity of DEAE-cellulose to polysaccharides is much larger than ion exchange resin. In addition, since ion exchange groups on cellulose are less, loose in arrangement and alkalescent, the adsorption of DEAE-cellulose to polysaccharides is weak and polysaccharides can be eluted out using salt solution of a certain ion concentration.

Anion exchange column chromatography is fit for separating various acidic polysaccharide, neutral polysaccharide and mucopolysaccharide. The separation mechanism of anion exchange column chromatography is not only ion-exchange, but also adsorption-desorption. So anion exchange column chromatography can be used in the separation of neutral and acidic polysaccharides, and the separation of different neutral polysaccharides as well. In general, when the pH value is 6.0, acidic polysaccharide can be adsorbed to the exchanger whereas neutral polysaccharide can not be adsorbed . Then the buffers which have same pH value and different ionic strength can be used to elute these acidic polysaccharides out respectively. The ability of polysaccharide to adsorb exchanger is related to polysaccharide structure. The adsorb ability usually increases with the increase of acidic groups in polysaccharide molecules. For linear molecules, larger MW neutral polysaccharide is easier to be adsorbed compared to smaller MW polysaccharide. The adsorb ability of straight chain polysaccharide is greater than that of branched chain polysaccharide. In most cases, 100 g of DEAE-cellulose can load 0.5⿿1.5 g of dry polysaccharide sample. The elution mode is usually to use buffers with different ionic strength to carry out gradient elution or stepwise elution. In addition, neutral polysaccharide can also form coordination compound with borax (sodium tetraborate). Based on this, DEAE-cellulose is sometimes processed into borax-type DEAE-cellulose. When the polysaccharide solution flows through the borax-type DEAE-cellulose column, polysaccharide will coordinate with borax and adsorb on the column. Then the column is eluted using borate solution of different concentrations. The eluate which flows out first is the polysaccharide fraction that does not coordinate with borax, and the eluate flowing out last is the polysaccharide fraction that coordinates with borax most strongly.

Besides DEAE-cellulose, the other two anion exchanger, i.e. DEAE-Sephadex and DEAE-Sepharose are also widely used. DEAE-Sephadex series has several products, such as DEAE-Sephadex A25 (often used for the MW < 30,000 polysaccharide purification) and DEAE-Sephadex A50. DEAE-Sepharose series also has several products, such as DEAE-Sepharose CL-6B (often used for the MW > 100,000 polysaccharide). Since these exchangers have three dimensional network structure, they possess not only ion exchange function but also molecular sieve effect. Compared with cellulose, they have higher charge density, and thus they have larger exchange capacity and better separation effect. However, when the pH value or ionic strength of the eluent changes, the two exchangers (DEAE-Sephadex and DEAE-Sepharose) will change much in volume and thus the flow rate will be affected. The process method of regeneration of DEAE-Sephadex and DEAE-Sepharose is same as DEAE-cellulose.

In summary, the above three anion exchangers (DEAE-cellulose, DEAE-Sephadex and DEAE-Sepharose) are widely used in polysaccharide purification. Meanwhile, there exists some disadvantages, especially when they are used for mucopolysaccharide purification. For example, the flow rate is low, the height of column bed may change with the change of buffer concentration and pH value and thus is not steady, the service life of exchanger is short, etc. The three exchangers are gradually replaced by another kind of anion exchanger which backbone is Sepharose FF with good chemical stability and fast flow rate after 1990⿿s. The typical product model is DEAE-Sepharose FF. The usage method of DEAE-Sepharose FF is similar to DEAE-cellulose. The DEAE-Sepharose FF can not be stored in the form of dry powder and it must be suspended in water for preservation.

