Description of cation exchanger for liquid chromatography. HPLC media. Polymeric Stationary Phases. Non Leaching media of LC.

1. WEAK AND STRONG CATION
EXCHANGER’S DIFFERENT
BEHAVIOR.
COMPARISON OF STRONG CATION EXCHANGERS (SULFO PROPYL
AND SULFO ETHYL) WITH EACH OTHERS AND WITH WEAK CATION
EXCHANGER (CARBOXYL) IN THE SEPARATION OF 5 PROTEINS.
1

2. • The 5 proteins involved are
Myoglobin Ribonuclease A α-Chymotrypsinogen A
Cytochrome c
Lysozyme
2

3. 3
USING
• THE SAME SIZE COLUMNS,
• THE SAME BUFFERS,
• THE SAME pH’S,
• THE SAME LINEAR FLOW RATES,
• THE SAME DETECTION WAVELENGTHS AND
• THE SAME PROTEINS,
WE NOTICE CERTAIN REVERSAL OF THE PROTEIN ELUTION AS WELL
AS CERTAIN CHANGES OF THE ELUTION PROFILES.
PLEASE TAKE NOTE:

4. 4
Separation of
5 proteins on a
4.6 x 100 mm
STYROS™ SP/XH.
HPLC System. HP 1100 with thermostatted
column compartment
Columns STYROS™ SP/XH 4.6 x 100 mm
Mobile Phase A: 20 mM Phosphate, pH = 6.8
B: A + 1 M NaCl, pH = 6.8
Flow rate 1 ml/min (360 cm/hr)
Gradient 12 to 55 % B in 12 cv,
Temperature 30 C
Detection 214 nm
Injection volume 20 l
Pressure Drop 9 bar (131 psi)
Samples (1:3:3:3:3 mg/ml each) Myoglobin, -Chymotrypsinogen A,
Ribonuclease A, Cytochrome C from
equine, Lysozyme.
HPLC System. HP 1100 with thermostatted
column compartment
Columns STYROS™ SE/XH 4.6 x 100 mm
Mobile Phase A: 20 mM Phosphate, pH = 7
B: A + 1 M NaCl, pH = 7
Flow rate 1 ml/min (360 cm/hr)
Gradient 1 to 15 % B in 9 cv,
Temperature 30 C
Detection 214 nm
Injection volume 20 l
Pressure Drop 6 bar (87 psi)
Samples (2.5mg/ml each) Myoglobin, -Chymotrypsinogen A,
Ribonuclease A, Cytochrome C from
equine, Lysozyme.
Separation of
5 proteins on a
4.6 x 100 mm
STYROS™ SE/XH.

5. 5
THE CHARGE ON THESE SURFACES ARE BOTH SIMILAR
THAT IS A SULFONYL FUNCTION ( –SO3-) . HOWEVER
THESE CHARGES ARE THETHERED TO
• A PROPYL CHAIN, -CH2-CH2-CH2-SO3- IN THE CASE OF
STYROS™ SP AND TO
• AN ETHYL CHAIN, -CH2-CH2-SO3- IN THE CASE OF
STYROS™ SE.
THE SURFACES ARE BOTH CONSIDERED AS STRONG
CATION EXCHANGERS.

6. 6
THERE IS AN ELUTION REVERSAL OF RIBONUCLEASE A
AND α-CHYMOTRYPSINOGEN A AS WELL AS A CHANGE IN
THE ELUTION PROFILE OF CYTOCHROME C.
THIS WOULD CERTAINLY REQUIRE THE UNDERSTANDING
OF A MOLECULAR BIOLOGIST TO SHED SOME LIGHT TO
THE PHENOMENON AS WELL AS EXTEND SUCH
UNDERSTANDING TO THE BEHAVIOR OF PROTEINS
TOWARDS NATURAL CATIONIC ENTITIES WITHIN THE
LIVING SPECIES.

7. 7
COMPARED TO STYROS™ SE THE CHANGE IN THIS CASE IS LIMITED TO THE
ELUTION PROFILE OF α-CHYMOTRYPSINOGEN A ONLY.
HIGHER SALTS ARE ALSO REQUIRED FOR THE ELUTION.
HPLC System. HP 1100 with thermostatted
column compartment
Columns STYROS™ CM/XH 4.6 x 100 mm
Mobile Phase A: 20 mM Phosphate, pH = 6.8
B: A + 1 M NaCl, pH = 6.8
Flow rate 1 ml/min (360 cm/hr)
Gradient 3 to 35 % B in 9 cv,
Temperature 30 C
Detection 214 nm
Injection volume 20 l
Pressure Drop 6 bar (87 psi)
Samples (1;3;3;3;3 mg/ml each) Myoglobin, -Chymotrypsinogen A,
Ribonuclease A, Cytochrome C from
equine, Lysozyme.
Separation of
5 proteins on a
4.6x100mm
STYROS™ CM/XH
STYROS™ CM, HOWEVER AS A WEAK CATION EXCHANGER DOES
DISPLAY DIFFERENT BEHAVIOR AS WELL:Myoglobin
RibonucleaseA
α-ChymotrypsinogenA
Lysozyme
Cytochromec
mAU
0
500
1000
1500
2000
2500
0 2 4 6 8 10 12 14 min

8. 8
ADDITIONAL INFORMATION AS WELL AS A NUMBER OF
DETAILED APPLICATION NOTES CAN BE FOUND ON
OraChrom’s site at https://www.orachrom.com or
https://www.orachrom.net