Accucore HPLC Columns for Biomolecule ... - Thermo Fisher Scientific
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Ultimate Core Performance
to Maximize Your Investment
Core Enhanced Technology – for Biomolecules The range of Accucore™ HPLC columns packed with 150 Å pore diameter particles allow biomolecule separations to benefit from the superb resolution and high speed enabled by the Core Enhanced Technology™ that Accucore is based on.
The key components of Core Enhanced Technology are: Solid Core Particles
Tight Control of Particle Diameter
2.6 µm diameter particles with a solid core generate high speed, high resolution separations without excessive backpressure
Enhanced selection process keeps particle size distribution to a minimum and produces high efficiency columns
Automated Packing Process
Advanced Bonding Technology
Enhanced automated procedures ensure that all columns are packed with the highest quality
Optimized phase bonding creates a series of high coverage, robust phases
Fast Separations High Peak Capacity Increased Sensitivity Lower Pressure Good Loading Capacity Reproducible Chromatography Long Lifetime Peptide and Protein Analysis
150 Å Solid Core Particles
Tight Control of Particle Diameter
Advanced Bonding Technology
Automated Packing Process
• • • •
• • • • • • •
• • •
• • • • • • •
• •
• • • •
The table above shows how the components of Core Enhanced Technology with 150 Å pore diameter particles contribute to the outstanding features of Accucore HPLC columns for biomolecule separations.
Table of Contents Why Core Enhanced Technology Works
2
150 Å Pore Diameter Particles
3
Column Formats
4
Analytical and Micro Columns. . . . . . . . . . . . . . . . . . . . 4 Defender Guard Cartridges . . . . . . . . . . . . . . . . . . . . . 4 nanoViper Columns . . . . . . . . . . . . . . . . . . . . . . . . . 4 Biomolecule Phases 5 Accucore 150-C18 Column
6
Peptide Separations
6
High Peak Capacity . . . . . . . . . . . . . . . . . . . . . . . . . 7 Reproducible Separations . . . . . . . . . . . . . . . . . . . . . . 7 Excellent Loading Capacity . . . . . . . . . . . . . . . . . . . . . 8 9 Accucore 150-C4 Column Protein Separation
9
Excellent Resolution . . . . . . . . . . . . . . . . . . . . . . . . 10 Reproducible Results . . . . . . . . . . . . . . . . . . . . . . . 10 Proven Loading Capacity. . . . . . . . . . . . . . . . . . . . . . 11 Thermo Scientific nanoViper Column Separations 12 Ordering Information Accucore HPLC Columns. . . . . . . . . . . . . . . . . . . . . . 13 Accucore Defender Guard Cartridges . . . . . . . . . . . . . . . 13
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
1
Why Core Enhanced Technology Works Two factors that strongly affect chromatographic efficiency are mass transfer and eddy diffusion, the C and A terms respectively from the Van Deemter equation.
H=A+
B + Cu u
H Height equivalent to theoretical plate (column length/efficiency)
A B C u
Eddy diffusion Longitudinal diffusion Resistance to mass transfer
Resistance to mass transfer is minimized by the solid core design of Accucore particles as the diffusional path of analytes is limited by the depth of the outer porous layer.
Mobile phase linear velocity
Material
Accucore 2 6 μm
Particle Size Distribution (D90/10)
1 15
The tight control of particle diameter and automated packing process used for Accucore columns result in a tight, highly uniform packed bed that minimizes eddy diffusion.
Lower Backpressure L h F dp2 dc2
Column length (cm) Mobile phase viscosity (cP) Flow rate (mL/min) Particle diameter (μm)
DP ~
250LhF dp2dc2
Column diameter (cm)
The equation above shows how backpressure is related to particle diameter. The tightly controlled 2.6 µm diameter of Accucore particles also results in lower backpressures than typically seen with sub-2 µm materials.
2
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
150 Å Pore Diameter Particles The 150 Å pore diameter solid core particles used in the Accucore 150-C18 and 150-C4 columns are designed specifically to provide the optimum combination of retention and resolution for peptides and proteins. The chromatograms below show the improved resolution and peak shapes achieved with Accucore 150-C18 compared to a C18 solid core phase with an 80 Å pore diameter.
Accucore 150-C18
1.12
3.76
7.09
8.74
13.46
8.91 11.20
Conventional Solid Core C18
1.01 8.52 6.98
3.60
13.13 10.84
Improved resolution Improved peak shape
Peak Capacity
150
Peak Capacity Comparison
140 130 120 110 100 90
Sharper peaks result in a significantly higher peak capacity for 150-C18 compared to the conventional solid core C18 column.
