designed to take full advantage of low - mac-mod · 2011. 10. 10. · designed to take full...
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®
Designed to take full advantage of low
dispersion, ultra-high pressure UPLC®
and UHPLC instruments
The introduction of ACE Excel UHPLC
columns gives chromatographers more
choices to achieve better results with
their UPLC and UHPLC instruments.
M o r e c h o i c e S . B e t t e r r e S U Lt S .
&
Manufacturing UHPLC columns is tough, as is evidenced by the current offering of commercial UHPLC columns that haven’t lived up to their potential and suffer from the perception that they are not nearly as rugged and reliable as HPLC columns. By drawing on their vast experience in manufacturing the finest HPLC columns, ACE is able to produce UHPLC columns that offer a level of performance that is seldom achieved with other UPLC or UHPLC columns. Now, chromatographers will get even more from their UPLC and UHPLC instruments. Excellent peak shape for basic compounds, improved column-to-column reproducibility, additional stationary phases that leverage multiple mechanisms of separations, and improved column ruggedness are now available to UPLC and UHPLC users with ACE Excel UHPLC columns.
The introduction of ACE Excel UHPLC columns means chromatog-raphers now have more choices within their reach to achieve better results with their UPLC and UHPLC instruments.
Figure 1: ACE Excel columns provide high-speed, high- resolution separations
An ACE Excel C18 column provides baseline separation of these 9 analytes in less
than 60 seconds.
conditionsColumns: ACE Excel C18, 2 µm, 3.0 x 50 mmMobile Phase: 30%B to 100% B in 60 seconds A = Water + 0.1% TFA B = AcetonitrileFlow Rate: 2.5 mL/minColumn Temperature: 40ºCPressure: 703 bar (10,335 psi)Detection: PDA, 254 nmInstrument: Agilent 1290 UHPLC
compound1. Acetanilide2. Acetophenone3. Propiophenone4. Butyrophenone5. Benzophenone6. Valerophenone7. Hexanophenone8. Heptanophenone9. Octanophenone
0 12 24 36 48 60
Time (sec)
1
ACe excel 2µm uHPLC columns Fully compatible with all commercial UPLC and UHPLC instruments
Brings renowned ACE HPLC columns’ advantages to UHPLC columns
Provides users of UPLC/UHPLC with additional choices in stationary phases, including the powerful C18-AR and C18-PFP phases
Delivers a high level of reliability and ruggedness
Offers easy scalability from UHPLC to HPLC to preparative LC
12
34
5
6 78 9
High-speed, high resolution, UHPLC separations
Chromatographic data supplied courtesy of a west coast U.S. pharmaceutical company.
All the well known advantages of ACE HPLC columns are now available in UHPLC columns
Figure 2: ACE Excel delivers excellent peak shape
As with ACE HPLC columns, ACE Excel UHPLC columns deliver excellent peak shape
for basic and acidic compounds. In this example, the ACE Excel C18 UHPLC column
provided superior peak shape compared to the Waters BEH C18 column.
conditions Columns: ACE Excel C18, 2 µm, 2.1 x 100 mm Acquity BEH C18, 1.7 µm, 2.1 x 100 mm Mobile Phase: 5% B to 100% B in 8.7 minutes A = Water + 0.05% TFA B = Acetonitrile + 0.05% TFA Flow Rate: 0.5 mL/min Column Temperature: 45ºC Detection: MSD Instrument: Waters Acquity UPLC
Chromatographic data supplied courtesy of a northeast U.S. pharmaceutical company. NOTE: Competitive separations may not be representative of all applications.
USP tailing FactorAnalyte Ace excel Acquity Beh Aspartame 1.36 1.47 Cortisone 1.08 1.17 Amitriptyline 1.68 1.78 Reserpine 1.34 1.51
0 1 2 3 4 5 0 1 2 3 4
Time (min)
ACE Excel C18 Acquity BEH C18 1.7µm
ACe Advantages Excellent peak shape for basic and acidic compounds
Unsurpassed column-to-column reproducibility
Rugged and reliable day-to-day performance and exceptional column lifetime
Peak shape
It is well known that even small amounts of unwanted and uncon-trolled secondary interactions can cause unacceptable peak tailing and poor column-to-column reproducibility. However, recognizing the problem and correcting it are two very different things. ACE columns have a well deserved reputation for delivering excellent peak shape, even for problematic basic compounds.
