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
Not detector dependent
Detector dependentppp
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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.
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
7
Influence of mobile phase
I it
ACN 1
Impurity
ACN 2
November 17, 2009Confidentiality Label
8
Overview
Signal to noise
Signal heightBaseline noise Signal heightBaseline noise
Not detector dependent
Detector dependent
Not detector dependent
Detector dependentppp
November 17, 2009Confidentiality Label
9
Baseline noise
Detector related:
Optical unit design VWD DAD• Optical unit design, VWD , DAD
• Cell type and geometry
• Data rate
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
16
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
18
Overview
Signal to noise
Signal heightBaseline noise Signal heightBaseline noise
Not detector dependent
Detector dependent
Not detector dependent
Detector dependentppp
November 17, 2009Confidentiality Label
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
Peak width 0.037 min
min0.5 1 1.5 2 2.5
0
50
mAU Resolution 1 902mAU
250
300
350
Peak width 0.019 min
Resolution 1.902
Dispersion volume optimized
0
100
150
200
Peak width 0.018 min
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)
November 17, 2009Confidentiality Label
28
Overview
Signal to noise
Signal heightBaseline noise Signal heightBaseline noise
Not detector dependent
Detector dependent
Not detector dependent
Detector dependentppp
November 17, 2009Confidentiality Label
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_20HZ.D)
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
DAD1 A, Sig=245,10 Ref=360,100 (BROSCHURE7...HZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST_40HZ.D)
mAU
DAD1 A, Sig=245,10 Ref=360,100 (BROSCHURE7...HZTESTADVR\DADTESTHZADVR 2009-07-14 10-15-26\DADTEST_80HZ.D)
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
November 17, 2009Confidentiality Label
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 / / /
November 17, 2009Confidentiality Label
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
Signal Height: 0.714955mAUS/N: 51
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
Signal Height: 0.608286mAUS/N: 343
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
November 17, 2009Confidentiality Label
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?
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
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
November 17, 2009Confidentiality Label
41
November 17, 2009Confidentiality Label
42
November 17, 2009Confidentiality Label
43