Reversed Phase HPLC Solutions for Proteins and Peptides

Transcription

1 Reversed Phase HPLC Solutions for Proteins and Peptides id pu an al ri yz e en fy tif y

2 About Phenomenex Founded in 982, Phenomenex is extremely dedicated to the development, manufacturing, and supply of innovative separation and purification products for the life science/pharmaceutical industries. However, we are even more dedicated to providing the highest level of customer support and satisfaction in the separation science industry. By personalizing science, we are able to ensure that every customer has the opportunity to work with energetic and extremely knowledgeable Phenomenex employees and distributors to address day-to-day responsibilities such as method development, product evaluations, and troubleshooting. Our customer first policy means that we deliver the same degree of support and service to the small, independent lab or university as well as major corporations. An extensive global presence through offices located in 2 countries worldwide and a vast network of distributors in over 6 countries enables Phenomenex to successfully supply and support the work of scientists worldwide. No matter where you are located in the world, you can always count on Phenomenex.

3 T a b l e o f C o n t e n t s Identify, Purify, and Analyze... page Dependable Solutions... 2 Guaranteed Performance, Lifetime, and Quality Jupiter 3 for Intact Protein Separation and Purification...5- Jupiter Proteo for Peptide Mapping and Peptide Purification Material Characteristics... 4 Ordering Information... 4

4 Identify, Purify, and Analyze The Jupiter HPLC column portfolio, including Jupiter 3 and Jupiter Proteo, offers optimized reversed phase solutions for protein characterization and purification. With these columns, one can identify, purify, and analyze almost any protein. 3 Å column designed for intact protein purification and analysis Separation of proteins, MW Available with C8, C5, and C4 bonded phases Excellent peak shape and resolution of protein samples 9 Å column engineered for peptide mapping and peptide separations Separation of proteins and peptides, MW Identify post-translational modifications Increased peak capacity and resolution Purify Intact Proteins Peptide Maps of Cytochrome c Tryptic Digests BOVINE CANINE EQUINE App ID 875 App ID min The excellent resolving power of Jupiter 5 µm 3 C8 has the ability to separate proteins with very similar compositions and chemical properties. Identify differences between peptide maps of similar proteins, which are typically difficult to resolve, due to the high peak capacity feature of Jupiter Proteo. Jupiter 3 5 µm C8 3 Å Jupiter 4 µm Proteo 9 Å G-453-E G-4396-E A). % TFA in Water B). % TFA in Acetonitrile A).2 % TFA in Water B). % TFA in Acetonitrile Temperature: A/B (75:25) to A/B (45:55) in 5 min (2 % B/min) Ambient 22 nm. Equine Cytochrome c 2. Bovine Cytochrome c 3. Canine Cytochrome c Temperature: A/B (95:5) to A/B (45:55) in 5 min, then to A/B (5:95) in 5 min, then hold at A/B (5:95) for 5 min, then hold at A/B (95:5) for 5 minutes Ambient 2 nm Tryptic digest of Cytochrome c genetic variants see chromatogram for species

5 Dependable Solutions Jupiter HPLC columns and bulk material are used throughout the life science industry in a variety of departments and applications. Phenomenex offers support and solutions in all areas of protein research and manufacturing, especially in characterization, purification, and proteomics/biomarker discovery. Protein Characterization Identify post-translational modifications Analyze intact antibodies and fragments Study PEGylated proteins Analyze Reduced IgG Protein/Peptide Purification Separate target compound from impurities Purify antibodies Separate protein components from one another Easy, direct scale-up to preparative and process scales Purify Away Degradants App ID 499 App ID min min Baseline separation between heavy and light chains of IgG is achieved on Jupiter 5 µm 3 C4. Due to its unique C2, 9 Å chemistry, Jupiter Proteo is able to purify insulin from its degradants ensuring high sample purity. Jupiter 3 5 µm C4 3 Å 5 x 2. mm F-467-B A). % TFA in Water/Acetonitrile (95:5) B).85 % TFA in Acetonitrile/IPA/Water (75:2:5) A/B (8:2) to (5:95) in 2 minutes.25 ml/minute 22 nm IgG Dog Reduced Jupiter 4 µm Proteo 9 Å G-4396-E A). % TFA in Water B).85 % TFA in 95:5 Acetonitrile/Water A/B (8:2) to A/B (2:8) in 5 minutes 22 nm (ambient) Human Insulin Proteomics/ Biomarker Discovery Perform peptide mapping for differential proteomics Identify low level proteins using capillary columns with increased sensitivity Excellent for 2 nd dimension of 2D-HPLC Temperature: 4 C Jupiter 4 µm Proteo 9 Å 5 x.5 mm F-4346-AF A). % TFA in Water B).8 % TFA in Acetonitrile A/B (95:5) for 5 min, then to A/B (55:45) in 55 minutes 5 µl/min 2 nm (ambient) HSA Tryptic Digest Human Serum Albumin Jupiter Proteo resolves many peaks at higher efficiencies, which is critical for peptide maps with a large number of generated peptides. App ID 4335