80 70 60 Accucore 150-C18
Conventional Solid Core C18
Stationary Phase
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
3
Column Formats Accucore HPLC columns for biomolecules are offered in both analytical, micro and nano formats. Optimum conditions and backpressure ratings are shown in the table below.
Format
Column ID
Optimum Flow Rate
Optimum Backpressure Injection Volume Rating
Temperature Rating
Nano
75 μm
300 nL/min
1 μL
800 bar
70 °C
Micro
2.1 mm
400 μL/min
1 μL
1000 bar
70 °C
Analytical
3.0 mm
800 μL/min
3 μL
1000 bar
70 °C
Analytical
4.6 mm
1800 μL/min
5 μL
1000 bar
70 °C
Analytical and Micro Columns Accucore HPLC columns are packed into our high pressure hardware. These stainless steel columns are engineered to the highest quality and have a pressure rating of 1000 bar.
Thermo Scientific Defender Guard Cartridges Guard columns are designed to protect your column from particulates introduced from the matrix or instrument and from any strongly retained components in the injected sample. Defender™ Guard Cartridges have been designed specifically to work with high speed, high efficiency separations.
nanoViper Columns The nanoViper fingertight connection system for nanoLC connections eliminates the assembly of PEEK sleeve connections. It is preassembled and fingertight to maximize ease-of-use. The nanoViper fitting is capable of withstanding pressures up to a 1000 bar and is compatible with third party valves and unions. Accucore nanoViper columns are available in 150 and 500 mm lengths – for ultra-high peak capacity.
4
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Biomolecule Phases Like all other Accucore products, the 150 Å pore size particles and the phases based on them are fully characterized using the suite of tests shown below.
Silica Support Properties
Bonded Phase Properties
Hydrophobic Interactions
Secondary Interactions
Acidic Interactions
Surface Area
Carbon load
Hydrophobic retention
Base activity
Acid interaction
HR
BA
Hydrophobic selectivity
Pore Size
Particle Size
Steric selectivity
C
IEX(2.7)
Ion exchange capacity (pH 7.6)
SS Particle Size Distribution
Ion exchange capacity (pH 2.7)
Chelation
HS
AI
IEX(7.6)
Hydrogen bonding capacity
HBC
Details, including the phase characterization, are given below for the Accucore biomolecule phases. HR/10 AI
HS
IEX (2.7)
SS
C
HBC
Phase characteristics are designed for the separation of peptides
IEX (7.6)
BA
Accucore 150-C18
HR/10 AI
HS
IEX (2.7)
SS
Accucore 150-C4 C
HBC
BA
IEX (7.6)
Lower hydrophobic retention, optimal retention of proteins and larger peptides
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
5
Accucore 150-C18 HR/10
Hydrophobicity
AI
Low
HS
pH Range 0
1 to 11
14
IEX (2.7)
Pore Size 0
150 Å
SS
300
Carbon Load (%)
C 0
7
HBC
25
Particle Size 2.6 µm
USP L1
BA
•
Designed for the separation of peptides
•
Outstanding resolution
IEX (7.6)
Peptide Separations Accucore 150-C18
240000 220000 200000 180000 160000
mAU
140000 1
120000 100000 80000
2
60000
4
3
Mobile phase A: 0.1 % TFA in 10:90 acetonitrile:water
7
5
Mobile phase B: 0.1 % TFA in 70:30 acetonitrile:water
6
40000
Gradient:
0-50 % B over 15 min; hold for 2 min; drop to 0 % in 0.1 min; hold at 0 % B for 5 min
Flow:
300 µL/min
Temperature:
35 °C
Column:
Accucore 150-C18 2.6 µm, 100 x 2.1 mm
Injection:
5 µL
Detection:
UV (220 nm)
20000 0 -20000 -40000 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
Peak Number
6
15
tR/min
Peptide
MW
1
1.12
Glycine-Tyrosine
238.24 Da
2.0
2
3.76
Valine-Tyrosine-Valine
379.45 Da
17.0
3
7.09
Met-Enkephalin
573.66 Da
21.0
4
8.74
Angiotensin III
931.09 Da
15.0
5
8.91
Leu-Enkephalin
569.65 Da
21.0
6
11.20
Ribonuclease A
~ 13700 Da
42.5
7
13.46
Insulin
5733.49 Da
30.0
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Concentration (µg/mL)
High Peak Capacity Higher peak capacities facilitate increased peptide identifications. Accucore 150-C18 provides much narrower peak widths, therefore significantly higher peak capacity than a column packed with < 2 µm wide pore fully porous C18. Accucore 150-C18 Backpressure: 175 bar
146
Peak Capacity
Conventional Solid Core C18 Backpressure: 170 bar
Peak Capacity Comparison
144 142 140 138 136 134 132 Accucore 150-C18
< 2 µm Wide Pore Fully Porous C18
Stationary Phase
< 2 µm Wide Pore Fully Porous C18 Backpressure: 320 bar
uAU
100000
nc = 1 +
50000 0 -50000 2
4
6
8
10
12
14
Time (min)
tg
(w )
nc Peak capacity tg Gradient time w Average peak width 10% height
Reproducible Separations Precision of retention times is critical for reliable analysis. The Accucore 150-C18 column exhibits excellent retention time reproducibility.
