New HPLC Column Options for UHPLCOptions for UHPLC · New HPLC Column Options for UHPLCOptions for...

New HPLC Column Options for UHPLCOptions for UHPLC

Rapid Resolution High Definition Columns and More

Maureen JosephLC Product ManagerLC Product ManagerJuly 16, 2009

Group/Presentation TitleAgilent Restricted

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UHPLC – Why Use Different Columns?

UHPLC instruments include a variety of high pressure LC choices that operate at pressures >400 bar/6000 psi.psi.

Small particle size columns <2-3um provide improved efficiency, resolution and productivity on UHPLC instruments.• ZORBAX Rapid Resolution HT, 1.8um columns for use up to

600 bar one example of a column for UHPLC600 bar one example of a column for UHPLC

Agilent’s new 1290 Infinity LC can operate up to 1200 bar.

The new ZORBAX Rapid Resolution High Definition 1.8um columns are the only columns that can be

used up to 1200 barGroup/Presentation Title

Agilent RestrictedMonth ##, 200X

used up to 1200 bar.

RRHD/RRHT Short 50mm Columns with Typical ACN Gradient Condition Don’t Require P > 600 barACN Gradient Condition Don t Require P 600 bar

Sample:Octyl dimethyl para-aminobenzoic acid, Column: RRHD Eclipse Plus C18 3 x 50mm 1.8um, Temp: 40C, Flow Rate: 1.5 ml/min, Gradient: 40-90% ACN/water over 2 minutes. Pressure: Up to 460 barPressure: Up to 460 bar


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1290 Infinity Introduction, CASUAgilent Restricted

May 2009

The 1290 Infinity Has a Wide Useable UHPLC Range


Month ##, 200X June 06, 2005Page 4

LC Beyond 600 bar Requires New RRHD ColumnsAgilent ZORBAX RRHT Eclipse Plus C18 2 1 x 100 mm 1 8 um; PN 959764‐902Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN 959764‐902,

SN USUXU01664, LN B08101

1817 7 21 259

700

y = 935.21x + 7.1903

R2 = 0.9993

y = 1025.3x + 13.279

R2 = 0.9973

y = 1817.7x + 21.259

R2 = 0.9946

500

600

300

400

Pressure (b

ar)

Acetonitrile/Water(60:40)

200

300

Acetonitrile/Water(50:50)

Methanol/Water(60:40)

0

100

0 0.1 0.2 0.3 0.4 0.5 0.6


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Flow Rate (mL/min)

Fast Separation with High Resolution on RRHD Column and Agilent 1290 Infinity LCColumn and Agilent 1290 Infinity LC

Octyl dimethyl para-aminobenzoic acid, 2.1 x 100mm 1.8um, 40C, 0.74 ml/min, 40-90% ACN/water over 4 minutes. Up to 775 bar


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May 2009

Methanol Selectivity Can Be Explored When Operating Pressure is not a ProblemWhen Operating Pressure is not a Problem

Octyl dimethyl para-aminobenzoic acid, 2.1 x 100mm 1.8um, 40C, y y p , , ,0.52 ml/min, 50-100% MeOH/water over 5.7 minutes. Up to 845 bar


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May 2009

What Type of Productivity Can Be Achieved Using RRHD Columns with the 1290 Infinity LCRRHD Columns with the 1290 Infinity LC1. Fast analysis – especially with somewhat complex samples

1 Gradient analysis with 4 10 components or more!1. Gradient analysis with 4-10 components or more!2. For LC and LC/MS systems

2. More resolution in a limited amount of time1. Choosing different column lengths for the resolution desired2. Maximizing peak capacity in gradient separations of complex samples


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Sub 1 Minute Separations with RRHD Columns

10 Compound Pharma Test Mix

Agilent ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8 μm

H2O (0.05% Formic acid) / 2-98 % Acetonitrile T = 80°CT = 80°CV_i = 0.5 µl WL = 210 nm DR = 80 HzF 2 l/ iF = 2 ml/minP = 1085 bar 1.0min


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MS/MS

“This method is performance-based which means that youThis method is performance based which means that you may modify the method to improve performance (e.g., to overcome interferences or improve the accuracy or precision of the results) provided that you meet all performance ) p y prequirements in this method.”

