a comprehensive approach to fast high … throughput analysis pittcon 2008 march 2008 a...

High Throughput AnalysisPittcon 2008March 2008

A Comprehensive Approach to Fast High Throughput LC/MS Analysis

Tony Brand, Ph.D.ScientistAgilent Technologies, Inc.

March, 2008

High Throughput AnalysisPittcon 2008

Productivity in LC and LC/MS Analysis

LC: To produce sufficient resolution in the shortest timepossible with minimal instrument cycle time, maximum

reproducibility and minimal downtime.

To review data quickly and efficiently.

To produce reports which provide information.

March, 2008


The Agilent 1200 Rapid Resolution LCHighest Performance RRLC/UV/MS and Conventional HPLC – All on one System!

PerformanceUp to 60% higher resolution compared to HPLCUp to 20x faster than HPLC

With same or better performanceCompliant with strictest regulatory performance requirements2000 Samples/Day on HT-configuration with ACR

Flexibility and Investment ProtectionRRLC and HPLC on standard delay volume configurationSupport of RRLC and HPLC on Std and Narrow bore columnsVery broad range of RRHT columnsSame chemistry easy, fast and secure method transfer1100 Compatibility and Emulation mode for non-Agilent softwareStepwise transition to RRLC, minimum training & switching costs

March, 2008


µ-Degasser• Maximum baseline stability and minimum noise by continues vacuum source

Binary Pump SL• RRLC support by 0.05 - 5ml/min flow rate range and 600bar • Support of 2.1– 4.6mm ID by configurable delay volume (120ul and 600-800ul)• Highest RT precision and lowest mixing noise

NEW High-Performance ALS SL+• RRLC support by new high-speed, 600bar injector• Higher robustness by new valve design, new quality needle and needle seat• Lowest carry over

Thermostatted Column Compartment SL• RRLC support by peltier-controlled column thermostatting up to 100°C• Minimized peak broadening on 2.1mm ID columns by low volume heat exchanger• Minimum cycle times by ACR (2Ps/10Pt valve saves wash & regeneration time)• Minimized UV noise by post-column cooling and improved T-control (< +/- 0.05°C)

DAD/MWD SL• 80Hz sampling rate• Highest sensitivity in UFLC by new cell design*, electronics and ETC (< 50uAU noise)• Support of 2.1, 3 and 4.6mm columns by 3 cells (2ul/3mm, 5ul/6mm, 13ul/10mm)

1200 RRLC System Overview Benefits of Key Modules Features

March, 2008


Area Precision (n=10)

Inj. Vol.: 3.0µl 0.307% RSD

Inj.Vol.: 1.0µl 0.569% RSD

Inj.Vol.: 0.5µl 0.672% RSD

min1.27 1.28 1.29 1.3 1.31

mAU

0

20

40

60

80

100

120

140

Buty

roph

enon

eBe

nzop

heno

ne

Vale

roph

enon

e

Hex

anop

heno

ne

Hep

tano

phen

one

Oct

anop

heno

neAcet

anilid

e

Acet

ophe

none

Prop

ioph

enon

e

min0.2 0.4 0.6 0.8 1 1.2 1.4

Agilent 1200 RRLC: Speed and ReproducibilityMaintaining Area Precision at High Analysis Speed

March, 2008


The New Agilent 1200 Hi-P ALS SL+

A Quantum Leap in reducing carryover

New lower delay volume in MAINPASS mode for RRLC

Higher precision (<1% for 0.1uL injections)

Injection volume: 0.1 to 40µl (optional, 100µl kit)

March, 2008


Agilent 1200 HiP-ALS SL+: Simple Superior Design

March, 2008


The Agilent 1200 HiP-ALS SL+: Single Draw Flow Through Design

Bypass ModeSample Loading

Mainpass ModeRunning Sample

March, 2008


The New Agilent 1200 Hi-P ALS SL+Cleaning Procedures

Inside of the needle is continuously flushed during the complete analysis

Outside of the needle is flushed prior to injection with fresh solvent. This outside wash of the needle is done using the flush port of the ALS and prevents contamination of the needle seat.

