For the HPLC separation of proteins and enzymes, usually reversed phase chromatography, gel permeation chromatography or ion-exchange chromatography are used. Because ion-exchange chromatography can achieve separation with a mobile phase of organic solvents, it is the preferred procedure. However for the separation of some enzymes which may lose their activity by contact with metals like, stainless steel or titanium columns, glass packed columns may be more appropriate. The COSMOGEL glass column consists of a hydrophilic polymer packed in a glass column, for separations requiring non-metallic contact. Furthermore, the high-pressure resistance of the glass makes it possible to use a 10mm polymer particle as the packing material. This way the COSMOGEL glass column can provide equivalently high-resolution to stainless steel columns. For ion exchange chromatography solvents containing halogens such as Cl are often used. In such cases, stainless steel columns react with the mobile phase, while glass columns are completely resistant to salts and other corrosive mobile phases.

	DEAE	QA	CM	SP
Stationari phase	totally porous spherical hydrophilic polymer
Average particle size	5 µm
Average pore size	approx. 1000Å

Gel type and characteristics

Product name	type	Functional group	Counter ion	Capacity (meq/g)	pI
COSMOGEL DEAE	Diethylaminoethyl	N⁺H(C₂H₅)₂	Cl^-	0.6	10.8
COSMOGEL QA	Quarternary ammonium	N⁺(CH₃)₃	Cl^-	0.4	11.0
COSMOGEL CM	Carboxymethyl	COO^-	Na⁺	0.3	5.7
COSMOGEL SP	Sulfopropyl	SO₃^-	Na⁺	0.4	2.6

Product name	Column size	pH range	Flow rate(ml/min.)		Max pressure(MPa)	Temp.(°C)
			normal	max.
COSMOGEL DEAE GLASS	8mmI.D.×75mm	<11	0.5-1.0	1.5	1.5	10-50
COSMOGEL QA GLASS		1-14
COSMOGEL CM GLASS		>4
COSMOGEL SP GLASS		1-14

Selection of the mobile phase

Generally, a mobile phase within the pH range of +/-1 pH unit away from the isoelectric point (pI) of the sample is used. For anion exchange columns mobile phase with 1 pH above the isoelectric point of the sample, and for cation exchange columns mobile phase with 1 pH below that is used. When Bis-tris HCI salt of a bivalent ion and Tris HCI salt of a univalent ion are in the same concentration, the elution force of the former is greater due to its ionic strength.

Anion exchange (DEAE, QA)		Cation exchange (CM, SP)
Buffer	pH	Buffer
	4.0	Formic Acid
Piperazine HCl	5.0	Acetic Acid
Bis-Tris HCl	6.5	Phosphoric Acid
Tris HCl	8.0	HEPES Na
Monoethanolamine HCl	9.5

The initial mobile Phase (A) is 20-50mM of one of the above mentioned buffer solutions and the final mobile phase (B) is the mobile phase (A) with an addition of 20-600mM of salt.

Selection of salts

In general, a salt containing the same ion as the ion of mobile phases buffer solution and the counter ion of stationary phase is used. NaCl is suitable for any column, however when the elution force is weak, CaCl₂, MgCl₂ for DEAE and QA columns and Na₂SO₄ for CM and SP columns can be used.
Since the ionic strength of these salts is greater than that of HCI with the same concentration, the retention is consequently greater.

Selection of organic solvents

When the retention is not strong enough with only a chromatography buffer solution and salt, the addition of some water-soluble organic solvents might improve the retention. With COSMOGEL Ion Exchange Column up to 20% of acetonitrile or 2-propanol or similar of water soluble organic solvent can be used.

Isoelectric point and elution time of proteins

Generally, in cation exchange chromatography, retention increases as the isoelectric point increases. The opposite is true in anion exchange chromatography.

Separation of proteins on anion exchange column

With an anion exchange column having a positive charge, with higher negative charge of the sample the retention becomes longer. When using a weak alkaline mobile phase with lower isoelectric point the elution becomes longer.

Separation of proteins on cation exchange column

With a cation exchange column having a positive charge, with higher positive charge of the sample the retention is increasing. When using a weak acid mobile phase with higher isoelectric point the retention time becomes.

Difference in the separation between anion and cation exchange column

Lipoxytase is an acidic protein with an isoelectric point of 5. Lipoxytase can be purified by anion exchange columns because its negative charge in mobile phase at pH 8.2. Lypoxitase can be purified by cation exchange columns because in lower pH (e.g. pH 4.5) than its pI (pI 5) it is charged positively Efficient separation of crude lipoxytases is achieved by using both anion and cation exchange column to separate impurities.
As an example, the 50% dissociation pH of the functional group of the cation exchange column COSMOGEL SP is 2.6, which has sufficient exchange capacity in a 4.5 pH mobile phase. However the 50% dissociation pH of the same cation exchange column COSMOGEL CM is 5.7, sufficient exchange capacity is not available in a 4.5 pH mobile phase and the curve has broad peaks.

Recovery of protein using an ion-exchange column

The-COSMOGEL ion Exchange Packing material is basically composed of a hydrophilic polymer. Since there is no non-specific absorption occurring, a high recovery of proteins can be expected.

Injection volume and resolution

For ion-exchange gradient separations, even when a large volume of diluted sample is injected; sufficient separation can be obtained because the sample is concentrated at the head of the column. The injection of a large volume of diluted sample has no effect on the chromatographic separation.

Sample loadability and resolution

An 8mmI.D. column can resolve a sample up to 1mg in size without any deterioration of separation characteristics. If the resolution factor is greater than the example below, a sample load of 10mg might be achieved.

Preparation of proteins

The COSMOGEL 20mm I.D.×100mm for mass preparation can separate a large sample volume up to 10mg without deterioration of the separation characteristics. If the resolution factor is high enough a sample load of 100mg may be permissible.

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Ordering information

GLASS PACKED COLUMN

Product name	Size	Code No.	End-fitting
COSMOGEL DEAE Glass Packed Column	8mmI.D.×75mm	37845-81	1/4-28UNF
COSMOGEL QA Glass Packed Column		37846-71
COSMOGEL CM Glass Packed Column		37844-91
COSMOGEL SP Glass Packed Column		37847-61

STAINLESS PACKED COLUMN

Product name	Size	Code No.	End-fitting
COSMOGEL DEAE Packed Column	7.5mmI.D.×75mm	43371-91	Waters type
COSMOGEL QA Packed Column		43373-71
COSMOGEL CM Packed Column		43375-51
COSMOGEL SP Packed Column		43377-31

GLASS GUARD COLUMN KIT

Product name	Size	Code No.	End-fitting
COSMOGEL 30DEAE Glass Guard Column Kit	Kit ontents: empty column, 10ml of packing material, nut, ferrule, tube	37854-61	1/4-28UNF
COSMOGEL 30QA Glass Guard Column Kit		37855-51
COSMOGEL 30CM Glass Guard Column Kit		37856-41
COSMOGEL 30SP Glass Guard Column Kit		37857-31

STAINLESS GUARD COLUMN KIT

Product name	Size	Code No.	End-fitting
COSMOGEL 30DEAE Guard Column Kit	Kit contents: empty column, 10ml of packing material, connecting tube	37928-01	Waters type
COSMOGEL 30QA Guard Column Kit		37926-21
COSMOGEL 30CM Guard Column Kit		37924-41
COSMOGEL 30SP Guard Column Kit		37922-61