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The effect of the amount of DEAE-cellulose on the gold recovery efficiency [1]
The first experimental series was performed by varying the amount of DEAE-cellulose from 10 to 400 mg while keeping the other parameters constant. Fig. 2 presents the variation of the gold recovery efficiency with an increasing amount of DEAE-cellulose. It is apparent from the figure that the recovery efficiency increases with an increasing amount of DEAE-cellulose.
The increase in gold recovery efficiency with an increasing amount of DEAE-cellulose can be easily explained by the fact that recovery reactions are thermodynamically favored by a high ratio of the sorbent to the metal to be recovered (Matsubara et al., 2000).
When the gold chloride solution is contacted by DEAE-cellulose, gold in the trivalent (Au3+) form is reduced to the metallic form (Auo) whereas hydroxyl groups in DEAE-cellulose are oxidized (Ogata and Nakano, 2005). This was proven by XRD analysis. The net reaction takes place as follows:
The oxidation of hydroxyl groups in DEAE-cellulose is expressed as follows
And the overall reaction is expressed as follows
When the reaction was allowed to run for 30 min, the recovery efficiency reached over 90% with DEAE-cellulose amounts of 50 mg and above, and started to level off at approximately 100% beyond 100 mg. Thus, the 10–60 mg DEAE-cellulose was chosen for further experiments.

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The product characterization [1]
To determine whether recovered gold is in the metallic form, XRD analysis was performed on samples after the DEAE-cellulose was burned off at 800 °C, after which pure gold was attained. The purity of the gold was found to be 99.8% by cupellation method. This indicates that gold can easily be isolated from DEAE-cellulose, resulting in pure gold powder. In the diffraction pattern, the peaks are clearly observed at 2 theta = 38, 44.4, 64.6, 77.6, 81.5 which are in agreement with the metallic gold peaks. The peaks clearly confirm that the trivalent gold ions present in the solution were reduced to metallic gold (Nakajima et al., 2003, Ogata and Nakano, 2005). Fig. 8a and b displays SEM images of the recovered gold particles before and after DEAE-cellulose was burned off at 800 °C. SEM images show that gold atoms agglomerated and were deposited on some regions of the DEAE-cellulose. After it was burned off at 800 °C, a porous structure was attained.

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The present work has demonstrated that gold recovery using DEAE-cellulose is effective for recovering gold from diluted gold chloride solutions. Using excessive amounts of DEAE-cellulose (at DEAE-cellulose/Au weight ratios of 400 and above), a gold recovery efficiency of over 99% is easily attainable at 130 rpm after 30 min, even at room temperature, whereas increasing the temperature from 30 to 60 °C enables an efficiency above 99% with a significantly smaller amount of DEAE-cellulose (at a DEAE-cellulose/Au weight ratio of around 120) under the same conditions. By optimizing process parameters, gold recovery approaching the theoretical maximum should be feasible with relatively small DEAE-cellulose additions. Increasing temperature caused an increase in gold recovery efficiency, in accordance with the published literature.
The gold recovery was found to occur via the reduction of trivalent gold ions present in the solution to metallic gold, which was proven by SEM images and XRD patterns. It has also been demonstrated that increasing the shaking rate and contact time leads to an increase in the gold recovery efficiency. Considering 10 mg DEAE-cellulose quantity, by increasing the shaking rate from 20 to 120 rpm, the gold recovery efficiency increased by around 50%, which indicates that shaking is necessary for gold recovery from diluted gold-bearing solutions. Among all parameters investigated in this study, the shaking rate was found to be the most effective in terms of recovery efficiency. The gold recovery by DEAE-cellulose is an intermediate-controlled process with an activation energy of 37.11 kJ/mol and obeys first order kinetics, in accordance with the published literature.
This study demonstrates that gold recovery can successfully be achieved using DEAE-cellulose as an alternative to traditional sorbents used in industrial gold recovery. In addition, the outstanding characteristics of DEAE-cellulose for gold recovery offer the possibility of efficient recovery of other precious metals. [1]

