• Fast separations • High resolution & high peak capacity • Low back pressure
MARCH 2014
• High temperature stability • Rugged, reliable performance • Use with UHPLC or HPLC equipment
Defining a new direction in HPLC & UHPLC separation
of Peptides & Proteins.
HALO Peptide and HALO Protein columns are packed with “core-shell” type particles, but not ordinary core-shell particles. HALO particles are produced with groundbreaking Fused-Core particle technology that allows for the production of superficially porous particles with carefully controlled pore size, particle size and porous layer thickness. Control over these parameters is essential for producing columns with high efficiency and dependable, reproducible performance.
HALO ProteinShell with 400 Å pores Shell with 160 Å pores Shell with 160 Å pores
HALO Peptide HALO-5 Peptide
0.6 µm3.4 µm
As research in proteomics has intensified, more demands have been placed on HPLC/UHPLC methods of analysis. Improved resolution to better analyze complex samples is required, as is faster separation to speed-up research and improve lab productivity. Fused-Core® particle technology used in the manufacture of HALO columns has facilitated the development of a class of columns specifically engineered to meet these demands. HALO BioClass columns not only provide higher resolution and faster separations than traditional HPLC columns, they also provide a durability that is unexpected from columns with such a high level of performance.
HALO BioClass columns are packed with “core-shell” type particles with the expected advantages of high resolution, fast separations, and low back-pressure. HALO began the revolution in “core-shell” types of columns and the proven value of groundbreaking Fused-Core particle technology continues to drive the rapid growth in the use of these types of UHPLC and HPLC columns.
HALO Protein and HALO Peptide columns are specifically designed for fast, high-res-olution separation of peptides and proteins. Specifically selected pore sizes, optimized porous shell thickness and particle size, and the use of extraordinary stable bonding chem-istry provides optimum separation of peptides up to about 5–10 kDa (HALO Peptide) and proteins and polypeptides in the range of 2 kDa up to 500 kDa (HALO Protein). The low back-pressure of these columns permits them to be used with either UHPLC or conventional HPLC equipment.
Introduction
4.6 µm
Solid CoreSolid Core
Solid Core
1 HALO Fused-Core particles
0.5 µm1.7 µm
2.7 µm
0.2 µm3.0 µm
3.4 µm
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HALO-5 Peptide ES-C18 provides improved resolution compared to a typical totally-porous wide-pore column
Achieve unusually high peak capacity by connecting HALO Peptide columns in series.
A HALO-5 Peptide ES-C18 column provides significantly sharper peaks than a typical totally porous “wide-pore” column (nominal 300 Å pore size). The thin porous shell allows solute molecules to quickly move in and out of particles, resulting in sharper peaks compared to totally porous particles of the same overall diameter.
HALO Peptide columns provide high peak capacity because of reduced band broadening resulting, in part, from short diffusion paths in the porous shell. Where unusually high peak capacity is needed, HALO Peptide columns can be connected together in series. In the example here, three 150 x 2.1 mm HALO Peptide ES-C18 columns were connected together to achieve a peak capacity of 560 for this mixture of α-1-glycoprotein tryptic digest and apotransferrin tryptic digest. The low back pressure of the HALO Peptide columns and the tolerance to high temperatures of the steric-protected C18 bonded phase permit two or more columns to be connected together without exceeding the pressure limit of an HPLC system.
2 3Sample:1. Asp-Phe2. Tyr-Tyr-Tyr3. Bradykinin4. Angiotensin II
5. Neurotensin6. β-endorphin7. Sauvagine8. Mellitin
Sample:Tryptic digest of α-1-glycoprotein and apotransferrin
Conditions: Column Dimensions: 4.6 x 150 mmMobile Phase: A = Water with 0.1% TFA, B = Acetonitrile with 0.1% TFAGradient: 5 to 60% B, 5 minutesFlow Rate: 4.0 mL/minTemperature: 40 ºC
Conditions: Column Dimensions: 2.1 x 450 mm (Three HALO Peptide ES-C18 columns connected in series)Mobile Phase: A = Water with 0.1% formic acid/20 mM ammonium formate, pH 3.6B = 20% A, 80% Acetonitrile Gradient: 5 to 55% B, 150 min.Flow Rate: 0.5 mL/min.Temperature: 70 ºC
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Time (minutes)
Time (minutes)
Time (minutes)0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
HALO-5 Peptide ES-C18 Peak capacity: 94Pressure: 313 bar
Totally Porous 5 μm, 300 Å, C18Peak capacity: 37Pressure: 331 bar
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peak width=0.015 2
Peak capacity: 560
peak width=0.022 2
peak width=0.022 8
peak width=0.064 8
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High resolution and high peak capacity
Optimum pore size, particle size, porous layer thickness and bonded phase chemistry provides high resolution separations.
