Which UHPLC for LC-MS? - Thermo Fisher Scientific · 2017-08-29 · Proprietary & Confidential The...

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Dr. Matthias Krajewski Product Specialist HPLC Thermo Fisher Scientific, Germering, Germany

Which UHPLC for LC-MS?

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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?



To achieve: • Faster separation • Better resolution • Less waste

5

What is UHPLC?



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

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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

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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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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)

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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.

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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.

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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;

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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


18


144 111

81 53

14 0

50

100

150

200

250

300


mpl

es p

er 2

4 hr

s


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

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144 111

81 53

14 0

50

100

150

200

250

300


mpl

es p

er 2

4 hr

s


199

142

97 59

14

144 111

81 53

14 0

50

100

150

200

250

300


sam

ples

per

24

hrs

• Fast runs (< 10min) • Throughput increase from 10-30% →Vanquish HPG systems for throughput in LC-MS


• LC runs (> 20min) • Long reconditioning phase required → Tandem UHPLC setups as alternative ways to achieve throughput

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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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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


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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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


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


Sam

ples

per

24

hrs


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

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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 tandem LC-MS workflow

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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

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1. With an UHPLC system you can focus on peak capacity and sample throughput.


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2. The system gradient delay volume (GDV) is the crucial parameter for your sample throughput.


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3. The operation principle of the pump and the pump mixer configurations contribute significantly to the GDV of your UHPLC system.


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4. Low pressure gradient (LPG) or high pressure gradient (HPG) pump technology is chosen in alignment with your application requirements.


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5. With HPG pump technology you can achieve maximum sample throughput.


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6. With LPG pump technology you can use ternary or quaternary gradients and advanced column chemistries for e.g. method development.


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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.


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Do you want to know more?

Contact us: [email protected]

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Thank you very much for your attention!

Questions?

Do you have additional questions or do you want to talk to an expert from Thermo Fisher Scientific? Please send an E-Mail to [email protected] and we will get back to you.

mailto:[email protected]

Which UHPLC for LC-MS? - Thermo Fisher Scientific · 2017-08-29 · Proprietary & Confidential The...

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