the 1290 infinity lc - 10x higher uv sensitivity 1290... · 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5...

Agilent 1290 Infinity LC:10x higher UV sensitivityg ythat makes the difference

How to improve sensitivity

A.G.HuesgenApplication ChemistLiquid Phase Separations Business/LPAD-LSG

November 2009

UV detector sensitivity

• Sensitivity depends on the signal height and the baseline noise

• The sensitivity is typically calculated as ratio of signal height to noise level (S/N)noise level (S/N).

Thi th iti it b i d ith b• This means the sensitivity can be improved either by increasing the signal height or by decreasing the noise level.

November 17, 2009Confidentiality Label

2

Overview

Signal to noise

Signal heightBaseline noise Signal heightBaseline noise

Not detector dependent

Detector dependent


Detector dependentppp


3

Baseline Noise

Not detector related:

Pump performance: pressure ripple mixing of solvents• Pump performance: pressure ripple, mixing of solvents

• Mobile phase chemistry: absorbance of the solvent, TFA problem quality e g new and old THF quality of modifiersproblem, quality, e.g new and old THF, quality of modifiers

• Instrument: cleaness and maintenance


4

1290 Infinity pump

Complete new design

Lowest Delay for ultra fast gradients• Lowest Delay for ultra fast gradients(10 µl w/o mixer, 40 µl with Jet Weaver)

• Active Damping for „ripple free“ pumpingLowest UV-noise

Active Damping for „ripple free pumping

• Agilent Jet Weaver for high efficient mixing


5

New mixer design

• Innovative Jet weaver- based on multi-layer micro fluidic channels (120 μm x 120 μm) to insure the flow is thoroughly mixed.

• Combines highest mixing efficiency with lowest delay volumes to virtally eliminate UV detector noiseUV detector noise.

• V35< 40µl and V100<75µl (for TFA applications) mixing volume are possible, just by turning the mixer around.


6

Comparison of noise with and without mixer

mAU

Mobile phase: 35/65% ACN+0.08TFA/Water+0.1TFA

mAU

0.8

0.9

1

No mixer on 1290 Infinity pump

0.6

0.7

0.3

0.4

0.5

With 35µl mixer on 1290 Infinity pump

min0.02 0.04 0.06 0.08 0.1

0.2

y p p


7

Influence of mobile phase

I it

ACN 1

Impurity

ACN 2


8

Overview

Signal to noise



Detector dependent




9

Baseline noise

Detector related:

Optical unit design VWD DAD• Optical unit design, VWD , DAD

• Cell type and geometry

• Data rate


10

Why do we need a new cell technology?10 mm pathlength10 mm pathlength

13 µl volumeShort 2.1 mm ID column

+ =

How to achieve smaller cell volume?

short path-length (3 long path-length (10 mms o pa e g (3mm, 2 µl)

long path-length (10 mm, 0.5 µl)

High light transmission => low noise

Low S/N Low light transmission=> high noise

Low S/N

Optofluidic Waveguides- Long path length- Small cell volume- High light transmission

Highest S/N

1290 Infinity Diode Array DetectorOptofluidic Waveguidesp g

Non-coated fiber (fused silica)

Advantages:

High light transmission by „Total-Internal Reflection“ (TIR) Principle

Advantages:

• Highly sensitive with small cell volume at the same time

• No RI (Refractive Index) and thermal effectsNo RI (Refractive Index) and thermal effects

• Data rate up to 160 Hz (multi wavelength + spectra)

