CM Biopharm · Product Support

CM-HA Resin Protocol Guide

Hydroxyapatite chromatography resin, dry powder · Made in Korea

Version 1.2.1 Updated 2026-11-12 For Research Use Only

Application notes Lysozyme →

Overview

CM-HA Resin is a ceramic hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂, supplied as a dry powder for packing your own chromatography column. It binds proteins through two kinds of sites: calcium sites (C-sites), which hold acidic groups, and phosphate sites (P-sites), which act like a cation exchanger for basic groups. Bound proteins are usually eluted with a rising sodium phosphate gradient.

The type, particle size, lot number and dates for your bottle are printed on the bottle label.

The conditions below are starting points. Adjust pH, salt and the gradient for your own sample. We update this page as we collect more data; check the version number at the top.

Buffers

Choose the equilibration buffer by your target protein. Both protocols use the same elution buffer B. Quantities are for 500 g of finished buffer (about 500 mL for the dilute buffers), using anhydrous phosphate salts.

Buffer	Composition	Use	NaH₂PO₄	Na₂HPO₄	NaCl	CaCl₂·2H₂O
A-II	5 mM sodium phosphate, 150 mM NaCl, 0.5 mM CaCl₂, pH 6.8	General proteins, basic proteins (e.g. lysozyme)	0.153 g	0.174 g	4.383 g	0.037 g
A-IV	5 mM sodium phosphate, 75 mM NaCl, 0.4 mM CaCl₂, pH 6.7	Acidic proteins (e.g. BSA)	0.170 g	0.154 g	2.192 g	0.029 g
B	500 mM sodium phosphate, pH 6.8	Elution and strip	15.297 g	17.390 g	–	–
Cleaning	1 M NaOH	Cleaning in place	20.0 g NaOH, water to 500 g
Storage	0.1 M NaOH	Column storage	2.0 g NaOH, water to 500 g

How to prepare

Weigh the salts into a tared bottle. Add CaCl₂ last.
Add purified water to about 90 % of the final mass and dissolve.
Adjust pH at room temperature with 1 M NaOH or 1 M H₃PO₄, drop by drop. A 5 mM buffer moves quickly.
Bring to the final mass with water, record pH and conductivity, and filter through 0.22 µm.

If you use NaH₂PO₄·2H₂O instead of the anhydrous salt, multiply its mass by 1.300 and reduce the water by the same amount. Keep buffer B at room temperature: Na₂HPO₄ crystallizes in the cold.

Column packing

Weigh the dry resin you need. Suspend it in equilibration buffer A (about 3 volumes of buffer per volume of powder) and let it hydrate for 15–30 minutes with gentle swirling. Do not use a magnetic stirrer; it grinds the particles.
Let the slurry settle, then pour off the cloudy top layer to remove fines. Repeat 2–3 times until the top layer is clear.
Make a 40–50 % slurry in buffer A and pour it into the column in one continuous motion, down a glass rod or the column wall, to avoid trapping air.
Pack at a flow rate somewhat higher than you will use for runs, until the bed height stops changing. Lower the flow adapter onto the bed.

The packed bed volume per gram of dry powder will be added in a later version.

Run method

Example for a 5 mL column. Scale flow rate with column cross-section.

Step	Buffer	Volume	Notes
Equilibrate	A-II or A-IV	5–10 CV	Until pH and conductivity of the outflow match buffer A
Load	Sample in buffer A	–	Dissolve or exchange the sample into buffer A. Filter 0.22 µm
Wash	A	3–5 CV	Until UV returns to baseline
Elute, step	A + B mixed to 50, 150, 300 mM phosphate	5 CV each	Good for first scouting runs
Elute, linear	5 → 500 mM phosphate	10–20 CV	For acidic proteins with A-IV, a gradient to 120 mM is often enough
Strip	B (500 mM)	3–5 CV	Removes tightly bound material

Flow rate: start at 1 mL/min for a 5 mL column. Mixing ratio for a step: %B = (target mM − 5) ÷ (500 − 5) × 100. For example, 150 mM is 29.3 % B.

Cleaning & storage

After each run: strip with buffer B, then wash with 3–5 CV of water.
Cleaning: 1 M NaOH, 3–5 CV at low flow, then water until neutral.
Storage of a packed column: 0.1 M NaOH. Re-equilibrate with buffer A before the next run.
Dry powder: keep the bottle tightly closed at 4–30 °C, away from moisture.

Cautions

Keep pH at 6.5 or above. Hydroxyapatite dissolves in acid.
Avoid chelating agents such as EDTA and citrate; they strip calcium from the resin.
Do not let the bed run dry.
Wear gloves and eye protection when handling the powder and NaOH.

Application notes

Conditions and results for specific target molecules. We add notes as data become available.

Target	Type	Elution	Status
Lysozyme	Basic protein	50 mM phosphate step (with 150 mM NaCl)	Preliminary

FAQ

My protein does not bind. What should I check first?

Dissolve or exchange the sample into the same buffer A used for equilibration. Water or other buffers change the binding conditions.
For acidic proteins (for example BSA), use buffer A-IV, which contains Ca²⁺ and less NaCl. For basic proteins, use A-II.
Check that the column outflow matches buffer A in pH and conductivity before loading.
Lower the flow rate during loading. For a 5 mL column, start at 1 mL/min.

My protein comes off too early, or not at all.

Change the phosphate gradient. If it elutes in the wash or the first step, lower the starting NaCl or use a shallower gradient. If it stays bound, extend the gradient up to 500 mM phosphate (buffer B). Basic proteins usually need higher phosphate than acidic ones.

Which buffers and additives should I avoid?

Avoid chelating agents such as EDTA and citrate, and any buffer below pH 6.5; both damage hydroxyapatite. Sodium phosphate buffers as listed above are recommended.

How many times can I reuse a packed column?

It depends on your sample and how well the column is cleaned. Strip with buffer B and clean with 1 M NaOH after use, and store in 0.1 M NaOH. We will add measured reuse data in a later version of this page.

How much powder do I need for my column?

The packed bed volume per gram of dry powder will be published in a later version. Until then, pack a small test amount first and measure the settled bed height in your column.

How should I store the dry powder, and how long does it keep?

Keep the bottle tightly closed at 4–30 °C, away from moisture. Use it before the expiry date printed on the label (Exp.), which is set at 5 years from manufacture.

Where can I get a Certificate of Analysis?

Contact us

Questions about this resin, a lot, or your results are welcome. Please include the lot number printed on the label (for example HA2612-A01) so we can check that batch.

Emailinfo@cmbiopharm.com Write an email

Phone (KST, weekdays)+82-43-716-0373 Call

We collect common questions on this page. Your question may help other users, and we will add the answer to the FAQ without your name.

Version history

Version	Date	Changes
1.2.1	2026-11-12	Added shortcut buttons to application notes at the top.
1.2.0	2026-11-12	Added Application notes; first note: lysozyme.
1.1.0	2026-11-12	Added FAQ and Contact us.
1.0.0	2026-11-12	First release: buffers A-II, A-IV and B with masses, packing, run, cleaning and storage.