new advances in hplc - agilent · (with flexible cube for lowest carry-over) 1290 infinity...

New Advances in UHPLC-Resolution, Speed & Sensitivity

Enhancing Productivity with Agilent’s Newest Instrumentation

Patrick CronanLC Applications Scientist

Boston, MA

September 14, 2012

The 1290 Infinity Quaternary LC SystemHow binary pump-like performance can enhance your lab efficiency

1290 Infinity DAD with 60mm cell (& HDR optional)

1290 Infinity TCC with broadest

September 24, 2012Page 2

1290 Infinity TCC with broadest valve selectivity

1290 Infinity Autosampler (with Flexible Cube for lowest carry-over)

1290 Infinity Quaternary Pump

Innovation: 1290 Infinity Quaternary PumpQuaternary Pump with Binary Pump-like Performance

1290 Infinity Quaternary Pump

• Boost PerformanceHighest accuracy and precision for composition and flow, with exceptionally low delay volume

• Save TimeAccelerates transfer of existing HPLC methods to UHPLC

• Reduce CostsOutstanding UHPLC performance at HPLC-like costs of ownership

New Inlet-Mixer - based on JetWeaver technology

Oct. 2011

4

1290 Infinity Quaternary PumpVarious tools enabling the high performance

Multi-Purpose-

Switchable Jet Weaver Mixer

5

4-channel Degasser

MCGV (Multi-Channel-Gradient-Valve) Inlet-Weaver Pump Head

Multi-Purpose-Valve

In-line Filter

Multipurpose valve functions for highest comfortStandard Application for standard Delay Volume

6

Multipurpose valve functions for highest comfortExtra Mixing Volume for lowest baseline ripple (TFA applications)

7

Multipurpose valve functions for highest comfortAutomatic Purging

8

Multipurpose valve functions for highest comfortAutomatic filter backflush

9

Power Range� For any kind of analysis

Composition Accuracy and Precision< 0.15 % RSD or 0.02 min SD

� 0.4 % (1-99 % Composition B)

� High RT precision in gradient runs

Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits

gradient runsFlow Accuracy and Precision

< 0.07 % RSD or 0.01 min SD

� 1.0 % or 10 µL

� High RT precision in isocratic runs

Composition Range1-99 %

� Wide analytical range

Delay Volume< 350 µL

� For fast quaternary gradients

� Different mixtures (H2O, MeOH, ACN) at � different pressures and � different flow rates

Full application range!

Composition Accuracy and PrecisionStep Gradient Analysis with Tracer

11 January, 26, 2012

Flow and Composition Accuracy and PrecisionIsocratic analysis of Glucocorticoids

3: Hydrocortisoneacetate

1: Hydrocortisone 2: Beclomethasone-di-proprionate

Column: Agilent ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 5 µm (p/n 959996-902)

Mobile phase: Water/ Acetonitrile = 30/70Flow rate: 1 mL/min

Stop time: 5 min

Injection vol.: 3 µL (needle wash for 3 sec)

Column Temp.: 30 °C

DAD: 254/10 nm, (ref 400/ 80 nm), 20 Hz, slit 4 nm

Flow and Composition Accuracy and PrecisionIsocratic analysis of Glucocorticoids

mAU

25

30

1

2

RT of compounds very sensitive regarding changes in

mobile phase composition!

min1 1.5 2 2.5 3 3.5

0

5

10

15

20

Water/ACN 29:71

3

Water/ACN 31:69

Water/ACN 30:70

Accuracy and PrecisionIsocratic analysis of Glucocorticoids

mAU

35

1: Hydrocortisone

Specification (< 0.07 %)

min0 1 2 3 4

0

5

10

15

20

25

30

35

3: Hydrocortisoneacetate

2: Beclomethasone-di-proprionate

Power Range� For any kind of analysis

Composition Accuracy and Precision< 0.15 % RSD or 0.02 min SD

� 0.4 % (1-99 % Composition B)