13.0
Peak Number 7
13.5
14.0
Peptide
tR (min)
% RSD
Insulin
13.58
0.11
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
7
Excellent Loading Capacity The data below demonstrates the impact on peak capacity of increasing the load of peptides on the Accucore 150-C18 column. Only for loads exceeding 2 µg on the column are significant decreases in peak capacity observed. 2200 2000 1800 1600
mAU
1400 1200 1000
13.1 µg
800
6.7 µg
600
3.4 µg
400
1.7 µg
200
0.8 µg
0 -200 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
Loading Capacity
200
Peak Capacity
150
100
50
0 0
2
4
6
8
10
Total Load on Column /µg
8
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
12
14
Total Load on Column (µg)
Peak Capacity
0.8
159.24
1.7
152.75
3.4
142.31
6.7
128.87
13.1
107.76
15
Accucore 150-C4 HR/10
Hydrophobicity
AI
Low
HS
pH Range 0
2 to 9
14
IEX (2.7)
Pore Size 0
150 Å
SS
300
Carbon Load (%)
C 0
2
HBC
25
Particle Size 2.6 µm
USP L26
BA
•
Significantly lower hydrophobic retention than C18
•
Ideal for retention of proteins and larger peptides
IEX (7.6)
Intact Protein Separation 130000 120000 110000
5
100000 90000 3
80000
mAU
70000
Mobile phase A: 0.1 % TFA in 30:70 acetonitrile:water Mobile phase B: 0.1 % TFA in 98:2 acetonitrile:water
60000 2
50000
6
Gradient:
0-30 % B over 8 mins 30-95 % B over 2 mins 95 % B hold for 1 min 0 % B hold for 4 mins
Flow:
400 µL/min
Temperature:
40 °C
Column:
Accucore 150-C4 2.6 µm, 100 x 2.1 mm
Injection:
2 µL 10 pmol/µL solution
Detection:
UV (214 and 280 nm)
40000 1
30000
*
20000 10000
4
0 -10000 0
1
2
3
4
5
6
7
8
9
10
Time (min) Peak Number
tR/min
Protein
MW
Concentration (µg/mL)
1
1.54
Insulin
6 kDa
40
2
2.66
Cytochrome C
12 kDa
80
3
4.42
Lysozyme
14 kDa
100 120
4
7.38
Myoglobin
18 kDa
5
7.88
Carbonic anhydrase
30 kDa
200
6
7.88
Ovalbumin
45 kDa
300
*
Carbonic anhydrase impurity
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations | Technical Guide
9
Excellent Resolution Accucore 150-C4 provides significantly sharper and higher peaks than a column packed with 5 µm wide pore fully porous C4 – thus offering better resolution and sensitivity. The Accucore 150-C4 also performs better than a column packed with < 2 µm wide pore fully porous C4 and generates only a fraction of the backpressure.
uAU
Accucore 150-C4
Accucore 150-C4 Backpressure: 185 bar
100000
100000
80000
80000
60000
60000
*
40000
20000
0
0 0
2
4
*
40000
20000
6
8
0
10
2
uAU
5 µm wide pore fully porous C4 80000
80000
60000
60000
40000
40000
20000
20000
0
0 0
2
4
6
8
6
8
*
2
4
6
8
Time (min)
Time (min)
Reproducible Results The Accucore 150-C4 column exhibits excellent peak shape and retention time reproducibility.
Protein
9.2
9.4
Time (min)
10
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Ovalbumin (45 kDa)
Mean tR / min % RSD 9.30
Data From Six Injections
10
< 2 µm wide pore fully porous C4 Backpressure: 320 bar
0
10
4
0.06
Mean peak width at half height / min
Mean asymmetry
0.05
1.14
10
Proven Loading Capacity As the load on the Accucore 150-C4 column increases up to 1200 pmoles (25 µg) peak shapes remain very sharp as shown by the peak capacity plot.
