API Optimization, Source Design, and Other Considerations of LC/MS

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1 API Optimization, Source Design, and Other Considerations of LC/MS

2 Instrument Design Considerations: The Atmosphere-Vacuum Interface and Pumping System

3 The hows and whys of the vacuum? We ve got to get from 760 torr to 10-6 Torr We need vacuum to focus and transport ions efficiently without scattering losses Profound statement: You can never have too much pumping Two approaches to vacuum pumping: Single stage pumping Differential (multi-stage) pumping A number of different approaches to atm-vac interface design are currently in production Heated capillary, on-axis sprayer (Finnigan API-1, API-2 sources) 90º off axis sprayer (AQA, Z spray, Agilent, TSQ Quantum) Different interface designs have different analytical and operational characteristics This presentation will focus on the heated capillary interface, which is used in the majority of Finnigan LC/MS interfaces

4 Single stage pumping Basis of PE-Sciex TAGA and API-III Design Plate orifice interface characteristics Interface cannot tolerate high liquid flows along the spray axis a gentle source Lots o pumping!! Advantages: Pumping!! Simple design High transport efficiency Disadvantages Must recycle (thaw) cryo surfaces daily Small pinhole Noisy pump operation Pump is expensive if it breaks

5 Schematic of a differentially-pumped, heated-capillary API/MS system Heated capillary interface characteristics Can tolerate the widest range of liquid flows Excellent for biomolecules Thermal degradation is possible with certain compounds but can be minimized ESI or APCI (1 atm) desolvation, ion evaporation 1 Torr (rough vacuum) mechanical pump Ion focusing 1mTorr (med vacuum) turbomolecular pump 1 Mass Analysis 3x10-6 Torr (vacuum) turbomolecular pump 2 Advantages: Larger inlet orifices possible 24/7 system Turbos are relatively reliable (technology improving and becoming lower cost) Disadvantages Must manage ion transport losses in various stages $ increase with # of stages

6 Cross-section of a turbomolecular pump Turbo pump characteristics: Sounds like a jet engine when spinning up or down Operation at high speed krmp Newer designs have multiple inlet stages and are very low maintenence

7 Example: Typical Operating Pressures in an LCQ Deca 760 torr 1.3 torr 1.7x10-3 torr 2.0 x10-5 torr (1.0x10-5 torr He) 3.5x10-3 torr He 60 m 3 /hr 100 L/sec 220 L/sec

8 Most API sources operate off-axis with respect to ion sampling to minimize spray-related noise The heated capillary is off-axis with respect to the skimmer in API-1, API-2, and all LCQ source designs Free-jet expansion skimmer 1 atm Heated Capillary Tube lens Vacuum Particles and droplets move to the center of the flow in the jet expansion, impinge on skimmer and are not transmitted, reducing noise dramatically

9 Design Considerations: on-axis vs. off-axis ion sampling API-1 TSQ, capillary-skimmer on-axis, Reserpine loop inj., 50 ul/min MeOH/H 2 O Sprayrelated noise

10 Design Considerations: on vs. off-axis ion sampling API-1 TSQ, capillary-skimmer off-axis, Reserpine loop inj., 50 ul/min MeOH/H 2 O

11 API Interface Optimization

12 Optimizing Parameters for API/LC/MS Interface Parameters Heat (desolvation) Heated capillary, interface plate, etc. N 2 nebulizer or sheath gas (droplet formation) N 2 auxiliary or bath gas (humidity control) Voltages Spray (HV for droplet formation in ESI) Corona (discharge in APcI) Interface (used to optimize ion transport) HPLC Parameters Flow rate Buffer (use volatile!) Column diameter Column chemistry

13 Interface Optimization Rules of Thumb Refer to manufacturers tip sheet as a starting point Some parameters are dependent on the input liquid flow rate Nebulizer gas ( set it and forget it above 10 ul/min) Auxiliary gas (increase with flow rate, off at low liquid flows) Interface temperature (increase with liquid flow rate) Interface voltages may need to be increased with flow rate, but temperature may accomplish the same task ESI spray voltage (only critical for low flow or nanospray applications) ESI electrode capillary entrance distance (move closer in low flow operation) Corona voltage (set it and forget it)

