Nylon-6 Capillary Channeled Fibers Nylon-6 Capillary Channeled Fibers as a Stationary Phase for Ion-as a Stationary Phase for Ion-Exchange Chromatography Exchange Chromatography

Separations of Proteins. Separations of Proteins.

1 1 Department of Chemistry, The College Of New Rochelle, New Rochelle, NY 10805Department of Chemistry, The College Of New Rochelle, New Rochelle, NY 108052 2 Department of Chemistry, Clemson University, Clemson, SC 29634-0973Department of Chemistry, Clemson University, Clemson, SC 29634-0973

Michelle Permaul1,2, Christine Straut2, and R. Kenneth Marcus1

ChromatographyChromatography Is a method of separation like an extraction except one Is a method of separation like an extraction except one

phase is held in place while the other moves past it.phase is held in place while the other moves past it.

The mobile phase (the solvent that moves up the column) is The mobile phase (the solvent that moves up the column) is either a liquid or a gaseither a liquid or a gas

The stationary phase (the solvent that stays in place on the The stationary phase (the solvent that stays in place on the column) is usually a viscous liquid that is either bonded to column) is usually a viscous liquid that is either bonded to the capillary tube or onto the surface of the column.the capillary tube or onto the surface of the column.

There are different types of chromatography.There are different types of chromatography.

High Performance Liquid High Performance Liquid Chromatography (HPLC)Chromatography (HPLC)

A form of liquid chromatography that is A form of liquid chromatography that is used to separate compounds that are used to separate compounds that are dissolved in a solution.dissolved in a solution.

Uses high pressure to force the solvent Uses high pressure to force the solvent through closed columns containing very fine through closed columns containing very fine particles containing high resolution particles containing high resolution separations.separations.

Non-Porous Capillary-Channel Non-Porous Capillary-Channel Polymer (C-CPTM) FibersPolymer (C-CPTM) Fibers

Polymer fibers developed by Tennessee Eastman, Polymer fibers developed by Tennessee Eastman, Kingsport, TNKingsport, TN

35-100 35-100 µµm wide, 20 m wide, 20 µµm thick with 8 capillary channels m thick with 8 capillary channels running entire length of the fiberrunning entire length of the fiber

Extraordinarily high ‘wicking’ actionExtraordinarily high ‘wicking’ action

Comparatively high surface area/volume ratios to Comparatively high surface area/volume ratios to circular cross-section fibers circular cross-section fibers

Employed in textile and personal care applications to Employed in textile and personal care applications to transport liquids transport liquids

Scanning Electron Micrograph of Scanning Electron Micrograph of Polyester C-CPPolyester C-CPTMTM Fiber Fiber

Polymer Fibers as Stationary PhasesPolymer Fibers as Stationary Phases

Polypropylene (PP) Polyester (PET)

n

CH2CH

CH3

C

O

C NH

n

O

NH

Nylon

O C

O

C O

n

CH2CH2

O

Ion Exchange ChromatographyIon Exchange Chromatography

Stationary Phase

+

+ + + + +++

-

Cation-Exchange Anion-Exchange

Why Ion-Exchange Chromatography?Why Ion-Exchange Chromatography?

Ion-exchangers are popular in pharmaceutical Ion-exchangers are popular in pharmaceutical industryindustry

Allows for biological activity of proteins and Allows for biological activity of proteins and peptides to be maintained during the separation peptides to be maintained during the separation process process Due to the use of salts (i.e., buffering)Due to the use of salts (i.e., buffering)

Typical IEC PhasesTypical IEC Phases

Polymer beadsPolymer beads Cation-exchangeCation-exchange Anion-exchangeAnion-exchange

Mixed-mode interactionMixed-mode interaction Hydrophobic and IECHydrophobic and IEC

FiberSpool

Rotary Counter

Fiber Loop

Monofilament

Column

Marcus research group : 1)Nelson, D. K.; Marcus, R. K. Journal of Chromatographic Science 2003, 41, 475-479. 4) Marcus, R. K.; Davis, W. C.; Knippel, B. C.; LaMotte, L.; Hill, T. A.; Perahia, D.; Jenkins, J. D. Journal of Chromatography A 2003, 986, 17-31.