Separation and Purification of PNLP [2]
PNLP was isolated and purified following the methods reported by Zhang et al. [Carbohydr. Polym. 2021, 251, 117078] and Li et al. [Int. J. Mol. Sci. 2023, 24, 15904]. A 10 mg/mL polysaccharide solution was slowly applied along the wall of a DEAE-cellulose column (26 mm × 50 cm). The sample was then sequentially eluted with distilled water and NaCl solutions of concentrations 0.1, 0.3, and 0.5 M. The elution was conducted at a flow rate of 0.5 mL/min, with the eluate collected in 12 min intervals per tube. Absorbance was quantified at 490 nm using the phenol-sulfuric acid assay, and the resulting elution profile was subsequently plotted. Based on the elution profile, four distinct fractions were obtained: PNLP-1 (eluted with distilled water), PNLP-2 (eluted with 0.1 M NaCl), PNLP-3 (eluted with 0.3 M NaCl), and PNLP-4 (eluted with 0.5 M NaCl).

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Isolation and Purification of Bamboo Shoot Polysaccharides [3]
Raw polysaccharide was extracted from bamboo shoot through hot water extraction methods, after which it was treated by deproteinization, water dialysis, DEAE-cellulose, and Sephadex-50 column chromatography grading. The results showed that papain combined with Sevag presented the optimum deproteinizing condition. The bamboo shoot polysaccharides were eluted with water, 0.05, 0.1, and 0.2 and 0.5 mol/L NaCl salt solution by DEAE-Cellulose 52 column grading; finally, the main components were eluted with water by Sephadex-50 grading.

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Isolation and purification of ZMP (a polysaccharide)[4]
The crude polysaccharides, named ZMP, were extracted from Z. Jujuba cv. Muzao fruit by ethanol precipitation, lyophilization, and then deproteination by Sevag reagent. The total yield rate of ZMP in the isolation procedure was 4.31% (Fig. 1), which is similar to that reported in the previous research. In order to increase the purity and obtain homogeneous polysaccharide products, ZMP were purified using an anion-exchange chromatography of DEAE-cellulose column (2.6 cm × 40 cm). The four fractions, eluted with deionized water, 0.05, 0.10, and 0.30 M NaCl, were named ZMP-1, ZMP-2, ZMP-3, and ZMP-4, respectively (Fig. 2). ZMP-1, which was eluted with deionized water, may be a neutral polysaccharide, whereas ZMP-2, ZMP-3, and ZMP-4, which were eluted with the 0.05, 0.10, and 0.30 mol/L NaCl solutions, respectively, are acidic polysaccharides. Each of the four fractions were further purified by gel filtration chromatography of Sephadex G-100 using distilled water as an eluent at a flow rate of 1.0 mL/min. The eluate (8 mL/tube) was collected automatically, resulting in GZMP-1, GZMP-2, GZMP-3, and GZMP-4 were obtained (Fig. 3). The four fractions were recovered at rates of 0.209%, 0.093%, 0.418%, and 0.255%, respectively, based on the original amount of ZMP. All four of these elution curves had a single, symmetrical peak, which indicates that all of the purified products were homogeneous polysaccharides.

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Gold recovery from dilute gold solutions using DEAE-cellulose. [1]
The study was conducted in a batch system by varying one recovery parameter at a time. For each experiment, 10 mL of the synthetically prepared gold solution of 50 ppm was contacted with DEAE-cellulose in a falcon tube to avoid exposure to air. In this manner, air was not able to diffuse into the system. Experiments were conducted in a temperature-controlled shaking water bath to ensure uniform heat convection at the surface of the falcon tube. The first experimental series was performed by varying the DEAE-cellulose amount while keeping the other parameters constant. In the second experimental series, the influence of the shaking rate was studied in the range of 20 rpm to 140 rpm. Shaking was performed in a temperature-controlled shaking water bath with a manually adjusted shaking rate. The third experimental series was conducted to investigate the effect of varying the reaction time from 30 to 120 min. In the final experimental series, the influence of temperature was investigated in the range of 30–60 °C.
Solid/liquid separation was performed following each run. For the ICP analyses, the 10 mL filtered solution was introduced into the machine following an appropriate dilution. The reaction time and solution pH were kept constant at 30 min and 4.07, respectively. After the amount of DEAE-cellulose was optimized, it was kept constant at 10 mg for the kinetic study. Thus, the DEAE-cellulose/gold weight ratio (10/0.5) was chosen to be around 20.
The efficiency of this recovery process was calculated from the percentage of gold recovery using the following equation:
where Co is the initial gold concentration (50 ppm) and Ct is the concentration at the end of the experiment[1].