� High RT precision in gradient runs

Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits

gradient runsFlow Accuracy and Precision

< 0.07 % RSD or 0.01 min SD

� 1.0 % or 10 µL

� High RT precision in isocratic runs

Composition Range1-99 %

� Wide analytical range

Composition RangeShallow gradient at low organic concentration

1: Theophylline

N

N

N

N

CH3

O

CH3

O

H

N

N

N

N

CH3

O

CH3

O

CH3

2: Caffeine

Column: Agilent Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm (p/n 699975-902)

Mobile phase: A = Water / B = Acetonitrile

Gradient: 2 – 12 % B in 5minFlow rate: 2 mL/min

Stop time: 5 min

Injection vol.: 5 µL

Column Temp.: 40 °C

DAD: 254/10 nm, (ref. 360/ 100 nm), 20 Hz


mAU

80

100

1: Theophylline

N

N

N

N

CH3

O

CH3

O

H

N

N

N

N

CH3

O

CH3

O

CH3

2: Caffeine

1

min2 2.5 3 3.5 4 4.5

0

20

40

60

2


0.3

0.4

0.5

RS

D R

T (%

)Retention time precision

0

0.1

0.2

0.3

1290 InfinityQuat. System

RS

D R

T (%

)

Theophylline

Caffeine



0.3

0.4

0.5

RS

D R

T (%

)Retention time precision

2.4

0

0.1

0.2

0.3



Quat. UHPLCOther vendor

RS

D R

T (%

)

Theophylline

Caffeine



mAU

200

250

Step gradient1 % steps from 0 to 10 %

0 10 20 30 40 50

0

50

100

150

1290 Infinity Quaternary system

Quaternary UHPLC from other vendor

Ternary/Quaternary GradientsBlend Assist

You need different concentrations of modifiers in your analysis, would like to have just one stock-solution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool – BlendAssist!

Water Water 1% TFA

Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in

Water and 0.08% TFA in ACN2. 20 – 80% gradient of ACN with 0.5% TFA in

1% TFA

ACNACN

1% TFA

Water and 0.4% TFA in ACN

Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program complex gradients (%A, B, C, D).

With BlendAssist: just program your binary organic/aqueous gradient and define the dilution factor!

Ternary/Quaternary GradientsBlend Assist - simple Tool for Online-Dilution of Modifiers and Gradient Set-Up

BlendAssist - Solvent/modifier channel definition

Buffer Advisor SoftwareQuaternary mixing

September 24, 201223

1260 Infinity Bio-inert HPLC

What is similar compared to standard1260 Infinity Quaternary LC?

� Pumping performance, gradient performance

� Pumping Specifications� Detector, Autosampler Performance and

� Detector, Autosampler Performance and Specs

� Overall System Robustness� TCC specs

600 bar also for fast or high resolution Bioseparations

Modular flexibility maintained


1260 Infinity Bio-inert HPLC

What is different compared to standard 1260 Infinity Quaternary LC

� Pump titanium based, no SST �Autosampler metal-free�Capillaries and connections metal-free�Detector Flow Cell metal-free

�Detector Flow Cell metal-free�pH-range 1-13, short term pH 14� Active Seal wash included in pump� Low pressure is possible

•Wide pH range•High salt tolerance•Lowest surface activity� Bio-Inertness


New Capillary Design Ensures Bio-inertnessCapillaries:

Metal cladded PEEK capillary

New capillary technolgy enables 600 bar

AND is completely metal free !!


Needle:

SST housingfor robustness

PEEK and ceramic for metal free sample path

Buffer Advisor SoftwareWorkflow

1: Select Buffer and Gradient Solution

2: Enter Gradient Timetable

3: Review Composition


3: Review Composition of Stock Solutions and Adjust

4: Gradient Display Section: Process and Review Gradient

Experimental Situation

Buffer Advisor SoftwareWorkflow

Buffer Advisor SoftwareTimetable import into Chemstation

32

Buffer Advisor SoftwarepH scouting

33

Buffer Advisor Softwarestable pH

34

30x wider linear UV range -Quantification of widely different concentration levels in one single run