ACE HPLC columns, and now ACE Excel UHPLC columns, are manu-factured using a proprietary process to produce ultra-inert stationary phases that yield the excellent peak shape for basic and acidic compounds for which ACE columns are so well known. This process starts with the production of ultra-pure, ultra-inert silica stationary phase supports and continues with an advanced bonding technology that yields a densely-bonded stationary phase. Leverage the power of bonded phase selectivity and combine it with uHPLC efficiency and speed
C18 bonded phases are the most popular for HPLC/UHPLC because they offer good retention and selectivity for a wide variety of sample types. In addition, they are perceived to be more rugged and reliable than other bonded phases. However, C18 bonded phases depend only on hydrophobic binding interaction, and occasionally shape selectivity, to achieve satisfactory separations. As versatile as C18 phases are, the limited number of mechanisms of separation they offer may lead to less than optimum separations in some cases and complete lack of resolution of important peak pairs in others.
ACE columns have led the way in offering C18 bonded phases with the advantage of extra selectivity. C18-AR and C18-PFP bonded phases have proved to be extremely powerful in leveraging selectivity to achieve separations not possible with C18 bonded phases. They aren’t meant to supplant C18 bonded phases, but rather complement them in providing additional mechanisms of separation that can be used to achieve better overall chromatographic results. It is fitting, then, that C18, C18-AR and C18-PFP bonded phases will be the first phases available as ACE Excel UHPLC columns. Additional bonded phases will follow shortly.
2
3
Dionex UltiMate 3000
Figure 3: ACE Excel UHPLC columns are compatible with all commercial UPLC and UHPLC instruments
ACE Excel UHPLC columns are designed to be fully compatible with all commercial UPLC or UHPLC instruments and engineered to ben-efit from the high flow rate, ultra-high pressure and low dispersion of these systems. ACE Excel columns provide chromatographers with more column choices so that even more value can be obtained from their UPLC/UHPLC instruments.
Fully compatible with all commercial UPLC and UHPLC instruments
Thermo Scientific Accela Waters Acquity UPLC
Acquity UPLC H–Class
Acquity UPLC I–Class
Agilent Technologies 1290 Infinity
1220 Infinity
1260 Infinity
1200 Infinity * Plus many other brands of
UHPLC instrumentation
Shimadzu
Nexera
Prominence
ACe excel uHPLC columns are compatible with:
• Waters Acquity UPLC, Acquity UPLC H-Class, Acquity UPLC I-Class • Agilent 1290 Infinity, 1220 Infinity, 1260 Infinity, 1200 Infinity • Thermo Scientific Accela • Shimadzu Nexera, Prominence • Dionex UltiMate 3000 • Plus many other brands of UHPLC instrumentation
Column-to-column reproducibility is affected by the production of the silica stationary phase support, the bonding of the stationary phase to the stationary phase support, and the packing of the stationary phases into the column. The better each stage of the column manufacturing process is controlled, the better will be the quality of the column.
ACE columns are subjected to extensive controls at each stage of the column manufacturing process. These controls ensure that ACE columns produce reliable, predictable performance from column-to-column and lot-to-lot.
Some UHPLC columns have gotten a reputation for lack of durability. Unfortunately, this reputation seems deserved. It is difficult to pack a column that will withstand the high flow rates and pressures of UHPLC. Add to this the increased rate of column inlet frit plugging and you are faced with columns that are, indeed, less durable than HPLC columns.
