**2. Ion exchange chromatograpy applications**

**Substance pKa Working pH** N-Methyl-piperazine 4.75 4.25-5.25 Piperazine 5.68 5.2-6.2 Bis-Tris 6.5 6.0-7.0 Bis-Tris propane 6.8 6.3-7.3 *Triethanolamine* 7.8 7.25-8.25 Tris 8.1 7.6-8.6 *N*-Methyl-diethanolamine 8.5 8.0-9.0 Diethanolamine 8.9 8.4-9.4 Ethanolamine 9.5 9.0-10.0 1,3-Diaminopropane 10.5 10.0-11.0

Conductivity detector is the most common and useful detector in ion exchange chromatogra‐ phy. However UV and other detectors can also be useful [10]. Conductivity detection gives excellent sensitivity when the conductance of the eluted solute ion is measured in an eluent of low background conductance. Therefore when conductivity detection is used dilute eluents should be preferred and in order for such eluents, to act as effective competing ions, the ion

Although recorders and integrators are used in some older systems, generally in modern ion exchange chromatography results are stored in computer. Retention time and peak areas are the most useful information. Retention times are used to confirm the identity of the unknown peak by comparison with a standard. In order to calculate analyte concentration peak areas

Direct detection of anions is possible, providing a detector is available that responds to some property of the sample ions. For example anions that absorb in the UV spectral region can be detected spectrophotometrically. In this case, an eluent anion is selected that does not absorb

or no absorbance in the UV spectral region can be detected spectrophotometrically by choosing a strongly absorbing eluent anion. An anion with benzene ring would be suitable [10]. Usually

detection method used. For conductivity the detection E should have either a significantly lower conductivity than the sample ions or be capable of being converted to a non-ionic form by a chemical suppression system. When a spectrophotometric detection is employed, E will often be chosen for its ability to absorb strongly in the UV or visible spectral region. The concentration of E- in the eluent will depend on the properties of the ion exchanger used and

. Anions with little

. The eluent anion must be compatible with the

are compared with the standards which is in known concentration [10].

UV. The eluent used in anion chromatography contains an eluent anion, E-

**Table 3.** Commonly used buffers for anion-exchange chromatography

exchange capacity of the column should be low [1].

will be the cation associated with E-

on the types of anions to be separated [10].

**1.5. Detection**

48 Column Chromatography

Na+

or H+

Ion exchange chromatography can be applied for the separation and purification of many charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc. from natural sources or synthetic origin. Examples in which ion exchange chromatography was used as a liquid chromatograpic technique for separation or purification of bioactive molecules from natural sources can be given as below.



solution) and the second step (with 500 mM NaCl solution) was further used for ion exchange chromatography to separate other egg white proteins. Separation proteins from 100 mM supernatant were allowed to pass through an anion exchange chromatographic column to separate different fractions. The unbound fractions were then passed through a cation exchange chromatographic column to separate further.

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injectionAfter sample injection flow-through fraction was collected using water as the eluent, followed by isocratic elution of the sample using 0.14 M NaCl. Finally the bound fraction was eluted using gradient elution (0.14-0.5 M) of NaCl- Anion

The unbound fraction was collected and used as starting material for cation exchange chromatography. The column was equilibrated with 10 mM citrate buffer, which was used as the starting buffer. After sample injection the column was eluted by isocratic elution using 0.14 M NaCl solution followed by gradient elution from 0.14 M to 0.50 M NaCl solution. The fractions were collected and freeze dried-Cation Exchange

The seeds were blended in a blender to extract the proteins followed by centrifugation (30,000g) at 4ºC. Then 450 g/l of ammonium sulphate were added to the supernatant

of the bound fraction was carried out by using 1 M NaCl in the equilibration buffer. All chromatographic steps were performed at the flow rate of 100 ml/h. Further separation selected fraction Q1, which was lyophilised and dissolved in 100 mM Tris– HCl (pH 7.6) buffer was performed onto a FPLC Superdex 75 column at a flow rate 0.5

reverse osmosis to increase the solids content from approximately 5.5% (w/w) to

pH and salt concentration were necessary to carry out the anion-exchange separation. A 0.01 *M* sodium acetate buffer, pH 5.8, was used for the starting state or feed loading buffer. After the whey feed was loaded onto the column, one column volume of this

to 70% saturation. The precipitate was removed by centrifugation and the supernatant was extensively dialysed against distiled water. The dialysed protein

extract was freeze dried and used for chromatographic separation.