New post-run automated cleaning procedure for the needle seat. Seat is flushed with mobile phase. The injection needle is elevated from the seat, but remains in mainpass and the pump delivers flow through the needle onto the seat.

March, 2008


Carry-over Chlorhexidine<0.003 % with MSD in SIM mode

min0.6 0.8 1 1.2 1.4 1.6 1.8

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1000

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2500

3000

3500

MSD1 TIC, MS File (D:\DATA\CARRYMSHEX24JAN\CARRYHEX24JAN2-57\HEX_240NGCARRY.D) ES-API, Pos, SIM, Frag: 70

chl

orhe

xidi

MSD1 TIC, MS File (D:\DATA\CARRYMSHEX24JAN\CARRYHEX24JAN2-57\BLANK_AFTER1.D) ES-API, Pos, SIM, Frag: 70 240ng Chlorhexidine injected

SIM ion :505.30 m/z

Carry over after injection of 1µl mobile phase: 0.0028%

240ng Chlorhexidine

Carry over peak

Former results: 0.041% carry over

No carry over in the UV could be observed under these conditions

Column: 2.1x50mm Zorbax SB C18, 1.8µmSample: Chlorhexidine dissolved in Water + 0.1%TFAMobile Phases: water+0.05%TFA, Acetonitrile+0.045%TFAFlow rate: 0,5ml/minGradient: at 0 min 30%B, at 3min 60%BInjection volume: 1µl, external needle wash with water + 0.1%TFA, draw and inject speed= 100µl/minColumn temperature: 50°CMSD API-ES positive: SIM ion 505.30, Fragmentor: 70, gas temp: 250°C, drying gas =12L/min , Vcap = 3000V

Early R&D data

March, 2008


• 100°C for higher speed and resolution in RRLC

• New low volume heat exchanger for minimized peak broadening on 2.1mm ID columns.

• 600bar 2Ps/10Pt micro valve for minimum cycle times by ACR (alternating column regeneration, which saves wash & regeneration time)

• PCC (post-column cooling) and improved peltier temperature control (< +/- 0.05°C) for minimized UV noise under most demanding conditions (< 50µAU at high flow rates and temperatures)

The Agilent 1200 RRLC System Thermostatted Column Compartment SL

March, 2008


Pre-Column heating/cooling

Post-Column heating/cooling

Integrated Column Selection or Column Regeneration Valve

Beyond Column Temperature, Managing Temperature Before the Detectors

The Agilent 1200 Thermostated Column Compartment SL

Column temperatures to 100oCHigher column temperatures

- reduce viscosity and back pressure- improve peak shape- improve recovery and reproducibility

March, 2008


Alternating Column Regeneration (ACR) saves up to 50% of cycle time

Throughput of > 2000 sample/day can be achieved in conjunction with

Automatic Delay Volume Reduction

Overlapped Injections

Offline Data Analysis

External Needle Wash

Sequential analysis (vs. parallel) ensures performance, flexibility, robustness and ease-of-use of std LC instruments

RRLC Speed & Throughput2000 Samples/Day using ACR

March, 2008


Post-column CoolingNoise reduction at high flow rates and high temperatures

0.685 - Anthracene

min0.5 1 1.5 2 2.5

mAU

-0.05

0

0.05

0.1

0.15

0.2

0.25

• Column Temperature: 80°C • Detector-in Temperature: 50 °C• Noise = 90 uAU• S/N = 3.3

• Column Temperature: 80°C• Detector-in Temperature: 24 °C• Noise = 30 uAU• S/N = 10• No Peak Broadening

Column: 4.6 x 50mm, 1.8um

Flow Rate: 4ml/min

March, 2008


High Temperature LC-MS/MS (HTLC-MS/MS) for High Throughput BioanalysisDaniel Tang, PhD, PDM, Michigan Laboratories, PGRD, Pfizer Pharmaceuticals

Post-Column Heating Temperature Effect on LC/MS Sensitivity

March, 2008


Agilent 1200 DAD and MWD:Flow Cells Recommendations in RRLC

13µl Standard Flow Cell: For highest sensitivityHigh-demanding quantitative work, e.g. analytical method development, QA/QC