ACE Excel will change your perception about durability of UHPLC columns. Not only are the ACE Excel columns packed extremely well, they incorporate an inlet frit design that is much less prone to plugging than other UHPLC columns. It is still recommended that you filter samples and mobile phases as you would with any UHPLC column, but ACE Excel columns are much more forgiving than other UHPLC columns and provide the same durability and lifetime that you would expect from HPLC columns.
Figure 4: ACE Excel are exceptionally durable UHPLC columns
A 2.1 x 100 mm ACE Excel C18 UHPLC column was subjected to over 2,000 gradient runs at an average pressure of 1,000 bar (14,500 psi). The column efficiency (plate number) and retention times were essentially unchanged at the end of this stability study.
Time (min)
Number of Injections
% I
niti
al E
ffici
ency
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 500 1000 1500 2000 2500
Initial injection at 1000 bar
2020th injection at 1000 bar
120
100
80
60
40
20
0
Column-to-column reproducibility and rugged, reliable performance
4
5
Figure 5: ACE stationary phases provide the identical selectivity whether they are UHPLC, HPLC or preparative HPLC columns
These chromatograms of a test sample run under the same mobile phase conditions on an ACE Excel C18 2 μm UHPLC column, an ACE C18 3 μm HPLC column and an ACE C18 5 μm HPLC column illustrates the consistent selectivity inherent in ACE stationary phases, regardless of the packing particle size. This permits easier transfer of methods from UHPLC to HPLC or HPLC to UHPLC. It also makes it easier to scale-up from analytical to preparative applications.
conditionsColumns: 2.1 x 50 mmMobile Phase: 30% B to 90% B in 1.4 minutes A = 20 mM Ammonium Formate + 0.1% Formic Acid B = Acetonitrile + 0.1% Formic AcidGradient: Hold at 30% B for 0.30 minutes 30 to 90% B in 1.10 minutesHold at 90% B for 0.40 minutesFlow Rate: 0.5 mL/minColumn Temperature: 30ºCDetection: AB Sciex Triple Quad(TM) 5500 MS operating in ESI (+) modeInstrument: Shimadzu Prominence UFLC system
Analytes1. Mebendazole 2. Protriptyline 3. Amitriptyline 4. Loperamide
Selectivity (α)Peak Pair Ace excel c18-Ar,2 µm Ace c18-Ar, 3 µm Ace c18-Ar,5 µm 1,2 1.07 1.07 1.072,3 1.05 1.05 1.053,4 1.11 1.12 1.13
Chromatographic data supplied courtesy of a northeast U.S. pharmaceutical company.
The same controls that ensure highly reproducible columns from lot-to-lot also ensure that you can more easily transfer methods from UHPLC to HPLC, or vice versa, or scale-up to preparative applica-tions when needed to isolate or purify a target compound. The column efficiency will, of course, change, but the band spacing (selectivity) will be predictably the same.
Easy scalability from UHPLC to HPLC to Preparative HPLC
0 0.5 1 1.5 2
ACE C18-AR, 5µm
0 0.5 1 1.5 2
ACE C18-AR, 3µm
Time (min)0 0.5 1 1.5 2
ACE Excel C18-AR, 2µm
1 1
22
33
4 4
Target Analytes
Analytes differing in hydrophobicity
Polar, moderately polar, and nonpolar analytes
Uncharged acids and bases
Ionized acids or bases using ion-pairing
recommended Applications
Analytes differing in hydrophobicity
Homologous compounds differing by –CH2
Figure 6: Comparison of Selectivity of ACE Excel C18 to Acquity BEH C18
The ACE Excel C18 will provide retention and selectivity similar to other C18 UHPLC columns, although occasionally there may be slight selectivity differences among C18 phases. However, you can depend on getting the same excellent peak shape for your analytes from ACE Excel C18 UHPLC columns as you've come to expect from ACE HPLC columns.
conditionsColumns: ACE Excel C18, 2 um, 2.1 x 100 mm Acquity BEH C18, 1.7 um, 2.1 x 100 mmMobile Phase: 5% B to 100% B in 8.7 minutes A = Water + 0.05% TFA B = Acetonitrile + 0.05% TFA
conditionsColumns: ACE Excel C18, 2 um, 3.0 x 50 mm Zorbax Eclipse Plus RRHD C18, 1.8 um, 2.1 x 50 mmMobile Phase: 5% B to 100% B in 8.7 minutes A = Water + 0.05% TFA B = Acetonitrile + 0.05% TFA
Analytes:1. Aspartame2. Cortisone3. Amitriptyline4. Reserpine
Chromatographic data supplied courtesy of a west coast biotechnology company. NOTE: Competitive separations may not be representative of all applications.