Agilent 1200 Infinity High Dynamic Range (HDR) Solution

History of DAD Linearity Gain The last 30 years

> 30

20.0

25.0

30.0

35.0

40.0

45.0

Line

arity

Gai

n

1200 Infinity HDR-DAD

With HDR

>30X Linearity

Detector Model (Intro Year)

1.4 1.7 2.0 1.4 2.9

0.0

5.0

10.0

15.0

20.0

1040 to 1090 1090 to 1050 1050 to 1100 1110 to 1200 1200 to 1290/60 1200 to HDR

Line

arity

Gai

n

(1988)(1986) (1995) (2006) (2010) (2011)


Programmable

GratingMirror

Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber

1260 / 1290 Infinity DAD

Deuterium Lamp

Max-Light Cartridge Cell10 mm or 60 mm path length

Programmableor fixed slit

1024 elementdiode-array


Effects of path length increase

Peak Dispersion

60 mm pathlengthlarge geom. cell volume (78 µL)

- Loss of resolution- Loss of signal height

10 mm pathlengthsmall geom. cell volume (13 µL)

high light transmission

Conventional flow cells:

large geom. cell volume (78 µL)lower light transmission

60 mm pathlength4 µL �V dispersion volume

Max-Light High Sensitivity cell:

Optofluidic waveguides (total internal reflection)


10x Higher Sensitivity 1290/1260 Infinity DAD compare to 1200 Series DAD SL

mAU

20

25

30

Columns: 150 x 4.6mm Zorbax SB C18, 5µm

Sample: Anthracene: 835 pg/µL

Mobile phase: A: Water, B: Acetonitrile

Elution: isocratic 80 % B

Injection volume: 5 µL

Flow: 1.5 mL/min

DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm

1260/1290 Infinity DAD

1260/1290 DAD60 mm

1200 DAD SL 10mm

Height [mAU] 28.876 4.938

Noise [mAU] 0.0098 0.0190

Signal/ noise 2944 259

Increase 11.4 xmin0 1 2 3 4 5

0

5

10

15

20 DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm

1200 Series DAD / DAD SL


30x Wider Linear Range with HDR-DAD3.7 mm and 60 mm and Max-Light flow cell

3.7 mm path-length

Detector Signal[AU/cm]

60 mm path-length

Computing the signals from60 mm path-length for the low concentration3.7 mm path-length for the high concentration

Conc [µg/mL]


1290/1260 High Dynamic Range – HDR Increasing Linear UV Range by >30x

Technology

� Cluster of two DADs

� DAD 1: 60 mm Cell – for low concentrations

� DAD 2: 3.7 mm Cell – for high concentrations

� Output: One, combined signal, normalized to 10 mm path length (HDR range: 0.6 x 10-6 to 6.7 AU/cm) vs for 1200 Series: 7 x 10-6 to 2 AU/cm)

60mmDAD 1:

Control / Usability

� Like standard 1290/1260 DAD: Control, data analysis, reporting

Investment Protection

� Existing 1290/1260 DAD can be upgraded (by 2nd DAD)

� Existing 1100/1200 Systems can be upgraded (by two 1290 or 1260 DAD)3.7mm

Clustered DADDAD 2:


Fixed Dose CombinationsFrom Roche Switzerland

Fixed dose combinations:

A combination of two or more

APIs in a fixed ratio of dosesAgilent 1200 (black): API 2: 10 µl injected

Agilent 1200 (red): API 1: 100 µl injected

Infinity HDR-DAD (blue): API 1 and 2 simultaneously: 70 µl injected

API 1

API 2


Fixed Dose CombinationsFrom Roche Switzerland

“With the Agilent 1200 Infinity HDR-DAD we significantly decreased our turnaround time. We also get lower

LODs and better area precision”

Dr. Rose-Marie Meier F. Hoffmann - La Roche AG

Infinity HDR-DAD

Agilent 1200 DAD


2 Injections needed if a conventional DAD is used

1µl injection to determine the first 3 peaks

min0 1 2 3 4 5

mAU

0

200

400

600

800

1000 Vitamin C

Paracetamol

Caffeine Chlorphenamine ?