Loading Capacity 140
120
100
Peak Capacity
1200 pmoles 600000 600 pmoles
mAU
500000 400000
300 pmoles
300000
120 pmoles
200000
80
60
40
60 pmoles
20
100000 30 pmoles 0
0
0
1
2
3
4
5
Time (min)
6
7
8
9
10
0
200
400
600
800
1000
1200
Total Load on Column (total pmoles)
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
11
nanoViper Column Separations Protein separation using formic acid as an MS compatible mobile phase additive 20 4
15
5 2
Mobile phase A: 0.1 % formic acid in water Mobile phase B: 0.1 % formic acid in acetonitrile
mAU
1 10
Gradient:
3
0-30 % B over 1 min 30-60 % B over 10 mins 60-95 % B over 1 min 95 % B hold for 3 mins
5
* 0
7
6
8
9
10
11
12
13
Flow:
300 nL/min
Temperature:
40 °C
Column:
Accucore 150-C4, 2.6 µm, 150 x 75 µm
Backpressure:
204 bar (100 % A)
Injection:
0.25 µL 2 pmol/µL solution
Detection:
UV (214 nm)
14
Time (min) Peak Number 1
tR /min
Protein
6.89
Cytochrome C (12 kDa)
2
7.34
Insulin (6 kDa)
3
8.77
Myoglobin (18 kDa)
4
9.57
Carbonic anhydrase (30 kDa)
5
11.02
Ovalbumin (45 kDa)
*
Carbonic anhydrase impurity
Base peak 50 fmol loading of BSA digest 18.40
100 90 80
Relative Abundance
Mobile phase A: 0.1 % formic acid in water
22.56 23.81
18.02
70
Mobile phase B: 90 % acetonitrile in water
19.10
16.14
Gradient:
60 22.43
50
24.98
27.39
95 % B hold for 2 mins
26.68
40
28.77
30
20.05
14.80
20.16
14.36
20 6.42
10
2.00
3.89
31.64
35.33
22.18
32.10
5.74
37.80
0 0
2
4
6
8
10
12
14
16
18
20
Time (min)
12
Flow:
300 nL/min
Temperature:
not controlled
Column:
Accucore 150-C18, 2.6 µm, 150 x 75 µm
Backpressure:
198 bar (100 % A)
Injection:
Direct on-column loading of 1 µL of BSA digest, 50 fmol/µL in water + 0.1% formic acid
Detection:
Thermo Scientific LTQ Orbitrap XL Mass Spectrometer coupled with a Proxeon Nano Spray Flex Ion Source
36.03
6.60 6.69 9.55 11.21 11.49 7.57 11.92
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
22
24
26
28
4-40 % B over 30 mins 40-95 % B over 2 mins
30.07
30
32
34
36
Ordering Information Accucore HPLC Columns Description
Particle Size
Accucore 150-C18
2.6 µm
Accucore 150-C4
2.6 µm
Length (mm)
2.1 mm ID
3.0 mm ID
4.6 mm ID
30 50 100 150 30 50 100 150
16126-032130 16126-052130 16126-102130 16126-152130 16526-032130 16526-052130 16526-102130 16526-152130
16126-033030 16126-053030 16126-103030 16126-153030 16526-033030 16526-053030 16526-103030 16526-153030
16126-034630 16126-054630 16126-104630 16126-154630 16526-034630 16526-054630 16526-104630 16526-154630
Accucore Defender Guard Columns (4/pk) Description Accucore 150-C18 Accucore 150-C4
Particle Size
Length (mm)
2.1 mm ID
3.0 mm ID
4.6 mm ID
2.6 µm 2.6 µm
10 10
16126-012105 16526-012105
16126-013005 16526-013005
16126-014005 16526-014005
UNIGUARD Direct-Connection Guard Cartridge Holders Description UNIGUARD Drop-In Guard Cartridge Holder Standard Replacement Tip
2.1 mm ID
3.0 mm ID
4.6 mm ID
852-00 850-RT
852-00 850-RT
850-00 850-RT
Accucore nanoViper Columns Description
Particle Size
Accucore 150-C18
2.6 µm
Accucore 150-C4
2.6 µm
Length (mm)
75 µm ID
150 500 150 500
16126-157569 16126-507569 16526-157569 16526-507569
Ultimate Core Performance
to Maximize Your Investment • 150 Å pore size solid core particles for fast biomolecule separations • Superb resolution of peptides and intact proteins at low backpressures • Exceptionally rugged and easy to use analytical and nano scale columns
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
13
Resources
for Chromatographers Thermo Scientific Chromatography Columns and Consumables Catalog This extensive catalog offers 600 pages of proven chromatography tools and product selection guides. Available online, with a robust search tool and optimized for your iPad®. Visit www.thermoscientific.com/catalog
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Ultimate Core Performance
to Maximize Your Investment
Core Enhanced Technology – for Biomolecules The range of Accucore™ HPLC columns packed with 150 Å pore diameter particles allow biomolecule separations to benefit from the superb resolution and high speed enabled by the Core Enhanced Technology™ that Accucore is based on.
