zenix sec-80 - obrnutafaza.hr · 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min absorbance at 214 nm 50mm...

www.sepax-tech.com Toll Free: 1-877-SEPAX-US 1

Having trouble applying size exclusion

chromatography to peptides under

10,000 Da? What if there was a size

exclusion column specifically

designed for small protein and

peptide separations?

Sepax Zenix™ SEC-80

provides a valuable solution

Separation parameters for peptide mixture using 75%

acetonitrile with 0.1% TFA in water

Peak Protein MW (Da)

Retention time (min)

Resolution Plate

counts

1 Insulin (porcine) 5778 7.75

16711

2 Glucagon 3483 8.03 1.07 12132

3 Angiotensin I 1297 8.46 1.58 19741

4 Bradykinin 1060 8.86 1.65 21060

NOTES Zenix™ SEC-80

Effect of Acetonitrile Mobile Phase Concentration

Column: Zenix™ SEC-80 (3 Pm, 7.8x300 mm) Mobile phase: 0.1% TFA with the indicated percentage of acetonitrile Flow rate: 0.8 mL/min; Injection volume: 5 µL Detection: UV214 nm; Temperature: Ambient (~25 ℃) Sample: 1. Insulin (0.5mg/mL) 2. Glucagon (0.5mg/mL) 3. Angiotensin I (0.5mg/mL) 4. Bradykinin (0.5mg/mL)

Ab

sorb

an

ce a

t 2

14

nm

min6 8 10 12 14

1

1

1

1

2

2

2

2

3

3

3

3

4

4

4

4

55% ACN

65% ACN

75% ACN

85% ACN

3um and 80Å: high efficiency 3um, 80Å pore for high resolution separation of peptides with molecular weights in the range of 1kD to 6kD.

Mobile Phase Compatibility: compatible with different mobile phases, including TFA/acetonitrile and methanol additives for peptide separation.

Peptides: insulin from porcine pancreas, glucagon, Angiotensin I and Bradykinin are separated on Zenix™ SEC-80 with good resolution.

Complex Peptide Mixtures: E. coli tryptic digests are fractionated successfully on Zenix™ SEC-80.

Highlighted FACTS:

http://www.sepax-tech.com/


Sepax Technologies, Inc. 5 Innovation Way

Newark, Delaware 19711, USA Tel: (302) 366-1101 | Fax: (302) 366-1151

E-mail: [email protected]

Sepax Zenix™ SEC-80 (Size Exclusion):

Ultra-high efficiency and resolution SEC

column. Zenix™-80 SEC 3um is specifically

designed for small protein and peptide

separations. Delivering unrivaled resolving

power and reproducibility. Made of uniform,

hydrophilic, and neutral nanometer thick

proprietary surface coating chemically

bonded on silica, offers long column lifespan

and negligible non-specific interactions.

What is Zenix™ SEC-80

Technical Specifications:

Phase Zenix™ SEC-80

Material Neutral, hydrophilic film bonded silica

Particle size (Pm) 3 Pore size (Å) ~ 80 Protein MW range (native) Up to 50,000

pH stability 2 – 8.5 (pH 8.5-9.5 can be tolerated temporarily)

Backpressure (psi) for 7.8x300 mm (1.0 mL/min)

~ 1,500

Maximum backpressure ~ 4,500

Salt concentration range 20 mM - 2.0 M

Maximum temperature ~ 80 oC Mobile phase compatibility

Aqueous and organic

50mM phosphate, 30% MeOH + 0.1% TFA

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

Abs

orba

nce

at 2

14 n

m50mM phosphate, 30% MeOH + 0.1% TFA

0.1% TFA, 75% ACN

25mM sodium acetate, 300mM NaCl, pH 4.5

Effect of Salt and Organic Additives on Peptide Separation

Peptide separation on Zenix™ SEC-80 (7.8 x 300mm). Panel A was with mobile phase 25mM sodium acetate and 300mM NaCl, pH 4.5.

Panel B was with mobile phase 50mM phosphate, 30% MeOH and 0.1% TFA. Peak elution order: 1. Insulin, 2. Bradykinin, 3. Angiotensin I, 4. Glucagon. The flow rate was

0.8 ml/min.

min6 8 10 12 14 16 18

Abs

orba

nce

at 2

14 n

m


1

23

4

25mM sodium acetate/300mM NaCl, pH 4.5A

B

NOTES Zenix™ SEC-80

Separation of E. coli digests on Zenix™ SEC-80 (7.8x300mm)

For complex E. coli tryptic digest, a mobile phase of 25 mM sodium acetate/300 mM NaCl gave the best separation. The bottom chromatogram was run with 50mM

phosphate/30%MeOH/0.1% TFA. The middle one was run with 0.1% TFA/75% ACN. The top chromatogram represents the best degree of peptide separation and was run

with 25mM sodium acetate/300mM NaCl, pH 4.5.


mailto:[email protected]


► Sepax’s 80 Å pore size was developed for analyzing small peptides and proteins with molecular weights under 50,000 Daltons.