6 Guaranteed Performance, Lifetime, and Quality It is difficult to compete with Jupiter standards. Jupiter is an extremely robust column with extended ph stability that undergoes rigorous quality testing and has extensive QC documentation. Each column has consistent specifications and thus consistent performance. Long Lifetime and Method Development Opportunities ph.5 stability Stable for over 25 hours at ph extremes Compatible with various LC/MS buffers Excellent resolutions down to. % TFA Effect of LC/MS Modifiers on Selectivity Stability of Jupiter 3 C8 at ph.5 and tfa teaf App ID 439 App ID 4389 Hours of Exposure Test Conditions: Column flushed in 2 mm Na 2 HPO 4 (ph.) in Water/Acetonitrile (5:5) Hours of Exposure Test Conditions: Column flushed in. % TFA (ph.5) in Water/Acetonitrile (5:5) Utilize ph for Method Development of Protein Separations formic acid ph 2. App ID 439 ph 4. Jupiter 4 µm Proteo 9 Å G-4396-E ph 9.5 App ID 8724 A) 5 mm modifier in Water B) 5 mm modifer in Acetonitrile (see chromatograms for modifiers) A/B (95:5) for 5 min, then to A/B (55:45) in 55 minutes Temperature: 22 C 2 nm Myoglobin Tryptic Digest Temperature: Jupiter 3 5 µm C8 3 Å G-453-E Ambient 25 nm. Bradykinin 2. Bombesin 3. Neurotension 4. Eledoisin

7 Guaranteed Performance, Lifetime, and Quality (cont d) Easy Scale-up to Preparative Columns and Bulk Material Identical bonding and base silica technology used in both analytical and preparative materials Large loading capacity Resistance to silica sheering and fine formation at high packing pressures High Mechanical Strength Silica Resists Sheering Jupiter 3 µm C8 3 Å Virgin Bulk Media After Packing in a DAC System Quality Proven Traceability assured throughout the manufacturing process Over 25 individual quality control tests performed on every batch of Jupiter material Materials Validation Document (MVD) accompanies every Jupiter column Material Characterization and Control Reproducibility Assured Guaranteed batch-to-batch and columnto-column reproducibility Tight control maintained over silica particle consistency, size, and smoothness Batch-to-Batch Reproducibility of Jupiter 3 5 µm C8 Silica Physical Tests and Specifications Pore size, particle size and distribution, metal content, surface area, carbon load and surface coverage specifications and results are all reported. Batch 6 Batch 5 Diagnostic Chromatography Tests Monitoring chromatographic specifications for silanol activity, hydrogen bonding capacity, hydrophobicity and peptide standards. ph Stability Every batch goes through ph.5 and. testing before release, the results of which are reported on each MVD. SEM Analysis Scanning Electron Microscopy (SEM) photos show surface smoothness and particle consistency as well as a visual representation of particle size distribution. Temperature: Jupiter 3 5 µm C8 3 Å G-453-E A). % TFA in Water B). % TFA in Acetonitrile A/B (75:25) to A/B (45:55) in 5 minutes Ambient 22 nm (ambient). Equine Cytochrome c 2. Bovine Cytochrome c 3. Canine Cytochrome c Batch 4 Batch 3 Batch 2 Batch