Other multiple-dose data (oral/rat) Lowest published toxic dose: 159 g/kg/90 days - Intermittent nutrition and overall metabolism: weight loss or weight gain decreased June 2017
Acute toxicity data (oral/rat) Lowest published toxic dose: 120 g/kg Gastrointestinal tract: hypermotility, diarrhea; Gastrointestinal tract: other changes June 2017
Acute toxicity data (skin/rabbit) Lethal dose (50% lethal): >2 g/kg June 2017
Other multiple-dose data (oral/rat) Lowest published toxic dose: 56000 mg/kg/8 weeks - Intermittent blood: thrombocytopenia; Blood: other changes; Blood: other cell count changes (not specified) June 2017
Acute toxicity data Inhalation/rat lethal concentration (50% lethal): >5800 mg/m3/4H June 2017

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LC50 (rat)> 5,800 mg/m3/4h

[1]. Gold recovery from dilute gold solutions using DEAE-cellulose. Hydrometallurgy. Volume 96, Issue 3, April 2009, Pages 253-257.

[2]. Separation, Purification, Structural Characterization, and In Vitro Hypoglycemic Activity of Polysaccharides from Panax notoginseng Leaves. Molecules. 2025 Feb 11;30(4):830.

[3]. Purification and Structural Identification of Polysaccharides from Bamboo Shoots (Dendrocalamus latiflorus). Int J Mol Sci. 2015 Jul 9;16(7):15560–15577.

[4]. Isolation, purification, and antioxidant activities of polysaccharides from Ziziphus Jujuba cv. Muzao. INTERNATIONAL JOURNAL OF FOOD PROPERTIES 2018, VOL. 21, NO. 1, 1–11.

[5]. Diethylaminoethyl cellulose (DEAE-C): applications in chromatography and organic synthesis. Arkivoc 2020, part i, 153-179.

[6]. Bioactivities, isolation and purification methods of polysaccharides from natural products: A review. Int J Biol Macromol. 2016 Jul 1;92:37–48.

