column selection for uhplc c - chemical analysis, life ... · time 0.1 95/5 a/b 225 nm (0.8 min)...

Group/Presentation TitleAgilent Restricted

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Tips and Tricks of UHPLC Column Technology and

Selection

Rapid Resolution High Definition Columns and More

August 20, 2009


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What is Different About Column Selection for UHPLC?

Only 2 Things!

1. Column Particle Size1. Smaller particle sizes are used (<3um)

2. Column Operating Pressure Limits1. Pressure limits are > 400 bar (600 – 1200 bar is typical)

Everything else about column selection remains the same!


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Agilent Has Three Types of Columns for UHPLC

There are 3 column types for UHPLC – choice depends on your requirements

RRHT columns – 1.8um columns for fast LC up to 600 bar • Designed for compatibility with the Agilent RRLC • Compatible with some UHPLC’s with pressure limits up to 800 bar

RRHD columns – 1.8um columns for up to 1200 bar• Designed for compatibility with the new Agilent 1290 Infinity LC• Useable up to 12000 bar• Fully UHPLC compatible

Upcoming Poroshell 120 columns• 600 bar pressure limit for use with HPLC/UHPLC• UHPLC type performance but good fit for 400 bar LC’s


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Rapid Resolution HT (1.8um) Columns for the Agilent 1200 RRLCOver 140 RRHT,1.8um 600 bar column choices available• 15 Different Column Chemistries – 14 bonded phases and silica for HILIC

use• 6 column lengths (150, 100, 75, 50, 30 and 20 mm long)• 3 internal diameters (4.6, 3.0 and 2.1 mm ID)• Each ZORBAX phase available in matching 3.5um and 5um choices, most

in 7um!• 2 Recommended Starting Choices:

– 959941-902 - Eclipse Plus C18 4.6 x 50mm, 1.8um for 1200 RRLC used for general applications

– 959741-902 – Eclipse Plus C18 2.1 x 50mm, 1.8um for the 1200 RRLC with an MS detector

– Short columns great for fast analysis and useable on non UHPLC instruments


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RRHT – Delivers Maximum Speed for HPLC or UHPLC

min0 1

1

2Peak TR N Rs1 0.40 2991 02 0.69 4025 6.9

Rapid Resolution HT Column (L1)ZORBAX Eclipse XDB-C18: 4.6x30mm, 1.8um

P/N 923975-902Eluent: 60% Methanol:40% Water,

Flow: 1.5 mL/minInjection: 1uL, Temp: 25 C

RRHT provides max speedfor ultra-fast analysis!


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1290 Infinity Introduction, CASUAgilent Restricted

May 2009

RRHD/RRHT Short 50mm Columns with Typical ACN 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 bar


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What Can I do Beyond This with UHPLC?RRHD Columns for Use with the Infinity 1290 LCUse a wider range of mobile phase choices

More viscous solvents – methanol, other alcoholsMobile phase additives to optimize separations (they increase viscosity)

Go faster – wider flow rate range!

Use longer columns – maximize resolution!

Optimize methods faster, improve productivity!


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

SN USUXU01664, LN B08101

y = 935.21x + 7.1903

R2 = 0.9993

y = 1025.3x + 13.279

R2 = 0.9973

y = 1817.7x + 21.259

R2 = 0.9946

0

100

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300

400

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600

700

0 0.1 0.2 0.3 0.4 0.5 0.6

Flow Rate (mL/min)

Pressure (b

ar)

Acetonitrile/Water(60:40)

Acetonitrile/Water(50:50)

Methanol/Water(60:40)


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The 1290 Infinity Has a Wide Useable UHPLC Range

June 06, 2005Page 9


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May 2009

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

Octyl dimethyl para-aminobenzoic acid, RRHD Eclipse Plus C18, 2.1 x 100mm 1.8um, 40°C, 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 Problem

Octyl dimethyl para-aminobenzoic acid, RRHD Eclipse Plus C182.1 x 100mm 1.8um, 40C, 0.52 ml/min, 50-100% MeOH/water over 5.7 minutes. Up to 845 bar


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Tips for Maximum Performance with UHPLC

Take advantage of the limits of your UHPLC to maximize your results!

