Promix TM. Enter a New Era in Biomolecule Analysis with. Columns. Unsurpassed Selectivity and Peak Capacity for Peptides and Proteins

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1 Promix TM Enter a New Era in Biomolecule Analysis with Promix TM Columns Unsurpassed electivity and Peak Capacity for Peptides and Proteins

2 Applications: Proteomics Peptide/Protein Analysis Peptide/Protein Purification Features & Benefits: 2-D HPLC with a ingle Column Ionic Interactions Reversed-Phase eparate peptides and proteins that differ only by a single ao acid. Enhanced selectivity for closely related peptides and proteins. Increased peak capacity compared to reversed-phase and ion exchange. Increased LC-M sensitivity using formic acid and ammonium acetate. Completely calable from capillary to prep. Polypeptide 2. Polypeptide impurity 2 Promix TM Technology Ionic Group: Retention and selectivity are controlled by analyte pi and mobile phase ionic strength. Independent control of acid/buffer concentration and organic modifier offers almost limitless control of retention and selectivity! Isocratic eparation of ynthetic Peptide and Impurity 250 x 4.6 mm 300 Å Flow rate:.0 ml/ Detection: UV 20 nm Mobile phase: MeCN 20% AmAc ph mm, 2 Hydrophobic Chain: Retention and selectivity are controlled by analyte hydrophobicity and mobile phase % organic Column: Vydac TM Protein & Peptide C8 50 x 4.6 mm 300 Å Flow rate:.0 ml/ Detection: UV 20 nm Mobile phase: MeCN 20% with AmAc ph mm

3 eparation of Insulin Analogs: Closely related peptides can be difficult to separate by reversed-phase. By combining ionic and hydrophobic interactions, Promix MP is able to separate these analogs, differing in sequence by a single ao acid pair, using the slight differences in pi as illustrated below. Gly Ile ValGluGlnCysCysThrerValCyserLeuTyrGlyLeuGluAsnTyrCysAsn PheValAsnGlyHisLeuCysGlyerHisLeuValGluAlaLeuTyrLeuValCysGlyGlu Arg ThrLysProThrTyrPhePheGly Gly Ile ValGluGlnCysCysThrerValCyserLeuTyrGlyLeuGluAsnTyrCysAsn PheValAsnGlyHisLeuCysGlyerHisLeuValGluAlaLeuTyrLeuValCysGlyGlu Arg ThrProLysThrTyrPhePheGly Gly Ile ValGlnGlnCysCysThrerIleCyserLeuTyrGlnLeuGluAsnTyrCysGly PheValAsnGlnHisLeuCysGlyerHisLeuValGluAlaLeuTyrLeuValCysGlyGlu Arg ArgArgThrLysProThrTyrPhePheGly Normal human insulin ynthetic insulin Humalog ynthetic insulin Lantus. Normal human insulin 2. ynthetic insulin Humalog 3. ynthetic insulin Lantus Buffer: AmFm 25 mm ph 3.0 Resolution: ize: 250 x 4.6 mm 300 Å Flow rate:.0 ml/ MeCN: gradient 20% to 40 % in 30 Injection: 5 ul ample: 30 units/ml each protein in water ml Buffer: AmAc 25 mm ph 3.5 Resolution: Buffer: AmAc 25 mm ph 4.0 Resolution:

4 eparation of a Complex Mixture containing very small peptides and very large peptides can be achieved using two different Promix columns. Very small peptides eluted in first.5 on Promix TM MP column were collected and resolved using Promix P column. ize: 50 x 3.0 mm 300 Å Mobile phase: MeCN gradient from 0 to 70% in 20, TFA 0.% Flow rate:.0 ml/ Column: Promix TM P ize: 50 x 4.6 mm 00 Å Mobile phase: MeCN gradient from 2 to 50% in 30, AmFm ph 3.0 gradient from 5 to 30 mm in 30 Flow rate:.0 ml/ Column temperature: 40 o C MeCN: gradient 2% to 40 % in 30 Buffer: gradient AmFm 25 mm to 5 mm ph 3.0 MeCN: gradient 2% to 50 % in 30 Buffer: AmFm 25 mm ph 3.0 Albu Digest electivity Affected by Buffer Concentration: Complex mixtures such as protein digests have different profiles at different ionic strengths. Multiple gradients can be applied to complex samples: organic gradient buffer gradient or both Each mode provides a different selectivity and elution profile x 4.6 mm 300 Å Flow rate:.0 ml/ Injection: 50 ul ample: 0.3 mg/ml