8 Jupiter 3 for Intact Protein Separation and Purification Reversed phase chromatography is a widely used HPLC technique for the separation, purification, and study of proteins as well as the discovery and development of biopharmaceuticals. The popularity of the method can be attributed to the speed and efficiencies typically achieved in its use. Jupiter 3 has proven its performance to chromatographers worldwide as a leading 3 Å solution. Achieve Baseline Resolution Between Proteins of Interest Super-smooth, high-mechanical strength silica reduces silica fine formation during the packing process ensuring highly efficient columns, thus improving resolution Ability to eliminate subsequent purification steps Easier method validation Sharp, Symmetric Peaks Shape for Easier Quantitation Ultra-pure (99.99 %, metal-free) silica and dense bonded phase coverage provides sharp peaks for your sample by decreasing the number of non-specific interactions High peak efficiency, which enables separation of more components Purify Key Proteins from One Another 4 4 Separate Key Components min Jupiter 3 5 µm C8 3 Å 2 Jupiter 3 5 µm C8 has excellent resolving power as seen above. Resolution is extremely important, especially when impurities can differ by only a few amino acids min Vydac 5 µm C8 3 Å App ID 4924 App ID 4925 Separation of Insulin Genetic Variants Bovine, human, and porcine insulin, proteins with very similar structures, are purified away from each other due to the strong resolving power of Jupiter 3. Temperature: Jupiter 3 5 μm C8 3 Å G-453-E A). % TFA in Water B). % TFA in Acetonitrile A/B (7:3) to A/B (68:32) in 2 minutes Ambient 2 nm. Bovine Insulin 2. Human Insulin 3. Porcine Insulin min App ID 5372 Temperature: Jupiter 3 5 µm C8 3 Å Vydac 5 µm C8 3 Å (238EV52-Everest) 25 x 2. mm A). %TFA/ 95 % Water/ 5 % Acetonitrile B).85 % TFA/ 95 % Acetonitrile/ 5 % Water A/B (8:2) to A/B (5:85) in 5 minutes.2 ml/min Ambient 22 nm. Aprotinin 2. Ribonuclease 3. Acid Glycoprotein 4. Fibrinogen 5. Leptin

9 Jupiter 3 for Intact Protein Separation and Purification (cont d) Proven Performance Sharp peaks enable improved resolution for easier quantitation Rugged material ensures long column lifetime and excellent reproducibility Easily separate complex protein mixtures Separation of Protein Mix 35 2 Wheat Protein Extract App ID 537 App ID min Jupiter 3 is capable of separating samples with a wide mixture of proteins that vary in size, chemistry, and concentration. Temperature: Jupiter 3 5 μm C4 3 Å 5 x 4.6 mm B-467-E A:. % TFA in Water B:. % TFA in Acetonitrile a) A/B (:) to A/B (8:2) in min (2 % B/min) b) A/B (8:2) to A/B (65:35) in.5 min ( % B/min) c) A/B (65:35) to A/B (53.5:46.5) in.5 min (7.67 % B/min) d) A/B (53.5:46.5) to A/B (53.5:46.5) for 2 min (constant B) Ambient 22 nm. Alkaline Phosphatase 2. Cyanocobalamin 3. RNase 4. Insulin 5. Transferrin 6. Trypsin Inhibitor (Data supplied by customer. Conditions proprietary.) 5 µm C8, 3 Å,. We purchased the Jupiter 3 C8 3 Å column a few months ago and have been quite impressed with its performance. The Jupiter 3 column provides better separation of the proteins. As for reproducibility, the control profiles have not changed since day one of its use. Recombinant Proteins App ID 74 (Data supplied by customer. Conditions proprietary.) 5 µm C4 3 Å, In comparison to another C4 column for the analysis of a recombinant protein, the Jupiter was much more rugged: typically hundreds of injections.

10 Jupiter 3 for Intact Protein Separation and Purification (cont d) Proven Performance (cont d) Compare PEGylated vs. Native Forms of Proteins Protoporphyrins β-lactoglobin A 8 PEGylated β-lactoglobin A Carbonic Anhydrase II 4 PEGylated Carbonic Anhydrase II Insulin PEGylated Insulin App ID 69 App ID min Reversed phase separation of PEGylated and native proteins on a Jupiter 3 C4 column. Note the good resolution of multiple PEGylated forms for all proteins tested. (Data supplied by customer. Conditions proprietary.) µm C4, 3 Å, 5 x 4.6 mm I found significant improvement in peak shape and symmetry. This was true not only for small peaks, but also for peaks 3 times larger as well. Jupiter 3 5 μm C4 3 Å 5 x 4.6 mm F-467-EO A) 2 % Acetonitrile /. % TFA in Water B) 7 % Acetonitrile /2 % IPA /.8 % TFA in Water A/B (85:5) to A/B (3:7) in 25 min Temperature: 45 C 24 nm PEGylated and Native Proteins Protein Mix Purification 4 First Peak Elutes Away from Void Volume App ID min Aprotinin is typically very difficult to retain. Due to the fact that Jupiter 3 is a high surface material, it has the hydrophobic ability to pull proteins away from the void volume. Temperature: Jupiter 3 5 μm C4 3 Å 25 x 2. mm G-467-BO A:. % TFA/ 95 % Water/ 5 % Acetonitrile B:.85 % TFA/ 95 % Acetonitrile/ 5 % Water A/B (85:5) to A/B (5:85) in 2 minutes.2 ml/min Ambient 22 nm. Aprotinin 2. Ribonuclease 3. Lysozyme 4. Lactalbumin 5. Leptin