2µl Micro Flow Cell: For highest resolutionUltra-fast semi-quantitative work, e.g. Screening Experiments, HT LC/MS/UV

5µl Semi-micro Flow Cell: Best compromise of sensitivity and resolutionFor good quantitative and qualitative results, e.g. Screening, HT LC/MS/UV, Early Formulation Studies

* Depends on analytical conditions and column dimension

+++

+++

Sensitivity* Resolution*Dimension

+++2 µl / 3mm++5 µl / 6mm+13 µl / 10mm

March, 2008


The New Agilent 1200 Series ELSD

• Proven detector for highest sensitivity and reliability

• Suitable for LC and RRLC

• Reliable and Robust

• Minimal maintenance

• System Support from a single vendor

March, 2008


Why ELSD? “Detection insurance”

min0 1 2 3 4 5 6 7 8

mAU

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5.42

7 min1 2 3 4 5 6 7 8

mV

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1.89

2 3.88

3

4.87

2

5.56

4

ELSD ~12.8 mg/l5 μl injection

DAD~ 12.8 mg/l5 μl injection

Prol

ine

Valin

e

Isol

eucin

e

Phen

ylal

anin

ePh

enyl

alan

ine

March, 2008


3. Detection

Agilent ELSD Technology: Simple, Reliable1. Nebulization

2. LT-Evaporation

March, 2008


Why ELSD?

ELSD sees any compound less volatile than the eluent

Analysis of biologically active compounds from soya

March, 2008


The New Agilent 1200 Series ELSD

Advanced technology by optimized nebulizers and Gas Supported Focusing in optical head

High and low (ambient to ~60°C) temperature evaporation

Low peak dispersion by optimizednebulizers, evaporator and head

4 nebulizers available for optimized sensitivity and peak width at different flow rates

Convenient front access for inspection and maintenance

Best sensitivity and peak shape and width

Applicable to non- and semi-volatile compounds

RRLC enabled for peak width >/= 0.8 sec

To cover all HPLC techniqiues from low µl- to semi-prep at 5 ml/min

User friendly, low maintenance

March, 2008


Low Temperature ELSD for lower limit of detection of semivolatiles ?

30° C evaporation

50° C evaporation

Analysis of caffeine with30° and 50°C evaprationtemperature

March, 2008


1200 Series ELSD in RRLC w/ peak width 0.8 sec

min0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

mV

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600

ELS1 A,Voltage (C:\WORK\ELSD\DATA FILES CHEMST\RRLC_PARABENS_3 2007-09-14 17-25-43\001-0101.D)

0.133

0.173

0.242

0.357

Analysis of 4 semi-volatile parabens in less than 0.4 minutes,data rate 30 Hz results in 25 data points for 0.8 sec peak

Evaporation: 26° C

0.8 sec

March, 2008


The ELSD Value Proposition

• For samples without chromophore, fluorescence, or ionizability: natural products, unknowns, combinatorial chemistry libraries ...

• Univeral Detection (well almost)

• Frequently used as complementary detection mode to UV and to LC/MS

• State of the art ELSD w/ excellent sensitivity and minimal peak width

• Low temperature evaporation enables detection of semi-volatiles, for example, ibupofen, parabens, caffeine

March, 2008


The Agilent 6000 LC-MS Detectors

March, 2008


The Agilent 6100 Series LCMS Quadrupole

March, 2008


Lens2RF – A Patented Agilent Solution

Issue: As m/z value goes up, amplitude of quadrupole RF increases, more difficult to inject ions into the quadrupole and ion transmission drops.

Octopole

Quad Mass Filter

Lens 1 Lens 2

Implemented on the 6130 and 6140 models

Solution: ramping the DC and RF amplitudes with mass on lens 2 and offsetting the phase relative to the quadrupole, ion transmission into the quadrupoleincreases.

Result: Ion transmission increased by ~3X at m/z 600 and up to 10X at m/z 3000

March, 2008


TIC Chromatogram: Customer Sample #48

min0.5 1 1.5 2 2.5 3 3.5

0

200000

400000

600000

800000

1000000

0.10

60.

128

0.26

3

1.79

81.