Prep 16/10 column (SP Sepharose FF)-Cation Exchange Chromatography

**Stationary Phase:** High-Prep 16/10 column (Q Sepharose FF)-Anion Exchange Chromatography High-

**Extraction procedure:** Seeds were grounded and soaked in 20 mM Tris-HCl buffer (pH 7.6) at 4 ºC for 24 h.

**Eluent:** The column was equilibrated and initially eluted with 20 mM Tris–HCl (pH 7.6). Elution

**Extraction procedure:** After the cheese making process the sweet whey is produced, it is further processed by

**Eluent 1:** For the anion-exchange process; it was found that two step changes, simultaneous in

**Stationary phase:** Pharmacia's Q- and S-Sepharose anion- and cation-exchange resins

**Eluent:** The column was equilibrated with water and the pH was adjusted to 8.0 before

Exchange Chromatography.

Chromatography.

**Analyte(s):** Ovalbumin, ovotransferrin, lysozyme, ovomucin [23].

**Stationary Phase:** Q-Sepharose Column (3 cm x 7 cm), anion-exchange

ml min-1.

14.6% (w/w).

**Analyte(s):** A 5447 Da antifungal peptide [24].

**Detection:** UV detector, 280 nm

**Source:** Sweet dairy whey

**Detection:** MS Detector

**Source** : *Phaseolus vulgaris*

**Sample 6:**

**Sample 7:**


precipitation of the 11S fraction was adjusted to pH 4.8 with 2M HCl and centrifuged (16.250 x g for 20 min at 2-5 ºC). The supernatant was stored at low temperature and the precipitate was dissolved in Tris-HCl buffer (pH 8). The process was repeated to

binary gradient where mobile phase was a buffer solution at a certain pH (always pHs higher than the isoelectric pH of soybean proteins, p*I* = 4.8–6.4) and mobile phase B was the same buffer solution containing as well Msodium chloride. The buffer solutions tried were: phosphate ffer (pH 7 and 12), Tris–HCl buffer (pH 8), borate buffer (pH 9), and carbonate buffer (pH 10). In all cases, the buffer concentration was 20 mM. For every buffer, different gradients were tried. The best separation for ybean proteins was obtained with the borate buffer (pH 9) and gradient starting with an isocratic step at 0% B for 2.5 min and from 0 to 70% B in 14 min (gradient slope, 5%B/min). A fine optimization of the selected gradient enabled a reduction of the analysis time keeping the separation. The final gradient was: 0% for 2 min and from 0 to 50% B in 10 min.

soxhlet apparatus to remove low molecular weight compounds. Extraction procedures continue until no color could be observed in the ethanol. The residue was extracted with water at 50 ºC, 2 hour for two times. Obtained extract was filtered through gauze and Whatman GF/A glass fiber filter and then concentrated at 40 ºC in vacuum and dialysed at cut-off 3500 Da to give a 50 ºC crude extract. The extracts was kept at -18

fractions were obtained by elution of linear NaCl gradient (0-1.4 M) in water. The carbohydrate elution profile was determined using the phenol-sulphiric acid method. Finally two column volumes of a 2 M sodium chloride solution in water were eluted to obtain the most acidic polysaccharide fraction. The relevant fractions based on the

glucuronic acid), rhamnose, galactose, arabinose and glucose (in acidic fraction) [22].

obtained using isoelectric precipitation of egg white in the presence of 100 mM NaCl solution. The dispersion was kept overnight at 4 ºC and separated by centrifugation at 15.300 x g for 10 min at 4 ºC. The precipitate was further suspended in 500 mM NaCl

centrifugation at 15.300 x g for 10 min at 4 ºC, the precipitate was freeze dried and stored at -20 ºC. The supernatants obtained during the first step (with 100 mM NaCl

solution while stirring for 4 h followed by overnight settling at 4 ºC. After

obtain a purified precipitated fraction containing the 7S globulin.

**Stationary Phase:** Anion exchange perfusion column POROS HQ/10 packed with cross-linked

**Eluent:** The starting point for the separation of soybean proteins by HPIEC was the use of a

**Extraction procedure:** The powdered roots of *C. tinctorium* were extracted with ethanol (% 96, v/v) using a

**Eluent:** For obtaining neutral fraction the column was eluted with water firstly. The acidic

carbohydrate profile were collected, dialysed and lyophilized.

**Analyte(s):** Glucose, galactose, arabinose (in neutral fraction) Uronic acids (Both galacturonic and

**Extraction procedure:** Fresh eggs were collected and the same day extract was obtained. Ovomucin was

polystyrene-divinylbenzene beads.

**Detection:** UV detector at 254 nm

**Detection:** UV detector, 490 nm

**Source:** Hen egg

**Sample 4:**

50 Column Chromatography

**Sample 5:**

**Analyte(s):** 11S globulin or glycinin and 7S globulin [21].

ºC or lyophilized. **Stationary Phase:** Anion exchange-DEAE-Sepharose column

**Source:** *Cochlospermum tinctorium* A. Rich.


**Extraction:** Fruits of the plant extracted with hot water yielded a crude polysaccharide sample,

subfractions LRP1, LRP2, LRP3, LRP4, and LRP5.