11 Jupiter 3 for Intact Protein Separation and Purification (cont d) Selecting the Appropriate 3 Å Phase Jupiter 3 C4 This low hydrophobicity phase is less likely to cause irreversible adsorption of sticky proteins and allows the use of shallow gradients along with lower concentrations of organic solvent. For proteins, MW For highly hydrophobic proteins Large Proteins on Jupiter 3 5 µm C4 Jupiter 3 C5 This bonded phase imparts greater ph stability compared to traditional C4 phases. One can expect longer column lifetimes and more stable, reproducible retention times because of the bonded phase s increased stability to hydrolysis. For proteins, MW For highly hydrophobic proteins More retentive than C4, offering slightly different selectivity App ID 476 Jupiter 3 C8 Excellent for polar as well as non-polar proteins; most retentive of Jupiter 3 phases, allowing one to separate proteins with slight differences in hydrophobicity For proteins, MW For hydrophilic proteins Most retentive phase Temperature: Injection Volume: Jupiter 3 5 μm C4 5 x 4.6 mm F-467-E A:. % TFA in Water B:.8 % TFA in Acetonitrile A/B: (95:5) to A/B: (2:8) in 2 minutes 22 C 28 nm 25 μl. Bovine Serum Albumin 2. Glutamic Dehydrogenase 3. b-galactosidase 4. Ovalbumin Separation on Jupiter 3 5 µm C Temperature: min Jupiter 3 5 µm C8 3 Å 5 x 2. mm F-453-B App ID 4937 A:. %TFA/ 95 % Water / 5 % Acetonitrile B:.85 % TFA/ 95 % Acetonitrile/ 5 % Water A/B (88:2) to A/B (5:85) in 2 minutes.2 ml/min Ambient 2 22 nm. Aprotinin 2. Ribonuclease 3. Lysozyme 4. Lactalbumin 5. Leptin

12 Jupiter 3 for Intact Protein Separation and Purification (cont d) Selecting a Suitable Particle Size Jupiter 3 3 µm Most efficient material currently available in C8 phase Ensures sharp, symmetric peaks resulting in excellent resolution Recommended when 5 µm materials don t give adequate baseline separation or peak shape Separation of Protein Mixture on 3 µm and 5 µm Materials Insulin Purification on 3 µm and 5 µm Materials App ID App ID min min App ID 6449 App ID min min Though the Jupiter 5 µm material provides good separation and peak shape, the 3 µm material dramatically improves resolution, sensitivity, and peak shape. This is critical when proteins closely elute. The highly efficient 3 µm material is able to fully resolve genetic variants of insulin, which is sometimes difficult to resolve on 5 µm materials. Jupiter 3 5 µm C8 3 Å Jupiter 3 3 µm C8 3 Å 5 x 4.6 mm F-453-E F-4263-E A). % TFA and 5 % Acetonitrile in Water B).8 % TFA and 9 % Acetonitrile A/B (8:2) to A/B (5:85) in 5 min (2 % B/min) Temperature: 4 C 24 nm. Ribonuclease A 2. Bovine Insulin 3. Lysozyme 4. Trypsin Inhibitor 5. β-lactoglobulin A Jupiter 3 5 µm C8 3 Å Jupiter 3 3 µm C8 3 Å 5 x 4.6 mm F-453-E F-4263-E A). % TFA and 5 % Acetonitrile in Water B).8 % TFA and 9 % Acetonitrile in Water A/B (7:3) to A/B (68:32) in 5 min 24 nm. Bovine Insulin 2. Human Insulin 3. Porcine Insulin

13 Jupiter 3 for Intact Protein Separation and Purification (cont d) Selecting a Suitable Particle Size (cont d) Jupiter 3 5 µm Designed for optimized combination of efficiency and low backpressure High efficiency materials yields good resolution for most assays Recommended for majority of separations on analytical and capillary scale Scale Up Quickly Between Particle Sizes 5 µm C8 Jupiter 3 µm Designed for preparative applications Highest efficiency for preparative applications Recommended when maximum resolution is required on the prep scale µm C8 Jupiter 3 5 µm Designed for preparative applications Best combination of performance and economy Recommended for most preparative/process applications 5 µm C8 Conditions same for all columns: A:. % TFA in Water B:. % TFA in Acetonitrile A/B (75:25) to A/B (45:55) in 5 min 22 nm. Equine Cytochrome c 2. Bovine Cytochrome c 3. Canine Cytochrome c Scaling up from 5 µm to preparative grades is easier due to the fact that the larger particle sizes are identical to the 5 µm material and are not prep-variants.