Try different solventsUse longer columnsVary your flow ratesUse RRHD 1.8um columns on >600 bar UHPLC’s for maximum flexibilityUse RRHT 1.8um columns on UHPLC’s with pressure limits <800 bar


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

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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Fast Analysis with UHPLC

Sub one minute separations are possible on UHPLC• With standard UV detection modes• With LC/MS• For a few compounds (1-5 compounds)• For up to 10 – 20 compounds


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Sub 1 Minute Separations with RRHD Columns

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

H2O (0.05% Formic acid) / 2-98% Acetonitrile T = 80°CInj Vol = 0.5 µl WL = 210 nm DR = 80 HzF = 2 ml/minP = 1085 bar

10 Compound Pharma Test Mix

1.0min


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Sub 1 Minute Separations with RRHD Columns

June 06, 2005Page 16

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

0.5min

6 Compound Biocide Mix in 0.5min

F = 2.0 ml/minP = 975 bar

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

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

1

2

2

45

6

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


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


F = 2 ml/minP = 975 bar

F = 2.3 ml/minP = 1110 bar

0.5min

0.4minZORBAX RRHD SB-C18 2.1 x 50mm 1.8 µm1200 bar

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

mAU

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min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

mAU

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300

400

500

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


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Fast Analysis Isn’t Always Sub 1 Minute!

Separations of 2 -5 minutes are still fast!

UHPLC can speed up many analyses.

Evaluate your options• Develop your method• Optimize your method if needed or desired


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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

0

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

.556

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 2 minute separation!


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Fast Analysis on Agilent Zorbax Rapid Resolution 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% 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

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

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


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Evaluating Your Fast LC Options Seven Biocides Separated in 3.0 Minutes

A: H2O (0.05 v% TFA) B: ACN (0.04 v% TFA)Time 0: 95/5 A/BTime 2.9: 85/15 A/BTime 3.9: 85/15 A/BFlow: 1.0 mL/minTemp: 30oCInjection: 1 uLStd. Conc: 50 ppm

1

2

3

4

5

67

1. Kathon 1A 2. Kathon 1B 3. Carbendazim4. 1,2-Benzisothiazol-3(2H)-one 5. 2-Phenoxyethanol 6. Benzoic Acid 7. Methyl Paraben

Pressure 630 bar

min0.5 1 1.5 2 2.5 3

RRHD Eclipse Plus C18 1.8 um 2.1 x 50mm Column


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Optimize Method – Change GradientSeven Biocides Separated in 1.1 MinutesRRHD Eclipse Plus C18 1.8 um 2.1 x 50mm Column

1

2

3

4

6

7


A: H2O (0.05 v% TFA) B: ACN (0.04 v% TFA)Time 0.0 95/5 A/B DAD: 275 nm (0 min)Time 0.1 95/5 A/B 225 nm (0.8 min)Time 0.25 90/10 A/B 255 nm (1.08 min)Time 1.0 65/35 A/B Time 1.1 0/100 A/B1.0 mL/min1 uL injection50 ppm standard30oC

Pressure 800 bar

5

min0.2 0.4 0.6 0.8 1 1.2

Increase speed by changing to a linear, segmented gradient.


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Separation of Seven Biocides in 0.7 minRRHD Eclipse Plus C18 2.1 x 50mm, 1.8 um Column

A: H2O (0.05 v% TFA) B: ACN (0.04 v% TFA)Time 0.0 95/5 A/B DAD: 275 nm (0 min)Time 1.0 55/45 A/B 225 nm (0.46 min)Time 1.1 0/100 A/B 255 nm (0.67 min)1.7 mL/min1 uL injection50 ppm std.30oC

1

2

3

4

67


Pressure 1017 bar

5

min0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Increase Flow Rate to Maximize Speed!


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Where Does this Example Fall in the 1290 Infinity Range


Both apps fall in the “Power Range”


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Tips for Fastest Analysis

Design your analysis time to meet your needs!

If doing gradient analysis - optimize your gradient for fast analysis.

Increase flow rate to achieve analysis time desired.


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Maximizing Productivity by Maximizing Resolution

Sometimes resolution is more important than speed!

UHPLC offers many alternatives for increasing resolution.

Speed can be maintained for some separations – will depend on sample complexity, flow rate etc.


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Choose Column Length for Analysis Time and Rs



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 40

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



α3,2: 1.20Rs3,2: 8.76N6 : 50,200Pw2: 0.018 minPw6: 0.037 min

α3,2: 1.19Rs3,2: 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

mAU

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300

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300


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Where Does this Example Fall in the 1290 Infinity Range


This app falls in the “Power Range”


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High Resolution Separation with Long 1.8um Columns



min0 2 4 6 8 10 12 14

mAU

0

50

100

150

200

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 barLow 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 ChoicesColumns 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 • ΔΦ • 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



min0 2 4 6 8 10 12 14 16 18

mAU

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min0 2 4 6 8 10 12 14 16 18

mAU

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min0 2 4 6 8 10 12 14 16 18

mAU

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200

RRHD SB-C18 2.1 x 50 mm, 1.8umPmax=366 barnc = 424

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

RRHD SB-C18 2.1 x 150 mm, 1.8umPmax=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 UV1290 Infinity


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



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 UV1290 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

589483386150

485418343100

42435831350

30 minutes20 minutes10 minutes

Licorice Root sampleGradient 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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Why Two Different C18 Columns? Eclipse Plus C18 and StableBond C18Different selectivity• Eclipse Plus C18 vs. StableBond C18

