Next Generation of HPLC

Curtis R. Campbell, Ph.D.Shimadzu Scientific Instruments, Inc.

The BasisProminence HPLC (2005)– World’s 1st Web Enabled Controller

– World’s Fastest Autosampler

– World’s Cleanest Autosampler

The ContinuationProminence UFLCXR (2008)

– Enhanced Pressure Tolerance

– Reduced delay volume

– Advanced Materials

– NO compromise in basic performance

The Next GenerationNexera UHPLC (2010)– Widest Pressure and Flow Range

– Enhanced Autosampler Performance

– Unique Engineering and Materials

– NO compromise in basic performance

A rose by any other name…

• Chromatography– Regardless of the acronyms thrown about, that is what we are talking about here.

– The ability to reliably and reproducibly separate, detect, and quantitate a liquid mixture

– The results of which are used to make innumerable decisions

• Crucial not to sacrifice basic performance in exchange for pressure tolerance

Qualities of a Good System

• Speed– Cycle Time

– Carryover

Regardless of System Pressure

Cycle Time

Injection Analysis Injection Analysis Injection Analysis

Start0 min 0 sec

Injection Analysis Injection Analysis Injection Analysis

Other UHPLC30sec for injection

Simadzu UFLC10sec for injection

Injection Analysis

Injection Analysis

Injection Analysis

Finish3 min 0 sec

Finish2 min 0 sec

Finish1 min 40 sec

33% Faster

44% FasterNexera with overlapping injection

Carryover

• Two ways to attack carryover– Innovative engineering and proper material choices

– Provide effective countermeasures

Minimized Carryover

Flow‐through needle design to prevent carryover

Thorough rinse of sample path by multiple rinse

Dip wash and active wash of needle surface

HighHigh‐‐strength seal strength seal materialmaterial

To achieve zero carryover and high‐pressure capability •Fine‐tipped needle•Reduced sealing area•Smooth inner surface of needle•High‐strength seal material

Newly Designed Needle / Injection Port

‐‐ 60%60%

SIL‐20AXR SIL‐30AReduced Reduced Sealing areaSealing area

Qualities of a Good System

• Speed– Cycle Time

– Carryover

• Precision– Gradient Reproducibility

– Injection Accuracy and Reproducibility

Regardless of System Pressure

Gradient Reproducibility‐

Zorbax Eclipse plus C18, 2.1mm, 50 mm, Water / Acetonitrile 15 → 95% (0.80min) 1.2 mL/min20 uL Shimadzu MIRC mixer40C245nm2 uL injection, 25ppm

R.T.

%RSD

Area R.T.

Acetophenone 0.51 min 0.156 0.068

Propiophenone 0.62 min 0.278 0.039

Butyrophenone 0.70 min 0.108 0.034

Valerophenone 0.77 min 0.259 0.045

Hexanophenone 0.84 min 0.196 0.042

Heptanophenone 0.90 min 0.260 0.034

Octanophenone 0.96 min 0.220 0.0330.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

uV

N=6 (overlay)

Excellent gradient reproducibility even in the ultra fast analysis.

Injection volume precision & linearity in the range of 1 to 50 μL @ 100 MPa

N=6 x 5set %RSD (Area) Spec.

1 μL x 6 0.17 %

2 μLx 6 0.23 %

5 μLx 6 0.06 % < 0.25 %

10 μLx 6 0.11 %

20 μLx 6 0.12 %

50 μLx 6 0.10 %

Injection volume

Peak Area (n=6) Linearity

1 μL 63857 3.4 %

2 μL 129668 1.9 %

5 μL 329103 0.4 %

10 μL 664574 - 0.5 %

20 μL 1328747 - 0.5 %

50 μL 3305754 ±0.0 %

Correlation Coefficient 0.9999960.999996 > 0.999

Sample : Caffeine 20 mg/L

Precision Linearity

Injection Precision and Linearity

Qualities of a Good System

• Speed– Cycle Time

– Carryover

• Precision– Gradient Reproducibility

– Injection Accuracy and Reproducibility

• Reliability

Regardless of System Pressure

Ω shaped rotor

New High Pressure Valve DesignTo withstand operation @ 130 MPa

•Minimized friction by specially coated stator•Ω‐shaped rotor seal (Patent pending) → to equalize pressure

Distribution of force eliminates rotor tilt →longer consumable life

High Pressure area @High Pressure area @ Run and Load/INJ modeRun and Load/INJ mode