1,2 2.25 2.562,3 1.46 1.533,4 1.26 1.274,5 1.08 1.095,6 1.10 1.106,7 1.15 1.157,8 1.12 1.118,9 1.10 1.10
C18
Mechanisms of Separation
Strength of interaction
Hydrophobic binding interactions
Shape selectivity
Very strong
Weak
Flow Rate: 0.5 mL/minColumn Temperature: 45ºCDetection: MSDInstrument: Waters Acquity UPLC
1,2 1.61 1.622,3 1.30 1.333,4 1.03 1.03
Chromatographic data supplied courtesy of a northeast U.S. pharmaceutical company.
0 1 2 3 4 5 0 1 2 3 4Time (min)
ACE Excel C18 Acquity BEH C18
Flow Rate: 2.5 mL/min (ACE Excel) 1.5 mL/min (Zorbax Eclipse Plus)Column Temperature: 40ºCDetection: PDAInstrument: Agilent Infinity 1290
0 0.2 0.4 0.6 0.8 1 1.2 1.4
ACE Excel C18
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Time (min)
Eclipse Plus RRHD C18
Peak Pair
Peak Pair
ACE Excel
ACE Excel
Eclipse Plus
Acquity BEH
Selectivity (α)
Selectivity (α)
6
Figure 7: Leveraging ACE Excel C18-AR Selectivity
These two chromatograms illustrate how the additional mechanisms of separation provided by the ACE Excel C18-AR compared to the Acquity BEH C18 generates a better separation for peak pairs 2/3 and 5/6.
conditionsColumns Dimensions: 2.1 x 50 mmMobile Phase: 20% B to 70% B in 5 minutes A = 20 mM KH2PO4, pH 2.8; B = 1:1 20mM KH2PO4:CH3OHFlow Rate: 0.31 mL/minColumn Temperature: 40ºCDetection: UV, 205 nm
Analytes1. Pyridoxine2. P-Aminobenzoic acid3. Pantothenic acid4. Folic acid5. Cyanocobalamin6. D-Biotin7. Riboflavin
0 1 2 3 4 5
0 1 2 3 4 5
1
2
2
3
3
4
5
6
7
Time (min)
1
4 5,6
7
ACE Excel C18-AR, 2 µm
Acquity BEH C18, 1.7µm
Target Analytes
Analytes with π bonding
Analytes with electron delocalization and electron- withdrawing groups, such as halogens, nitro groups, ketones, esters, and acids
Analytes with different dipole moments
Analytes differing in hydrophobicity
Polar, moderately polar, and nonpolar analytes
Uncharged acids and bases
recommended Applications
Analytes differing in hydrophobicity, homologous compounds differing by –CH2
Stereoisomers
Steroids
Taxanes
Substituted aromatics
Highly aqueous conditions
Particularly recommended for applications where a typical C18 does not provide an adequate separation
C18-AR(C18 with Phenyl group)
Mechanisms of Separation
Strength of interaction
π-π interactions
Dipole-dipole interactions
Hydrophobic binding interactions
Shape selectivity
Strong
Strong
Strong
Moderate
7
Figure 6: Comparison of Selectivity of ACE Excel C18 to Acquity BEH C18
NOTE: Competitive separations may not be representative of all applications.