► Organics in the mobile phase are an alternative additive for the analysis of hydrophobic, or sticky, sample types.

► The Zenix™-C SEC phase has a coating chemistry with a lay-down monolayer on porous silica which was developed for the analysis of hydrophobic, or sticky, sample types.

► A short, 2.1 x 50 mm, column is advantageous for use during a fast mass spec analysis of large biomolecules and small molecule drugs.

Highlighted FACTS:

NOTES

Similarities between Zenix™ and Zenix™-C SEC-80 P h a s e s f o r t h e A n a l y s i s o f A p r o t i n i n Column: Zenix SEC-80 7.8x300 mm, Flow rate: 1 mL/min, Detection: UV 214 and 280 nm, Mobile phase: 150 mM Sodium Phosphate Buffer pH 7.0, Injection Volume: 5 µL

Zenix™ SEC-80 and Zenix™-C SEC-80 for Peptide Analysis

Having trouble applying size exclusion chromatography to peptides under 50,000 Da? What if there was a size exclusion column specifically designed for small protein and peptide separations?

Sepax’s Zenix™ and Zenix™-C SEC-80 provide a valuable solution

Overlay of Peptide Runs on Zenix™ SEC-80 7.8 x 300 mm

Overlay of Peptide Runs on Zenix™-C SEC-80 7.8 x 300 mm

Differences between Zenix™ and Zenix™-C SEC-80 Phases for the Analysis of Angiotensin I Acetate Column: Zenix SEC-80 7.8x300 mm, Flow rate: 1 mL/min, Detection: UV 214 and 280 nm, Mobile phase: 150 mM Sodium Phosphate Buffer pH 7.0, Injection Volume: 5 µL

Column: Zenix SEC-80 7.8x300 mm, Flow rate: 1mL/min, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with 0.1% TFA, Injection Volume: 5 µL

Column: Zenix-C SEC-80 7.8x300 mm, Flow rate: 1mL/min, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with 0.1% TFA, Injection Volume: 5 µL



Sepax Technologies, Inc. 5 Innovation Way

Newark, Delaware 19711, USA

Tel: (302) 366-1101 | Fax: (302) 366-1151

E-mail: [email protected]

Sepax Zenix™ and Zenix-C SEC-80 (Size Exclusion): Ultra-high efficiency and resolution SEC column.

Zenix™-80 SEC 3um is specifically designed for small

protein and peptide separations. Delivering

unrivaled resolving power and reproducibility.

Made of uniform, hydrophilic, and neutral

nanometer thick proprietary surface coating

chemically bonded on silica, offers long column

lifespan and negligible non-specific interactions.

Technical Specifications:

Phase Zenix SEC-80 and Zenix-C SEC-80

Material Neutral, hydrophilic film bonded silica

Particle size (Pm) 3

Pore size (Å) 80

pH stability 2 – 8.5 (pH 8.5-9.5 can be tolerated temporarily)

Backpressure (psi) ~ 1,500

Maximum backpressure ~ 4,500

Maximum temperature ~ 80 oC

Mobile phase compatibility

Aqueous and organics

NOTES Calibration Curve for Zenix™ and Zenix™ -C SEC-80 What are Zenix™ SEC-80 and

Zenix™-C SEC-80?

Zenix™ SEC-80 and Zenix™-C SEC-80 for Peptide Analysis

™

Mobile Phase Comparison on Zenix™ SEC-80 7.8 x 300 mm

Column: Zenix™ SEC-80 and Zenix™-C SEC-80 7.8x300 mm, Flow rate: 1 mL/min, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with 0.1% TFA

C o l u m n : Z e n i x™ S E C - 8 0 7 . 8 x 3 0 0 m m , F l o w r a t e : 1 m L / m i n , Detection: UV 214 nm, Injection Volume: 5 µL, Sample: Insulin (1 mg/mL)

Column: Zenix™ SEC-80 7.8x300 mm, Flow rate: 0.2 mL/min, Detection: UV 228 nm, Mobile phase: 50 mM NH4Ac : ACN = 70:30 (v/v), Injection Volume: 0.1 µL


mailto:[email protected]

Authors Haiying Chen Katherine McLaughlin Sepax Technologies, Inc. 5 Innovation Way Newark, DE 19711 USA

Peptide Separations Using Size Exclusion Chromatography

Peptide Separation

Abstract Size exclusion chromatography (SEC) has been widely applied in

protein separation based on their molecular sizes. Here we

present a peptide mixture from 1kD to 6 kD separation on size

exclusion chromatography column Zenix™-80. The effect of

different mobile phases on the peptides’ separation performances

was also explored. In the separation of insulin, glucagon,

angiotensin I and bradykinin, 0.1% TFA/75% acetonitrile/H2O

proved to be the optimum mobile phase. Peptide SEC can be

applied to fractionate complex peptide mixtures as a first

dimension chromatography in a multidimensional

chromatography scheme. For complex E. coli tryptic digest, a

mobile phase of 25 mM sodium acetate/300 mM NaCl gave the

best separation.

