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Dr. Matthias Krajewski Product Specialist HPLC Thermo Fisher Scientific, Germering, Germany
Which UHPLC for LC-MS?
2
Content of the Webinar
Throughput in LC-MS
Gradient delay volume
Pump technologies
Thermo Scientific™ Vanquish™ UHPLC systems for throughput
Additional ways for throughput
3
What is UHPLC?
• System back pressure above 600 bar
• Analytical column with sub-2 micron particles
4
What is UHPLC?
• System back pressure above 600 bar
• Analytical column with sub-2 micron particles
To achieve: • Faster separation • Better resolution • Less waste
5
What is UHPLC?
• System back pressure above 600 bar
• Analytical column with sub-2 micron particles
To achieve: • Faster separation • Better resolution • Less waste
Up to 1000 bar 1500 bar
• Highest pressure capability up to 1500 bar
• Flow rates up to 5 mL/min
• Lowest system dispersion and GDV
• Unmatched detection sensitivity and linearity
• Biocompatible
Quaternary system • Quaternary solvent blending
• Pressures up to 1000 bar
• Flow rates of up to 8 mL/min
• Biocompatible
Binary system • Binary high pressure solvent mixing
• Pressures up to 1000 bar
• Flow rates of up to 8 mL/min
• Biocompatible
Vanquish Flex systems
Vanquish Horizon systems
6
Why is Throughput in LC-MS Important?
We require sophisticated LC-MS technology.
We must manage regulatory demands.
We need systems with high-resolution, selectivity, sensitivity, robustness, large number of molecules to be discovered,
identified and quantified in one single run.
We require high throughput capabilities at the same time.
Which UHPLC system is suitable for our high throughput LC-MS analysis?
Vanquish UHPLC system Thermo Scientific™ Q Exactive™ hybrid quadrupole Orbitrap™ MS
Vanquish UHPLC system Thermo Scientific™ TSQ Quantiva™ triple quadrupole MS
7
Why is the GDV of your “Front-End” Important for Throughput?
Gradient delay volume (GDV)
Thermo SCIENTIFIC
Thermo SCIENTIFIC
Thermostatted column
compartment
Autosampler Pump
Extra column volume (ECV)
Thermo SCIENTIFIC
Detector
• Gradient delay volume (GDV):Volume of fluid between mixingpoint of the gradient and columnhead
• Extra column volume (ECV):Volume of fluid between sampleinjection point and midpoint ofthe detector’s flow cell.
0 4 8 12 16 -40
310
mAU
Time (min)
WVL:265 nm
Flow: 1.0 mL/min Water + 0.1% acetone: 0%
100% GDV
8
Why is the GDV of your “Front-End” Important for Throughput? Short runtimes and higher throughput • Column equilibration time is
related to the GDV.
• Small GDV - shorter equilibration times
• Contribution of equilibration
time is relevant for short runs.
0,00 2,50 5,00 7,509,10350500625750
min
Gradient Variable
Equilibration Not variable
Pressure trace
Gradient delay
Retention time shift
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Why do UHPLC Systems have Different System GDVs?
Low pressure side
High pressure side
Point of mixing
Vanquish Flex Quaternary pump
By default, LPG pump systems have large GDVs.
UHPLC systems can use different pump technologies: Low pressure gradient (LPG)
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Why do UHPLC Systems have Different System GDVs?
Low pressure side
High pressure side
Point of mixing
Vanquish Flex Quaternary pump
By default, LPG pump systems have large GDVs.
pump GDV
UHPLC systems can use different pump technologies: Low pressure gradient (LPG)
11
Why do UHPLC Systems have Different System GDVs?
UHPLC systems can use different pump technologies: High pressure gradient (HPG)
Point of mixing
Vanquish Horizon pump
Low pressure side High pressure side
By default, HPG pump systems have small GDVs.
12
Why do UHPLC Systems have Different System GDVs?
UHPLC systems can use different pump technologies: High pressure gradient (HPG)
Point of mixing
Vanquish Horizon pump
Pump GDV
Low pressure side High pressure side
By default, HPG pump systems have small GDVs.
13
What UHPLC for LC-MS?