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Fast SeparationsExtraordinarily high column efficiency and the short diffusion path length of HALO Fused-Core particles facilitate ultra-fast separations.
Fast separation of peptides and small proteins
The extraordinary high efficiency and short diffusion path length of HALO columns permit the use of short columns and high mobile phase velocity to achieve ultra-fast separations. This example shows the sep-aration of 11 peptides and small proteins in less than 60 seconds using a 50 mm length column and a mobile phase velocity of 0.8 cm/sec. Although it is advisable that typical totally porous particle columns not be operated at mobile phase velocities greater than 0.5 cm/sec due to excessive loss in resolution, HALO columns, because of their Fused-Core particle technology, can be operated at high mobile phase velocities without sacrificing resolution.
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Sample:1. Gly-Tyr2. Val-Tyr-Val3. Angiotensin 1 and 2 (1-7) amide4. Met-enk5. Angiotensin 1 and 2 (1-8) amide
6. Angiotensin II7. Leu-enk8. Ribonuclease A9. Angiotensin (1-12) (human)10. Angiotensin (1-12) (mouse)11. Porcine insulin
Conditions: Column: HALO Peptide ES-C18, 4.6 x 50 mmMobile Phase: A = 90% Water with 0.1% TFA and 10% Acetonitrile, B = 70% Acetonitrile and 30% Water with 0.1% TFAGradient: 0 to 87.5% B, 1 minuteFlow Rate: 5.0 mL/minTemperature: 60 ºC
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Time (minutes)
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89
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Fast separation of proteins
This example shows a fast, high-resolution separation of seven proteins in less than 8 minutes on a 100 mm length HALO Protein C4. Note that the pressure is only 109 bar for this fast separation due to the optimized 3.4 μm particle size.
5Sample:1. Cytochrome c (12.4 kDa)2. Lysozyme (14.3 kDa)3. α-Chymotrypsin (25 kDa)4. Catalase (250 kDa)5. Carbonic anhydrase (29 kDa)6. Enolase (46.7 kDa)7. β-Amylase (200 kDa)
Conditions: Column: HALO Protein C4, 2.1 x 100 mmMobile Phase: A = Water with 0.1% TFA, B = 80% Acetonitrile and 20% Water with 0.1% TFAGradient:Time %B0 min. 352 min. 47.58 min. 60Flow Rate: 0.5 mL/minTemperature: 60 ºC Pressure: 109 bar
0 1 2 3 4 5 6 7 8Time (minutes)
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HALO Protein columns provide fast separations at low back pressure
The low pressure of HALO Protein columns permit them to be operated at high flow rates to achieve fast, high-resolution separations without incurring unacceptably high pressure. In this example, a typical UH-PLC-type column run at a flow rate of 1.1 mL/min generates 570 bar pressure. The HALO Protein column on the other hand, is operated at twice the flow rate, 2.2 mL/min, and accomplishes the separation in half the time while generating a more comfortable pressure of 455 bar.
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Low back pressureHALO UHPLC columns, by virtue of their optimized particle sizes, generate significantly less back pressure compared to sub-2 μm UHPLC columns. This lower pressure puts less stress on equipment and facilitates more trouble free operation. It is this moderate back pressure of HALO columns that also permits them to be used with conventional HPLC equipment as well as UHPLC equipment.
Sample:1. Ribonuclease A2. Cytochrome c3. Lysozyme4. α-Lactalbumin5. Catalase6. Enolase
The different selectivities of the HALO Peptide ES-C18 and HALO Peptide ES-CN columns can be leveraged to achieve better separations.
A HALO Peptide ES-C18 column is generally the column to choose for separating peptides. However, there may be occasions, as these comparison chromatograms illustrate, where the different selectivity offered by the HALO Peptide ES-CN will make it the best choice for a separation. Peak pairs 2/3 and 5/6 are poorly separated on the HALO Peptide ES-C18, but are well separated on the HALO Peptide ES-CN.