2

Introduction Size exclusion chromatography (SEC) has been applied successfully to separate different sizes of proteins under native conditions. Different pore sizes have been developed to accommodate different ranges of molecular weight of biological samples. In order to apply the size exclusion chromatography to peptides under 10,000 Da, a few limitations have to be overcome. Even very small peptides can exist in different conformations and exhibit secondary structures.1 Therefore, peptides tend to adsorb to column matrixes by ionic and hydrophobic interactions.2 High salt concentrations, denaturing agents, and organic additives will minimize such interactions, thus enabling the separation of peptides according to their molecular weights. Sepax Zenix™ SEC columns are based on uniform, hydrophilic, and neutral nanometer thick films chemically bonded on high purity and mechanically stabilized silica. Zenix™ SEC-80 is specifically designed for small protein and peptide separations. Its phase is similar to the other Zenix™ SEC with the same particle size 3µm, but different pore size at 80 Å. In this application note, we present separation of four peptides bradykinin (1,060 Da), angiotensin I (1,297 Da), glucagon (3,483 Da) and insulin (5,778 Da) under different separation conditions. Separation of E. coli tryptic digest on Zenix™-80 is also investigated with different mobile phase conditions. Experimental HPLC system:

Agilent 1200 HPLC with binary pump

SEC column and LC method:

Zenix™-80 (3 PM, 80 Å, 7.8x300 mm) was used for the peptide size exclusion separations. Mobile phases include different percentages of acetonitrile with 0.1% TFA and water, high salt and methanol additives. The flow rate was at 0.8 mL/min. Chemicals and Reagents: Bradykinin acetate salt, MW 1,060 Da Angiotensin I Acetate, MW 1,297 Da Glucagon, MW 3,483 Da Insulin from Porcine Pancreas, MW 5,778 Da

All four peptide samples were purchased from Sigma-Aldrich. 5 mg/mL stock solutions were made with 50 mM acetic acid. For sample injections, stock solutions were further diluted with 50 mM acetic acid to desired concentration. Sequencing grade modified trypsin was purchased from Promega. E. coli lysate tryptic digestion: E. coli lysate tryptic digestion was performed according to the procedure described previously.3, 4 Briefly 0.5 mg dried lysate were reconstituted in 100 Pl 6 M urea, 50 mM Tris-HCl, pH 8.0. A 0.2 M dithiothreitol (DTT) stock solution was added to obtain 10 mM concentration and the protein mixture was incubated at room temperature for 1 hour. A final concentration of 30 mM iodoacetamide (IAM) was reached by adding a stock solution of 0.2 M IAM. The alkylation was performed at room temperature for 1 hour. The final urea concentration was reduced to 0.6 M with the addition of 50 mM Tris-HCl, 1 mM CaCl2, pH 7.6. The protein mixture was then digested with trypsin overnight at 37 oC with a trypsin: protein ratio of 1:50 (w/w). 1 Pl of formic acid was added to stop the digestion. The digest was maintained at 4 oC in an auto sampler for SEC-HPLC runs or frozen at -20 oC for future analysis. Results Figure 1A showed the separation profile of four peptides with mobile phase 25 mM sodium acetate/300 mM NaCl, pH 4.5. Four peptides did not get separated with the addition of high salt. Based on the individual peptide injections (data not shown), the wide peak between 14.5 and 16.5 minutes in figure 1A was glucagon and insulin. Interaction between the peptides and the solid phase was the reason that glucagon and insulin were eluted later than angiotensin I and bradykinin (the main peak at 10.2 minutes). When the mobile phase was switched to 50 mM phosphate, 30% MeOH and 0.1% TFA, four peptides were separated, but not according to their molecular weight order. According to the molecular weights, the order of elution should be insulin, glucagon, angiotensin I and bradykinin.