• Cartridge design for ease of use

12

g g

Optical design of 1290 Infinity detector

GratingMi

Programmable

Mirror

D t i

Slit

Deuterium Lamp

Diode array

„Max-Light“ Cartridge Cell

10mm and a 60mm path length cells are available

13

p g

Noise: DAD SL vs.VWD D vs.1290Infinity DAD

Noise: +/-5µAU-0.04

mAU

-0.06

-0.02

1200RR DAD SL

Noise: +/-2.8µAU

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

-0.1-0.08

mAU

-0 06-0.04-0.02

1200RR DAD SL

1290 Infinity DAD 10mm cell

Noise: +/-2µAU

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

-0.08-0.1

-0.06

mAU

-0.04-0.02

1290 Infinity DAD, 10mm cell

A il hi h d VWD

N i / 0 6 AU

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

-0.08

0 04

-0.1

-0.06

mAU-0.02

Agilent high end VWD

Noise = +/-0.6µAU

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

-0.08

-0.04

-0.1

-0.06 1290 InfinityDAD, 60mm cell


14

(ASTM conditions)

Detector: Influence of data rate

Parameter DAD 129060mm cell

DAD1290 10mm cell

Noise PtoP(mAU) at 10 Hz 0.0008412 0.006271

Noise PtoP(mAU) at 20 Hz 0.005241 0.02214


15

The new 1290 Infinity detector design offers

Programmable slit (1 to 8 nm)—provides optimum incident light conditions for rapid optimization of sensitivity linearity and

Ultra low noise—with unique 60 mmAgilent Max-Light high sensitivitycartridge cell optimization of sensitivity, linearity and

spectral resolution

RFID tags for flow cell and lamps ensure d t t bilit d t ki

cartridge cell

More reliable and robust peak integration process due to less

data traceability and usage tracking

Engineered for simplicity and ease of use—cartridge design allows fast, easy exchange

f fl ll N t d f d ili fib ll

baseline Drift

Multiple wavelength and full spectraldetection at a highest sampling rate of flow cell. Non-coated fused silica fiber cell

optics for robust performance and handling

detection at a highest sampling rate(160 Hz)—keeps pace with the analysis speed of ultra-fast LC


16


17

Signal height

Non detector related:

Column: diameter and length e g 4 6 vs 2 1 solumn id• Column: diameter and length, e.g. 4.6 vs. 2.1 solumn id,

• Post-column dispersion

• Delay volume: isocratic step in the beginning of a chromatogram

Detector related:

• Pathlength, cell volume, cell geometry, optical slit width, response time, date rate


18

Overview

Signal to noise



Detector dependent




19

Column diameter

Smaller internal diameter of column increases sensitivity?

This is only true if the same amount of sample can be introduced onto the column with smaller internal diameter!

4.6 mm column Injection vol. 5 μL DMSO OK!

2.1 mm column Injection vol. 5 μL DMSO OK???

20

Shorter columns

Faster runs result in more sensitivity?

Faster runs result in higher peaks with smaller peak width but with the same area. Higher peaks mean improved signal to noise ratio and consequently more sensitivitynoise ratio and consequently more sensitivity.

150 mm 5 µm 50 mm 1 8 µm40 mAU

150 mm, 5 µm

olue

ne

120 mAU50 mm, 1.8 µm

olue

ne

To To

21

Higher flow rates

Faster runs result in more sensitivity?

The response of the detector depends on the concentration. Higher flow rates dilute the sample (same sample amount) and consequently the response is reduced.

2 4 mL/min0 6 mL/min95 mAU

2.4 mL/min

olue

ne

120 mAU0.6 mL/min

Tolu

ene

ToT

22

Dispersion volume( t di i )Dispersion volume (system dispersion)

• “Dispersion is the sample bandspreading or dilution which occurs in connecting tubing, sample valves, flow cells and in column end-fittings.”

• Capillary connections (internal diameter, length)Capillary connections (internal diameter, length)• Connections (Unions, T-pieces, Fittings)• Addional valves (Alternating column regeneration)

Peak dispersion depends on Detector:• Inlet heat exchangerInlet heat exchanger• Volume of the detector cell• Cell geometry

23

Dispersion volume after the column( t di i )Dispersion volume (system dispersion)

• “Dispersion is the sample bandspreading or dilution which occurs in connecting tubing, sample valves, flow cells and in column end-fitting.”