The key components of Core Enhanced Technology are: Solid Core Particles
Tight Control of Particle Diameter
2.6 µm diameter particles with a solid core generate high speed, high resolution separations without excessive backpressure
Enhanced selection process keeps particle size distribution to a minimum and produces high efficiency columns
Automated Packing Process
Advanced Bonding Technology
Enhanced automated procedures ensure that all columns are packed with the highest quality
Optimized phase bonding creates a series of high coverage, robust phases
Fast Separations High Peak Capacity Increased Sensitivity Lower Pressure Good Loading Capacity Reproducible Chromatography Long Lifetime Peptide and Protein Analysis
150 Å Solid Core Particles
Tight Control of Particle Diameter
Advanced Bonding Technology
Automated Packing Process
• • • •
• • • • • • •
• • •
• • • • • • •
• •
• • • •
The table above shows how the components of Core Enhanced Technology with 150 Å pore diameter particles contribute to the outstanding features of Accucore HPLC columns for biomolecule separations.
Table of Contents Why Core Enhanced Technology Works
2
150 Å Pore Diameter Particles
3
Column Formats
4
Analytical and Micro Columns. . . . . . . . . . . . . . . . . . . . 4 Defender Guard Cartridges . . . . . . . . . . . . . . . . . . . . . 4 nanoViper Columns . . . . . . . . . . . . . . . . . . . . . . . . . 4 Biomolecule Phases 5 Accucore 150-C18 Column
6
Peptide Separations
6
High Peak Capacity . . . . . . . . . . . . . . . . . . . . . . . . . 7 Reproducible Separations . . . . . . . . . . . . . . . . . . . . . . 7 Excellent Loading Capacity . . . . . . . . . . . . . . . . . . . . . 8 9 Accucore 150-C4 Column Protein Separation
9
Excellent Resolution . . . . . . . . . . . . . . . . . . . . . . . . 10 Reproducible Results . . . . . . . . . . . . . . . . . . . . . . . 10 Proven Loading Capacity. . . . . . . . . . . . . . . . . . . . . . 11 Thermo Scientific nanoViper Column Separations 12 Ordering Information Accucore HPLC Columns. . . . . . . . . . . . . . . . . . . . . . 13 Accucore Defender Guard Cartridges . . . . . . . . . . . . . . . 13
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
1
Why Core Enhanced Technology Works Two factors that strongly affect chromatographic efficiency are mass transfer and eddy diffusion, the C and A terms respectively from the Van Deemter equation.
H=A+
B + Cu u
H Height equivalent to theoretical plate (column length/efficiency)
A B C u
Eddy diffusion Longitudinal diffusion Resistance to mass transfer
Resistance to mass transfer is minimized by the solid core design of Accucore particles as the diffusional path of analytes is limited by the depth of the outer porous layer.
Mobile phase linear velocity
Material
Accucore 2 6 μm
Particle Size Distribution (D90/10)
1 15
The tight control of particle diameter and automated packing process used for Accucore columns result in a tight, highly uniform packed bed that minimizes eddy diffusion.
Lower Backpressure L h F dp2 dc2
Column length (cm) Mobile phase viscosity (cP) Flow rate (mL/min) Particle diameter (μm)
DP ~
250LhF dp2dc2
Column diameter (cm)
The equation above shows how backpressure is related to particle diameter. The tightly controlled 2.6 µm diameter of Accucore particles also results in lower backpressures than typically seen with sub-2 µm materials.
2
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
150 Å Pore Diameter Particles The 150 Å pore diameter solid core particles used in the Accucore 150-C18 and 150-C4 columns are designed specifically to provide the optimum combination of retention and resolution for peptides and proteins. The chromatograms below show the improved resolution and peak shapes achieved with Accucore 150-C18 compared to a C18 solid core phase with an 80 Å pore diameter.
Accucore 150-C18
1.12
3.76
7.09
8.74
13.46
8.91 11.20
Conventional Solid Core C18
1.01 8.52 6.98
3.60
13.13 10.84
Improved resolution Improved peak shape
Peak Capacity
150
Peak Capacity Comparison
140 130 120 110 100 90
Sharper peaks result in a significantly higher peak capacity for 150-C18 compared to the conventional solid core C18 column.
80 70 60 Accucore 150-C18
Conventional Solid Core C18
Stationary Phase
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
3
Column Formats Accucore HPLC columns for biomolecules are offered in both analytical, micro and nano formats. Optimum conditions and backpressure ratings are shown in the table below.