3

Figure 1B showed the separation profile with the elution in the order of insulin, bradykinin, angiotensin I and glucagon. The combination of methanol and TFA was not strong enough to disrupt the interactions between the column matrix and peptides. Thus, it did not achieve separation of four peptides according to their molecular sizes. When the mobile phase was switched to a TFA/acetonitrile system, all four peptides were eluted at earlier retention times, and separation resolutions were improved dramatically. The solid phase and peptide interaction was minimized with the addition of high concentration acetonitrile. As seen in Figure 2, the separation of the four peptides was closely associated with the percentage of acetonitrile in the mobile phases. To achieve the best separation of the peptide mixture, the optimal acetonitrile concentration was at 75% in aqueous 0.1% TFA. Table 1 summarized the separation parameters for four peptides on Zenix™-80. Angiotensin I and bradykinin achieved baseline separation. Furthermore, the elution order of the four peptides was related to their molecular weights or sizes under the denaturing 0.1% TFA/75% acetonitrile/H2O. Size exclusion chromatography can be used as a pre-fractionation first dimension separation for complex peptide mixtures. Figure 3 showed the separation profiles of E. coli digest under different mobile phase conditions. Under acidic salt condition with 25 mM sodium acetate/ 0.3 M NaCl, pH 4.5, the chromatogram exhibited a higher degree of peptide separation than the ones with the other two mobile phases - 0.1% TFA/75%acetonitrile/H2O and 50 mM phosphate/ 30% MeOH/0.1% TFA. Conclusion Zenix™-80 successfully separated four peptides whose molecular weights range from 1 kD to 6 kD. Organic additives help disrupt the column-peptide interaction. There is an optimum concentration of acetonitrile at 75% in aqueous 0.1% TFA as mobile phase to give the best separation of the four peptides. Two 7.8x300 mm Zenix™-80 columns in tandem will improve separation with wider retention time between peptides.

Rarely, a complex protein digest can be resolved in one single reversed phase LC run. With Zenix™-80 SEC, E. coli lysate can be pre-fractionated and the fractions can be further subjected to ion exchange or C18 reversed phase separation. SEC fractions can be directly applied to 2D LC/MS/MS as well for peptide mapping and identification purpose. Thus sample complexity can be greatly reduced with Zenix™-80 SEC pre-fractionation. For more information on ZenixTM- SEC products, please visit our website: http://www.sepax-tech.com/Zenix.php or contact us at 1-877-SEPAX-US.

4

min6 8 10 12 14 16 18

Abs

orba

nce

at 2

14 n

m

50 mM phosphate, 30% MeOH + 0.1% TFA

12

3

4

25 mM sodium acetate/300 mM NaCl, pH 4.5A

B

Figure 1. Peptide separation (bradykinin, angiotensin I, glucagon and insulin) on Zenix™-80 (7.8x300 mm). Panel A was with mobile phase 25 mM sodium acetate and 300 mM NaCl, pH 4.5. Panel B was with mobile phase 50 mM phosphate, 30% MeOH and 0.1% TFA. Peak elution order: 1. Insulin, 2. Bradykinin, 3. Angiotensin I, 4. Glucagon. The flow rate was 0.8 mL/min.

Abs

orba

nce

at 2

14 n

m

min6 8 10 12 14

1

1

1

1

2

2

2

2

3

3

3

3

4

4

4

4

55% ACN

65% ACN

75% ACN

85% ACN

Figure 2. Effect of mobile phase acetonitrile concentration on the separation of the four peptide mixtures on Zenix™-80 (7.8x300 mm) (Peak 1, insulin, Peak 2, glucagon, Peak 3, angiotensin I, Peak 4, bradykinin). The mobile phases contained 0.1% TFA with the indicated percentage of acetonitrile. The flow rate was 0.8 mL/min. 5 PL of peptide mixture (0.5 mg/mL concentration for each peptide) was injected.

5

Table 1. Separation parameters for the four peptide mixture with mobile phase 75% Acetonitrile in 0.1% TFA with water. Peak Protein MW (Da) Retention

time (min) Resolution Plate counts

1 Insulin (porcine) 5,778 7.75 16,711 2 Glucagon 3,483 8.03 1.07 12,132 3 Angiotensin I 1,297 8.46 1.58 19,741 4 Bradykinin 1,060 8.86 1.65 21,060


min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

Abs

orba

nce

at 2

14 n

m

50 mM phosphate, 30% MeOH + 0.1% TFA

0.1% TFA, 75% ACN

25 mM sodium acetate, 300 mMNaCl, pH 4.5

Figure 3. Separation of E. coli digests on Zenix™-80 (7.8x300mm) under indicated mobile phases. Bottom chromatogram was for the run with 50mM phosphate/30%MeOH/0.1% TFA. The middle one was for the run with 0.1% TFA/75% acetonitrile. The top chromatogram represented the run with 25 mM sodium acetate/300 mM NaCl, pH 4.5. The inset was for the zoom view for the part of the chromatogram circled. Reference:

1. Irvine G. J. Biochem. Biophys. Methods, (2003) 56 233-242. 2. Swergold G., and Rubin C. Anal. Biochem. (1983) 131, 295-300 3. Tryptic digestion protocol: http://ipmb.sinica.edu.tw/proteomics/Documents/In-Sol-Digest.pdf 4. Kinter, M., and Sherman, N. E. 2000, Protein sequencing and identification using

tandem mass spectrometry. JohnWiley & Sons, Inc. pp.161-163

http://ipmb.sinica.edu.tw/proteomics/Documents/In-Sol-Digest.pdf

6

Order information

Part Number Particle Size Pore Size IDxLength 213080-2105[1] 3 µm 80 Å 2.1x50mm 213080-2130 3 µm 80 Å 2.1x300mm 213080-4605[1] 3 µm 80 Å 4.6x50mm 213080P-4605[1][2] 3 µm 80 Å 4.6x50mm 213080-4615 3 µm 80 Å 4.6x150mm 213080-4625 3 µm 80 Å 4.6x250mm 213080-4630 3 µm 80 Å 4.6x300mm 213080P-4630[2] 3 µm 80 Å 4.6x300mm 213080-7805[1] 3 µm 80 Å 7.8x50mm 213080-7815 3 µm 80 Å 7.8x150mm 213080-7820 3 µm 80 Å 7.8x200mm 213080-7830 3 µm 80 Å 7.8x300mm 213080-10005[1] 3 µm 80 Å 10.0x50mm 213080-1010 3 µm 80 Å 10.0x100mm 213080-10015 3 µm 80 Å 10.0x150mm 213080-10025 3 µm 80 Å 10.0x250mm 213080-10030 3 µm 80 Å 10.0x300mm 213080-21205[1] 3 µm 80 Å 21.2x50mm 213080-21225 3 µm 80 Å 21.2x250mm 213080-21230 3 µm 80 Å 21.2x300mm

[1] Guard column [2] Column packed with PEEK tubing

AE1001

© Sepax Technologies, Inc.

Columns: Antibodix™ NP10 (10 µm,4.6x250 mm) Mobile Phases: A, 3 mM phosphate buffer, pH 5.0; B, 20mM phosphate buffer, pH 7.5 Gradient: as indicated on the chromatogram Flow Rate: 1.0 mL/min Detection: UV 214 nm Temperature: Ambient Concentration: 2.0 mg/mL Injection Volume: 5 µL Sample: a peptide drug (pI=7~8)

Analysis of Peptide on Antibodix™ NP10 (10 µm, 4.6x250 mm)

Keywords: Weak cation exchange, Antibodix, peptide

Min2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Gradient：0% - 50% B (20 min)

Gradient：0% - 20% B (20 min)20%B after 20 min

BE1001


Column: Bio-C18, 5 Pm, 300 Å, 4.6×250 mm Mobile Phase: A: 10% ACN (0.1% TFA); B: 90% ACN (0.1% TFA) Flow Rate:

Time (min) 0 10 1.0 mL/min

B (%) 10 50

Temperature: Ambient Detection: UV 214 nm Injection Volume: 20 µL Samples: 1. Peptide

Keywords: Bio-C18, reverse phase, peptide, biochemistry

Analysis of a Peptide on Bio-C18 (5µm, 300 Å, 4.6x250mm)

BE1002


Column: Bio-C18, 5 Pm, 300 Å, 4.6x250 mm Mobile phase: CH3OH: Buffer (0.05 M KH2PO4, pH 3.0) =57: 43 Flow rate: 0.8 mL/min Temperature: Ambient Detection: 238nm Injection Volume: 20 µL Sample: 1. Microcystin

Keywords: Bio-C18, reverse phase, microcystin, environmental

Analysis of Microcystin on Bio-C18 (5µm, 300 Å, 4.6x250mm)

Column: Zenix SEC-300, 3Pm, 7.8x300mm and Zenix-C SEC-300, 3Pm, 7.8x300mm

Mobile Phase: 150 mM Sodium Phosphate Buffer, pH 7.0 Flow Rate: 1.0 mL/min Temperature: 25 oC Detection: UV 214nm Injection Volume: 20 µL Sample: 4kD peptide and 20kD mPEG-MAL

(methoxy-PEG-Maleimide) (6mg/mL)


Analysis of mPEG and mPEG-peptide on Zenix and Zenix-C SEC Phases

EE1001

mAU

0

40

80

120

min2 4 6 8 10 12

mAU

0

100

200

300

400

Zenix-300

Zenix-C 300

2xmPEG-peptide

mPEG-peptide

methoxy-PEG-Maleimide

Zenix SEC-300

Zenix-C SEC-300

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

20

40

60

80

100

120

140Zenix-300

Zenix-C 300


Mobile Phase: 150 mM Sodium Phosphate Buffer, pH 7.0 Flow Rate: 1.0 mL/min Temperature: 25 oC Detection: UV 214nm Injection Volume: 20 µL Sample: 20kD mPEG-MAL (methoxy-PEG-Maleimide) (1mg/mL)

Zenix SEC-300

Zenix-C SEC-300

OH1003


Column: HP-C18, 5 µm, 120 Å ,4.6×150 mm Mobile phase: ACN: H2O: HAC = 500: 500: 1 (v/v) Flow rate: 1.0 mL/min Temperature: Ambient Detection: 273 nm Injection Volume: 10 µL Sample: Valsartan

