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Chelating Resins

Chelating Sepharose FF and Chelating Captarose HF are metal chelate affinity chromatography media prepared by coupling iminodiacetic acid (IDA) to agarose.
Chelating Captarose HF is characterized by greater rigidity, higher pressure resistance, and faster flow rates compared to Chelating Sepharose FF.
Iminodiacetic acid (IDA) provides three coordination sites for chelating metal ions, which in turn offer three binding sites for the specific adsorption of target proteins.
Under identical ligand density and metal ion conditions, both Chelating Sepharose FF and Chelating Captarose HF exhibit stronger affinity interactions compared to IMAC (Immobilized Metal Affinity Chromatography) Sepharose FF.
Once charged with metal ions such as Cu(2+), Zn(2+), Ni(2+), Co(2+), etc., these media can be used for the purification and separation of recombinant and natural proteins.

Product Type Prepacked columns, Resins

Pack Size 1mL 5mL 10mL 25mL 100mL 500mL 1L 5L 10L

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DetailOrder InformationDocuments

Product	Chelating Singarose FF	Chelating Captarose HF
Features	Stronger affinity
Application	Purification of histidine-tagged proteins
Matrix	6% highly cross-linked agarose	High-rigidity agarose
Particle size	90 μm	75 μm
Functional group	Iminodiacetic Acid
Copper capacity	30-41 μmol Cu²⁺/mL resin
Binding capacity	/
Flow velocity	＜250 cm/h	＜500 cm/h
Max. pressure	＜0.3 MPa	＜0.5 MPa
pH stability	3-12(operational), 2-14(CIP, short term, when removing metal ions)
Chemical stability	Stable in commonly used buffer solutions: 1.0M NaOH, 20% ethanol, 6M guanidine hydrochloride, 8M urea；Avoid using chelating agents: EDTA, EGTA, citrates, etc.; avoid using reducing agents: β-mercaptoethanol, DTT, etc.
Storage conditions	20% ethanol; 4-30℃

Pack size	Cat. No.
Pack size	Chelating Singarose FF	Chelating Captarose HF
1ml XTrap Prepacked columns	HC1050111	HC1050311
5ml XTrap Prepacked columns	HC1050112	HC1050312
5ml XScreen Prepacked columns	HC1050113	HC1050313
10ml	HC1050100	HC1050300
25ml	HC1050101	HC1050301
100ml	HC1050102	HC1050302
500ml	HC1050103	HC1050303
1L	HC1050104	HC1050304
5L	HC1050105	HC1050305
10L	HC1050106	HC1050306

Operating Instructions

1. Column Packing

1.1 Packing Buffer Preparation

Purified water; degas byultrasonication for 15 min.

1.2 Resin Preparation

Calculate the required amount ofresin using a compression factor of approximately 1.15, and weigh out theresin. Exchange the storage solution for packing buffer using a vacuumfiltration flask. Add sufficient packing buffer to prepare a resin slurry ofapproximately 50%.

1.3 Column Preparation

Inspect the chromatography column toensure that all components are intact and clean. Install the bottom adaptor andtighten the O-ring. Secure the column vertically on a laboratory stand; checkand adjust its alignment with a spirit level. Fill a syringe with packingbuffer, connect it to the bottom outlet, and slowly expel the buffer to removeair bubbles from the bottom screen. Remove the syringe and install the bottomplug. Add packing buffer to a height of approximately 2 cm in the column.

1.4 Column Packing (example: 16 mm column diameter, 10 cm bedheight)

Mix the resin slurry thoroughly and pour it slowly into the columnalong a glass rod. If the liquid level remains below the top of the column, addpacking buffer to fill the column. Connect the adaptor to the chromatographysystem, start the pump, and remove air from the tubing and top screen at anappropriate flow rate; then pause the system. Insert the adaptor into thecolumn at a 45° angle, secure it, and tighten the sealing ring while preventingair from entering. Remove the bottom plug and direct the bottom tubing into awaste container. Pack at 60 cm/h until the resin-bed interface stabilizes.Increase the linear velocity to 600 cm/h, then gradually reduce it to 300 cm/hand maintain the final rate for 45 min. Mark the resin-bed interface and stopthe system. Reinstall the bottom plug and disconnect the top of the column fromthe pump. Loosen the adaptor sealing ring slightly, lower the adaptor to 3 mmbelow the resin surface, and retighten the seal. Connect the top and bottomcolumn tubing to the chromatography system and perform a column-efficiencytest.