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HALO-5 Peptide ES-CN
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Sample:1. Asp-Phe2. Angiotensin (1–7) amide3. Tyr-Tyr-Tyr4. Bradykinin
5. Leu-Enk 6. Angiotensin II7. Neurotensin8. β-endorphin 9. Sauvagine 10. Mellitin
Conditions: Column Dimensions: 4.6 x 150 mmMobile Phase: A=Water with 0.1% TFA, B=Acetonitrile with 0.1% TFAGradient: 5 to 50% B, 30 minutesFlow Rate: 1.0 mL/minTemperature: 40 ºC
0 5 10 15 20 25 30
0 5 10 15 20 25 30
Time (minutes)
Time (minutes)
HALO-5 Peptide ES-C18
Conditions: Column Dimensions: 2.1 x 100 mmMobile Phase: A = Water with 0.1% TFA, B = Acetonitrile with 0.1% TFAGradient: As labeled on the chromatogramsFlow Rate: As labeled on the chromatogramsTemperature: 60 ºC
HALO Protein C4Flow rate: 2.2 mL/minGradient: 23 to 52% B, 2.5 minutes Pressure: 455 bar
Totally Porous 1.7 μm, 300 Å, C4Flow rate: 1.1 mL/minGradient: 23 to 52% B, 5 minutes Pressure: 570 bar
0.0 0.5 1.0 1.5 2.0 2.5
Time (minutes)
peak width=0.010 2
peak width=0.014 5
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0.0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Time (minutes)
peak width=0.018 2
peak width=0.030 5
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Effect of temperature on protein recovery and peak shape HALO Peptide and HALO Protein columns demonstrate outstanding temperature stability
These overlaid chromatograms illustrate the dramatic improvement in the peak shape and recovery of proteins that takes place as the column temperature is increased. The benefits of operating at elevated tempera-ture are only valuable if the column is stable. As shown in Figure 8, the HALO Protein columns are extremely stable at elevated temperature.
In a test designed to observe the effect of high temperature on bonded phase stability, both the HALO Protein C4 and the HALO Protein ES-C18 columns demonstrated outstanding durability. Similar temperature stability tests performed on HALO Peptide ES-C18 and HALO Peptide ES-CN columns also showed outstanding durability for these columns.
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High temperature stability
The temperature stability of HALO Peptide and HALO Protein columns allows them to be used at elevated temperature to improve resolution or to provide better protein recovery and peak shape. Operating at elevated column temperature is espe-cially valuable when analyzing large hydrophobic proteins.
Sample:1. Lysozyme2. BSA3. α-Chymotrypsinogen A4. Enolase5. Ovalbumin
Conditions: Column: HALO Protein C4, 2.1 x 100 mmMobile Phase: A = Water with 0.1% TFA, B = Acetonitrile with 0.1% TFAGradient: 28 to 58% B, 10 minutesFlow Rate: 0.45 mL/minDetection: uv, 215 nm
Conditions: Column Dimensions: 2.1 x 100 mmMobile Phase: A = Water with 0.1% TFA, B = Acetonitrile with 0.1% TFATemperature: 90 ºCGradient for HALO Protein C4: 25 to 40% B, 10 minutesFlow Rate for HALO Protein C4: 0.5 mL/minGradient for HALO Protein ES-C18: 25 to 45% B, 5 minutesFlow Rate for HALO Protein ES-C18: 1.0 mL/min
Column Volumes
EnolaseEnolaseCatalase
CatalaseApomyoglobin
α-Lactalbumin
Lysozyme Lysozyme
Cytochrome cCytochrome c
Column Volumes
Ret
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(min
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Ret
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(min
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5HALO Protein C4 HALO Protein ES-C18
0 2500 5000 7500 10000 12500 15000
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Time (minutes)
30 °C
60 °C
90 °C
HALO Peptide Specifications HALO Protein Specifications
STATIONARY PHASE SUPPORT, PEPTIDE • 2.7 µm or 4.6 µm diameter spherical, ultra-pure, “Type B” silica • Porous silica layer fused to the surface of a solid core • 160Å pore size
STATIONARY PHASE SUPPORT, PROTEIN • 3.4 µm diameter spherical, ultra-pure, “Type B” silica • Porous silica layer fused to the surface of a solid core • 400Å pore size