Peak height: less sensitivityDispersion

Peak height: less sensitivity

Peak width: less resolution

• Capillary connections (internal diameter, length)

LF4

DLFr

⋅⋅⋅⋅

=24

42 πσ

24

Aris-Taylor equationmD24

Influence of dispersion volume after the column

mAU

200

Dispersion volume not optimized

Peak width 0 037 min

Resolution 0.961

50

100

150

Peak width 0.038 min


min0.5 1 1.5 2 2.5

0

50

mAU Resolution 1 902mAU

250

300

350


Resolution 1.902

Dispersion volume optimized

0

100

150

200


25

min0.5 1 1.5 2 2.50

Influence of Pre-column delay volume

Delay volume• “System volume from the point of mobile phase mixing to the

l h d” (W D l LCGC 2006 V l 24 N 5 458 466)column head” (W. Dolan LCGC 2006 Vol 24, No 5, 458-466)

Delay volume

• A gradient change reaches the top of the column with delay• Isocratic step in the beginning influences peak width of first fast

eluting peaks

26

eluting peaks

Influence of Delay VolumeColumn: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm

Delay volume ~ 700 μL

mAU

300

400

Co u O S C 8 50 , 8 μFlow: 0.42 mL/min

Delay volume 700 μL

100

200

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

0

mAU400

Delay volume ~120 μL200

300

400

0

100

200

27

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

Effects of pre and post column non-optimized dispersion volumedispersion volume

mAU

140

40

60

80

100

120

min0 0.1 0.2 0.3 0.4

0

20

mAU

140

40

60

80

100

120

min0 0.1 0.2 0.3 0.40

20

Column switching valve with bad capillary connections (10port 2position valve)


28

Overview

Signal to noise



Detector dependent




29

Sensitivity, Lambert-Beer law

dcIA ⋅⋅=⎟⎟⎞

⎜⎜⎛

−= ε1logA: Absorptionε: Extinction coefficient

C idcI

A ⎟⎟⎠

⎜⎜⎝

ε0

log c: Concentrationd: path length

Absorption

30

Absorption coefficient

Optical programmable slit for more light transmissiontransmission

Programmable slit to optimize sensitvity and spectral resolution

31

Influence of optical slit width

Optical slit width 8 nm vs.4nm vs.1nm

mAU160

120

140

para

ben

Noise between0.02 – 0.06 min

60

80

100

Eth

yl

20

40

60

min0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0

20

32

Influence of slit width on sensitivity

Sli id h [ ] S/N (6 SD) S/N (P P)Slit width [nm] S/N (6 SD) S/N (PtoP)

1 392 476

4 567 830

8 924 10858 924 1085

A wider slit results in more sensitivity!

33

Influence of data rate on peak heightDAD1 A Sig=245 10 Ref=360 100 (BROSCHURE7 HZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST 25HZ D)

min0 0.1 0.2 0.3 0.4 0.5

mAU

0

DAD1 A, Sig 245,10 Ref 360,100 (BROSCHURE7...HZTESTADVR\DADTESTHZADVR 2009 07 14 10 15 26\DADTEST_25HZ.D)

mAU

DAD1 A, Sig=245,10 Ref=360,100 (BROSCHURE7...DHZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST_5HZ.D)

2.5Hz

5Hz

min0 0.1 0.2 0.3 0.4 0.50

mAU

0

DAD1 A, Sig=245,10 Ref=360,100 (BROSCHURE7...HZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST_10HZ.D)

5Hz

10Hzmin0 0.1 0.2 0.3 0.4 0.5

0

min0 0.1 0.2 0.3 0.4 0.5

mAU

0


DAD1 A Sig=245 10 Ref=360 100 (BROSCHURE7 HZTESTADVR\DADTESTHZADVR 2009 07 14 10 15 26\DADTEST 40HZ D)

20Hz

min0 0.1 0.2 0.3 0.4 0.5

mAU

0


mAU


40Hz

80Hz

min0 0.1 0.2 0.3 0.4 0.5

mAU

0

mAU

0

DAD1 A, Sig=245,10 Ref=360,100 (BROSCHURE7...ZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST_160HZ.D)