Low pressure area @ Load modeLow pressure area @ Load mode

Qualities of a Good System

• Speed– Cycle Time

– Carryover

• Precision– Gradient Reproducibility

– Injection Accuracy and Reproducibility

• Reliability

Regardless of System Pressure

••System FlexibilitySystem Flexibility

Unsurpassed Flexibility

•First and foremost the system has to be able to run current methods•Must have the ability to adapt for system variations•Capable of growing with the application demands

Pumping Configuration

• Two ways to form a gradient– Binary High Pressure Mixing

– Low Pressure Gradient Formation (LPGE)

Pumping Configuration

AbsorbanceDetector

LC/MS

• Neither is “correct”

• Combination of the two is optimal

• Modular system allows the ability to do even more– 2 Dimensional system

– Multi‐plex system

Multiplexed System for Higher Throughput

The MPX‐2 is a dual stream multiplexing solution to increase LCMS/MS throughput. The samples are alternately injected from the two UPLC lines without dead time.

Injector Capabilities

• Minimized carryover

• Multiple injection modes

• Flexible format– Different sample vial types

– Expandability for high throughput labs

Loop injection

A

B

C

D

E

Direct injection

B

CD

E

Tube B LPV ‐ HPVTube C Pump ‐ HPVTube D HPV ‐ column

Tube E HPV ‐ drain valve

Tube A Sample loop (1.5uL/5uL/20uL)

Tube B LPV ‐ sample loop

Tube C Pump – HPVTube D HPV ‐ column

Tube E HPV ‐ drain valve

Flow Diagram for Fixed Loop Injection

HPV

LPV

Drainvalve

Column

Pump

HPV

LPV

Drainvalve

Column

Pump

134 uL 18 uL

Rack Changer for More Sample Capacity

Provides over 4600 consecutive sample analysis96 well, 384 well, and vial platescoolable rack changer (4 to 40 ºC)

Summary

• Prominence HPLC– Great All Round HPLC

• ProminenceXR– Fantastic All Round Enhanced HPLC

• Nexera UHPLC– The next era of HPLC– Widest Pumping Range– Fastest, Cleanest Autosampler– Most Flexible

Summary• Wide Range of S/W Control

– Shimadzu Lab Solutions– Atlas by ThermoFisher– Chromeleon by Dionex– Clarity by Data Apex– Empower by Waters– EZChrom Elite by Agilent

• MS S/W Control– Analyst by AB Sciex– Xcalibur by ThermoFisher– Hystar by Bruker

THANK YOU

I look forward to the discussion and your questions.

For more go to Keyword: Nexera

Carryover

Chlorhexidine 2,000 ng/uL

Post-blank 1 0.0006%0.0006%Post-blank 2 0.0003%

Post-blank 3 0.0004%

Carryover of chlorohexidine @ 100 MPa

Results : 0.0006 % Without injection port rinse, needle internal rinse

With needle outer surface rinse

Carryover

Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um)Mobile Phase: : Methanol, Water= 2/8Flow rate : 0.4 mL/minTemperature : 30 Injection volume : 5 μLRinse solution : 0.05 % formic acid in methanolNeedle wash : outer surface flush by rinse pump (1 second flush)

: needle soak wash (0 second)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 mV

50

60

70

80

90

100

110

MPa

Detection :260nm

Carryover

Chlorhexidine 2,000 ng/uL

Post-blank 1 N.D.N.D.

Carryover Carryover of chlorohexidine @ 100 MPa

Specification : < 0.0015< 0.0015%,

Results : Not detected With injection port rinse, needle internal rinse and needle outer surface rinse

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-1

0

1

2

3

4mV

50

60

70

80

90

100

110

MPaDetection :260nm

Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um)Mobile Phase: : Methanol, Water= 2/8Flow rate : 0.4 mL/minTemperature : 30 Injection volume : 5 μLRinse solution : 0.05 % formic acid in methanolNeedle wash : outer surface flush by rinse pump (1 second flush)

: needle soak wash (0 second): needle internal rinse

Injection port rinse : performed

Rinse Function Test 1 Test 2 Test 3 Test 4

Injection Port Rinse New

Needle Internal Rinse New

Needle Internal Flush–Back & Forth Rinse- New

Needle Outer surface rinse-Needle Dip-

Needle Outer surface rinse-Flush by Rinse pump- 1sec

Carryover ( % ) 0.0106 %0.0106 % 0.0006 %0.0006 % 0.0015 %0.0015 % N.D.N.D.

Carryover Summary ‐Chlorohexidine‐

Chlorohexidine 2000 ng/μL, 5 μL injection

Binary Pumping system