Optimum performance at high pressure• Greater stability and pressure range with ZORBAX

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 umSample: 1. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

Eclipse Plus C181 23

4

1 2 3 4 5

1st choiceBest Resolution& Peak Shape

StableBondSB-C18

1 234

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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RRHD Column Pressure Similar to AcquityRRHD Eclipse Plus C18 vs. Acquity BEH C18 (2.1 x 50mm)

A: H2O (0.05 v% TFA) B: ACN (0.04 v% TFA)Time 0: 95/5 A/BTime 2.9: 85/15 A/BTime 3.9: 85/15 A/BFlow: 1.0 mL/minTemp: 30oCInjection: 1 uLStd. Conc: 50 ppm


RRHD Eclipse Plus C18 Pressure 630 bar

12

3

4

56

7

min0.5 1 1.5 2 2.5 3

35

6 7

min0.5 1 1.5 2 2.5

Acquity BEH C18Pressure 690 bar

1

2

4

Seven Biocides Separated in 3.0 Minutes


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Seven Biocides Separated in 1.1 Minutes Comparison of RRHD Eclipse Plus C18 and Acquity BEH C18


A: H2O (0.05 v% TFA) B: ACN (0.04 v% TFA)Time 0.0 95/5 A/B DAD: 275 nm (0 min)Time 0.1 95/5 A/B 225 nm (0.8 min)Time 0.25 90/10 A/B 255 nm (1.08 min)Time 1.0 65/35 A/B Time 1.1 0/100 A/B1.0 mL/min1 uL injection50 ppm standard30oC

Pressure 800 bar

1

2

3

4

5

67

min0.2 0.4 0.6 0.8 1 1.22

RRHD Eclipse Plus C182.1 x 50mm, 1.8um

1 3

4

5

6

7

min0.2 0.4 0.6 0.8 1 1.2

Acquity BEH C18 2.1 x 50mm, 1.7um

At lower pressure, RRHD Eclipse Plus C18 and Acquity BEH C18 look comparable.But as pressure increases and gradient speed increases, RRHD is more stable.


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What are the Additional Concerns About Using RRHD UHPLC ColumnsAre the columns rugged and reliable?• Does the small particle size make these columns difficult to use?• Small particle size means small frit size. Does this limit my sample types?

How many injections can be done on a column and how long will it last?• Do I need to replace columns frequently?


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Sample Types for RRHD Columns – Tips for Optimal Column LifetimeMinimal sample prep for completely soluble/clean samples• Use HPLC grade solvents for sample prep

Filter dirty samples for longest lifetime• Minimize particulates with 0.2um filter instead of 0.45um filter

Follow recommended directions in the column box• Data Sheet• Additional instructions on insert card (also provided in Appendix here)• Expect lifetime to be impacted by bonded phase, mobile phase, other

operating condition choices – just as with normal column selection


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Use RRHD Eclipse Plus C18 Columns with a Variety of Samples

Biocides Standards

Hand Sanitizer Sample - Injected Neat


1

2

3

4

5

6 7

min0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

2-Phenoxyethanol Methyl Paraben

min0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Identification of 2-Phenoxyethanol & Methyl Paraben in a Hand Sanitizer Sample in 0.7 minutes

Some samples can be injected onto sub 2-micron columns with limited sample prep.This is a liquid sample.


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Protein Precipitated Plasma Can be Injected on RRHD Columns

Sample was plasma, protein precipitated, centrifuged, filtered through 0.2um filter.Columns have 0.5um frit. If you do not filter the samples shorter lifetime would be expected.

Rapid Injections of Protein Precipitated Plasma on RRHD Column

200300400500600700

120

040

060

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010

0012

0014

0016

0018

0020

0022

0024

0026

0028

00

Injection #

Pres

sure

(bar

)


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RRHD Columns Have Good Lifetime at >1000 bar

Lifetime Test of RRHD Column at 1100 bar

8000

1000012000

14000

1600018000

20000

3

251

503

750

1000

1250

1500

1750

2000

2250

2500

2750

3000

3250

Injection #

N

In Progress

In Progress

Clean sample injected 5000 times showing good bed stability. Actual user lifetime may be influenced by operating conditions.


