new hplc column options for uhplc.ppt - agilent · 2016-08-30 · • these new columns will have...
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![Page 1: New HPLC Column Options for UHPLC.ppt - Agilent · 2016-08-30 · • These new columns will have new part numbers (table below) • The column shipping with the 1290 Infinity LC](https://reader034.vdocument.in/reader034/viewer/2022042923/5f7068042a07f030c75ff5b9/html5/thumbnails/1.jpg)
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New HPLC Column Options for UHPLC
Rapid Resolution High Definition
Columns and More
Chor Koon Tan, PhD
Business Development Manager
Columns & Supplies Division
Asia-Pacific
Agilent Technologies
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UHPLC – Why Use Different Columns?
UHPLC instruments include a variety of high pressure LC choices including those designed for high throughput/fast LC.
These instruments operate at pressures >400 bar/6000 psi.
The desire is to have small particle size columns <2-3um for improved efficiency, resolution and productivity on these instruments.
These provide the flexibility to operate above 400 bar for many applications – for speed or for enhanced resolution with long 1.8um columns
Agilent’s new 1290 Infinity LC can operate up to 1200 bar.
This requires columns designed for up to 1200 bar operating conditions – the new RRHD Columns!
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The 1290 Infinity Has a Wide Useable UHPLC Range
June 06, 2005Page 3
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Productivity that Can Be Achieved Using RRHD Columns with the 1290 Infinity LC
1. Fast analysis – especially with somewhat complex samples
1. Gradient analysis with 4-10 components
2. More resolution in a limited amount of time
1. Choosing different column lengths for the resolution desired
2. Maximizing peak capacity in gradient separations of complex samples
RRHD – Rapid Resolution High Definition
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Fast Analysis on ZORBAX Rapid Resolution High Definition Eclipse Plus C18
min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
mAU
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100
200
300
400
500
600
DAD1 D, DAD1C, DAD: Signal C, 280 nm/Bw:10 nm Ref 400 nm/Bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D)
0.2
72
0.4
15
0.4
80
0.7
57
0.8
83
1.0
11
1.0
55
1.5
24
1.5
56
DAD1 E, DAD1D, DAD: Signal D, 255 nm/Bw:10 nm Ref 400 nm/Bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D)
1.6
84
PG
THBP
TBHQNDGA
BHA
Ionox-100
OGDG
BHT AP
Data Courtesy of: Gerd Vanhoenacker Research Institute for ChromatographyKennedypark 26 8500 Kortrijk Belgium
RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um, Pmax 1070 barThis 10 component sample requires a gradient that goes over 1000 bar.This is a sub 2minute separation!
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Fast Analysis on Agilent Zorbax Rapid Resolution High Definition Eclipse Plus C18
Method
Column ZORBAX RRHD Eclipse Plus C18 2.1 x 50 mm, 1.8 µm
(SN USDAY01001)
Mobile phase A=0.02% H3PO4, B=acetonitrile/methanol 3/1
Flow rate 1.6 mL/min
Gradient 0-1.85 min 30-100% B
Injection volume 1 µL
Detector Sig=280/10 nm, Ref=400/50, 80 Hz
Sig=255/10 nm, Ref=400/50, 80 Hz
Temperature 45°C
Sample Antioxidant mixture, ca. 100 ppm
Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography
Kennedypark 26 8500 Kortrijk Belgium
A 1.6 mL/min flow rate is very fast, and allows a fast separation.
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Where Does this Example Fall in the 1290 Infinity Range
June 06, 2005Page 7
This app falls in the “Power Range”
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Choose Column Length for Analysis Time and Rs
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RRHD 1.8um 1200 bar
Mobile Phase: 40:60 water:acetonitrile, Flow Rate: 0.5 mL/min, Temp: ambient, Columns: RRHD SB-C18, 2.1 mm ID, Lengths as noted Peaks: 1: uracil, 2 acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone
Rs3,2: 5.64N6 : 10,700Pressure: 350 bar
Rs3,2: 11.98N6 : 34,100Pressure:750 bar
Rs3,2: 9.02N6 : 21,100Pressure: 566 bar
50 mm
100 mm
150 mm
min0 1 2 3 4
min0 1 2 3 4
min0 1 2 3 4
0
The 150mm length RRHD column becomes a good option for high resolution with the 1290 Infinity LC.