Nylon 6 FiberNylon 6 Fiber

H[NH(CH2)5CO]nOH

pH < 4

H3N+-Nylon-COOH H3N+-Nylon-COO- H2N-Nylon-COO-

pH~ 4.0-8.0 pH > 8

Separation of Proteins by Ion Exchange on Nylon-6

Weak cation-exchange separation on a 1.5 x 250 mm nylon-6 column (flow rate = 4 mL/min, H2O:1.0 M NaCl gradient (10 mM tris buffer, pH 8) = 100:0 to 0:100 in 5 min.

0.0 0.5 1.0 1.5 2.0-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035Holotransferrin

Ribonuclease A

Lysozyme

Time (min)

Abs

orb

an

ce

R. D. Stanelle, C.M. Straut, R.K. Marcus, J. of Chrom. Sci., accepted Jan. 2006

ExperimentalExperimental 250 x 2.1 ID stainless steel column250 x 2.1 ID stainless steel column

74 rotation, 3848 fibers, 25% interstitial fraction74 rotation, 3848 fibers, 25% interstitial fraction

5 protein suite with a range of pI’s from 4.9-11.05 protein suite with a range of pI’s from 4.9-11.0 Bovine Serum Albumin (4.9), Insulin (5.4), Myoglobin Bovine Serum Albumin (4.9), Insulin (5.4), Myoglobin

(6.7), Ribonuclease A (8.7), and Lysozyme (11.0)(6.7), Ribonuclease A (8.7), and Lysozyme (11.0)

Effect of pHEffect of pH pH 4-8pH 4-8 10mM K10mM K22POPO44 Gradient conditions: 0-100% 0.5M Gradient conditions: 0-100% 0.5M NaCl in 20 min at 1.0 mL/minNaCl in 20 min at 1.0 mL/min

Measured ParametersMeasured Parameters

Retention TimeRetention Time

SelectivitySelectivity

ResolutionResolution

Effect of pH on Retention of a Protein Effect of pH on Retention of a Protein SuiteSuite

0-3 min loading at 10mM K0-3 min loading at 10mM K22POPO44 Gradient conditions: 0-100% 0.5M NaCl in 20 min at 1.0 mL/min Gradient conditions: 0-100% 0.5M NaCl in 20 min at 1.0 mL/min

H3N+-Nylon-COO-

pH~ 4.0-8.0

11.6411.859.338.110.6011.00Lysozyme

5.746.256.336.500.608.70RibonucleaseA

0.600.550.710.610.627.0Myoglobin

0.598.110.480.480.635.40Insulin

0.590.590.610.620.614.90BSA

87654

pHpIProtein

11.6411.859.338.110.6011.00Lysozyme

5.746.256.336.500.608.70RibonucleaseA

0.600.550.710.610.627.0Myoglobin

0.598.110.480.480.635.40Insulin

0.590.590.610.620.614.90BSA

87654

pHpIProtein

Effect of pH on RetentionEffect of pH on Retention Unretained retention time: 47 minsUnretained retention time: 47 mins

Least retained proteins: acidic proteins, lowest pILeast retained proteins: acidic proteins, lowest pI

Rnase, Lys had the highest retention times except at pH 4Rnase, Lys had the highest retention times except at pH 4

Insulin did not follow the trend, negligibly retained except for Insulin did not follow the trend, negligibly retained except for pH 7.pH 7.