2. Column Efficiency Test

Test column efficiency immediatelyafter packing. Column performance is typically assessed using the number oftheoretical plates per meter (N/m) and the peak asymmetry factor (As). Highercolumn efficiency indicates greater separation capability. For a more accurateassessment, use a test-sample volume of 1.0% of the column volume and a linearvelocity of 15-30 cm/h. Inject the sample as close to the column inlet aspossible to minimize dilution.

Table 2. Column EfficiencyTest Methods

Item	Contents
Test Method	Acetone Method	NaCl Method
Sample	1.0% (v/v) acetone in water	0.8 M NaCl solution
Sample Volume	1.0%CV*	1.0%CV*
Mobile Phase	Water	0.4 M NaCl solution
Flow Rate	30 cm/h	30 cm/h
Detection	UV 280	Conductivity

Note*: CV (Column Volume) refers tocolumn volume and is abbreviated as CV below.

Column Efficiency Calculation

Calculate N/m and As from the UVcurve (or conductivity curve) using the following equations:

N/m = N/L

N = 5.54(VR/Wh)²

As=b/a

Note: In the equations, VR is theretention volume; Wh is the peak width at half height; L is the bed height inmeters; and N is the number of theoretical plates. VR and Wh must use the sameunits. In Figure 1, a is the leading half-width of the peak at 10% peak height,and b is the trailing half-width at 10% peak height.

Figure 1. SchematicDiagram for Column Efficiency Calculation

Column efficiency test chromatogram showing retention volume and peak widths

The peak should be symmetrical, withan asymmetry factor (As) as close to 1.0 as possible; values between 0.8 and1.8 are generally acceptable. N/m > 3,000.

3. Separation andPurification

Charge Chelating Singarose FF withmetal ions before separation and purification. For detailed metal-charginginstructions, see Section 4, Regeneration.

Metal-ionselection: Cu²⁺, Zn²⁺, and Ni²⁺ are commonlyused. Cu²⁺ and Zn²⁺ are used most often to purifyuntagged proteins. Cu²⁺ binds strongly to certain proteins, and someproteins bind only to Cu²⁺. Zn²⁺ binds more weakly tosome proteins and binds only a limited range, making it useful for purificationof specific proteins. Ni²⁺ is commonly used to purify His-taggedproteins. Co²⁺, Fe³⁺, and Ca²⁺ are additionaloptions.

ForChelating Singarose FF, phosphate buffer at neutral to mildly alkaline pH (pH7.0-8.0) is preferred. Avoid EDTA, citrate, and similar reagents. Tris-HCl mayalso be used, but should be avoided when metal-protein affinity is very weak.Tables 3 and 4 list common additives and concentrations.

Toreduce nonspecific binding of host-cell proteins, a low concentration ofimidazole (20-40 mM) is typically added to the binding buffer and sample. Thebuffer must contain 0.15-0.5 M NaCl to suppress ionic interactions.

Table 3. Additives that do not interfere with protein binding toChelating Singarose FF

Additive	Typical Concentration	Additive	Typical Concentration
Phosphate, Tris, borate, and HEPES	20-100 mM	Nonionic detergent	≤2%
NaCl	≤2 M	Triton X-100	≤2%
KCl	≤1 M	Tween-20	≤2%
guanidine hydrochloride	≤6 M	Octyl glucoside	≤2%
urea	≤8 M	Dodecyl maltoside	≤2%
Glycerol	≤50%	C12E8、C10E6	≤2%
isopropanol	≤60%	PMSF (protease inhibitor)	1 mM
ethanol	≤30%	Pepsin inhibitor[TR1]	1 μM
1% benzamidine (protease inhibitor)	≤1 mM	Leupeptin (protease inhibitor)	0.5 μg/mL

Table 4. Additives that may disrupt protein binding to ChelatingSingarose FF (avoid whenever possible)

Additive	Typical Concentration	Additive	Typical Concentration
β-mercaptoethanol	—	Citrate	Low concentrations tolerated
reduced glutathione	—	Histidine	Can replace imidazole
Strong reducing agents (DTT, TCEP, DTE)*	—	glycine	—
Metal chelators (EDTA, EGTA)**	—	glutamine	—
Ionic detergents (cholate, SDS)	—	Arginine	—
Sodium azide	3 mM	NH₄Cl	—

Note*:Avoid strong reducing agents whenever possible to prevent reduction of themetal ions and consequent impairment of protein binding. The maximum toleratedconcentration depends on the sample. Reducing agents may be present only in thesample, not in the buffer.

Note**:Avoid metal-ion chelators whenever possible to prevent metal ions from beingstripped from the resin. The maximum tolerated concentration depends on thesample. Chelators may be present only in the sample, not in the buffer.