Analysis of Valsartan on HP-C18

Keywords: HP-C18, reverse phase, valsartan, antihypertensive drugs, pharmaceuticals

PE1001


Column: Proteomix® SX-NP3 (3Pm, 4.6x50mm) Mobile Phases: A, 5 mM CH3COONH4; B, 0.5 M CH3COONH4:ACN=4:1 (v/v) Gradient: 0-50% B (20 min) Flow Rate: 0.6 mL/min Detection: ELSD SofTA & UV 280 nm Injection Volume: 5 µL Concentration: 0.1 mg/mL Samples: Peptide Sequence Net Charge C1 Ac-Gly-Gly-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide ＋1 C2 Ac-Lys-Tyr-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide ＋2 C3 Ac-Gly-Gly-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide ＋3 C4 Ac-Lys-Tyr-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide + 4 (Courtesy of Miyako Kawakatsu, M&S Instruments Inc.)

Separation of Peptide on Proteomix® SCX-NP3 (3 µm, 4.6x50 mm) – volatile buffer

SofTA

@280nm

C1 C2

C3C4

Pressure

Keywords: Ion-exchange, Proteomix, strong cation exchange, peptide, volatile buffer

ZE1002


Column: Zenix™-100 (3 µm, 100 Å, 7.8x300 mm) Mobile Phase: ACN:H2O:TFA=65:35:0.1(v/v) Flow Rate: 1.0 mL/min Temperature: Ambient Detection: UV 214 nm Injection Volume: 10 µL Samples: Ribonuclease A (5.931 min), human insulin (6.936 min), thymosin α1

(7.252 min), somatostatin (8.089 min)

Analysis of Peptide Mixture on Zenix™-100 (7.8x300)

Keywords: Size exclusion, Zenix, peptide, thymosin α1, human insulin, Somatostatin

min0 2 4 6 8 10 12 14

mAU

0

100

200

300

400

5.93

1 6.93

67.

252

8.08

9

ZE1004


Mobile Phase: 150 mM Phosphate Buffer, pH 7.0; Flow Rates: 0.35 mL/min for 4.6x300 mm & 1.0 mL/min for 7.8x300 mm Temperature: Ambient Detection: UV 214 nm Injection Volumes: 5 µL for 4.6x300 mm & 20 µL for 7.8x300 mm Sample: Peptide mixture (provided by a customer)

SEC Resolution Comparison for Peptide Separation

Keywords: Size exclusion, Zenix, peptide, high resolution

Min1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0

Zenix SEC-150 (3 µm, 150 Å, 4.6x300mm)

Zenix SEC-150 (3 µm, 150 Å, 7.8x300mm)

Brand X (5 µm, 150 Å, 4.6x300mm)

ZE1005


Column: Zenix™ SEC-150 (3 µm, 150 Å, 7.8x300 mm) Mobile Phase: 150 mM Phosphate Buffer, pH 7.0; Flow Rate: 1.0 mL/min Temperature: Ambient Detection: UV 220 nm Injection Volume: 10 µL Sample: Salcatonin-type peptide mixture with MW of 8 kD and 5kD

Analysis of 5kD and 8kD Peptides by Zenix™ SEC-150

Keywords: Size exclusion, Zenix, peptide, salcatonin

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

Min1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

ZE1006


min0 2 4 6 8 10 12 14 16

mAU

0

200

400

600

800

1000

7.74

98.

034

8.45

78.

858

Zoom

Column: Zenix™ SEC-80 (3 µm, 80 Å, 7.8x300 mm) Mobile Phase: ACN:H2O:TFA=75:25:0.1(v/v) Flow Rate: 0.8 mL/min Temperature: Ambient Detection: UV 214 nm Injection Volume: 5 µL Sample: Insulin from Porcine pancreas (7.749min, MW 5,778), Glucagon (8.034min,

MW 3,483), Angiotensin I (8.45min, MW 1,297), Bradykinin (8.858, MW 1,060)

Analysis of Four Peptide Mixture from 1kD to 6kD on Zenix™-80 (7830)

Keywords: Size exclusion, Zenix, peptide, 80 Å pore size, insulin, bradykinin, glucagon, angiotensin I


Mobile Phase: 150 mM Sodium Phosphate Buffer, pH 7.0Flow Rate: 1.0 mL/minTemperature: 25 oCDetection: UV 214nmInjection Volume: 20 µLSample: 4kD peptide and 20kD mPEG-MAL

(methoxy-PEG-Maleimide) (6mg/mL)