5 eparation tudy of Lantus and Humalog imilarly to a traditional ion separation, the buffer concentration plays an important role in the mixed-mode technology altering the degree of ionic interaction of the biomolecules with the stationary phase. The amount of the organic modifier is also important in changing the degree of hydrophobic interaction. Independent adjustment of the amount of buffer and organic modifier creates infinite number of separation conditions that are suitable for many types of biomolecules. Generally three types of gradient separation mode available on Promix columns. Gradient of MeCN with isocratic buffer concentration, gradient of buffer concentration with isocratic MeCN concentration, and dual gradient of both MeCN and buffer. MeCN gradient 0% to 35% in 2 TFA gradient 0.05% to 0.4% in 2 MeCN gradient 0% to 35% in 2 TFA gradient 0.4% to 0% in 2 Column : Promix MP Column size: 3.0 x 50 mm 300 Å Flow rate:.0 ml/ Injection : 5 ul ample: Mixture of Humalog and Lantus 6 units/ml MeCN gradient 25% to 20% in 2 TFA gradient 0.4% to 0% in 2 MeCN gradient 0% to 35 % in 2, TFA 0.05% MeCN gradient 0% to 35 % in 2, TFA 0.% MeCN gradient from 0% to 35 % in 2, TFA 0.2% MeCN 25% TFA gradient 0.4 % to 0.05% in 2 MeCN 22.5% TFA gradient 0.4% to 0% in 2 MeCN 20% TFA gradient 0.4% to 0% in

6 Alternative electivity of Promix TM vs. RP 300Å Columns: An indication of the alternative selectivity of Promix TM columns can be observed when the digest fraction collected from a Reversed-Phase column is reinjected onto a Promix TM column or visa versa. The elution time usually expands providing additional resolution and peak capacity. Column: Vydac ize: 50 x 4.6 mm 300 Å Mobile phase: MeCN gradient from 0 to 70% in 30, TFA 0.% Flow rate:.0 ml/ Column temperature: ambient ize: 250 x 4.6 mm 300 Å Mobile phase: MeCN gradient from 0 to 70% in 30, TFA 0.% Flow rate:.0 ml/ Column temperature: 40C Digest fraction collected on Vydac column at 0-5 Digest fraction collected on Vydac column at 5-20 Fraction collected on Vydac column at 0-5 re-injected in Promix TM MP column Fraction collected on Vydac column at 5-20 re-injected in Promix TM MP column Column: Promix MP ize: 50 x 4.6 mm 300 Å Flow rate:.0 ml/ MeCN: gradient 0% to 50 % in 30 Buffer: AmFm 25 mm ph 3.0 Injection: 50 ul Digest fractions collected on Promix MP column from 5 to 8 and from 8 to 2 Column: ACE 3 C8-300 ize: 00 x 2. mm 300 Å Flow rate: 0.3 ml/ MeCN: gradient 0% to 50 % in 30 Buffer: TFA 0.% Injection: 75 ul Fraction collected on Promix MP column at 5-8 re-injected in ACE 3 C8-300 column Fraction collected on Promix MP column at 8-2 reinjected in ACE 3 C8-300 column

7 mall Peptides Test Mixture (5 Angiotensins). ARG-VAL-TYR-VAL-HI-PRO-ILE 2. ARG-VAL-TYR-VAL-HI-PRO-PHE 3. ALA-PRO-GLY-AP-ARG-ILE-TYR-VAL-HI-PRO-PHE 4. AP-ARG-VAL-TYR-VAL-HI-PRO-PHE-HI LEU 5. AP-ARG-VAL-TYR-ILE-HI-PRO-PHE-HI-LEU Flow rate:.0 ml/ Injection: 40 ul, 4.6 x 250 mm 300 Å Mobile phase: MeCN gradient 5-35 % in 5 Gradient TFA % in 5 ;, 4.6 x 50 mm 300 Å Mobile phase: MeCN gradient 0-40 % in 30 Gradient TFA % in 30 ; Large Peptides Mixture. Ribonuclease A (mw 3,500) 2. Insulin (mw 5,700) 3. Lysozyme (mw 4,300) 4. B-Lactalbu (mw 4,200) 5. Carbonic Anhydrase (mw 29,000) ize: 4.6 x 50 mm 300 Å Mobile phase: MeCN gradient % in 30 Gradient TFA % in 30 ; Flow rate: ml/ Injection: 00 ul

8 Column election and Use. Use the following chart to select a column based on the analyte s properties 2. elect the column length based on sample complexity mm for proteomics, protein digests, and complex samples mm for synthetic analysis and purification 3. elect the column i.d. based on sample loading and sensitivity needs 4. Mobile Phase should be ph 2-4 with: TFA ( %) Formic Acid (0.-0.9%) Ammonium Acetate or Ammonium Formate 5. Organic Modifier should be Acetonitrile 6. Ionic strength, ph, and organic modifier gradients can be used to optimize the separation Molecular Weight (hydrophobicity) kda Promix TM Column election Chart Promix LP: 800Å Promix MP: 300 or 800 Å Promix AP: 00 or 300Å Promix P:00Å pi To order: By phone By fax By mail mail@sielc.com Create your Part Number: We recommend to select stationary phase with pore size 00 Å for Promix LP; pore size 00 or 300 Å for Promix AP; pore size 300 or 800 Å for Promix MP and pore size 800 Å or none porous for Promix LP. Type of packing Promix LP Promix MP Promix AP Promix P LP MP AP P Column ID 22 mm 4.6 mm 3.0 mm 2.0 mm.0 mm 0.5 mm 0.25 mm 0. mm MP Column length 250 mm mm mm mm mm mm 00 Packing size 3 um 5 um 0 um 20 um Pores size 00 Å 300 Å 800 Å Noneporous