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Gain Efficiency and Resolution with Long Length 150 + 100 mm = 250 mm 1.8um @ 1100 bar
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α3,2: 1.20
Rs3,2: 8.76N6 : 50,200Pw2: 0.018 minPw6: 0.037 min
α3,2: 1.19
Rs3,2: 6.42N6 : 31,400Pw2: 0.016 minPw6: 0.029 min
150 mm (P:582 bar)
150 +100 mm (P:1095 bar)1. Uracil2. Acetophenone3. Propiophenone4. Butyrophenone5. Valerophenone6. Hexanophenone
Mobile Phase 25:75 water:acetonitrile, Flow Rate: 0.5 mL/min, ambient temperature,
ZORBAX RRHD SB-C18, 1.8um columns
High Rs in 3.5 minutes, 250mm, 1.8um at 1100 barResolution is fine on 150mm column, but for even more Rs longer column is used.
min0 0.5 1 1.5 2 2.5 3 3.5
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Where Does this Example Fall in the 1290 Infinity Range
June 06, 2005Page 10
This app falls in the “Power Range”
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High Resolution Separation with Long 1.8um Columns
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min0 2 4 6 8 10 12 14
mAU
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DAD1 A, Sig=254,4 Ref=off (E:\090422JWH\PRESSURE000027.D) PMP1, PMP1B, Pressure
Mobile Phase A: water B:acetonitrile, F=0.25 mL/min, ambient temperature,
Columns: ZORBAX RRHD, SB-C18, 2.1 x 150+100 mm, 1.8um
Gradient 10-100% B/15 min.
Pressure 800- 900 bar
Low point of gradient about 480 bar
Long column or columns in series provide max Rs with this complex
sample – smoked food flavoring.
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Achieving Optimal Resolution for a Gradient with RRHD Column Choices
Columns range in length from 50 – 150mm.
Column length and gradient time can be changed to optimize the resolution and retention of the analytes.
k* =
S • ∆Φ • Vm
tG • F
These 3 parameters canbe easily changed.tG = gradient timeF = flow rate Vm = column volume
(change by changing column length)
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Separation of Licorice Root on RRHD Columns –30 minute gradient on each length
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min0 2 4 6 8 10 12 14 16 18
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min0 2 4 6 8 10 12 14 16 18
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min0 2 4 6 8 10 12 14 16 18
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RRHD SB-C18 2.1 x 50 mm, 1.8um
Pmax=366 bar
nc = 424
RRHD SB-C18 2.1 x 100 mm , 1.8um
Pmax=595 bar
nc = 485
RRHD SB-C18 2.1 x 150 mm, 1.8um
Pmax=768 bar
nc = 589
Rs: 2.40
Rs: 1.37
Rs: 0
Mobile Phase: 10-100% B /30 min A: 0.1% formic acid (fa) B: acetonitrile with 0.1% faF=0.4 mL/min Ambient temperature 280nm UV
1290 Infinity
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Separation of Licorice Root on RRHD Columns –30 minute gradient on each length
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2 4 2 4 6
Rs: 2.40Rs: 1.37
2 4
Rs: 0
Mobile Phase: 10-100% B /30 min A: 0.1% formic acid (fa) B: acetonitrile with 0.1% fa F=0.4 mL/min Ambient temperature 280nm UV
1290 Infinity
50 mm 100 mm 150 mm
7 peaks 8 peaks 9 peaks
Increasing peak capacity led to increased Rs for this sample.
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Peak Capacity Comparisons – Length and Gradient Time
10 minutes 20 minutes 30 minutes
50 313 358 424
100 343 418 485
150 386 483 589
Licorice Root sample
Gradient Time Column
Length
Shaded boxes indicate comparable peak capacities
nc= tg/wbaseline of last eluting peak1
1. Neue, U.D., et. al, Advances in Chromatography, 41, 93 (2001)
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RRHD 1.8um Columns
The part numbers!