Vanquish Flex Quaternary
Vanquish Flex Binary
used mixers: 25 + 10 = 35 µL
used mixer: 25 + 10 = 35 µL
used mixer: 25 + 10 = 35 µL
Vanquish Horizon
Low pressure gradient (LPG)
1034 bar 1500 bar 1034 bar
High pressure gradient (HPG)
High pressure gradient (HPG)
Tested Vanquish systems LC-MS analysis of pesticides
0
20
40
60
80
100
0 5 10 15 20 25 30
sol
vent
B [%
]
LC gradients [min]
2 min 5 min 10 min 20 min
100 min
2 min
5 min
10 min
100 min
20 min
2,250 2,500 2,750-2,4e52,5e6
5,5e6
min
counts
time aligned
0.0 12.5 25.0 37.5 50.0 Time [min]
-1.0e3
1.3e6
2.5e6
3.8e6
5.0e6
6.3e6
7.0e6
I n t e
n s i t y
[ c o u
n t s ]
min
counts
RT shift; decreased intensity;
14
Total Analysis Time (TAT) of Vanquish UHPLC Systems
Pressure traces (2 min gradient)
pum
p pr
essu
re (b
ar)
Overlay (n=5); w/ signal offset
350
400
500
600
700
750
350
400
500
600
700
750
0,00 2,00 4,00 6,00 8,00 9,10 350
400
500
600
700
750
min
Gradient Variable
Reconditioning Not variable
TAT: 9.10 min
Gradient delay
Extracted ion chromatograms
Vanquish Flex Quaternary Vanquish Flex Binary Vanquish Horizon
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
0 5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
Rel
ativ
e Ab
unda
nce
RT: 0.00 - 4.80
Overlay; w/o signal offset
Retention time shift
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TAT Optimization of Vanquish UHPLC Systems
Pressure traces (2 min gradient)
pum
p pr
essu
re (b
ar)
Overlay (n=5); w/ signal offset
350
400
500
600
700
750
350
400
500
600
700
750
0,00 2,00 4,00 6,00 8,00 9,10 350
400
500
600
700
750
min
Gradient Variable
Reconditioning Not variable
TAT: 6.55 min
+5 GDV
TAT: 6.55 min
TAT: 9.35 min
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TAT Optimization of Vanquish UHPLC Systems
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Throughput Considerations
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min sa
mpl
es p
er 2
4 hr
s
Vanquish Flex Quaternary
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Throughput Considerations
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min sa
mpl
es p
er 2
4 hr
s
Vanquish Flex Quaternary
199
142
97 59
14
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min
sam
ples
per
24
hrs
• Fast runs (< 10min) • Throughput increase from 10-30% →Vanquish HPG systems for throughput in LC-MS
Vanquish Flex Quaternary Vanquish HPG systems
19
Throughput Considerations
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min sa
mpl
es p
er 2
4 hr
s
Vanquish Flex Quaternary
199
142
97 59
14
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min
sam
ples
per
24
hrs
• Fast runs (< 10min) • Throughput increase from 10-30% →Vanquish HPG systems for throughput in LC-MS
Vanquish Flex Quaternary Vanquish HPG systems
• LC runs (> 20min) • Long reconditioning phase required → Tandem UHPLC setups as alternative ways to achieve throughput
20
14
0
10
20
30
100 min sa
mpl
es p
er 2
4 hr
s
Vanquish Flex Quaternary Vanquish tandem
UHPLC setup
*
* analytical gradient: 70min; reconditioning phase: 30min
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Vanquish Flex Binary and Horizon Systems – What‘s the difference?
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Vanquish Flex Binary and Horizon Systems – What‘s the difference?
2
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time (min)
0 10 20 30 40 50 60 70 80 90
100
Rel
ativ
e Ab
unda
nce
11
polar
non-polar
Methamidophos
Quinoxyfen RT precision for 199 consecutive injections
60 ms ≡ 0.001 min
2 min
Vanquish Flex Binary
11
2
1.080 1.180 -5.0e5
0.0e0
1.0e6
2.0e6
3.0e6
min
counts
SD: 158 ms
2.850 2.900 2.950 -1.0e6
0.0e0
2.0e6
4.0e6
6.0e6
8.0e6
9.0e6
min
counts
SD: 109 ms
Vanquish Horizon
1.040 1.100 1.140 -5.0e5
0.0e0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
min
counts
SD: 40 ms
2.700 2.800 -1.0e6
0.0e0
2.5e6
5.0e6
7.5e6
1.0e7
1.1e7
min
counts
SD: 35 ms
22
Vanquish Flex Binary and Horizon Systems – What‘s the difference?
Higher RT precision of the Vanquish Horizon system can reduce your number of technical replicates, one important aspect to be considered for high-throughput.