Cellulose is a tasteless, white, powdery fiber. Density: 1.5 g/cm³. It is a biopolymer that constitutes the cell walls of plant tissues. Its preparation involves treating cotton with an organic solvent to remove wax and extracting with sodium hydroxide solution to remove pectic acid. It is the main fiber constituting the cell walls of plant tissues (wood, cotton, flax, grass, etc.). Its technical applications depend on the strength and flexibility of the fiber. It is insoluble in water. It can be dissolved in sulfuric acid through chemical degradation and is also soluble in concentrated zinc chloride solution. It is soluble in copper ammonium hydroxide (Cu(NH₃)₄(OH)₂) aqueous solution. DEAE-cellulose is a glycoside. DEAE-cellulose has been found in Aronia melanocarpa and Hyphaene thebaica, and relevant data have been reported. It is a polysaccharide whose glucose units are linked in a manner similar to cellobiose. It is a major component of plant fibers, with cotton being the purest natural form of this substance. As a raw material, it forms the basis for various products, including chromatography, ion exchange materials, explosives manufacturing, and pharmaceutical formulations. This study investigated a method for recovering gold from a synthetically prepared diluted gold-containing solution with a concentration of 50 ppm using DEAE-cellulose, a common biopolymer derivative. The effects of different recovery parameters on the gold recovery rate were investigated in detail. The results showed that the gold recovery rate increased with increasing adsorbent dosage and contact time. At room temperature, with shaking at 130 rpm for 30 minutes and using 20-40 g/L DEAE-cellulose, a gold recovery rate of 99% was achieved. On the other hand, when the reaction temperature was increased to 60 °C, even with a smaller amount of adsorbent (6 g/L), gold could be recovered from the solution with an efficiency of 99%. Shaking rate and temperature were shown to play a crucial role in the recovery process. The study also found that the DEAE-cellulose method for gold recovery is an intermediate-controlled process with an activation energy of 37.11 kJ/mol. XRD patterns and SEM images show that the recovered gold exists in a metallic state. [1] Natural cellulose has poor adsorption capacity and physical stability because the three hydroxyl groups are all attached to the same ring, which may cause steric hindrance. Moreover, due to the presence of crystalline regions in the polymer matrix, the hydroxyl groups do not easily participate in chemical reactions (O'O et al., 2008; Mark et al., 1967). By modifying it through chemical reactions such as etherification, esterification, halogenation and oxidation, the adsorption capacity and structural stability of natural cellulose for heavy metal ions can be improved (O'Connell et al., 2008). When cellulose beads are mainly treated with 2-(diethylamino)ethyl chloride hydrochloride solution and other steps are performed, DEAE-cellulose can be obtained (Ishimura et al., 1998). The molecular structure of DEAE-cellulose is shown in Figure 1b. DEAE-cellulose is one of the most commonly used resins in ion exchange chromatography. It contains ionizable tertiary amine groups and has a lower hydroxyl content than natural cellulose. The counterion of DEAE-cellulose is Cl⁻. This study used the weakly basic cellulose anion exchanger DEAE-cellulose (Matsubara et al., 2000) to recover gold from dilute gold chloride solutions. This study aimed to elucidate an efficient recovery process using DEAE-cellulose (a biopolymer derivative) as an adsorbent to efficiently recover gold from dilute gold-containing solutions, and to describe the optimal conditions and parameters of this recovery process. To this end, the effects of the following parameters on gold recovery efficiency were investigated: DEAE-cellulose dosage, reaction time, oscillation rate, and temperature. Furthermore, the activation energy of the recovery process was calculated through kinetic studies. This study optimized the extraction process of crude polysaccharide (PNLP) from Panax notoginseng leaves using an ultrasound-assisted dual-enzyme method combined with single-factor response surface methodology. Subsequently, the crude polysaccharide was purified and separated using DEAE-cellulose 52 column chromatography, and structural analysis, antioxidant activity evaluation, and digestive enzyme inhibitory activity detection were performed. The hypoglycemic effect of the purified fraction was further elucidated. Results showed that the optimized crude polysaccharide extraction rate was 17.13 ± 0.29%. The purified fraction PNLP-3 (eluted with 0.3 M NaCl) was obtained by DEAE-cellulose 52 column chromatography, with a total sugar content of 81.2% and a molecular weight of 16.57 kDa. PNLP mainly consists of arabinose, galactose, and galacturonic acid, with molar percentages of 20.24%, 33.54%, and 24.27%, respectively. PNLP-3 mainly consists of arabinose and galactose, with molar percentages of 29.97% and 49.35%, respectively. In this study, the IC50 values of PNLP-3 against α-glucosidase and α-amylase were 1.045 mg/mL and 9.53 mg/mL, respectively, indicating its hypoglycemic activity. Molecular docking results confirmed that PNLP-3 has stronger inhibitory activity against α-glucosidase. In addition, PNLP-3 alleviated hyperglycemia in insulin-resistant HepG2 cells by enhancing glucose consumption and glycogen synthesis. The antioxidant activity of PNLP-3 was positively correlated with its concentration, which may have played a hypoglycemic role by reducing oxidative stress. These findings highlight the potential application value of Panax notoginseng leaf polysaccharides in the treatment of type 2 diabetes and provide new ideas for regulating blood sugar using natural polysaccharides. [2] Three polysaccharides, BSP1A, BSP2A and BSP3B, were isolated from bamboo shoots (Dendrocalamus latiflorus). The separation methods included DEAE-cellulose-52 (ion exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A and BSP3B were determined by gel permeation chromatography (GPC) to be 10.2, 17.0 and 20.0 kDa, respectively. BSP1A was composed of arabinose, glucose and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contain arabinose, xylose, glucose, and galactose, respectively, with molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1. β-elimination reactions confirmed the presence of O-glycopeptide bonds in BSP1A, BSP2A, and BSP3B. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contain β-D-pyranose rings. However, BSP1A contains both β-D-pyranose and α-D-pyranose rings. Congo red staining results indicated that BSP1A and BSP2A possess a triple helix structure, while BSP3B does not. Nuclear magnetic resonance spectroscopy showed that BSP1A may be mainly linked by β-1,6-glucanpyran type, and a small amount of 1,6-glycosidic galactopyranose and arabinose bonds; BSP2A mainly showed →5)β-Ara(1→ and →3)β-Gal(1→) linkage. In addition, BSP3B mainly showed →3)β-Glu(1→ and →3)β-Gal(1→) linkage, and may also contain a small amount of other glycosidic bonds. [3]