14 Jupiter Proteo for Peptide Mapping and Peptide Purification Traditionally, 3 Å columns were used for peptide purification and mapping. 3 Å silica materials are excellent tools for intact proteins, but do not provide the column resolution and peak capacity required for peptide mapping. Jupiter Proteo breaks all traditions and optimizes column parameters to provide optimal performance for peptides. A 9 Å, high surface area silica maximizes stationary phase interaction, a 4 µm particle provides high column efficiencies, and a unique C2 bonded phase with proprietary endcapping sharpens peak symmetry and increases peak capacity. Identify More Peaks with Increased Peak Capacity 4 µm particles produce column efficiencies similar to 3 µm materials, but backpressure of 5 µm particles C2 phase bonded onto a ultra-high surface area (475 m 2 /g) silica increases bonded phase/sample interaction and column capacity Less peak tailing, due to endcapping, means sharper peaks and better separation between closely eluting peptides Myoglobin Tryptic Digest Jupiter Proteo 9 Å Avg peaks Vydac MS C8 3 Å Avg peaks App ID 448 App ID min Due to the unique material characteristics of Jupiter Proteo, peak capacity increases by 4-6 %. An increase in capacity is essential to identifying more peptides in enzymatic digest samples. Zorbax SB C8 3 Å Avg peaks Jupiter 4 µm Proteo 9 Å Vydac 5 µm MS C8 3 Å Zorbax 5 µm SB-C8 3 Å G-467-E A).2 % TFA in Water B). % TFA in Acetonitrile A/B (95:5) for 5 min, then to A/B (6:4) in 55 minutes Temperature: 22 C 2 nm Myoglobin Tryptic Digest App ID 44 Method Threshold Peak Width Min Area Min Height Determining peak counts - The large number of peaks in a given tryptic digest makes counting peaks visually both inaccurate and subjective. For a more accurate approach, peak counting was performed using Agilent Technologies (HP) ChemStation software. Four different integration parameters at different sensitivity settings were used in calculating the number of peaks and an average. The parameters changed within each method were: minimum peak area, minimum peak height, peak width, and threshold. The table describes the parameters used for each calculation.

15 Jupiter Proteo for Peptide Mapping and Peptide Purification (cont d) Better Identification of Post-translational Modifications (PTMs) Better resolution enables easier identification of shifts in peak location to determine if a PTM has occurred 9 Å, 4 µm, C2 Jupiter Proteo materials are engineered for peptide mapping and peak identification Oxidation of β-lactoglobulin Control Oxidation Product App ID 4394 App ID 4395 Jupiter 4 µm Proteo 9 Å G-4396-E A).2 % TFA in Water B). % TFA in Acetonitrile A/B (95:5) for 5 min, then to A/B (6:4) in 55 minutes Temperature: 22 C 2 nm Top Chromatogram β-lactoglobulin tryptic digest Bottom Chromatogram Oxidized β-lactoglobulin tryptic digest Oxidation is commonly seen with methionine due to its readily oxidized sulfur group. A tryptic digest of β-lactoglobulin on Jupiter Proteo easily reveals retention time changes due to the oxidation of methionine containing peptides. Deamidation of RNase Control Deamidation Product App ID 4392 Jupiter 4 µm Proteo 9 Å G-4396-E A).2 % TFA in Water B). % TFA in Acetonitrile A/B (95:5) for 5 min, then to A/B (6:4) in 55 min Temperature: 22 C 2 nm Top Chromatogram RNase tryptic digest Bottom Chromatogram Deamidated RNase tryptic digest App ID 4393 Deamidation of asparagine to aspartic acid and glutamine to glutamic acid can occur in protein products. A tryptic digest of Rnase shows several examples of deamidation. 2