2
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time (min)
0 10 20 30 40 50 60 70 80 90
100
Rel
ativ
e Ab
unda
nce
11
polar
non-polar
Methamidophos
Quinoxyfen RT precision for 199 consecutive injections
60 ms ≡ 0.001 min
2 min
Vanquish Flex Binary
11
2
1.080 1.180 -5.0e5
0.0e0
1.0e6
2.0e6
3.0e6
min
counts
SD: 158 ms
2.850 2.900 2.950 -1.0e6
0.0e0
2.0e6
4.0e6
6.0e6
8.0e6
9.0e6
min
counts
SD: 109 ms
Vanquish Horizon
1.040 1.100 1.140 -5.0e5
0.0e0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
min
counts
SD: 40 ms
2.700 2.800 -1.0e6
0.0e0
2.5e6
5.0e6
7.5e6
1.0e7
1.1e7
min
counts
SD: 35 ms
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What Level of Throughput is Possible with the Vanquish Horizon?
Column Thermo Scientific™ Hypersil GOLD™ 50 x 2.1 mm, 1.9 µm
567 samples per day
LC conditions
LC-MS analysis of pesticides Throughput maximization
methamidophos (2)
quinoxyfen (11)
fenuron (4)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
Rel
ativ
e Ab
unda
nce
1.0 mL/min
1.25 mL/min flow rate
gradient
2
11
4 TAT: 1.85 min
0
10
20
30
40
50
60
70
80
90
100
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 200 400 600
RT
in [m
in]
Sample injection # At least 10 Hz are required to obtain RT SD = 0.001 min for peak width at 50% of 0.025 min (1.5 s).
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Additional Ways For Throughput Shallow LC gradient
Reconditioning time may take more than 20 min until next sample injection.
Reconditioning of >20 min Not variable
Gradient of >70 min Variable
TAT: 100min
0
50
100
0 20 40 60 80 100
Elue
nt B
[%]
Time [min]
Analytical gradient 70%
Reconditioning 30%
Discussion starter
Target of tandem UHPLC setups!!!
199
142
97 59
14
144 111
81 53
14 0
50
100
150
200
250
300
2 min 5 min 10 min 20 min 100 min
Sam
ples
per
24
hrs
Vanquish Flex Quaternary Vanquish HPG systems
20
14
0
10
20
30
100 min
Sam
ples
per
24
hrs
Vanquish Flex Quaternary Vanquish tandem
UHPLC setup
*
* Analytical gradient: 70min; reconditioning phase: 30min
25
Vanquish UHPLC Tandem LC Setup to Increase Productivity
Pump 2
Pump 1
Vanquish tandem LC and LC-MS workflow
2-position/6-port (2p6p) valve
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Vanquish UHPLC Tandem LC Setup to Increase Productivity
Vanquish tandem LC-MS workflow
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Vanquish UHPLC Tandem LC Setup to Increase Productivity
Vanquish tandem LC-MS workflow
Overlay of 5 total ion current chromatograms Vanquish tandem setup: 2x Thermo Scientific™ Acclaim™ VANQUISH™ column C18 2.1x250 mm, 2.2 µm, Lot No. 1425071; A: 0.1/100 FA/Water (v/v); B: 0.1/100 FA/Acetonitrile (v/v) Gradient: 1-45% B in 40 min, 60 °C; 400 µL/min; Injection volume 5 µL, 1 µg Infliximab SMART digest; Reconditioning: Two times 90% B for 5 min, 24 min equilibration at 1% B; Detection: Q Exactive HF MS, R=15k, mass range 140-2000; UV detection@214 nm
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Summary of the Webinar
29
1. With an UHPLC system you can focus on peak capacity and sample throughput.
Summary of the Webinar
30
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
Summary of the Webinar
31
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.
Summary of the Webinar
32
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.
4. Low pressure gradient (LPG) or high pressure gradient (HPG) pump technology is chosen in alignment with your application requirements.
Summary of the Webinar
33
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.
4. Low pressure gradient (LPG) or high pressure gradient (HPG) pump technology is chosen in alignment with your application requirements.
5. With HPG pump technology you can achieve maximum sample throughput.
Summary of the Webinar
34
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.
4. Low pressure gradient (LPG) or high pressure gradient (HPG) pump technology is chosen in alignment with your application requirements.
5. With HPG pump technology you can achieve maximum sample throughput.
6. With LPG pump technology you can use ternary or quaternary gradients and advanced column chemistries for e.g. method development.
Summary of the Webinar
35
1. With an UHPLC system you can focus on peak capacity and sample throughput.
2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.
3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.
4. Low pressure gradient (LPG) or high pressure gradient (HPG) pump technology is chosen in alignment with your application requirements.
5. With HPG pump technology you can achieve maximum sample throughput.
6. With LPG pump technology you can use ternary or quaternary gradients and advanced column chemistries for e.g. method development.
7. For LC-MS applications with very long run times, you can increase your productivity with tandem UHPLC system setups.
Summary of the Webinar
37
Thank you very much for your attention!
Questions?
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