EPA Method 1694 section 1 6 allows method flexibilityGroup/Presentation Title


1290 Infinity Applications

June, 2009

EPA Method 1694, section 1.6 allows method flexibility

Ultrafast LC/MS Analysis for 15 Analyte Subset

RRHD Eclipse Plus C182.1x 50 mm, 1.8 um750 bar1 minute

Peak Width 0.7 secTime Composition

0.0 10% ACN1.5 100% ACN

Ulti t d h t l ith b lli ti di tGroup/Presentation Title


1290 Infinity Applications

June, 2009

Ultimate speed on a short column with ballistic gradient

Sub 1 Minute Separations with RRHD ColumnsColumn: ZORBAX RRHD SB-C18 2 1 x 50mm 1 8 µm

6 Compo nd Biocide Mi in 0 5min

Column: ZORBAX RRHD SB C18 2.1 x 50mm 1.8 µmGradient: H2O (0.05% trifluoroacetic acid) / 10-40 % ACN/1min

21 2-methyl-4-isothiazolin-3-one 6 Compound Biocide Mix in 0.5min

F = 2.0 ml/minP = 975 bar

T = 60°C

1. 2 methyl 4 isothiazolin 3 one2. 5-chloro-2-methyl-4-isothiazolin-3-one3. Carbendazim4. Benzisothiiazol-3(2H)-one5. 2-phenoxyethanol6. Methylparaben

0.5min

T = 60 CV_i = 0.5 µl x 100ppm eachWL = 275 nm DR = 160 Hz

1

26

45

6

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45


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Month ##, 200XPage 12 June 06, 2005Page 12

Increase Flow Rate for Fastest Separation on RRHD Use Complete Pressure Range of 1290 Infinity UHPLC

F = 2 ml/minP = 975 barmAU

T = 60°CV_i = 0.5 µl x 100ppm eachWL 275

0.5min

200

300

400

500 WL = 275 nm DR = 160 Hz

F 2 3 l/ i

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

100

mAU500 F = 2.3 ml/min

P = 1110 bar0.4min

ZORBAX SB-C18 2.1 x 50mm 1.8 µm

200

300

400

500

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

100


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Month ##, 200XPage 13 June 06, 2005Page 13

Fast Analysis on ZORBAX Rapid Resolution High Definition Eclipse Plus C18Definition Eclipse Plus C18

mAU

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)

0.4

15

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)

THBP RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um, Pmax 1070 bar

500

Pmax 1070 barThis 10 component sample requires a gradient that goes over 1000 bar.This is a sub 2minute separation!

300

400

0.2

72PGOG

Data Courtesy of: Gerd Vanhoenacker Research Institute for ChromatographyKennedypark 26 8500 Kortrijk Belgium

200

80

0.7

57

83

1.0

55

1.5

24

4

TBHQNDGA

BHA

OGDG

BHT AP

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

0

100

0.4

8

0.8

8

1.0

11 1.5

56 1.6

84

Ionox-100BHT AP


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Fast Analysis on Agilent Zorbax Rapid R l ti Hi h D fi iti E li Pl C18Resolution High Definition Eclipse Plus C18MethodColumn ZORBAX RRHD Eclipse Plus C18 2.1 x 50 mm, 1.8 µm

(SN USDAY01001)Mobile phase A=0.02% H3PO4, B=acetonitrile/methanol 3/1Flow rate 1.6 mL/minGradient 0‐1 85 min 30‐100% BGradient 0‐1.85 min 30‐100% BInjection volume 1 µLDetector Sig=280/10 nm, Ref=400/50, 80 Hz

Sig=255/10 nm, Ref=400/50, 80 HzTemperature 45°CSample Antioxidant mixture, ca. 100 ppm

A 1 6 mL/min flow rate is very fast and allows a fast separation

Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography

A 1.6 mL/min flow rate is very fast, and allows a fast separation.