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Agilent Poroshell 120 Columns for HPLC and UHPLC

Porous Shell, 0.5um 120Å, pores

2.7um particles

Poroshell 120 columns have: 1. sub 2um like high efficiency2. At ~40-50% lower pressure3. A 2.7um particle size4. A column for HPLC and UHPLC5. A “standard” frit to reduce clogging

The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path

Solid Core

1.7um

} 0.5um

} 0.5um


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Fast Analysis of Analgesics on Poroshell 120 –Demonstrating “Fitness” for 400 bar LCs

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

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20

40

min1 2 3 4

mAU

0

20

40

min1 2 3 4

mAU

0

20

40

min1 2 3 4

mAU

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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 3 4 56

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


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Poroshell 120 Operates at Lower Pressure than Sub 2-micron

min0 2 4 6 8 10

min0 2 4 6 8 10

RRHT Eclipse Plus C181.8 umP = 510 Bar

Poroshell 120 EC‐C18 2.7 um P = 332 Bar

Columns: 4.6 x 100 mmA: Water 0.1% Formic AcidB: Acetonitrile 0.1% formic AcidGradient 2 ml/minInitial 8 % B10 min 30% B275 nm 2mm flow cellInjection: 10 uLAgilent 1200 SL 40 °C

1. Hydroquinone2. Resorcinol3. Catechol4. 4‐Nitrophenol5. p‐cresol6. o‐cresol7. 2‐Nitrophenol8. 2,3 Dimethyl phenol9. 2,5 Dimethyl phenol10. 1‐Naphthol

10

4 56 7

8

9

10

12

3

12

34 5

678

9

Poroshell 120 Provides sub 2-micron like efficiency and Rs at lower pressure.

Agilent RRHT has lower pressure than most sub 2um columns, but Poroshell is even lower.


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Agilent UHPLC Column Selection Guidelines

With 3 choices available for UHPLC how do you choose the right column for your application?

There may be more than 1 right choice or a different choice for each application.

The 3 choices are:

ZORBAX Rapid Resolution HD 1.8um, 1200 bar

ZORBAX Rapid Resolution HT, 1.8um, 600 bar

Poroshell 120, 2.7um, 600 bar


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Agilent UHPLC Column Selection Guidelines

1000+ bar UHPLC

“Fitness”

2um frit

Dirty Samples?

X (3.0 & 2.1)

XRRHD,

1200 bar

<50mm

RRHT, 600 bar

X only 2.7um

Poroshell120, 600

bar

ID’s4.6, 3.0 & 2.1 mm

Scalability

1.8, 3.5etc.

400 bar LC “Fitness”

High Rs (N)

Fast Analysis


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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 to be used over >1000 bar.

RRHT 600 bar UHPLC columns with over 140 choices and are ideal with the Agilent 1200 RRLC.

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


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Appendix

A few additional points are included in the appendix.

Column matches to other UHPLC instruments

Recommendations for maximum lifetime with RRHD/RRHT columns


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Which Columns Are Recommended with Which LC

YESYESAgilent 1200 RRLC (600 bar)

YESAgilent 1290 Infinity LC (1200 bar)

YESYESYes*Dionex RSLC (800 bar)

YESYESHitachi LaChrom Ultra

(600 bar, 8700 psi)

YESYESShimadzu

Prominence UFLC-xr(660 bar)

Yes*YESJasco Xtreme-LC (1000 bar)

Yes*YESThermo Accela (1000 bar)

Yes*YESWaters Acquity (1000 bar)

Poroshell 120 (600 bar)

RRHD (1200 bar)RRHT (600 bar)

* Must adhere to pressure limits of column, not instrument.


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Basic Instructions for Using RRHD/RRHT Columns Effectively1. Install and run the column only in the flow direction marked

on the column

2. Use only high quality, chromatography grade solvents3. Filter all aqueous buffers and all samples through a mobile

phase appropriate 0.2um filter before use.

4. Replace bottles of mobile phase buffer every 24-48 hours –do not add mobile phase to the bottle.

5. Use an in-line filter to prolong column life. 1. 5067-1551 for 2.1 or 3.0mm ID RRHT columns or 5067-1553 for 4.6

mm ID RRHT columns2. In-line filter ships with 1290 pump for use with RRHD columns and can

be ordered separately – part number 5067-4638.


Month ##, 200X

Abstract

UHPLC is a high productivity tool in the analytical lab. Modern LC column technologies such as sub 2-micron particle columns are key to maximizing productivity with these powerful LC instruments. This presentation will provide information on new Rapid Resolution High Definition; 1.8um columns manufactured with new hardware and improved packing processes to deliver superior, rugged and reliable performance with the 1290 InfinityLC. In addition, this presentation will provide some information on the soon to be available Poroshell 120 superficially porous particles as these provide an interesting alternative to sub 2-micron columns for high productivity solutions.


Month ##, 200X

Resolution on ZORBAX RRHD SB C18, 2.1 x 150mm, 1.8µm, 1200bar

August 31, 2009Confidentiality Label

54

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

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DAD1 B, DAD1A, DAD: Signal A, 210 nm/Bw:4 nm Ref 360 nm/Bw:80 nm (F:\001-0102.D)

130 Peaks> 30 minutes

column selection for uhplc c - chemical analysis, life ... · time 0.1 95/5 a/b 225 nm (0.8 min)...

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