- Isopropyl alcohol experiments carried out as - Isopropyl alcohol experiments carried out as follow up and retention time decreased.follow up and retention time decreased. - Suggests hydrophobic activities- Suggests hydrophobic activities

Effect of pH on the retention of a five protein mixture of BSA (A), Insulin (B), Myo (C), RNase (D), and Lyso (E), Loading:0-5 min. 100% sodium phosphate at 1.0 mL/min. Gradient conditions: 0-100% 0.5M NaCl in 20 min at 1.0 mL/min

A,B,C

-0.001

0.004

0.009

0.014

0.019

0.024

0.029

0 5 10 15 20 25 30Time (min)

pH 4pH 5pH 6

pH 8pH 7

A,C

A,B,C

A,B,C,D

A,B,C,D,E

D

D

E

EB,D

E

E

AU@280 nm

Effect of pH on Retention

Effect of pH on Retention TimeEffect of pH on Retention Time

RT Increased with increasing pHRT Increased with increasing pH

Cationic properties of the nylon-6 fiber Cationic properties of the nylon-6 fiber increase with increasing pHincrease with increasing pH

Anionic properties decrease with increasing Anionic properties decrease with increasing pHpH

0

1

2

10

20

30

40

50

60

70

80

3 4 5 6 7 8 9

pH

Sel

ecti

vity

BSA/Myo

BSA/Insulin

Myo/RNase

Ribo/Lys

Selectivity of the Proteins

Effect of pH on SelectivityEffect of pH on Selectivity Generally increased as pH increasedGenerally increased as pH increased

Myoglobin did not follow the trendMyoglobin did not follow the trend - drop in selectivity at pH 6- drop in selectivity at pH 6 - caused by increase in retention of myo - caused by increase in retention of myo and decrease in retention of ribo.and decrease in retention of ribo.

Large increase for BSA/ Ins at pH 7Large increase for BSA/ Ins at pH 7 - caused by the increased retention of - caused by the increased retention of insulininsulin

Effect of pH on ResolutionEffect of pH on Resolution Significantly impacted by the variations in pHSignificantly impacted by the variations in pH

As pH increased, resolution increased, similar to As pH increased, resolution increased, similar to that of selectivitythat of selectivity

BSA/ Ins similarly increased resolution at pH 7 BSA/ Ins similarly increased resolution at pH 7 because of increased retention of ins.because of increased retention of ins.

BSA/ Myo also showed an increase in resolution BSA/ Myo also showed an increase in resolution at pH 6 because of the increased retention of myo.at pH 6 because of the increased retention of myo.

0

1

2

3

4

5

8

3 4 5 6 7 8 9pH

Resolution BSA/

Myo

BSA/Insulin

Myo/RNase

Ribo/Lys

Resolution of the different Proteins.

ConclusionConclusion Surface of Nylon-6 proves to be zwitterionicSurface of Nylon-6 proves to be zwitterionic - Selectivity for both acidic and ionic proteins - Selectivity for both acidic and ionic proteins - minimal retention of acidic proteins - minimal retention of acidic proteins suggest minimal effect of anionic interactionssuggest minimal effect of anionic interactions

BSA retained by anion-exchangeBSA retained by anion-exchange

Ins has mixed-mode retentionIns has mixed-mode retention IEC and hydrophobicIEC and hydrophobic

RNase and Lyso retained by cation-exchangeRNase and Lyso retained by cation-exchange

pH 6 provides best resolution and selectivitypH 6 provides best resolution and selectivity

Future DirectionsFuture Directions

Optimization of gradient conditions and Optimization of gradient conditions and flow rateflow rate

Investigation of other salt types at different Investigation of other salt types at different pH’spH’s

Investigation of selectivity of other proteins Investigation of selectivity of other proteins on nylon-6 fiberon nylon-6 fiber

AcknowledgementsAcknowledgements Christine StrautChristine Straut

Dr. R. Kenneth MarcusDr. R. Kenneth Marcus Marcus Research GroupMarcus Research Group

NSF- REU ProgramNSF- REU Program Mr. Richard CassettaMr. Richard Cassetta

Clemson University SURPClemson University SURP