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y g p yKennedypark 26 8500 Kortrijk Belgium

Where Does this Example Fall in the 1290 Infinity Range

This app falls in the “Power Range”pp g



Choose Column Length for Analysis Time and RsMobile Phase: 40:60 water:acetonitrile, Flow Rate: 0.5 mL/min, Temp: ambient, Columns: RRHD SB-C18, 2.1 mm ID L th t d P k 1 il 2 t h 3 i h 4 b t h 5 l h 6

RRHD 1.8um 1200 bar

ID, Lengths as noted Peaks: 1: uracil, 2 acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone

Rs3,2: 5.64N6 : 10,700N6 : 10,700Pressure: 350 bar50 mm

min0 1 2 3 4

Rs3,2: 9.02N6 : 21,100Pressure: 566 bar

100 mm

Rs3,2: 11.98N6 : 34,100150 mm

min0 1 2 3 4

Pressure:750 bar

min0 1 2 3 40

The 150mm length RRHD column becomes a good option for high resolution with


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g g p gthe 1290 Infinity LC.

Gain Efficiency and Resolution with Long Length 150 + 100 mm = 250 mm 1.8um @ 1100 bar150 100 mm 250 mm 1.8um @ 1100 bar

α3,2: 1.196 42

150 mm (P:582 bar)

Mobile Phase 25:75 water:acetonitrile, Flow Rate: 0.5 mL/min, ambient temperature, ZORBAX RRHD SB-C18, 1.8um columns

mAU300

Rs3,2: 6.42N6 : 31,400Pw2: 0.016 minPw6: 0.029 min

50 ( 58 ba )

100

150

200

250

α3,2: 1.20Rs3 2: 8.76

150 +100 mm (P:1095 bar)1. Uracil2 Acetophenone

min0 0.5 1 1.5 2 2.5 3 3.5

0

50

mAU

250

300

Rs3,2: 8.76N6 : 50,200Pw2: 0.018 minPw6: 0.037 min

2. Acetophenone3. Propiophenone4. Butyrophenone5. Valerophenone6. Hexanophenone

50

100

150

200

250

High Rs in 3.5 minutes, 250mm, 1.8um at 1100 bar

min0 0.5 1 1.5 2 2.5 3 3.5

0

50


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Resolution is fine on 150mm column, but for even more Rs longer column is used.

Where Does this Example Fall in the 1290 Infinity Range

This app falls in the “Power Range”pp g



High Resolution Separation with Long 1.8um ColumnsColumns

mAU

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

200

Gradient 10-100% B/15 min.Pressure 800- 900 barLow point of gradient about 480 bar

100

150

50

min0 2 4 6 8 10 12 14

0

Long column or columns in series provide max Rs with this complex


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sample – smoked food flavoring.

Achieving Optimal Resolution for a Gradient with RRHD Column ChoicesRRHD Column ChoicesColumns range in length from 50 – 150mm.

Column length and gradient time can be changed to optimizeColumn length and gradient time can be changed to optimize the resolution and retention of the analytes.

k* =tG • F

These 3 parameters canbe easily changed.tG = gradient timek =

S • ΔΦ • Vm

G gF = flow rate Vm = column volume

(change by changing column(change by changing column length)


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Separation of Licorice Root on RRHD Columns –30 minute gradient on each length30 minute gradient on each lengthmAU

200 RRHD SB-C18 2.1 x 50 mm, 1.8umRs: 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 UV1290 Infinity

min0 2 4 6 8 10 12 14 16 180

50

100

150 Pmax=366 barnc = 424

min0 2 4 6 8 10 12 14 16 18mAU

100

150

200

RRHD SB-C18 2.1 x 100 mm , 1.8umPmax=595 bar nc = 485

Rs: 1.37

min0 2 4 6 8 10 12 14 16 180

50

mAU

200

nc 485

Rs: 2 40

0

50

100

150

200

RRHD SB-C18 2.1 x 150 mm, 1.8umPmax=768 bar nc = 589

Rs: 2.40


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min0 2 4 6 8 10 12 14 16 180

Separation of Licorice Root on RRHD Columns –30 minute gradient on each length30 minute gradient on each length

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 UV1290 Infinity

50 mm 100 mm 150 mm100 mm 150 mm

7 peaks 8 peaks 9 peaks

Rs: 2.40Rs: 1.37Rs: 0

2 4 2 4 62 4


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Increasing peak capacity led to increased Rs for this sample.