16 Jupiter Proteo for Peptide Mapping and Peptide Purification (cont d) Selectivity to Improve Resolution Jupiter Proteo is engineered with respect to efficiency, selectivity, and resolution to effectively purify peptides Resolve peptides of only -2 amino acid differences Purify contaminants from target peak Resolve Peptides with Similar Hydrophobicity Insulin Purified from its Degradants Jupiter 4 µm Proteo 9 Å Zorbax 5 µm SB-C8 3 Å Vydac 5 µm MS54 3 Å App ID 4428 App ID 4427 App ID ºC min 3 6 ºC App ID 495 App ID min Vydac 5 µm TP54 3 Å App ID ºC Jupiter Proteo is able to fully resolve peptides that differ in hydrophobicity by one methyl group. App ID min Jupiter 4 µm Proteo 9 Å Zorbax 5 µm SB-C8 3 Å Vydac 5 µm MS54 3 Å Vydac 5 µm TP54 3 Å G-467-E A). % TFA B).85 % TFA in Acetonitrile A/B (95:5) to A/B (55:45) in 2 minutes Temperature: 22 C 24 nm. NH2-Arg-Gly-Gly-Ala-Gly-Gly-Leu-Gly-Leu-Gly-Lys-Amide 2. Ac-Arg-Gly-Gly-Gly-Gly-Gly-Leu-Gly-Leu-Gly-Lys-Amide 3. Ac-Arg-Gly-Ala-Gly-Gly-Gly-Leu-Gly-Leu-Gly-Lys-Amide 4. Ac-Arg-Gly-Val-Gly-Gly-Gly-Leu-Gly-Leu-Gly-Lys-Amide 5. Ac-Arg-Gly-Val-Val-Gly-Gly-Leu-Gly-Leu-Gly-Lys-Amide HPLC separations on Jupiter Proteo at different time points of insulin incubated under basic conditions at 6 C. Time points were taken at 5, 3, and 6 minutes. As seen above, there was an increase of numerous degradation products with extended exposure at elevated temperature that Jupiter Proteo was able to separate. Jupiter 4 µm Proteo 9 Å G-4396-E A). % TFA in Water B).85 % TFA in 95:5 Acetonitrile/Water A/B (8:2) to A/B (2:8) in 5 minutes 22 nm (ambient) Human Insulin 3