Vitamin CParacetamol

Caffeine

Chlorphenamine

5µl injection to determine the last peak and further impuritiesThe first 2 peaks are out of linear range

min0 1 2 3 4 5

mAU

0

1000

2000

3000

3800Detector saturationOutside the linear range

2 Injections needed if a conventional DAD is used

ChlorphenaminemAU

15

17.5

20

DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_1UL09.D)

DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL10.D)

mAU

0

0.2

0.4

0.6

0.8

1

Chlorphenamine

Imp 3

Imp 4

September 24, 2012Confidentiality Label

45

Blue: 1µl injection with 3.7mAU peak height for Chlorphenamine

Red: 5µl injection with 21.8mAU peak height for Chlorphenamine

min4 4.5 5 5.5 6

0

2.5

5

7.5

10

12.5

15

Imp 3 Imp 4

min4.6 4.7 4.8 4.9 5 5.1 5.2 5.3

-0.2

0

Comparison of conventional DAD vs. HDR DAD, 5µl injection

mAU

2500

3000

3500

4000

Red conventional DAD: 2 peaks out of linear range with 5µl injection

Green HDR-DAD: All peaks within linear range

Paracetamol

Vitamin CDetector saturationOutside the linear range


min0 1 2 3 4 5

0

500

1000

1500

2000All peaks within linear range (up to 6AU)

Caffeine

Chlorphenamine

Comparison of LOD

mAU

0.8

0.9

1

1.1

1.2

1.3

HDR DAD

Conventional DAD

More reliable automated peak integration


Compound LOD with S/N=3 for Conventional DAD

LOD with S/N=3 for HDR- DAD solution

Chlorphenamine ~1ng ~0.1ng

min3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.80.5

0.6

0.7

0.8

Comparison of conventional DAD and HDR DAD, 5µl injection

mAU

15

20

DAD1 K, Sig=254,20 Ref=380,80 (SOP 98 GRI...B\HDR_GRIPPO_HDR_21FEB 2012-02-21 07-51-02\GRIPPO_HDR5UL09.D)DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL09.D)

Red conventional DAD: determination of impurities and chlorphenamine in a second run

Blue HDR DAD: dermination of all peaks in one run

ParacetamolCaffeine

ChlorphenamineVitamin C

48

min1 2 3 4 5

0

5

10

Imp 1 Imp 2 Imp 3 Imp 4

Comparison of area precision

14.908 13.013 5.37 5.723

1

1.5

2

2.5

3

RS

D A

rea

(%)

Comparison of Area Precision

HDR Signal

Conv.DAD signal (1µl)

Conv.DAD (5µl)


HDR DAD 5µl injection volume for detemination of all compounds (blue) Conventional DAD: 1µl injection volume for determination of Vit C, paracetamol and Caffeine (orange)

5µl injection volume for determination of Chlorphenamine and further impurities (green)

0

0.5

Intelligent System Emulation Technology

(ISET)


What is ISET?- Two components

A. Best in class performance of the 1290 Infinity LCBroad power range, ultra-low delay volume, superior sensitivity, unmatched flow and composition accuracy

Peak 3Overlay of 10 runs

RSD = 0.005 %

Page 51

B. The revolutionary ISET emulationalgorithmFull characterization of target LC systemsCreation of emulation function

FA

Method Transferability: 1290 Infinity LC- System Emulation Technology

Concept

EmulatedSystem A

FA

Page 52

Select System to be emulated by a simple mouse click:

Agilent 1100 Series

Agilent 1200 Series LC

Agilent 1260Infinity LC

Agilent 1220Infinity LC

Alliance(2012)

Intelligent System emulation technology- How it works (1290 to 1200)

250

300

350

400

mAU

Injection

Same gradient conditions by moving the gradient and by emulating the gradient mixing behavior.

Page 53

Programmed gradient

1290 gradient

1200 gradient

0 0.5 1 1.5 2 2.5 3 3.5

0

50

100

150

200

min

gradient mixing behavior.