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In this study, the crude polysaccharide of jujube (Ziziphus Jujuba cv. Muzao) was separated and purified by DEAE-cellulose-52 and Sephadex G-100 gel filtration chromatography. Four components were collected and named GZMP-1, GZMP-2, GZMP-3 and GZMP-4, respectively. The molecular weights of the four components were determined to be 111.2, 95.1, 84.2, and 571.4 kDa, respectively, using high-performance gel permeation chromatography (HPLC). Gas chromatography analysis of the monosaccharide composition confirmed that GZMP-1 consisted of rhamnose, arabinose, glucose, and galactose. Rhamnose, arabinose, and galactose were the main components of GZMP-2 and GZMP-3, while GZMP-4 consisted only of rhamnose and arabinose. Scanning electron microscopy showed that the surfaces of GZMP-1 and GZMP-4 were relatively smooth, while the surfaces of GZMP-2 and GZMP-3 had more wrinkles. Fourier transform infrared spectroscopy analysis indicated that GZMP-1 and GZMP mainly possessed α-glycosidic bonds. In vitro antioxidant activity of the polysaccharides showed that jujube polysaccharides possessed significant antioxidant activity, capable of scavenging DPPH and hydroxyl radicals in a concentration-dependent manner. These results indicate that jujube (Z. Jujuba cv. Muzao) polysaccharides have the potential as functional foods and carriers for developing natural antioxidant drugs. [4] This review aims to point out that… readers should note the applications of DEAE-cellulose/DEAE-C in organic reactions, which are not limited to the preparation of heterocyclic compounds but may extend to other types of organic compounds. DEAE-cellulose/DEAE-C is an ammonium salt commonly used in chromatographic applications and can therefore be considered a potential mild acid catalyst or proton donor, theoretically capable of catalyzing standard acid-catalyzed organic reactions. In addition, the resinous properties of DEAE-cellulose/DEAE-C may provide ideas for carrying out organic reactions in the solid phase. [5] Polysaccharides play a variety of roles in life processes, have a wide range of biological activities, and have great potential in the healthcare, food and cosmetic industries due to their therapeutic effects and relatively low toxicity. This article reviews the main functions of polysaccharides in antitumor, antiviral and anti-inflammatory biological activities. Due to their great structural heterogeneity, the methods for the isolation and purification of polysaccharides are quite different from those for other macromolecules (e.g.…). Similar to proteins, achieving homogeneity is the first step in studying the structure, pharmacology and structure-activity relationship of polysaccharides. Based on our laboratory's accumulated experience and published literature, this article also introduces methods widely used in polysaccharide isolation and purification. [6]

9013-34-7

16211032

Solid powder

1.1±0.1 g/cm3

519.1±50.0 °C at 760 mmHg

267.7±30.1 °C

0.0±3.1 mmHg at 25°C

1.518

0.68

DEAE-CELLULOSE; Cellulose DEAE; Diethylaminoethyl-cellulose; DEAE-Sephacel(R); Diethylaminoethyl-Sephacel(R); (6S)-2-(hydroxymethyl)-6-[(3S)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxane-3,4,5-triol; (5S)-6-(hydroxymethyl)-5-{[(2S)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxane-2,3,4-triol; ...; 9013-34-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

1. Operating Procedures

1.1 Column Packing

(1) Equilibrate all materials and reagents to the chromatography operating temperature. Prepare buffers and degas all buffer solutions.