17 Material Characteristics Packing Material Particle Shape/Size (μm) Pore Size (Å) Pore Volume (ml/g) Surface Area (m 2 /g) Carbon Load % Calculated Bonded Phase Coverage (µmole/m 2 ) End Capping C4 Spher. 5,, Yes C5 Spher. 5,, Yes C8 Spher. 3, 5,, Yes Proteo Spher. 4, Yes ORDERING INFORMATION 4 μm & 5 μm Capillary Columns (mm) Phases 5 x.3 5 x.3 25 x.3 5 x.5 5 x.5 25 x.5 5 μm C4 3 Å B-467-AC F-467-AC G-467-AC B-467-AF F-467-AF G-467-AF 5 μm C8 3 Å B-453-AC F-453-AC G-453-AC B-453-AF F-453-AF G-453-AF 4 μm Proteo 9 Å B-4396-AC F-4396-AC G-4396-AC B-4396-AF F-4396-AF G-4396-AF 3 μm, 4 μm & 5 μm Microbore and Minibore Columns (mm) SecurityGuard Cartridges (mm) Phases 5 x. 5 x. 25 x. 5 x 2. 5 x x 2. 4 x 2.* 5 μm C4 3 Å B-467-A F-467-A G-467-A B-467-B F-467-B G-467-B AJ μm C5 3 Å B-452-A F-452-A G-452-A B-452-B F-452-B G-452-B AJ μm C8 3 Å B-453-A F-453-A G-453-A B-453-B F-453-B G-453-B AJ μm Proteo 9 Å B-4396-A F-4396-A G-4396-A B-4396-B F-4396-B G-4396-B AJO μm C8 3 Å B-4263-B F-4263-B AJO-432 for ID: mm 3 μm, 4 μm & 5 μm Analytical and Preparative Columns (mm) SecurityGuard Cartridges (mm) Phases 5 x x x x 25 x x 3.* x 5 x 2.2** 5 μm C4 3 Å B-467-E F-467-E G-467-E B-467-N F-467-P AJO-433 AJO-7225 AJO μm C5 3 Å B-452-E F-452-E G-452-E B-452-N F-452-P AJO-4327 AJO μm C8 3 Å B-453-E F-453-E G-453-E B-453-N F-453-P AJO-432 AJO-7224 AJO μm Proteo 9 Å B-4396-E F-4396-E G-4396-E B-4396-N F-4396-P AJO-674 AJO-7275 AJO μm C8 3 Å F-4263-E G-4263-E AJO-432 for ID: mm 9-6 mm 8-29 mm μm Analytical and Preparative Columns (mm) SecurityGuard Cartridges (mm) Phases $ 56 $ 56 $ 56 $ 56 $ 56 $ 56 If Jupiter does not provide you with at least an equivalent separation as compared to a column of similar phase, particle size and dimension, send in your comparative data within 45 days and keep the Jupiter column for FREE. $ 435 $ 56 $ 595 $ 435 $ 56 $ 595 $ 299/ pk $ 445 $ 57 $ 299/ pk $ 435 $ 56 $ 595 $,28 $ 2,75 $ 299/ pk $ 439/3 pk $ 299/ea $ 57 $ 65 $ 299/ pk /3 pk /ea *SecurityGuard Analytical Cartridges require holder, KJ-4282 SemiPrep SecurityGuard Cartridges require holder, AJ-722 **PREP SecurityGuard Cartridges require holder, AJ-8223 t PREP SecurityGuard Cartridges require holder, AJ x x 25 x x 3 25 x 5 4 x 3.* x 5 x 2.2** 5 x 3. t $ 385 $ 95 $ 2,399 Inquire Inquire $ 299/ pk $ 435/3 pk $ 299/ea $ 325/ea C4 3 Å G-468-E G-468-N G-468-P G-468-U G-468-V AJO-433 AJO-7225 AJO-723 AJ-834 C5 3 Å G-454-E G-454-N G-454-P G-454-V AJO-4327 AJO-737 C8 3 Å G-455-E G-455-N G-455-P G-455-U G-455-V AJO-432 AJO-7224 AJO-723 AJ-833 Proteo 9 Å G-4397-E G-4397-N G-4397-P G-4397-U G-4397-V AJO-674 AJO-7275 AJO-7842 AJ-834 for ID: mm 9-6 mm 8-29 mm 3-49 mm 5 μm Analytical and Preparative Columns (mm) SecurityGuard Cartridges (mm) Phases 25 x x 25 x x 3 25 x 5 4 x 3.* x 5 x 2.2** 5 x 3. t C4 3 Å G-469-E G-469-N G-469-P G-469-U G-469-V AJO-433 AJO-7225 AJO-723 AJ-834 C5 3 Å G-456-E G-456-N G-456-P G-456-V AJO-4327 AJO-737 C8 3 Å G-457-E G-457-N G-457-P G-457-U G-457-V AJO-432 AJO-7224 AJO-723 AJ-833 for ID: mm 9-6 mm 8-29 mm 3-49 mm Other Dimensions available upon request. Bulk Material available. $ 289 $ 699 $,699 Inquire Inquire $ 299/ pk $ 439/3 pk $ 299/ea $ 325/ea 4