Peak Capacity Comparisons – Length and Gradient TimeTime

Licorice Root sample

nc= tg/wbaseline of last eluting peak1

10 minutes 20 minutes 30 minutesGradient Time Column

Length

50 313 358 424

100 343 418 485

150 386 483 589

Shaded boxes indicate comparable peak capacities

1 Neue U D et al Advances in Chromatography 41 93 (2001)


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1. Neue, U.D., et. al, Advances in Chromatography, 41, 93 (2001)

Why Two Different C18 Columns? Eclipse Plus C18 and StableBond C18Eclipse 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 formatRRHD format


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Different ZORBAX RRHT/RRHD C18 Bonded Phases for Maximum SelectivityyMobile 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 umSample: 1. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

Eclipse Plus C181 2 41st choiceBest Resolution

3

1 2 3 4 5

Best Resolution& Peak Shape

StableBondSB-C18

1 234

2nd choiceGood alternate selectivity due to non-endcapped

min1 2 3 4 5

M ltiple bonded phases for most effecti e method de elopment


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Multiple bonded phases for most effective method development.

Peak Shape of Comparison of sub 2 um columnsEclipse Plus is Superior to Acquity BEH C18Eclipse Plus is Superior to Acquity BEH C18

Mobile Phase = 80% 25 mM phosphate (pH 3): 20% ACN, Flow Rate = 0.3 mL/min Detection: 205nmAnalyte: Dextromethorphan

5.837ACQUITY BEH C18 2.1 x 50 mm, 1.7 umP = 228 Bar N = 10107, Peak Width = 0 137 min

Tf = 1.4

min1 2 3 4 5 6

5.503

Peak Width 0.137 min, Tf= 1.39

RRHT Eclipse Plus C18 Tf 1 2

min1 2 3 4 5 6

RRHT Eclipse Plus C18 2.1 x 50 mm, 1.8 umP = 202 Bar N = 10881,Peak Width = 0.124 min, Tf 1 23

Tf = 1.2

min1 2 3 4 5 6Tf= 1.23

Many columns are competitive and slight improvements in peak shape can be important for maximum sensitivity


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be important for maximum sensitivity.

Resolution on RRHD vs. Other Sub 2um on 1290 InfinityInfinity

mAU350 α : 1 36

RRHD SB-C18 2.1 x 50mm, 1.8um USCES1057

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

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80

50100150200250300 α3,2: 1.36

Rs3,2 5.64N6 : 10700TF6: 1.04

USCES1057

mAU

50100150200250300350

α3,2: 1.22Rs3,2 3.69N6 : 9400TF6: 1.17

Acquity HSS C18 SB 2.1 x 50 mm 1.8um

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80

50

mAU

200250300350

6

α3,2: 1.29Rs3,2 4.58

Acquity BEH C18 2.1 x 50 mm, 1.7um

2

65

43

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80

50100150 N6 : 11000

TF6: 1.1416

StableBond offers greatest Rs and is still the best column for low pH lifetime


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on the market!

Are There Other Column Types for UHPLC?Review of Van Deemter Curves for EfficiencyReview of Van Deemter Curves for Efficiency

There are lots of column types for UHPLC – depending on speed resolution and throughput requirementsspeed, resolution, and throughput requirements

RRHD columns – 1.8um columns for up to 1200 bar

RRHT l 1 8 l f f t LC d ith 600 bRRHT 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 limitIdeal 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 separations4 6 ID l id l f t thi l li it ith 3 0 d• 4.6 mm ID columns ideal for use at this lower pressure limit, with 3.0 and 2.1 mm ID columns also available

Upcoming Poroshell 120 columns


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p g

Van Deemters for RRHT and RRHD Columns RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um;

12

14

PN 959758-902, SN DAZ01002, LN B009013Naphthalene k=4.0

PN 959764-902, SN USUXU01664, LN B08101Heptanophenone k=10.8

Note pressure of more than 500 bar on 100mm length!With just acetonitrile and water – new 1290 Infinity LC ideal

10

12

DAZ01002 LFS Column performance report results

N=24900TF 1.08P=520F=0.489

N=24900TF 1.08P=550F=0.520

N=22300TF 1.01P=535F=0.571

j y

6

8

h

N=20600TF 1.02P=324F=0.3

2

4

Van Deemters are basically the same for RRHT and RRHD this is0

0 1 2 3 4 5 6 7 8

u (mm/s)

Van Deemters are basically the same for RRHT and RRHD, this is expected.