Format
Column ID
Optimum Flow Rate
Optimum Backpressure Injection Volume Rating
Temperature Rating
Nano
75 μm
300 nL/min
1 μL
800 bar
70 °C
Micro
2.1 mm
400 μL/min
1 μL
1000 bar
70 °C
Analytical
3.0 mm
800 μL/min
3 μL
1000 bar
70 °C
Analytical
4.6 mm
1800 μL/min
5 μL
1000 bar
70 °C
Analytical and Micro Columns Accucore HPLC columns are packed into our high pressure hardware. These stainless steel columns are engineered to the highest quality and have a pressure rating of 1000 bar.
Thermo Scientific Defender Guard Cartridges Guard columns are designed to protect your column from particulates introduced from the matrix or instrument and from any strongly retained components in the injected sample. Defender™ Guard Cartridges have been designed specifically to work with high speed, high efficiency separations.
nanoViper Columns The nanoViper fingertight connection system for nanoLC connections eliminates the assembly of PEEK sleeve connections. It is preassembled and fingertight to maximize ease-of-use. The nanoViper fitting is capable of withstanding pressures up to a 1000 bar and is compatible with third party valves and unions. Accucore nanoViper columns are available in 150 and 500 mm lengths – for ultra-high peak capacity.
4
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Biomolecule Phases Like all other Accucore products, the 150 Å pore size particles and the phases based on them are fully characterized using the suite of tests shown below.
Silica Support Properties
Bonded Phase Properties
Hydrophobic Interactions
Secondary Interactions
Acidic Interactions
Surface Area
Carbon load
Hydrophobic retention
Base activity
Acid interaction
HR
BA
Hydrophobic selectivity
Pore Size
Particle Size
Steric selectivity
C
IEX(2.7)
Ion exchange capacity (pH 7.6)
SS Particle Size Distribution
Ion exchange capacity (pH 2.7)
Chelation
HS
AI
IEX(7.6)
Hydrogen bonding capacity
HBC
Details, including the phase characterization, are given below for the Accucore biomolecule phases. HR/10 AI
HS
IEX (2.7)
SS
C
HBC
Phase characteristics are designed for the separation of peptides
IEX (7.6)
BA
Accucore 150-C18
HR/10 AI
HS
IEX (2.7)
SS
Accucore 150-C4 C
HBC
BA
IEX (7.6)
Lower hydrophobic retention, optimal retention of proteins and larger peptides
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
5
Accucore 150-C18 HR/10
Hydrophobicity
AI
Low
HS
pH Range 0
1 to 11
14
IEX (2.7)
Pore Size 0
150 Å
SS
300
Carbon Load (%)
C 0
7
HBC
25
Particle Size 2.6 µm
USP L1
BA
•
Designed for the separation of peptides
•
Outstanding resolution
IEX (7.6)
Peptide Separations Accucore 150-C18
240000 220000 200000 180000 160000
mAU
140000 1
120000 100000 80000
2
60000
4
3
Mobile phase A: 0.1 % TFA in 10:90 acetonitrile:water
7
5
Mobile phase B: 0.1 % TFA in 70:30 acetonitrile:water
6
40000
Gradient:
0-50 % B over 15 min; hold for 2 min; drop to 0 % in 0.1 min; hold at 0 % B for 5 min
Flow:
300 µL/min
Temperature:
35 °C
Column:
Accucore 150-C18 2.6 µm, 100 x 2.1 mm
Injection:
5 µL
Detection:
UV (220 nm)
20000 0 -20000 -40000 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
Peak Number
6
15
tR/min
Peptide
MW
1
1.12
Glycine-Tyrosine
238.24 Da
2.0
2
3.76
Valine-Tyrosine-Valine
379.45 Da
17.0
3
7.09
Met-Enkephalin
573.66 Da
21.0
4
8.74
Angiotensin III
931.09 Da
15.0
5
8.91
Leu-Enkephalin
569.65 Da
21.0
6
11.20
Ribonuclease A
~ 13700 Da
42.5
7
13.46
Insulin
5733.49 Da
30.0
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Concentration (µg/mL)
High Peak Capacity Higher peak capacities facilitate increased peptide identifications. Accucore 150-C18 provides much narrower peak widths, therefore significantly higher peak capacity than a column packed with < 2 µm wide pore fully porous C18. Accucore 150-C18 Backpressure: 175 bar
146
Peak Capacity
Conventional Solid Core C18 Backpressure: 170 bar
Peak Capacity Comparison
144 142 140 138 136 134 132 Accucore 150-C18
< 2 µm Wide Pore Fully Porous C18
Stationary Phase
< 2 µm Wide Pore Fully Porous C18 Backpressure: 320 bar
uAU
100000
nc = 1 +
50000 0 -50000 2
4
6
8
10
12
14
Time (min)
tg
(w )
nc Peak capacity tg Gradient time w Average peak width 10% height
Reproducible Separations Precision of retention times is critical for reliable analysis. The Accucore 150-C18 column exhibits excellent retention time reproducibility.