18 Phenex Syringe Filters & Membranes For Sample and Solvent Filtration Prior to Chromatography! Phenex Syringe Filters Rapid filtration of HPLC samples prior to analysis Particulate, PVC, and extractable-free filters Consistent, reliable performance Phenex Features Low absorption Particulate-free Maximum chemical compatibility Minimum extractables Excellent flow rate High total throughput Ordering Information Low hold-up volume Certified quality % integrity tested Easy pore identification PVC-free Bi-directional use Tip: Try a Sample Pack! The best way to determine if a specific Phenex membrane is suitable for your application. Request yours today by phone or visit Phenex Syringe Filters 4 mm Diameter mm Diameter mm Diameter for < 2 ml sample volumes for 2 - ml sample volumes for - ml sample volumes Membrane Type/Size Part No. Unit Price Part No. Unit Price Part No. Unit Price.45 µm Phenex-RC 3 AF-33-2 /Pk $ 35 AF-23-2 /Pk $ 55 AF /Pk $ 95 (Regenerated Cellulose) AF /Pk 54 AF /Pk 62 AF /Pk 78 Phenex-PES 3 AF /Pk 8 AF /Pk 25 (Polyethersulfone) AF /Pk 86 Phenex-PTFE 6 AF-32-2 /Pk 45 AF-22-2 /Pk 8 AF-2-2 /Pk 25 (Polytetrafluoroethylene) AF /Pk 58 AF /Pk 72 AF /Pk 86 Phenex-NY AF /Pk 4 AF /Pk 6 AF-7-2 /Pk 25 (Nylon) AF /Pk 56 AF /Pk 64 AF /Pk 82 Phenex-GF/CA 2,3,4 An integrated syringe filter unit containing an inert borosilicate glass fiber prefilter and a CA membrane. AF-8B9-2 /Pk 245 (Glass Fiber/Cellulose Acetate) Excellent for filtration of tissue culture media, general biological sample filtration and clarification. AF-8B9-52 5/Pk 98.2 µm Phenex-RC 3 AF /Pk $ 35 AF /Pk $ 55 AF /Pk (Regenerated Cellulose) AF /Pk 54 AF /Pk 62 AF /Pk Phenex-PES 3 AF /Pk (Polyethersulfone) AF /Pk Phenex-PTFE 6 AF /Pk 45 AF /Pk 8 AF-22-2 /Pk (Polytetrafluoroethylene) AF /Pk 58 AF /Pk 72 AF /Pk Phenex-NY AF /Pk 4 AF /Pk 6 AF-27-2 /Pk (Nylon) AF /Pk 56 AF /Pk 64 AF /Pk Phenex-GF/CA 2,3,4 An integrated syringe filter unit containing an inert borosilicate glass fiber prefilter and a CA membrane. AF-8A9-2 /Pk (Glass Fiber/Cellulose Acetate) Excellent for filtration of tissue culture media, general biological sample filtration and clarification. AF-8A9-52 5/Pk.2 µm Phenex-GF 2 Prefiltration of heavily contaminated or highly viscous samples. When used in-line preceding AF /Pk (Glass Fiber) a membrane filter, clogging of the membrane filter is prevented and sample clean up is optimized. AF /Pk $ $ mm diameter. 2. Glass fiber filters are 26 mm diameter and made of borosilicate. They will remove 9 % of all particles >.2 µm. 3. Housing material is methacrylate butadiene styrene (MBS) polymerisate. Also known as Cryolite. 4. Cellulose acetate is surfactant-free mm diameter. 6. Hydrophobic membrane. Can be made hydrophilic by pre-wetting with IPA. 7. Additional dimensions and membrane types are available. Please contact your local Phenomenex technical consultant or distributor for availability or assistance. 8. Larger quantity purchases at significant savings are available. If Phenex Syringe Filters do not perform as well or better than your current syringe filter product of similar membrane, diameter and pore size, send in your comparative data within 45 days and keep the Phenex products for FREE!

19 Jupiter is a registered trademark of Phenomenex, Inc. Phenex is a trademark of Phenomenex, Inc. Zorbax is a registered trademark of Agilent Technologies. Vydac is a registered trademark of Alltech Associates, Inc. Columns used for comparison were purchased directly from original manufacturer. Comparative separations may not be representative of all applications. 8 Phenomenex, Inc. All rights reserved. Subject to Phenomenex Standard Terms & Conditions which may be viewed at www. Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or international@. Australia PO Box 484 Lane Cove, NSW 266 Australia tel.: fax: auinfo@ Austria Zeppelinstr Aschaffenburg Germany anfrage@ Canada 4 Madrid Ave. Torrance, CA USA (8) (3) info@ Denmark Gydevang Allerød Denmark dkinfo@ France Parc des Grillons, Bat.3 6 route de Sartrouville Le Pecq Cedex France franceinfo@ Germany Zeppelinstr Aschaffenburg Germany anfrage@ Ireland Queens Avenue, Hurdsfield Ind. Est., Macclesfield, Cheshire SK 2BN, UK tel.: fax: eireinfo@ Italy Via Serenari, 5/D 43 Castel Maggiore (BO) Italy italiainfo@ New Zealand P O Box 3-6 Milford 74 North Shore City New Zealand nzinfo@ Puerto Rico 273 Sierra Morena, Suite #4 San Juan, Puerto Rico 926 (8) 54-HPLC (3) info@ United Kingdom Queens Avenue, Hurdsfield Ind. Est., Macclesfield, Cheshire SK 2BN, UK ukinfo@ USA 4 Madrid Ave. Torrance, CA USA (3) (3) info@

20 4 Madrid Avenue Torrance, CA USA PRSRT STD U.S. POSTAGE PAID TORRANCE, CA PERMIT NO. 85 i d e n t i f y P u r i f y A n a ly z e Reversed Phase HPLC Solutions for Proteins and Peptides 562_us