• These new columns will have new part numbers (table below)
• The column shipping with the 1290 Infinity LC will be the ZORBAX Rapid
Resolution High Definition (RRHD) Eclipse Plus C18, 2.1 x 50mm, 1.8um
PN Description
959759-902 RRHD Eclipse Plus C18, 2.1 x 150mm, 1.8um
959758-902 RRHD Eclipse Plus C18, 2.1 x 100mm, 1.8um
959757-902 RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um
859700-902 RRHD SB-C18, 2.1 x 150mm, 1.8um
858700-902 RRHD SB-C18, 2.1 x 100mm, 1.8um
857700-902 RRHD SB-C18, 2.1 x 50mm, 1.8um
859700-906 RRHD SB-C8, 2.1 x 150mm, 1.8um
858700-906 RRHD SB-C8, 2.1 x 100mm, 1.8um
857700-906 RRHD SB-C8, 2.1 x 50mm, 1.8um
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Why Two Different C18 Columns? Eclipse Plus C18 and StableBond C18
Different selectivity
• Eclipse Plus C18 vs. StableBond C18
Optimum peak shape
• Achieved with endcapped C18 – Eclipse Plus C18
Optimum stability at low pH
• Achieved with non-endcapped C18 – StableBond C18
Additional bonded phases will be converted to the RRHD format
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Different ZORBAX RRHT/RRHD C18 Bonded Phases for Maximum SelectivityMobile phase: (69:31) ACN: water Flow 1.5 mL/min. Temp: 30 °C Detector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um
Sample: 1. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)
Eclipse Plus C181 2
3
4
1 2 3 4 5
1st choiceBest Resolution
& Peak Shape
StableBond
SB-C181 23
4
min1 2 3 4 5
2nd choiceGood alternate selectivity due
to non-endcapped
Multiple bonded phases for most effective method development.
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Peak Shape of Comparison of sub 2 um columnsEclipse Plus is Superior to Acquity BEH C18
min1 2 3 4 5 6
5.837
min1 2 3 4 5 6
5.503
ACQUITY BEH C18 2.1 x 50 mm, 1.7 umP = 228 Bar N = 10107, Peak Width = 0.137 min, Tf= 1.39
RRHT Eclipse Plus C18 2.1 x 50 mm, 1.8 umP = 202 Bar N = 10881,Peak Width = 0.124 min, Tf= 1.23
Mobile Phase = 80% 25 mM phosphate (pH 3): 20% ACN, Flow Rate = 0.3 mL/min Detection: 205nm
Analyte: Dextromethorphan
Tf = 1.4
Tf = 1.2
Many columns are competitive and slight improvements in peak shape can be important to maximum sensitivity.
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Resolution on RRHD vs. Other Sub 2um on 1290 Infinity
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min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
mAU
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300
350
min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
mAU
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350
min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
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α3,2: 1.36
Rs3,2 5.64N6 : 10700TF6: 1.04
α3,2: 1.22
Rs3,2 3.69N6 : 9400TF6: 1.17
α3,2: 1.29
Rs3,2 4.58N6 : 11000TF6: 1.14
RRHD SB-C18 2.1 x 50mm, 1.8um USCES1057
Acquity HSS C18 SB 2.1 x 50 mm 1.8um
Acquity BEH C18 2.1 x 50 mm, 1.7um
1
2
65
43
Mobile Phase: 40:60 water:acetonitrile, 0.1% Formic Acid Flow Rate: 0.5 mL/min, Temp: ambient, Det: UV 254 nm Columns: RRHD SB-C18, 2.1 x 50mm, 1.8um and competitive Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone
StableBond offers greatest Rs and is still the best column for low pH lifetime on the market!
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Are There Other Column Types for UHPLC?Review of Van Deemter Curves for Efficiency
Column types for UHPLC – depending on speed, resolution, and throughput requirements
1. RRHD columns – 1.8um columns for high productivity up to 1200 bar
2. RRHT columns – 1.8um columns for fast LC and with a 600 bar pressure limit
• Ideal for fast LC and for working at the 600 bar pressure limit
• Available in lengths from 20mm to 150 mm, with 20, 30, 50 and 75mm lengths ideal for fast high resolution separations
• 4.6 mm ID columns ideal for use at this lower pressure limit, with 3.0 and 2.1 mm ID columns also available
3. Upcoming Poroshell 120 columns
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Van Deemters for RRHT and RRHD Columns
John W. Henderson JrGroup/Presentation
Title
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0
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DAZ01002 LFS Column performance report resultsN=20600TF 1.02P=324F=0.3
RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 um;
PN 959758-902, SN DAZ01002, LN B009013
Naphthalene k=4.0
RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um;
PN 959764-902, SN USUXU01664, LN B08101
Heptanophenone k=10.8
N=24900TF 1.08P=520F=0.489
N=24900TF 1.08P=550F=0.520
N=22300TF 1.01P=535F=0.571
2.1 x 100 RRHD Optimum flow rate is about 0.5 mL/min. (60% MeCN, Themostatted 26°C, ~LC Performance Report Conditions)
Note pressure of more than 500 bar on 100mm length!With just acetonitrile and water – new 1290 Infinity LC ideal
Van Deemters are basically the same for RRHT and RRHD, this is expected.