Figure 8: Leveraging ACE Excel C18-PFP Selectivity
Typical C18 UHPLC columns are unable to separate all 8 of these analytes under these conditions. In particular, hydrophobic binding interaction appears to offer no selectivity for separating peaks 1,2 and 3 and peaks 5 and 6. The ACE Excel C18-PFP, however, provides the selectivity needed to separate the co-eluting analytes. It is likely the additional selectivity provided by π–π and dipole–dipole interaction that is responsible for achieving the better overall separation.
conditionsColumns Dimensions: 2.1 x 50 mmMobile Phase: 1:1 CH3OH:H2OFlow Rate: 0.21 mL/minColumn Temperature: 40ºCDetection: UV, 254 nm
Analytes1. 1,2,3-trimethoxybenzene2. 1,2,4-trimethoxybenzene3. 1,2-dimethoxybenzene4. 1,4-dimethoxybenzene
ACE Excel C18-PFP 2µm
0 1 2 3 4 5 6 7
1
23 4 56
7
8
0 1 2 3 4 5 6 7
5,6
Superficially porous C18, 2.6µm
1,2,3
4
7
8
0 1 2 3 4 5 6 7
Superficially porous EC-C18, 2.7µm
1,2,3
4
5,6
7
8
0 1 2 3 4 5 6 7
Acquity BEH C18, 1.7µm
Time (min)
1,2,3
4
5,6
7
8
C18-PFP(C18 with pentafluorophenyl group)
Mechanisms of Separation
Strength of interaction
π-π interactions
Dipole-dipole interactions
Hydrophobic binding interactions
Shape selectivity
Target Analytes
Analytes with π bonding
Analytes with electron donating groups, such as phenols, aromatic ethers and amines
Analytes with proton donor groups
Analytes with different dipole moments
Analytes differing in hydrophobicity
Polar, moderately polar, and nonpolar analytes
Uncharged acids and bases
recommended Applications
Stereoisomers
Steroids
Taxanes
Substituted aromatics
Analytes differing in hydrophobicity, homologous compounds differing by –CH2
Highly aqueous mobile phase conditions
Particularly recommended for applications where a typical C18 does not provide an adequate separation
Strong
Strong
Strong
Strong
5. Methoxybenzene (anisole)6. 1,3-dimethoxybenzene7. 1,3,5-trimethoxybenzene8. toluene
8
NOTE: Competitive separations may not be representative of all applications.
Stationary Phase Support
Ultra-pure, spherical “Type B” silica 2 µm particle size 100 Å pore size 300 m2/gram Bonded Phase Carbon Load: C18: 15.5% C18-AR: 15.5% C18-PFP: 14.3% Densely bonded and exhaustively end-capped pH range of 2 to 8
Specifications
Part Numbers & PricesC18-PFPC18-ARC18
Dimensions Part Number Price150 x 2.1mm EXL-101-1502U $735.00
150 x 3.0mm EXL-101-1503U $735.00
150 x 4.6mm EXL-101-1546U $735.00
125 x 2.1mm EXL-101-1202U $695.00
125 x 3.0mm EXL-101-1203U $695.00
125 x 4.6mm EXL-101-1246U $695.00
100 x 2.1mm EXL-101-1002U $650.00
100 x 3.0mm EXL-101-1003U $650.00
100 x 4.6mm EXL-101-1046U $650.00
75 x 2.1mm EXL-101-7502U $620.00
75 x 3.0mm EXL-101-7503U $620.00
75 x 4.6mm EXL-101-7546U $620.00
50 x 2.1mm EXL-101-0502U $545.00
50 x 3.0mm EXL-101-0503U $545.00