Analysis of an mPEG-peptide on Zenix SEC Phases

Z�ϭϬϭϭ

mAU

0

40

80

120

min2 4 6 8 10 12

mAU

0

100

200

300

400

Zenix-300

Zenix-C 300

2xmPEG-peptide

mPEG-peptide

methoxy-PEG-Maleimide

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

20

40

60

80

100

120

140Zenix-300

Zenix-C 300


Mobile Phase: 150 mM Sodium Phosphate Buffer, pH 7.0Flow Rate: 1.0 mL/minTemperature: 25 oCDetection: UV 214nmInjection Volume: 20 µLSample: 20kD mPEG-MAL (methoxy-PEG-Maleimide) (1mg/mL)

ZE1008


min2 4 6 8 10 12 14 16

mAU

0

50

100

150

200

1

3

24

5

6

Column: Zenix™ SEC-80 (3 µm, 80 Å, 7.8x300 mm) Mobile Phase: ACN:H2O:TFA=75:25:0.1(v/v) Flow Rate: 0.8 mL/min Temperature: Ambient Detection: UV 214 nm Injection Volume: 5 µL Sample: 1.Ribonuclease A (13.7 kD); 2. Insulin from Porcine pancreas (5,778 Da);

3. Glucagon (3,84 3); 4. Angiotensin I (1,297 Da); 5. Bradykinin (1060 Da); 6. Uracil (120 Da)

Peptide Separations on ZenixTM-80 (7830) from 13.7 kD to 120 Da

Keywords: Size exclusion, Zenix, peptide, 80 Å pore size, insulin, bradykinin, glucagon, angiotensin I, ribonuclease A, Uracil, small peptide

ZE1002


Column: Zenix™-100 (3 µm, 100 Å, 7.8x300 mm) Mobile Phase: ACN:H2O:TFA=65:35:0.1(v/v) Flow Rate: 1.0 mL/min Temperature: Ambient Detection: UV 214 nm Injection Volume: 10 µL Samples: Ribonuclease A (5.931 min), human insulin (6.936 min), thymosin α1

(7.252 min), somatostatin (8.089 min)

Analysis of Peptide Mixture on Zenix™-100 (7.8x300)

Keywords: Size exclusion, Zenix, peptide, thymosin α1, human insulin, Somatostatin

min0 2 4 6 8 10 12 14

mAU

0

100

200

300

400

5.93

1 6.93

67.

252

8.08

9

1

Column: Zenix SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Flow rate: 0.8 mL/min, Detector: UV 214 nm, Injection volume: 10 µL , Column temperature: Room temperature, Samples: 1 mg/mL exenatide in water (MW= 4,187 Da) used for treatment of diabetes mellitus type 2

Exenatide Analysis on Zenix SEC-80

www.sepax-tech.com Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

EE1002

min 0 2 4 6 8 10 12 14

50% ACN with 0.1% TFA



mAU

0

500

1000

1500

2000

2500

3000

2

Column: Zenix SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Mobile phase: ACN : H2O : TFA =80 : 20 : 0.1 ( v/v ), Flow rate: 0.8 mL/min, Detector: UV 214 nm, Injection volume: 10 µL, Column temperature: Room temperature, Samples: 1 mg/mL exenatide in water


min 0 2 4 6 8 10 12

mAU

0

500

1000

1500

2000

7.6

07

8.2

68

9.4

50

min 7 8 9

mAU

0

50

100

150

200

7.6

07

8.2

68

9.4

50


EE1002

Pegylated Exenatide on Zenix and Zenix-C Column: Zenix SEC-300 and Zenix-C SEC-300 (3 µm, 300 Å, 7.8 x 300 mm), Mobile phase: 50 mM CH3COONH4 : ACN = 90 : 10 ( v/v ), Flow rate: 0.5 mL/min, Detector: UV 214 nm, Column temperature: 25 ℃, Injection volume: 15 µL, Pressure: 42 bar, Sample: 3.3 mg/mL PEG-Exanatide in water (PEG 23 kD)


min 10 12 14 16 18

mAU

100

150

200

250

300

350

400

450

Zenix-C SEC-300

Zenix SEC-300

Tailing 1.07

Tailing 2.37

EE1002

1

Transfer Factor Oral Solution SEC Separation


Column: SRT SEC-100, Zenix SEC-80 and Zenix-C SEC-100 7.8 x 300 mm, Mobile phase: 150 mM sodium phosphate buffer pH 7.0, Flow rate: 1.0 mL/min, Detector: UV 251 nm, Sample: 10 µL 1mg/mL transfer factor oral solution (dietary supplement/peptide mixture, immune molecules to boost immune response)

min 0 2 4 6 8 10 12 14 16 18

mAU

0 50

100 150 200 250

Zenix-C SEC-100

min 0 2 4 6 8 10 12 14 16 18

mAU

0 50

100 150 200 250

Zenix SEC-80

min 0 2 4 6 8 10 12 14 16 18

mAU

0 50

100 150 200 250

SRT SEC-100

Best separation

EE1003

Analysis of Peptide Digest on Proteomix SCX NP1.7

min 5 10 15 20

mAU

0

10

20

30

40

50

60 5 µg digested BSA 10 µg digested BSA

Column: Proteomix SCX NP1.7 2.1 x 30 mm without guard, Flow rate: 0.35 mL/min, Detection: UV 214 nm, Mobile phase: A: 10 mM phosphate buffer, pH 2.5, B: A + 1 M NaCl , C: 100% acetonitrile, Sample: trypsin digested BSA, Pressure: 380 bar