14 Cross-section of ESI source head Aux gas Spray voltage Sheath gas

15 Interface Optimization Rules of Thumb Some parameters are dependent on the compound or chemistry! Very thermo-labile or fragile molecules may require: Lower interface temperatures Lower interface voltages Molecules that have affinity for other solvent molecules may require: Higher interface temperatures Higher interface voltages Use of auxiliary gas

16 Pre-indexed ESI probe on API-2 Simple setting of probe position as a fxn of flow rate Ludicrous flow 1mL/min 200 ul/min 50 ul/min or less

17 ESI: Effect of Auxiliary N2 Flow on Solvent Background API-1 TSQ, Astemizole loop injections 200 ul/min 80/20 MeOH/H 2 O 10mM NH 4 OAc

18 ESI: Effect of Capillary Temperature on S/N at 50 ul/min API-1 TSQ, Astemizole loop injections 50 ul/min 80/20 MeOH/H 2 O 10mM NH 4 OAc

19 ESI: Effect of Capillary Temperature on S/N at 1 ml/min API-1 TSQ, Astemizole loop injections 1 ml/min 80/20 MeOH/H 2 O 10mM NH 4 OAc

20 Cross-Section of APcI source head Aux gas usually off Vaporizer temp C

21 Compound dependent optimization strategies For example, optimizing on a target compound for quantitation purposes Tee sample into HPLC stream using infusion pump Monitor analyte signal and optimize each MS parameter as desired 1-10uL/min vial 1 Your desired flow rate, e.g. 400ul/min

22 Optimization strategies, continued Teeing in: Advantages are extensive time for tuning and tweaking Disadvantage is possibility of interface contamination with your target compound Other options: DAC scan or parameter ramp. Finnigan TSQ allows for scanning parameters during loop injection. This is often faster and leads to less interface contamination.

23 Example Optimum ESI Parameter Matrix for API-1 Source Liquid Flow Rate Capillary Temp. Spray Voltage ESI-Cap Distance Sheath Gas Aux Gas <10 ul/min C < cm >40 off C 4.5 kv 1 cm 70 psig off- ul/min 10 units C 4.5 kv 1.5 cm 70 psig 20 units ul/min 1000 ul/min C 4.5 kv 1.5 cm 70 psig 30+ units

24 Optimizing HPLC Conditions for API Operation

25 HPLC buffer optimization notes Remember: ESI is a process that takes pre-formed ions in solution and pops them into the gas phase Try to pick a buffer that will promote the ionization of your compound The above does not always allow for optimum chromatography, there may be tradeoffs Stick with volatile buffers if possible Stick with low ionic strength buffers if possible Some modifiers suppress signal (e.g., TFA) New column technology is reducing the requirements of modifier use Modifiers in APCI have less of an effect since ions are formed from vaporized sample via chemical ionization

26 Solvent System Example of solution chemistry effects: Leu-enkephalin Signal with various solvent systems, ESI positive mode Tyr-Gly-Gly-Phe-Leu 50/50 ACN/H2O 0.1% NH4OH 50/50 MeOH/H2O 0.1% NH4OH 50/50 ACN/H2O 0.02% TFA 50/50 ACN/H2O 0.05% TFA 50/50 ACN/H2O 0.1% TFA 50/50 MeOH/H2O 0.02% TFA 50/50 MeOH/H2O 0.05% TFA 50/50 MeOH/H2O 0.1% TFA 50/50 MeOH/H2O 10mM NH4OAc 50/50 MeOH/H2O 5mM NH4OAc 50/50 ACN/H2O 0.1% Formic 50/50 ACN/H2O 1% Acetic 50/50 MeOH/H2O 0.1% Formic 50/50 MeOH/H2O 1% Acetic 100 ACN 100 MeOH 100 H2O 50/50 ACN/H2O 50/50 MeOH/H2O Ion Signal, Counts (protonated ion species)

27 Picking a column diameter Not sample limited (impurity profiling, process, QC) mm i.d. Somewhat sample limited (peptide digests, in vivo metabolite isolates, pharmacokinetics studies) 1-2 mm i.d. Sample limited (peptides, extensive multi-experiment studies on a single sample) mm i.d. Severely sample limited (in-gel protein digests, real-time invivo monitoring) mm i.d.