**Extraction:** Stipe powder of *C. comatus* (100 g) was extracted three times with 1 L 95% ethanol

applied to a DEAE-Sepharose CL-6B column.

M sodium phosphate buffer (pH 6.0). **Detection:** UV Detector, 490 nm (phenol–H2SO4) and 500 nm (Folin–phenol)

**Eluent:** 0.2 M sodium phosphate buffer (pH 6.0), and linear gradient of 0.3–1.5 M NaCl in 0.2

**Analyte(s):** Polysaccharides; disaccharide α,α-trehalose, α-D-glucan, β-D-glucan, α-L-fuco-α-D-

**Extraction:** The dried and defatted fruit calyx extracted with different enzyme Neutral proteinase,

**Eluent:** The column was eluted first with distilled water, and then with gradient solutions (0.1

**Stationary Phase:** DEAE-cellulose column

**Detection:** UV Detector, 280 nm

**Source:** *Coprinus comatus*

**Sample 10:**

**Sample 11:**

**Eluent:** Distilled water, 0.05–0.50 mol/L NaHCO3 -

**Analyte(s):** Glycoconjugate polysaccharide (LRGP1) [27]

**Stationary Phase:** DEAE-Sepharose CL-6B column (3.5 cm × 30 cm).

galactan [28].

**Source:** *Physalisalkekengi* var. *francheti*

**Stationary Phase:** DEAE anion-exchange column

CLRP. The carbohydrate of CLRP was 66.2% and protein content was 7.3%. CLRP was a black Polysaccharide sample in which the pigment could not be removed by colum chromatography. To avoid the influence of pigment on the structure analysis, decoloration was performed with 30% H2O. After decoloration, the carbohydrate content of decolored CLRP was 93.2% and protein content was 4.4%. Decolored CLRP was purified by anion exchange chromatography, yielding five polysaccharide

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solution

under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled water for 2 h at 80 °C with intermediate centrifugation (2000 × *g*, 15 min). After concentrating the collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried at 40 °C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100 mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was

Papain and alkaline protease, respectively, in their suitable pH and temperature and then each extract was centrifuged at 5000 rpm for 10 min. The supernatant was concentrated and then precipitated by the addition of ethanol in 1:4 (v/v) at room temperature. The precipitate was dissolved in distilled water and the solution was then washed with sevag reagent (isoamyl alcohol and chloroform in 1:4 ratio), which were centrifuged at 5000 rpm for 15 min and the protein was removed. The supernatant was dialyzed against deionized water for 24 h before concentration under vacuum evaporator at 55 °C. The mixture was precipitated by the addition of ethanol in 1:4 (v/v) at room temperature and the precipitate was freeze dried. Total sugars were

determined by the phenol–sulfuric acid assay using glucose as standard.

M, 0.25 M, 0.5 M NaCl and 0.5 M NaOH), at a flow rate of 0.6 mL/min. The major polysaccharidefractions were collected with a fraction collector and concentrated using a rotary evaporator at 55 °C and residues were loaded onto a Sephadex G-200


buffer was passed through to wash out any material that did not bind to the resin, including the IgG. Next, two column volumes of 0.05 *M* sodium acetate, pH 5.0, were passed through the column to desorb those proteins whose p*I* values were above 5.0. This includes the β-lactoglobulin and bovine serum albumin. This was then followed by

two column volumes of 0.1 *M* sodium acetate, pH 4.0, to finally desorb the αlactalbumin whose p*I* range is 4.2–4.5, and thus above that of the passing pH wave of 4.0. After this second step change, the cleaning cycle was then implemented to

appropriate to carry out the cation-exchange separation. The buffer used was 0.05 *M* sodium acetate, pH 5.5, as the starting state or feed loading buffer. One column volume loading of the anion-exchange breakthrough curve fraction was optimum for loading onto the cation-exchange column. After the anion-exchange breakthrough curve fraction was loaded onto the column, one column volume of the initial buffer was passed through to wash out any material that did not bind to the resin. Next a step change in pH was implemented to elute the bound IgG. This was accomplished by passing two column volumes of the buffer, 0.05 *M* sodium acetate, pH 8.5. As the pH wave of this buffer passed through the cation bed it initiated the elution of the IgG because the upper value of its p*I* range is 8.3. After this pH step change the cleaning