3.1 Column Equilibration

Connectthe chromatography column to the purification system. Equilibrate the columnwith at least 5 CV of binding buffer until the pH and conductivity of thecolumn effluent match those of the binding buffer. After equilibration, zerothe UV detector.

Note:To minimize the effect of metal-ion leakage on chromatography performance, washthe column before equilibration with 1 CV of 0.5 M imidazole containing 1 MNaCl, followed by 5 CV of purified water. Equilibrate the column with bindingbuffer before sample loading.

Note:When purifying inclusion bodies, add 8 M urea or 6 M guanidine hydrochloride toboth the binding buffer and the sample.

3.2 Sample Loading

Loadthe sample, filtered through a 0.2 or 0.45 μm membrane, onto the chromatographycolumn. The sample volume depends on the resin binding capacity, theconcentration of resin-binding molecules in the sample, and the otherchromatography conditions.

3.3 Column Washing

Washthe column with 5-10 CV of binding buffer to remove impurities that do not bindunder these conditions, until the UV signal returns close to baseline.Alternatively, use the impurity-washing conditions established duringpreliminary experiments.

3.4 Elution

Flushthe column with elution buffer and begin collecting eluate when the target UVpeak begins to rise.

Note:Select competitive elution, elution by lowering the pH, or chelator-mediatedelution as appropriate for the application.

Competitive elution: Increase the concentration of a substance withaffinity for the metal ions (e.g., Ni²⁺), such as 0-2 M NH4Cl, 0-0.5M imidazole, or 0-0.5 M histidine, using a linear or stepwise gradient.Gradient elution at the constant pH of the binding buffer is recommended.[TR2]

Elutionby lowering pH: As pH decreases, weakly bound proteins elute before stronglybound proteins. Below pH 4.0, metal ions such as Ni²⁺ dissociatefrom the resin, allowing elution. If the target protein is sensitive to low pH,neutralize the collected eluate with 1 M Tris-HCl at pH 9.0, using one-tenth ofthe collected volume.

Chelatorelution: A 50 mM solution of EGTA or EDTA can strip metal ions such as Ni²⁺from the resin and elute the bound material. This approach can also elutedenatured or precipitated proteins and is generally not recommended. Residualmetal ions in the eluate can be removed using Huachun Sindex G25 (M), catalognumber HC30802. Regenerate the resin before reuse.

3.5 Cleaning and Storage

Washwith 3-5 CV of 0.5 M imidazole containing 1 M NaCl, followed by purified wateruntil the conductivity of the column effluent reaches 0. Then wash with 2-3 CVof 20% ethanol. Store the column at 4-30°C.

Note:Resin reusability depends on the sample. Use a separate resin preparation foreach sample type to prevent cross-contamination.

4. Regeneration

Residualimpurities and metal-ion leakage impair chromatography performance and bindingcapacity. Regenerate the resin and recharge it with metal ions after 1-5 cyclesas recommended. Follow the regeneration procedure below:

4.1Wash the chromatography column with 10 CV of purified water.

4.2Wash with 3-5 CV of metal-stripping buffer: 20 mM phosphate buffer, 0.5 M NaCl,50 mM EDTA, pH 7.4.

4.3Wash the column with 3-5 CV of binding buffer without imidazole.

4.4Wash the column with 5 CV of purified water.

4.5Charge the resin with at least 0.5 CV of 0.1 M metal-ion solution, such asNiSO4, at 15 cm/h.

4.6Wash the column with 3 CV of 0.5 M sodium acetate-acetic acid buffer, pH 4.0.

4.7Equilibrate the column with 5 CV of binding buffer and use immediately.

4.8Wash the column with 5 CV of 20% ethanol before storage.

Note:Use a metal-ion concentration of 0.1 M. Maintain the solution at neutral ormildly acidic pH and filter through a 0.2 μm membrane before use.

Note:Avoid phosphate buffers when charging with Ca²⁺, as precipitateswill form.

Note:Because Fe³⁺ readily forms insoluble species in neutral solutions,remove Fe³⁺ by incubating the resin overnight with 50 mM EDTA. Whencharging the resin with Fe³⁺, perform chelation at a low pH(approximately pH 3.0) to prevent Fe³⁺ precipitation.

5. Cleaning in Place

Cleaning in place (CIP) removesstrongly bound, precipitated, or denatured material from the resin. Residualimpurities can impair column performance; substantial accumulation can clog thecolumn, increase backpressure, and reduce flow rate. Regular CIP preventscontaminants from accumulating in the bed and helps maintain resin bindingcapacity and flow properties.