(2) Weigh the required amount of media (packing material). Swell the media in purified water at room temperature for 4 hours, or in warm water for 1 hour (avoid water bath). After swelling, wash the media with 5 column volumes (CV) of purified water.

(3) Inspect all column components, ensuring the integrity of the frits, the tightness of O-rings and end plugs, and the cleanliness and integrity of the glass tube.

(4) Wet the bottom end-piece with water or buffer and maintain a small liquid head. Ensure no air bubbles are trapped at the bottom outlet.

(5) Pour the slurry into the column in one continuous motion along the inner wall using a glass rod as a guide, avoiding bubble formation. Open the column outlet to allow the gel to settle freely under gravity. Connect the top end-piece.

(6) Activate the peristaltic pump. Pass buffer through the column at a flow rate 1.33 times the operating flow rate to stabilize the bed (ensure pressure does not exceed the maximum pressure tolerance of the media).

1.2 Column Equilibration

Equilibrate the column with the equilibration buffer used for sample loading. The column is fully equilibrated when the effluent pH and conductivity match those of the starting buffer.

1.3 Sample Application

The salt concentration and pH of the sample should closely match those of the equilibration buffer. Excessive salt concentration or low pH may cause the sample to fail to bind. Perform buffer exchange via dialysis or desalting to transfer the sample into the starting equilibration buffer.

The most common procedure involves binding the target molecule to the ion exchange column while impurities flow through. However, in some cases, it is also feasible to bind impurities to the column while allowing the target molecule to flow through.

The ionic strength of the buffer should remain low to avoid interfering with sample binding. The recommended operating pH should be within 0.5 pH units of the buffer pKa and differ by at least one pH unit from the isoelectric point (pI) of the target molecule.

1.4 Protein Elution

For DEAE-cellulose media, elution is typically performed using either increasing salt concentration (ionic strength) or decreasing pH, applied via linear or step gradients.

1.5 Column Regeneration

Depending on sample properties, regenerate the column by washing with a high ionic strength buffer (e.g., 2 M NaCl) or by altering the buffer pH, followed by re-equilibration with the equilibration buffer. If the binding capacity changes, indicating the presence of substances such as denatured proteins or lipids not removed during regeneration, implement a Cleaning-in-Place (CIP) procedure.

1.6 Cleaning-in-Place (CIP)

If a significant change in binding capacity is observed after several runs, perform CIP. Clean the media in situ with 2-5 column volumes (CV) of 0.1 M NaOH solution to remove precipitated proteins, hydrophobically bound proteins, and lipoproteins. Immediately after CIP, rinse thoroughly with copious amounts of purified water until the effluent is neutral.

2. Storage

Unused Media: Store sealed at 4-25°C.

Used Media: Rinse thoroughly with purified water. Store in 20% ethanol at 4°C.

3. Precautions

(1) Prior to application, samples must be membrane-filtered and decolorized. Otherwise, impurities and pigments may be adsorbed onto the media, adversely affecting its performance. All buffers must be filtered through 0.45 µm membranes.

(2) Due to the relatively fine particle size of this media, ensure the column is equipped with appropriately sized frits to prevent media leakage.

(3) Avoid using high concentrations of strong acids or bases during operation. Acid and base concentrations should be kept below 0.15 M. Alkaline solutions may reduce flow rates.

(4) When selecting a column for ion exchange media, avoid long narrow columns as they increase operating pressure.

(5) Binding and elution methods vary significantly depending on the sample. Consult relevant literature for specific sample types.

 (Please use freshly prepared in vivo formulations for optimal results.)