80Hz

160Hz


34

min0 0.1 0.2 0.3 0.4 0.50

Influence of Data Rate on Performance

Data Rate (Hz) Resolution peak 5 Peak Width last peak (min)

Peak Height (mAU) of Peak 3

160 1.89 0.00307 1171.2

80 1.83 0.00323 1131.1

40 1.57 0.00381 1006.4

20 1.06 0.00565 738.6

10 0.56 0.0102 431.2

5 / 0.0203 217.15 / 0.0203 217.1

2.5 / / /


35

Influence of detector data ratemAU

80

100

80 Hz

20

40

60

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

0

mAU

30

40

50

10 Hz

min0 0 1 0 2 0 3 0 4 0 5

0

10

20

36

min0 0.1 0.2 0.3 0.4 0.5

1200 DADSL vs. 1200 VWD D vs.1290 DAD at 10Hz10pg Anthracene injected

1290Infinity DAD 60mm cellmAU

-1-0.5

0

LOD 87fgNoise: 0.002297mAUSignal Height: 0.055597mAU

10pg Anthracene injected Factor 4.7

min1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

-1.5

mAU

0

1290 Infinity DAD 10mm cell

Signal Height: 0.055597mAUS/N: 242

LOD 412fgNoise: 0.013970mAU

Agilent high end VWDmin*1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

-1.5-1

-0.5

mAU LOD 97fg


Agilent high end VWD

min*1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

-1.5-1

-0.50 Noise: 0.001775mAU


1200RR DADSL

min1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

mAU

1 5-1

-0.50

LOD: 637 fgNoise:0.01950 mAUSignal Height: 0.611923mAUS/N: 31 38


37

min*1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

-1.5 S/N: 31.38

Conventional method optimized for more sensitivity on the 1290 Infinityon the 1290 Infinity

Using:g

•1290 pump with lowest ripple and lowest delay volume

•Using modern DAD designg g

•High efficiency columns

Taking care of:

•Post column dispersion volume

•Using highly pure solvents

What factor can we gain?


38

Conventional LC, 1200 Standard LC

mAU

Conventional LC: 1200 Standard LC with 2

3

Co e t o a C 00 S a da d Cstandard autosampler and quaternary pumpSignal/noise: 76.5 (last Peak)

1

2

-1

0

min2 4 6 8 10

Column: 4.6x150mm, 5µm, flow rate:1.5ml/min, run time: 12min


39

More Sensitivity with UHPLC, 1290 Infinity

More speed and sensitivity with mAU

30

p yUHPLC, 1290 Infinity LC SystemSignal/noise: 740

15

20

25

Conventional LC with 1200 Standard LC

Factor 9.6 more sensitivity

5

10

15 Conventional LC with 1200 Standard LCSignal/noise: 76.5 (last Peak)

min0 0.2 0.4 0.6 0.8

0


40

Column: 2.1x50 mm RRHD Zorbax C18,1.8µm, DAD: 160Hz, Run time: 0.6min, flow rate: 2ml/min

Summary 1290 Infinity UHPLC

More Powerful

Power range 1200 bar (2 ml/min);800 bar 5 ml/min)Delay volume (Pump (w/o) mixer: 10 µl)

DAD: lowest detector noise typically < +/- 3 µAU/cm for10mm path length, <+/- 0.6µAU for 60mm path length cellMax-Light High Sensitivity cellMore

S iti it 10 times more sensitivity vs,conventional chromatographyl LC/MS 6000 Series + Jet-stream technology

Sensitivity

Injection principles (fixed loop and flow-through) Method transferability from all vendors (U)HPLC)TCC: column switching, easy valve exchange

More Flexibility

ALS precision for small volumes ( <0.7% @ 1-2ul)Pump: e.g RT stability < 0.2 % (1.5 min runs)DAD: more robust baseline (no RI effects)

More Precise


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the 1290 infinity lc - 10x higher uv sensitivity 1290... · 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5...

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