Time (min) %A %B %C 1.5 90 0 10 20 40 50 10

PE1002

Gradient Comparisons for the Analysis of Peptides on Proteomix SCX NP1.7

Column: Proteomix SCX NP5 4.6 x 100 mm without guard, Flow rate: 0.35 mL/min, Detection: UV 214 nm, mobile phase: A: 10 mM phosphate buffer, pH 2.5, B: A + 1 M NaCl , C: 100% acetonitrile, Sample: Peptide mixture, Pressure: 310 bar, Gradient: 1.5 - 10 min as indicated

min 2 4 6 8 10

mAU

0

100

200

300

400

500

600

700

800

0 - 90% B

2 - 90% B

3 - 90% B

5 - 90% B

1. AAAAL (1 mg/mL) 2. AAAKL (2mg/mL) 3. AAKKL (2mg/mL) 4. AKKKL (2mg/mL) 5. KKKKL (2mg/mL)

1 2

4 5

3

PE1003

Peptide Separation Comparison on Proteomix SCX NP5 and Proteomix SCX NP1.7 Column: Proteomix SCX NP5 4.6 x 100 mm without guard and NP1.7 2.1 x 30 mm, Detection: UV 214 nm, mobile phase: A: 10 mM phosphate buffer, pH 2.5, B: A + 1 M NaCl, C: 100% acetonitrile, Sample: 2 PL Peptide mixture, Pressure: 74 bar for NP5, 540 bar for NP1.7

Time (min) %A %B %C 1 89 1 10 6 0 90 10

min 0 1 2 3 4 5 6

mAU

0

50

100

150

200

250

300

350

5 µm 1.0 mL/min

1.7 µm 0.6 mL/min

PE1003

Time (min) %A %B %C 1 89 1 10 6 0 90 10

min 3 4 5 6

mAU

10

20

30

40

50

60

70

5 µm 1.0 mL/min

1.7 µm 0.6 mL/min

Zoomed View of Peptide Separation Comparison on Proteomix SCX NP5 and Proteomix SCX NP1.7

Column: Proteomix SCX NP5 4.6 x 100 mm without guard and NP1.7 2.1 x 30 mm, Detection: UV 214 nm, mobile phase: A: 10 mM phosphate buffer, pH 2.5, B: A + 1 M NaCl, C: 100% acetonitrile, Sample: 2 PL Peptide mixture, Pressure: 74 bar for NP5, 540 bar for NP1.7

PE1003

1

Column: Zenix SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Flow rate: 0.8 mL/min, Detector: UV 214 nm, Injection volume: 10 µL , Column temperature: Room temperature, Samples: 1 mg/mL exenatide in water, MW= 4,187 Da, used for treatment of diabetes mellitus type 2


min 0 2 4 6 8 10 12 14

mAU

0

500

1000

1500

2000

Mobile phase 1: ACN : H2O : TFA =50 : 50 : 0.1 ( v/v ) Mobile phase 2: ACN : H2O : TFA =70 : 30 : 0.1 ( v/v ) Mobile phase 3: ACN : H2O : TFA =80 : 20 : 0.1 ( v/v )

Mobile 3

Mobile 1

Mobile 2


ZE1009

2

Column: Zenix SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Mobile phase: ACN : H2O : TFA =80 : 20 : 0.1 ( v/v ), Flow rate: 0.8 mL/min, Detector: UV 214 nm, Injection volume: 10 µL, Column temperature: Room temperature, Samples: 1 mg/mL exenatide in water


min 0 2 4 6 8 10 12

mAU

0

500

1000

1500

2000

7.6

07

8.2

68

9.4

50

min 7 7.5 8 8.5 9 9.5

mAU

-100

-50

0

50

100

150

200 7

.607

8.2

68

9.4

50


Pegylated Exenatide on Zenix and Zenix-C

min 10 12 14 16 18

mAU

100

150

200

250

300

350

400

450

Zenix-C SEC-300

Zenix SEC-300

Column: Zenix SEC-300 and Zenix-C SEC-300 (3 µm, 300 Å, 7.8 x 300 mm), Mobile phase: 50 mM CH3COONH4 : ACN = 90 : 10 ( v/v ), Flow rate: 0.5 mL/min, Detector: UV 214 nm, Column temperature: 25 ℃, Injection volume: 15 µL, Pressure: 42 bar, Sample: 3.3 mg/mL PEG-Exanatide in water (PEG 23 kD)


Tailing 1.07

Tailing 2.37