50 x 4.6mm EXL-101-0546U $545.00
35 x 2.1mm EXL-101-3502U $510.00
35 x 3.0mm EXL-101-3503U $510.00
35 x 4.6mm EXL-101-3546U $510.00
30 x 2.1mm EXL-101-0302U $500.00
30 x 3.0mm EXL-101-0303U $500.00
30 x 4.6mm EXL-101-0346U $500.00
20 x 2.1mm EXL-101-0202U $470.00
20 x 3.0mm EXL-101-0203U $470.00
20 x 4.6mm EXL-101-0246U $470.00
Dimensions Part Number Price150 x 2.1mm EXL-109-1502U $735.00
150 x 3.0mm EXL-109-1503U $735.00
150 x 4.6mm EXL-109-1546U $735.00
125 x 2.1mm EXL-109-1202U $695.00
125 x 3.0mm EXL-109-1203U $695.00
125 x 4.6mm EXL-109-1246U $695.00
100 x 2.1mm EXL-109-1002U $650.00
100 x 3.0mm EXL-109-1003U $650.00
100 x 4.6mm EXL-109-1046U $650.00
75 x 2.1mm EXL-109-7502U $620.00
75 x 3.0mm EXL-109-7503U $620.00
75 x 4.6mm EXL-109-7546U $620.00
50 x 2.1mm EXL-109-0502U $545.00
50 x 3.0mm EXL-109-0503U $545.00
50 x 4.6mm EXL-109-0546U $545.00
35 x 2.1mm EXL-109-3502U $510.00
35 x 3.0mm EXL-109-3503U $510.00
35 x 4.6mm EXL-109-3546U $510.00
30 x x2.1mm EXL-109-0302U $500.00
30 x x3.0mm EXL-109-0303U $500.00
30 x 4.6mm EXL-109-0346U $500.00
20 x 2.1mm EXL-109-0202U $470.00
20 x 3.0mm EXL-109-0203U $470.00
20 x 4.6mm EXL-109-0246U $470.00
Dimensions Part Number Price150 x 2.1mm EXL-1010-1502U $735.00
150 x 3.0mm EXL-1010-1503U $735.00
150 x 4.6mm EXL-1010-1546U $735.00
125 x 2.1mm EXL-1010-1202U $695.00
125 x 3.0mm EXL-1010-1203U $695.00
125 x 4.6mm EXL-1010-1246U $695.00
100 x 2.1mm EXL-1010-1002U $650.00
100 x 3.0mm EXL-1010-1003U $650.00
100 x 4.6mm EXL-1010-1046U $650.00
75 x 2.1mm EXL-1010-7502U $620.00
75 x 3.0mm EXL-1010-7503U $620.00
75 x 4.6mm EXL-1010-7546U $620.00
50 x 2.1mm EXL-1010-0502U $545.00
50 x 3.0mm EXL-1010-0503U $545.00
50 x 4.6mm EXL-1010-0546U $545.00
35 x 2.1mm EXL-1010-3502U $510.00
35 x 3.0mm EXL-1010-3503U $510.00
35 x 4.6mm EXL-1010-3546U $510.00
30 x 2.1mm EXL-1010-0302U $500.00
30 x 3.0mm EXL-1010-0303U $500.00
30 x 4.6mm EXL-1010-0346U $500.00
20 x 2.1mm EXL-1010-0202U $470.00
20 x 3.0mm EXL-1010-0203U $470.00
20 x 4.6mm EXL-1010-0246U $470.00
Ace excel 2µm Ultra-inert UhPLc columns
Ace excel 2µm Ultra-inert UhPLc columns
Ace excel 2µm Ultra-inert UhPLc columns
Column Maximum temperature of 100 °C for pH 2–5, 60 °C for pH 6–8 Maximum pressure of 1,000 bar (15,000 psi) Note: Working at the limits of temperature and pH may result in shorter column lifetimes.
Contact
MAC-MOD Analytical Phone: 800-441-7508 Fax: 610-358-5993
Website: www.mac-mod.com Email: [email protected]
103 Commons Court, P.O. Box 587 Chadds Ford, PA 19317
Toll-Free: 1.800.441.7508 | Phone: 1.610.358.9696 | Fax: 1.610.358.5993 E-mail: [email protected] | Web Site: www.mac-mod.com
Address: 103 commons court, chadds Ford, Pennsylvania 19317
® ACE is a registered trademark of Advanced Chromatography Technologies (ACT).