NaCl, 20 mM diethyldithiocarbamic acid, 5% glycerol, and 2% polyvinylpyrrolidone. The buffer used was 3 ml g-1 of the fresh leaves. The homogenate was filtered through a layer of cheesecloth and stored at 20°C for 24 h. After thawing, it was centrifuged at 8000x*g*, for 40 min at 4°C. The supernatant was collected and ammonium sulfate was added to 70% saturation. The resulting precipitate was recovered by centrifugation at 8000x*g* for 40 min, redissolved in tris-buffered saline, TBS (50 mM Tris–HCl, pH 7.5 containing 0.3 M NaCl) and dialysed against the buffer overnight at 4°C. The solution was then centrifuged at 13,000x*g* for 15 min and the supernatant was collected and stored at -20°C. An aliquot of the dialysed ammonium sulfate fraction containing protein was applied to the affinity chromatography on the *N*-acetylgalactosamineagarose column. And then further separation was performed on Sephacryl S-200 column followed by anion exchange chromatography. Further purification was also

prepare the column for the next run.

cycle was then implemented.

**Detection:** UV Detector

**Sample 8:**

52 Column Chromatography

**Sample 9:**

[25].

**Source:** *Morus alba* (mulberry) leaves

**Detection:** UV Detector, 280 nm **Analyte(s):** Lectins, MLL 1 and MLL 2 [26]

**Source:** *Lycium ruthenicum* Murr.

**Eluent 2:** For the cation-exchange process, it was found that one step change in pH was

**Analyte(s):** α-lactalbumin, β-lactoglobulin, bovine serum albumin, immunoglobulin G and lactose

**Extraction procedure:** Fresh leaves were homogenized in ice-cold 50 mM Tris–HCl, pH 7.5, containing 0.3 M

performed by anion exchange and gel filtration chromatography

**Stationary Phase:** Anion-exchange chromatography, a DEAE-Sephacel column (2x9 cm)

stepwise with the buffer containing NaCl.

**Eluent:** Equilibrated with 20 mM Tris–HCl, pH 7.5 at flow rate 20 ml min-1 and then eluted


**Analyte:** Tannase [31]

**Source:** *Castanospermum australe* **Extraction:** 50% MeOH extract of seeds

**Eluent:** 0.5 M NH4OH, H2O **Detection:** UV Detection by HPTLC

**Stationary Phase:** (1) Amberlite IR-120B (500 mL H+ form), (2) Dowex 1-X2 column (3.8×90 cm, OH<sup>−</sup>form),

**Analyte(s):** Pyrolizidine alkaloids; fagomine; 6-epi-castanospermine; castanospermine; australine;

Since the isolation of pharmacologically active substances which are responsible for the activity became possible at the beginning of the 19th century drug discovery researches have increased dramatically [33]. Therefore within the last decade there has also been increasing interest in the liquid chromatographic processes because of the growing pharmaceutical industry and needs from the pharmaceutical and specialty chemical industries for highly specific and efficient separationmethods.Severaldifferenttypesofliquidchromatographytechniquesareutilizedfor isolation of bioactive molecules from different sources [25]. Ion exchange chromatography is probablythemostpowerfulandclassictypeofliquidchromatography.Itisstillwidelyusedtoday for the analysis and separation of molecules which are differently charged or ionizable such as proteins, enzymes, peptides, amino acids, nucleic acids, carbohydrates, polysaccharides, lectins byitselforincombinationwithotherchromatographictechniques[34].Additionallyionexchange chromatographycanbeappliedforseparationandpurificationoforganicmoleculesfromnatural sourceswhichareprotonatedbasessuchasalkaloids,ordeprotonatedacidssuchasfattyacidsor amino acid derivatives [35]. Ion exchange chromatography has many advantages. This method is widely applicable to the analysis of a large number of molecules with high capacity. The technique is easily transferred to the manufacturing scales with low cost. High levels of purifica‐ tion of the desired molecule can be achieved by ion exchange step. Follow-up of the nonsolvent extractable natural products can be realized by this technique [17,35]. Consequently ion ex‐ change chromatography, which has been used in the separation of ionic molecules for more than half a century is still used as an useful and popular method for isolation of natural products in modern drug discovery and it continue to expand with development of new technologies.

Ankara University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey

epi-australine-2-O--D-glucopyranoside and 1-epi-australine [32].

(3) Amberlite CG-50 column (3.8×90 cm, NH4 + form), (4) Dowex 1-X2 column (3.8×90

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cm, OH<sup>−</sup> form) (Repeated separation on different ion exchange columns).

3-epi-fagomine; 2,3-diepi-australine; 2,3,7-triepi-australine; 3-epi-australine; 2Rhydroxymethyl-3S-hydroxypyrrolidine; castanospermine-8-O--D-glucopyranoside; 1-

**Sample 14**

**3. Conclusion**

**Author details**

Özlem Bahadir Acikara