Clean the column after approximately3-5 cycles to restore resin performance. Recommended cleaning conditions fordifferent impurities and contaminants are provided below:

5.1 To remove proteins bound byionic interactions, wash the column with 2-3 CV of 2 M NaCl, followed bybinding buffer.

5.2 To remove precipitated proteinsand hydrophobically bound material, first strip the metal ions, then soak thecolumn in 0.2-1 M NaOH for at least 1 h.

5.3 To remove strongly boundhydrophobic proteins, lipids, and similar material, wash the column with 3-5 CVof 70% ethanol or 30% isopropanol for 15-20 min, then equilibrate with bindingbuffer.

Alternatively, soaking overnight in6 M guanidine hydrochloride can remove most contaminating proteins. If thecolumn will be used immediately after cleaning, wash with 3-10 CV of bindingbuffer.

6. Sanitization

Sanitization minimizes microbialcontamination of the chromatography column. Because 20% ethanol is neitherbactericidal nor depyrogenating, treat Chelating Singarose FF ChromatographyResin with 70% ethanol for 12 h, or soak it in 0.5-1 M NaOH for 0.5-1 h.

7. Storage

Store unused resin at 4-30°C in adry, well-ventilated, clean area. Keep the container tightly closed. Do notfreeze.

Store columns packed with ChelatingSingarose FF sealed and immersed in 20% ethanol or 2% benzyl alcohol to preventmicrobial contamination.

Replace the storage solution withfresh solution every 3 months to prevent microbial growth resulting fromethanol evaporation.

Note: Before using stored resin,perform CIP and equilibrate with at least 5 CV of binding buffer.

8. Linear Scale-up

A purification process optimized atlaboratory scale can be scaled linearly to pilot or production scale. Considerthe following during scale-up:

Keep the residence time constant tomaintain stable dynamic binding capacity.

Select the column volume based onthe required binding capacity. If the bed height changes, assess the effect onthe purification step.

Determine the column diameter fromthe required flow rate and the bed height from the established residence time.A bed height of 10-25 cm is generally recommended.

Ensure uniform sample concentrationand consistent elution conditions.

Troubleshooting

Table 3. TroubleshootingRecommendations

Issue	Possible Cause and Recommended Solution
Excessive pressure during use	Replace the inline filter.
Excessive pressure during use	The chromatography column is clogged; perform CIP cleaning.
Unstable pressure curve	Remove air bubbles from the sample loading pump.
Unstable pressure curve	Use a bubble trap to remove air bubbles from the sample.
Protein does not bind or binding capacity is low	Sonication denatures the protein: use milder sonication conditions during lysis.
	Protein binding is weak: select a resin with higher binding capacity.
	The tag is buried within the protein: optimize expression conditions or redesign the sequence.
	Protein aggregates during loading: add a nonionic detergent to the sample and buffer.
	Equilibration pH is unsuitable: adjust buffer pH to 6.5-8.0.
	The resin is no longer effective: replace it with fresh resin.
	Sample load exceeds capacity: reduce the sample load.
	Protein or lipid accumulation impairs binding: clean promptly or replace the resin.
	Loading flow rate is too high: reduce the loading flow rate.
Poor elution performance	No product is collected, or binding is low: confirm that the tagged protein binds to the resin.
	Elution flow rate is too high: reduce it.
	Elution-buffer volume is insufficient: increase it.
	Imidazole concentration is too low: try increasing it.
	Elution contact time is insufficient: reduce the flow rate to increase contact between the elution buffer and resin.
	Impurity-washing conditions are unsuitable: optimize imidazole and NaCl concentrations to improve purity and recovery.
	Impurities have high affinity for the metal ions: combine this step with another resin type.
	Elution buffer is ineffective: prepare fresh elution buffer.
	Nonspecific ionic binding: add 0.1-0.2 M NaCl to the elution buffer.
	Nonspecific hydrophobic binding: add 0.1% Triton X-100 to the elution buffer.
	Aggregation or precipitation occurs during elution: establish protein solubility and stability under the elution conditions.
Resin discoloration	The sample contains pigments or colored proteins: perform CIP or regeneration strictly after purification.
Resin discoloration	Reducing agents in the sample or buffer turn the resin brown: avoid or reduce their use.
Elution peak gradually broadens	The chromatography column may be contaminated. Optimize the elution conditions and perform CIP as specified.
Increased CIP peak	CIP strength is insufficient; optimize the CIP conditions.
Ligand leakage may be higher during initial use.	Before initial use, run one blank cycle and perform CIP.