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Why Is It Important to Know the Optimal Flow Rate?Efficiency is Highest at Optimal Flow Rate
John W. Henderson JrGroup/Presentation
Title
Agilent RestrictedMonth ##, 200X
min0.5 1 1.5 2 2.5 3
3.5
min0.25 0.5 0.75 1 1.25 1.5 1.752
DAZ01002 LFS Column performance report resultsN=20600TF 1.02P=324F=0.3
DAZ01002 N=25100TF 1.08P=550F=0.520
Sub-Optimal Optimal
0.3 mL/min 0.5 mL/min
N= 20,600 N= 25,100
P= 324 bar P = 550 bar
At sub optimal flow rates the efficiency is lower and all of the benefits of
a 1.8um columns or UHPLC technology may not be apparent.
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Van Deemter for Poroshell 120 and RRHT
H (cm) vs Flow rate (mL/min)
(60:40 ACN:H2O, all columns 4.6mm x 50mm)
0
0.0005
0.001
0.0015
0.002
0.0025
0 1 2 3 4 5 6
Flow Rate (mL/min)
Pla
te H
eig
ht
H (
cm)
Poroshell 120 SB-C18, 2.7µm
RRHT Zorbax SB-C18, 1.8 µm
1.8 µm totally porous particles2.7 µm Poroshell 120
H (cm) vs Flow rate (mL/min)
(60:40 ACN:H2O, all columns 4.6mm x 50mm)
0
0.0005
0.001
0.0015
0.002
0.0025
0 1 2 3 4 5 6
Flow Rate (mL/min)
Pla
te H
eig
ht
H (
cm)
Poroshell 120 SB-C18, 2.7µm
RRHT Zorbax SB-C18, 1.8 µm
1.8 µm totally porous particles2.7 µm Poroshell 120
Superficially porous particles and 1.8um particles have similar efficiencies and are both choices for UHPLC.
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Poroshell 120 Particle technology
Page 25
SOLID CORE < 3 µm < 2 µm
Poroshell 120 Superficially porous
Totally porousFully porous
sub-two-micron (STM)
T. Maloney, J. Cunliffe, J. Sep. Sci. 2007, 30, 3104 – 3109
G. Guiochon et al, Journal of Chromatography A, 1157 (2007) 289–303
G. Guiochon et al, Journal of Chromatography A, 1166 (2007) 30–46
Yu Zhang et al, Journal of Chromatography A, 1216 (2009) 4597–4605
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Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 26
Fast Analysis of Analgesics on Poroshell 120 –High Flow Rates, for Fast Analysis are Possible
Conditions: Column: Poroshell 120 SB-C18, 4.6 x 50mm, 2.7um Mobile Phase: 0.2% Formic Acid 80% water:20% ACN Temperature: 25 °C, Detection: 275 nm, Sample Injection: 2ul
2
min1 2 3 4
mAU
0
20
40
min1 2 3 4
mAU
0
20
40
min1 2 3 4
mAU
0
20
40
min1 2 3 4
mAU
0
20
40
1.0 mL/min 142 bar
1.5 mL/min 211 bar
2.0 mL/min 279 bar
2.5 mL/min 347 bar
1 34 5
6
1. Acetaminophen 2. Caffeine 3. 2-acetamidophenol 4. Acetanilide 5. Phenacetin 6. Salicylic acid
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Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 27
Conclusions
New Rapid Resolution High Definition (1.8um) columns are one solution for UHPLC systems and are unique in the marketplace because of their ability to be used over >1000 bar.
This makes them ideal for use with the 1290 Infinity LC.
Other UHPLC columns are also available including the RRHT, 600 bar columns with over 140 choices. The 4.6 mm ID columns represent a unique choice for easy to use UHPLC performance.
The upcoming release of the Poroshell 120 columns will provide a 3rd option for UHPLC type columns because a