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John W. Henderson JrGroup/Presentation Title

Agilent Restricted

2.1 x 100 RRHD Optimum flow rate is about 0.5 mL/min. (60% MeCN, Themostatted 26°C, ~LC Performance Report Conditions)

Why Is It Important to Know the Optimal Flow Rate?Efficiency is Highest at Optimal Flow RateEfficiency is Highest at Optimal Flow Rate

DAZ01002 LFS Column performance

Sub-Optimal Optimal0.3 mL/min 0.5 mL/minperformance

report resultsN=20600TF 1.02P=324F=0.3

N= 20,600 N= 25,100P= 324 bar P = 550 bar

min0.5 1 1.5 2 2.5 3 3.5DAZ01002 N=25100TF 1.08P=550F=0.520

min0.25 0.5 0.75 1 1.25 1.5 1.75 2

At sub optimal flow rates the efficiency is lower and all of the benefits of


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John W. Henderson JrGroup/Presentation Title


a 1.8um columns or UHPLC technology may not be apparent.

What is Poroshell 120

Porous Shell, 0.5um

120Å pores

A new HPLC column with: 1. sub 2um like high efficiency2. At ~40-50% lower pressure 120Å, pores

2.7um particlesp

3. A 2.7um particle size4. A “standard” frit to reduce

potential clogging

The particle has a solid core (1.7um) and porous outer layer with a 0 5 diff i th

} 0.5um

Solid Core

1.7um

0.5um diffusion path

Poroshell is the Agilent name used to describe superficially porous udescribe superficially porous particles.

} 0.5um


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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)

0.0025

H (cm) vs Flow rate (mL/min)(60:40 ACN:H2O, all columns 4.6mm x 50mm)

0.0025

0.0015

0.002

0 00 5

H (cm)

Poroshell 120 SB-C18, 2.7µm

RRHT Zorbax SB-C18, 1.8 µm0.0015

0.002

0 00 5

H (cm)

Poroshell 120 SB-C18, 2.7µm

RRHT Zorbax SB-C18, 1.8 µm

0 0005

0.001

0.0015

Plate Height

1.8 µm totally porous particles2.7 µm Poroshell 120

0 0005

0.001

0.0015

Plate Height

1.8 µm totally porous particles2.7 µm Poroshell 120

0

0.0005

0 1 2 3 4 5 6

Flow Rate (mL/min)

0

0.0005

0 1 2 3 4 5 6

Flow Rate (mL/min)( / )( / )

Superficially porous particles and 1.8um particles have similar efficiencies and are both choices for UHPLC


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similar efficiencies and are both choices for UHPLC.

Fast Analysis of Analgesics on Poroshell 120 –High Flow Rates, for Fast Analysis are PossibleHigh Flow Rates, for Fast Analysis are Possible

2

mAU

401.0 mL/min 142 bar1 3 4 5

1. Acetaminophen 2. Caffeine 3. 2-acetamidophenol 4. Acetanilide 5. Phenacetin 6. Salicylic acid

2

min1 2 3 40

20

mAU

40 1.5 mL/min 211 bar

4 56

min1 2 3 40

20

mAU40

1.5 mL/min 211 bar

2 0 mL/min 279 bar

min1 2 3 40

20

mAU40

2.0 mL/min 279 bar

2 5 L/ i 347 b

Conditions: Column: Poroshell 120 SB-C18 4 6 x 50mm 2 7um Mobile Phase: 0 2% Formic Acid 80% water:20% ACN

min1 2 3 40

202.5 mL/min 347 bar


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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

Conclusions

New Rapid Resolution High Definition (1.8um) columns are one solution for UHPLC systems – and the Agilent 1290 Infinity LC.

They are unique in the marketplace because of their ability toThey are unique in the marketplace because of their ability to be used over >1000 bar.

Other UHPLC columns are also available including the RRHT,Other UHPLC columns are also available including the RRHT, 600 bar columns with over 140 choices (ideal with the Agilent RRLC).

Th 4 6 ID RRHT l t i h i fThe 4.6 mm ID RRHT columns represent a unique choice for easy to use UHPLC performance.

The upcoming release of the Poroshell 120 columns will provideThe upcoming release of the Poroshell 120 columns will provide a 3rd option for analytical UHPLC type columns because they provide productivity – efficiency and speed similar to 1.8um.


Month ##, 200X

New HPLC Column Options for UHPLCOptions for UHPLC · New HPLC Column Options for UHPLCOptions for...

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