13.0
Peak Number 7
13.5
14.0
Peptide
tR (min)
% RSD
Insulin
13.58
0.11
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
7
Excellent Loading Capacity The data below demonstrates the impact on peak capacity of increasing the load of peptides on the Accucore 150-C18 column. Only for loads exceeding 2 µg on the column are significant decreases in peak capacity observed. 2200 2000 1800 1600
mAU
1400 1200 1000
13.1 µg
800
6.7 µg
600
3.4 µg
400
1.7 µg
200
0.8 µg
0 -200 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
Loading Capacity
200
Peak Capacity
150
100
50
0 0
2
4
6
8
10
Total Load on Column /µg
8
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
12
14
Total Load on Column (µg)
Peak Capacity
0.8
159.24
1.7
152.75
3.4
142.31
6.7
128.87
13.1
107.76
15
Accucore 150-C4 HR/10
Hydrophobicity
AI
Low
HS
pH Range 0
2 to 9
14
IEX (2.7)
Pore Size 0
150 Å
SS
300
Carbon Load (%)
C 0
2
HBC
25
Particle Size 2.6 µm
USP L26
BA
•
Significantly lower hydrophobic retention than C18
•
Ideal for retention of proteins and larger peptides
IEX (7.6)
Intact Protein Separation 130000 120000 110000
5
100000 90000 3
80000
mAU
70000
Mobile phase A: 0.1 % TFA in 30:70 acetonitrile:water Mobile phase B: 0.1 % TFA in 98:2 acetonitrile:water
60000 2
50000
6
Gradient:
0-30 % B over 8 mins 30-95 % B over 2 mins 95 % B hold for 1 min 0 % B hold for 4 mins
Flow:
400 µL/min
Temperature:
40 °C
Column:
Accucore 150-C4 2.6 µm, 100 x 2.1 mm
Injection:
2 µL 10 pmol/µL solution
Detection:
UV (214 and 280 nm)
40000 1
30000
*
20000 10000
4
0 -10000 0
1
2
3
4
5
6
7
8
9
10
Time (min) Peak Number
tR/min
Protein
MW
Concentration (µg/mL)
1
1.54
Insulin
6 kDa
40
2
2.66
Cytochrome C
12 kDa
80
3
4.42
Lysozyme
14 kDa
100 120
4
7.38
Myoglobin
18 kDa
5
7.88
Carbonic anhydrase
30 kDa
200
6
7.88
Ovalbumin
45 kDa
300
*
Carbonic anhydrase impurity
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations | Technical Guide
9
Excellent Resolution Accucore 150-C4 provides significantly sharper and higher peaks than a column packed with 5 µm wide pore fully porous C4 – thus offering better resolution and sensitivity. The Accucore 150-C4 also performs better than a column packed with < 2 µm wide pore fully porous C4 and generates only a fraction of the backpressure.
uAU
Accucore 150-C4
Accucore 150-C4 Backpressure: 185 bar
100000
100000
80000
80000
60000
60000
*
40000
20000
0
0 0
2
4
*
40000
20000
6
8
0
10
2
uAU
5 µm wide pore fully porous C4 80000
80000
60000
60000
40000
40000
20000
20000
0
0 0
2
4
6
8
6
8
*
2
4
6
8
Time (min)
Time (min)
Reproducible Results The Accucore 150-C4 column exhibits excellent peak shape and retention time reproducibility.
Protein
9.2
9.4
Time (min)
10
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
Ovalbumin (45 kDa)
Mean tR / min % RSD 9.30
Data From Six Injections
10
< 2 µm wide pore fully porous C4 Backpressure: 320 bar
0
10
4
0.06
Mean peak width at half height / min
Mean asymmetry
0.05
1.14
10
Proven Loading Capacity As the load on the Accucore 150-C4 column increases up to 1200 pmoles (25 µg) peak shapes remain very sharp as shown by the peak capacity plot.