BONDED PHASE: HALO PEPTIDE ES-C18 • Steric-protected C18, octadecyldiisobutylsilane • No endcapping • pH range: 1 to 8 • Maximum Temperature: 90 ºC
BONDED PHASE: HALO PROTEIN ES-C18 • Steric-protected, octadecyldiisobutylsilane • Fully endcapped • pH range: 1 to 9 • Maximum Temperature: 90 ºC
BONDED PHASE: HALO PEPTIDE ES-CN • Steric-protected CN, cyanopropyldiisopropylsilane • Fully endcapped • pH range: 1 to 8 • Maximum Temperature: 90 ºC
BONDED PHASE: HALO PROTEIN C4 • Butyldimethylsilane • Fully endcapped • pH range: 1 to 9 • Maximum Temperature: 90 ºC
Maximum Recommended Pressure: 9,000 psi, 600 Bar Maximum Recommended Pressure: 9,000 psi, 600 Bar
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Guard Columns, 3 pk
HALO BioClass Columns
HALO BioClass Part Numbers
Dimensions (mm) HALO-5 Peptide ES-C18
HALO-5 Peptide ES-CN HALO Peptide ES-C18 HALO Peptide ES-CN HALO Protein C4 HALO Protein ES-C18
0.075 x 50 91529-402 91529-404 91229-402 91229-404 94319-414 94319-402
0.075 x 150 91529-702 91529-704 91229-702 91229-704 94319-714 94319-702
0.10 x 50 91528-402 91528-404 91228-402 91228-404 94318-414 94318-402
0.10 x 150 91528-702 91528-704 91228-702 91228-704 94318-714 94318-702
0.20 x 50 91527-402 91527-404 91227-402 91227-404 94317-414 94317-402
0.20 x 150 91527-702 91527-704 91227-702 91227-704 94317-714 94317-702
0.30 x 50 91526-402 91526-404 91226-402 91226-404 94316-414 94316-402
0.30 x 150 91526-702 91526-704 91226-702 91226-704 94316-714 94316-702
0.50 x 50 91525-402 91525-404 91225-402 91225-404 94315-414 94315-402
0.50 x 150 91525-702 91525-704 91225-702 91225-704 94315-714 94315-702
1.0 x 30 95121-302 95121-304 92121-302 92121-304 93411-314 93411-302
1.0 x 50 95121-402 95121-404 92121-402 92121-404 93411-414 93411-402
1.0 x 100 95121-602 95121-604 92121-602 92121-604 93411-614 93411-602
1.0 x 150 95121-702 95121-704 92121-702 92121-704 93411-714 93411-702
2.1 x 20 95122-202 95122-204 92122-202 92122-204 93412-214 93412-202
2.1 x 30 95122-302 95122-304 92122-302 92122-304 93412-314 93412-302
2.1 x 50 95122-402 95122-404 92122-402 92122-404 93412-414 93412-402
2.1 x 75 95122-502 95122-504 92122-502 92122-504 93412-514 93412-502
2.1 x 100 95122-602 95122-604 92122-602 92122-604 93412-614 93412-602
2.1 x 150 95122-702 95122-704 92122-702 92122-704 93412-714 93412-702
2.1 x 250 95122-902 95122-904
3.0 x 30 95123-302 95123-304 92123-302 92123-304 93413-314 93413-302
3.0 x 50 95123-402 95123-404 92123-402 92123-404 93413-414 93413-402
3.0 x 75 95123-502 95123-504 92123-502 92123-504 93413-514 93413-502
3.0 x 100 95123-602 95123-604 92123-602 92123-604 93413-614 93413-602
3.0 x 150 95123-702 95123-704 92123-702 92123-704 93413-714 93413-702
3.0 x 250 95123-902 95123-904
4.6 x 30 95124-302 95124-304 92124-302 92124-304 93414-314 93414-302
4.6 x 50 95124-402 95124-404 92124-402 92124-404 93414-414 93414-402
4.6 x 75 95124-502 95124-504 92124-502 92124-504 93414-514 93414-502
4.6 x 100 95124-602 95124-604 92124-602 92124-604 93414-614 93414-602
4.6 x 150 95124-702 95124-704 92124-702 92124-704 93414-714 93414-702
4.6 x 250 95124-902 95124-904
10 x 50 95110-402 95110-404 92110-402 92110-404
10 x 100 95110-602 95110-604 92110-602 92110-604
10 x 150 95110-702 95110-704 92110-702 92110-704
10 x 250 95110-902 95110-904
Dimensions (mm) HALO-5 Peptide ES-C18
HALO-5 Peptide ES-CN HALO Peptide ES-C18 HALO Peptide ES-CN HALO Protein C4 HALO Protein ES-C18
2.1 x 5 95122-102 95122-104 92122-102 92122-104 93412-114 93412-102
3.0 x 5 95123-102 95123-104 92123-102 92123-104 93413-114 93413-102
4.6 x 5 95124-102 95124-104 92124-102 92124-104 93414-114 93414-102
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94900-001Guard Column Holder
® HALO and Fused-Core are registered trademarks of Advanced Materials Technology, Inc.
Achrom NV/SA Philippostraat 5, 9870 Machelen
Tel: +32/9-380 06 47 | Fax: +32/9-386 08 86
[email protected] | www.achrom.be