Select pump

Intelligent System Emulation Technology ISETImplementation

Select sampler

Page 54

PesticidesColumn: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min

mAU

500

600

700

800

Sim

azin

e

Chl

orid

azon

e

Chl

orto

luro

n

Diu

ron

Pro

pazi

neTe

rbut

ylaz

ine

Pro

met

ryn

1290 System with iSETM

etam

itron

Cya

nizi

ne

Rs= 1.87

55

min1 1.5 2 2.5 3 3.5

0

100

200

300

400

1290 System

Rs= 1.90

1100 Binary System

Rs= 1.15


56


57

Generic emulation- Limitations

Example: 1290 to Vendor X

Injection

250

300

350

Generic emulation: Move the gradient

mAU

Page 58

0 0.5 1 1.5 2 2.5 3 3.5

0

50

100

150

200

min

Programmed gradient

1290 gradient

Vendor X


Generic emulation- Limitations

Example: 1290 to Vendor X

Injection

250

300

350

mAU


Page 59

Programmed gradient

1290 gradient

Vendor x

0 0.5 1 1.5 2 2.5 3 3.5

0

50

100

150

200

min

Does not consider the gradient mixing behavior (different curves) ordelay volume changes because of pressure changes for pumps with damper.

Method Transfer Possibilities Today- A) Adding physical volume (plumbing solution)

1290 + 1000 �L dwell vol.

Example: 1290 Infinity LC

1100/1200 Quat Pump

Page 60

Advantages

• Works well for generic, slower analysis• No Method change

Disadvantages

• Requires hardware change, no flexibility

• Hardware change difficult in validated environment

• Requires manual determination of the dwell volume (Pumps with damper the dwell volume is not constant and depends on pressure)

Isocratic hold: move the gradient

Method Transfer Possibilities Today- B) Isocratic Hold

Example: 1290 to 1200

Injection

250

300

350

mAU

Isocratic hold: move the gradient

Page 61

Programmed gradient

1290 gradient

1200 gradient

0 0.5 1 1.5 2 2.5 3 3.5

0

50

100

150

200

min

Does not consider the gradient mixing behavior (different curves).



1200 Series LCBinary Pump

1290 Infinity LC+ 860 µl “Delay Volume”

Page 62





1260 Infinity LC

1290 Infinity LC+ 900 µl hold

Page 63

Advantages

• Flexibile, Isocratic Hold is part of the method

Disadvantages

• Works not in all cases• Requires manual determination of the dwell volume/

isocratic hold (Pumps with damper the dwell volume is not constant and depends on pressure)

• Requires modification of the methods (should be avoided in validated environment,but doesn’t require revalidation USP Chapter <621>)

Method transfer, pesticide example2.1x100 mm Zorbax Eclipse Plus, 1.8 µm column, flow: 0,8 mL/min

(A)1100

Quaternary400 bar

(B)

1 2,3 4 5 6,7 8 9 10,11,12 13 14 15 16


(C)1290 Infinity

1200 barwith ISET

(B)1290 Infinity

1200 bar

Summary1290 Quaternary Pump:� Full 1290 Infinity LC Power range

� Highest composition precision and accuracy across pressure and flow rate range

� Full application range with binary-like pump performance! 1290 Systems using ISET:• Achieves higher productivity for method development • Simplify your instrument to instrument method transfer while minimizing costs

• Run your legacy methods with ISET and use UHPLC speed for faster separations

• Run your legacy methods with ISET and use UHPLC speed for faster separations

HDR DAD Solution:• Determination of low and high dose drugs is possible in one run• Improved Precision and Lower Detection Limits as compared to convention DAD

Using Buffer Advisor with BioInert Quat System:• Shortens and simplifies the workflow for pH scouting by using pre-validated

buffer systems and dynamically mixed gradients• Excellent tool using for automated pH scouting and accurate ion exchange

chromatography

65

new advances in hplc - agilent · (with flexible cube for lowest carry-over) 1290 infinity...

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