Loading Capacity 140
120
100
Peak Capacity
1200 pmoles 600000 600 pmoles
mAU
500000 400000
300 pmoles
300000
120 pmoles
200000
80
60
40
60 pmoles
20
100000 30 pmoles 0
0
0
1
2
3
4
5
Time (min)
6
7
8
9
10
0
200
400
600
800
1000
1200
Total Load on Column (total pmoles)
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
11
nanoViper Column Separations Protein separation using formic acid as an MS compatible mobile phase additive 20 4
15
5 2
Mobile phase A: 0.1 % formic acid in water Mobile phase B: 0.1 % formic acid in acetonitrile
mAU
1 10
Gradient:
3
0-30 % B over 1 min 30-60 % B over 10 mins 60-95 % B over 1 min 95 % B hold for 3 mins
5
* 0
7
6
8
9
10
11
12
13
Flow:
300 nL/min
Temperature:
40 °C
Column:
Accucore 150-C4, 2.6 µm, 150 x 75 µm
Backpressure:
204 bar (100 % A)
Injection:
0.25 µL 2 pmol/µL solution
Detection:
UV (214 nm)
14
Time (min) Peak Number 1
tR /min
Protein
6.89
Cytochrome C (12 kDa)
2
7.34
Insulin (6 kDa)
3
8.77
Myoglobin (18 kDa)
4
9.57
Carbonic anhydrase (30 kDa)
5
11.02
Ovalbumin (45 kDa)
*
Carbonic anhydrase impurity
Base peak 50 fmol loading of BSA digest 18.40
100 90 80
Relative Abundance
Mobile phase A: 0.1 % formic acid in water
22.56 23.81
18.02
70
Mobile phase B: 90 % acetonitrile in water
19.10
16.14
Gradient:
60 22.43
50
24.98
27.39
95 % B hold for 2 mins
26.68
40
28.77
30
20.05
14.80
20.16
14.36
20 6.42
10
2.00
3.89
31.64
35.33
22.18
32.10
5.74
37.80
0 0
2
4
6
8
10
12
14
16
18
20
Time (min)
12
Flow:
300 nL/min
Temperature:
not controlled
Column:
Accucore 150-C18, 2.6 µm, 150 x 75 µm
Backpressure:
198 bar (100 % A)
Injection:
Direct on-column loading of 1 µL of BSA digest, 50 fmol/µL in water + 0.1% formic acid
Detection:
Thermo Scientific LTQ Orbitrap XL Mass Spectrometer coupled with a Proxeon Nano Spray Flex Ion Source
36.03
6.60 6.69 9.55 11.21 11.49 7.57 11.92
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
22
24
26
28
4-40 % B over 30 mins 40-95 % B over 2 mins
30.07
30
32
34
36
Ordering Information Accucore HPLC Columns Description
Particle Size
Accucore 150-C18
2.6 µm
Accucore 150-C4
2.6 µm
Length (mm)
2.1 mm ID
3.0 mm ID
4.6 mm ID
30 50 100 150 30 50 100 150
16126-032130 16126-052130 16126-102130 16126-152130 16526-032130 16526-052130 16526-102130 16526-152130
16126-033030 16126-053030 16126-103030 16126-153030 16526-033030 16526-053030 16526-103030 16526-153030
16126-034630 16126-054630 16126-104630 16126-154630 16526-034630 16526-054630 16526-104630 16526-154630
Accucore Defender Guard Columns (4/pk) Description Accucore 150-C18 Accucore 150-C4
Particle Size
Length (mm)
2.1 mm ID
3.0 mm ID
4.6 mm ID
2.6 µm 2.6 µm
10 10
16126-012105 16526-012105
16126-013005 16526-013005
16126-014005 16526-014005
UNIGUARD Direct-Connection Guard Cartridge Holders Description UNIGUARD Drop-In Guard Cartridge Holder Standard Replacement Tip
2.1 mm ID
3.0 mm ID
4.6 mm ID
852-00 850-RT
852-00 850-RT
850-00 850-RT
Accucore nanoViper Columns Description
Particle Size
Accucore 150-C18
2.6 µm
Accucore 150-C4
2.6 µm
Length (mm)
75 µm ID
150 500 150 500
16126-157569 16126-507569 16526-157569 16526-507569
Ultimate Core Performance
to Maximize Your Investment • 150 Å pore size solid core particles for fast biomolecule separations • Superb resolution of peptides and intact proteins at low backpressures • Exceptionally rugged and easy to use analytical and nano scale columns
Thermo Scientific Accucore HPLC Columns for Biomolecule Separations
13
Resources
for Chromatographers Thermo Scientific Chromatography Columns and Consumables Catalog This extensive catalog offers 600 pages of proven chromatography tools and product selection guides. Available online, with a robust search tool and optimized for your iPad®. Visit www.thermoscientific.com/catalog
Chromatography Resource Center Our web-based resource center provides technical support, applications, technical tips and literature to help move your separations forward. Visit www.thermoscientific.com/crc
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