28 Column Arsenal for LC/MS Qualitative Small Molecule Analysis You may want to choose a variety of chemistries Stable column for low ph (TFA) work (e.g., Zorbax SB-C18) Polar compounds in highly aqueous mobile phases (e.g., YMC-AQ) Negative ion work stable column at ph 6-7 for (e.g., Luna C18) Base-deactivated for low or no TFA work (e.g., MAGIC C18) High-throughput and/or quantitation Choose columns of short length (5 cm or less) and small particle diameter (3-5 um) (e.g., MAGIC bullets) Use in-line filters! As in qualitative analysis, may be useful to have a variety of column chemistries on hand Peptide Use a good base deactivated 300A C18 column, leave out the TFA if you need ultimate sensitivity (e.g., MAGIC C18) Capillary columns for proteomics applications (often home-packed) Protein large pore polymeric, 5 cm length (e.g., PLRP-4000A)

29 The Issue of Flow Splitting Pre-Column Post-Column

30 For ESI, do you need to split the LC flow to obtain optimum performance? But wait there are 2 kinds of splits Pre-column, pre-injector (to use a smaller i.d. column) Post-column (to reduce the total flow into the MS) Need for post-column split is dependent on the source design Heated capillary sources offer compatibility of the entire liquid flow range (< ul/min) Post-column splitting is often recommended for other designs (consult the manufacturer) Maybe you want to split! Post-column: data-dependent fraction collection or purification Post-column: since ESI is roughly concentration-dependent you won t loose a significant amount by splitting Pre-column: you want to do capillary or nano LC

31 How do you split if you need to? Example: Pre-column splitting for micro-column operation Columns of the same length and packing will give ~the same pressure drop if operated at their optimum flow rates. Therefore a split based on the cross-sectional areas of the columns will be created. E.g., for 4.6:1 mm ~ 20:1 split old clunky HPLC that only likes 1 ml/min Injector or AS analytical column e.g., 1 x 100 mm 50 ul/min balance column e.g., 4.6 x 100 mm 950 ul/min to waste

32 Variable pre-column splitters Designed to give a particular pressure drop at a given input flow rate. Select a resistor to give you the right flow. Example: Michrom Variable Pre-column Splitter Assembly. These are very convenient, particularly for capillary column applications. Input flow R1 Tee Injector or AS R2 R3 To Waste To micro Column And MS R4

33 ESI: Effect of Post-Column Flow Splitting on Performance API-1 TSQ, Astemizole tablet extract 2.1x100 mm C18, 400 ul/min 80/20 MeOH/H 2 O 10mM NH 4 OAc 400 ul/min no split 200 ul/min 1:1 split 100 ul/min 3:1 split 50 ul/min 7:1 split 10 ul/min 39:1 split

34 Effect of post-column splitting on peak areas API-1 source on TSQ shows fairly constant signal, regardless of flow to ESI/MS, but response is higher without splitting? API-1 TSQ, Astemizole tablet extract 2.1x100 mm C ul/min 80/20 MeOH/H 2 O 10mM NH 4 OAc P e a k % A r e a % 51% 45% 53% Flow Rate to ESI (ul/min)

35 Effect of post-column splitting on peak areas API-1 TSQ, Adenosine (MW 267), 4.6 mm column at 1 ml/min 90/10 H 2 O /MeOH 25 mm NH 4 OAc % P e a k A r e a % 32% Flow Rate to ESI (ul/min)

36 Effect of Column Diameter on Peak Areas TSQ w/api-1 ESI

37 Effect of Column Diameter on Peak Areas Smaller column diameters result in higher concentrations at the detector and increased peak areas, but not as much as expected? ul/min to ESI 100% 200 ul/min to ESI 1mL/min to ESI P e a k % 54% A r e a mm 2.1 mm 4.6 mm Column inner diameter

38 Possible explanation for not-so-concentration-dependent behavior of Finnigan Source Design ±5kV Nozzle Gas Sheath Higher liquid flows Result in more dense plume and concentration of aerosol toward the center of the plume Needle Aerosol Heated Capillary An-off axis sampling of the spray would likely result in a purely concentration-dependent device

39 Splitting issues Pre-column, pre-injector splitting can be used to couple conventional HPLCs to small bore columns. In an optimized heated-capillary API interface, there is no need to flow-split post column unless sample collection is desired. Response in ESI is a fxn of analyte concentration. Better mass sensitivity will be obtained with smaller column diameters and lower flow rates. On-axis heated-capillary interface has interesting response at high flow rates. It appears that aerosol density may result increased sampling efficiency at higher flow rates.

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