835

1.99

3

2.20

3

Chromatographic parameters: Column: Zorbax SB C-18 RR 2.1x50mmGradient: 5% ACN to 95% over 3 min at 1.0 ml/min.

Peak #5 Integration Parameters:Width: .0239 min (1.43 sec)

# Spectra across peak: 46

MS Acquisition parameters: Scan range: m/z 100 to 900Cycle time: 0.085 sec. (~9400 u/sec)

m/z420 425 430 435 440 4450

20

40

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80

100Max: 357248

436.

243

7.2 43

8.2

[M +H]+

[M +Cl35]+ [M +Cl37]+

Chlorine isotopes of Peak #5

March, 2008


Simultaneous Electrospray and APCI: Pos/Neg

NH

+

NN

min0.5 1 1.5 2 2.5

1.10

1

2.51

7

min0.5 1 1.5 2 2.5

2.50

9

APCI

ESI, usually ionizes >80% of analytes

Indole

Crystal Violet

2.49

2

min0.5 1 1.5 2 2.5

1.10

8

Simultaneous ESI/APCI, Pos

N

min0.5 1 1.5 2 2.5

1.98

0

APCI

1.98

2

min0.5 1 1.5 2 2.50.

991

Simultaneous ESI/APCI, Neg

min0.5 1 1.5 2 2.5

0.94

6

ESI

HexaneSulfonic Acid

F

FF

NO- O

NO

O

SO

OOH

Positive Ion Mode Negative Ion Mode

Dinitrobenzo-trifluoride

March, 2008


The Agilent Multi-Mode Source Design: InnovationTruly Simultaneous Electrospray and APCI

Nebulizer (grounded)

Reversing electrode (+ve)

APCI counter electrode pin

Gas temperature controllerIR emitter power to controlled by feedback from temperature sensor in vapor stream exiting source

ES Charging electrode (-ve)

Infra-red heaterTuned to H2O IR band

Infra-red heaterTuned to H2O IR band

MS capillary inlet with shield and countercurrent N2 drying gas flow

ES Field shaping electrodes

Thermal Container

Drying gas consumption is reduced ca. 50% compared to dedicated ESI source because heat is supplied by IR emitters

APCI corona needle (+ve)Separator

Isolates ions generated by ES and APCI

March, 2008


Vaporization Using Infrared Emitters:Optimal APCI Conditions for Reserpine (10 pg)

min10 20 30 40 50

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3500Vaporizer

temperature

250 °C

225 °C

200 °C

175 °C

150 °C

125 °C

Relativepeak area

16.8

15.1

12.7

8.3

3.7

1

Rel. std dev.4 replicates

2.0%

1.7%

2.1%

2.9%

1.5%

13.4%

75:25 MeOH:water, 5 mM ammonium formate1 mL/min

March, 2008


Beyond browsing to reviewing…..

Analytical Studio Review

A simple, clean interface for reviewing small to large to very large data sets.Comprehensive data displayPeak purity, sample purity, target massesA New Reporting Engine: Clean, Simple & Brief

March, 2008


Analytical Studio Reviewer (ASR)Fast Data Review on Everyone’s Desktop

CLND Chromatogram

MS Spectrum

MS TIC

UV Chromatogram

Sample Purity ELSD Chromatogram

March, 2008



March, 2008


Analytical Studio Review: Report Versatility

ChemStation ASR File


Raw Data

Report

Report

March, 2008


Analytical Studio ReviewA New Reporting Engine: Clean, Simple & Brief

March, 2008


Analytical Studio Review – Flexible Reporting

March, 2008


ASR – Editing Feature

March, 2008


SQ on Mass Hunter Migration

Instrument Control

Real-time monitoring

Method set-up

Autotune

Qualitative Analysis

Chromatographic results

Spectral results

Find compounds

Quantitative Analysis

User filters

Compound results

Calibration curve

March, 2008


Now Single Quad Users get Powerful and Easy Report Customization

Drag and drop columnsfrom XML results

Add tables and graphics using tool buttons

Print Preview in Excel

March, 2008


The Agilent 1200 Series LC and 6000 Series LC-MS

Thanks for listening!

a comprehensive approach to fast high … throughput analysis pittcon 2008 march 2008 a...

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