Mass Spectrometry. Fundamental LC-MS. Vacuum Systems
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1 Mass Spectrometry Fundamental LC-MS Vacuum Systems i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.
2 Aims and Objectives Aims and Objectives Aims Introduce students to vacuum technology Explain the importance of vacuum in LC/MS Introduce the equipment and technology associated with foreline and high vacuum pumping equipment Explore the fundamental incompatibility between vacuum mass analysers and the flow rates used in common HPLC techniques Explain the function of vacuum at various stages of the common mass analysing devices Outline common vacuum system troubleshooting & maintenance operations Objectives At the end of this Section you should be able to: List the advantages of maintaining good vacuum within the mass analyser Identify the reasons for vacuum within the mass analyser and how vacuum levels effects ion trajectories within each major analyser stage Identify and understand the technology and function of the various vacuum pumps used with LC/MS analysers Understand the need for good vacuum equipment maintenance and be able to carry out basic vacuum system troubleshooting
3 Content Introduction 3 Vacuum Systems 4 Effects of Vacuum Systems on Analyte Ions 4 Rotary Pumps 5 Foreline Pumps 6 Turbomolecular Pumps 7 Diffusion Pumps 8 Vacuum and Flow Rate Incompatibility 9 Establishing Vacuum and Transmission 10 The Sampling Orifice Plate 11 Nozzle Skimmer Region Analyte Enrichment 12 Vacuum Systems 13 Molecular Beam Theory in the Nozzle Skimmer Region 14 Practical Implication of the Skimmer Position 15 Vacuum System Troubleshooting & Maintenance 16 Vacuum leaks 18 Vacuum Gauges 18 Foreline Pumps Gas Ballasting 19 Foreline Pumps Exhaust Filters 21 Foreline Pumps Rotary Pump Oil 23 High Vacuum Pumps 24 References 25 Crawford Scientific 2
4 Introduction Mass analysers require high levels of vacuum in order to operate in a predictable and efficient way. The processes of ion isolation and separation on the basis of mass to charge ratio require the analyte ions under investigation to behave in a way that can be predictably managed and influenced by the instrument electrostatic components. Ions need to be guided on a specific pathway through the spectrometer, influenced only by the imposition of electric, magnetic and / or radio frequency fields, which can only be practically realized when vacuum technology is used to remove the majority of background (air) molecules, which might, for example, cause deviation of ions through collision. The vacuum systems of most modern LC-MS systems consist of a differentially pumped system, usually with a foreline pump establishing a rough vacuum and a high vacuum pump or pumps situated on the analyser body to establish the high levels of vacuum required for effective mass to charge ratio measurement. Crawford Scientific 3
5 Vacuum Systems A high level of vacuum within the instrument prevents deviation of the analyte ions from the required path and assists the processes of ion movement and separation in the following ways: By providing an adecuate mean free path for the analyte ions By providing collision free ion trajectories By reducing ion-molecular reactions By reducing background interference i At low vacuum levels, analyte ions collide against background gas molecules, certain reactions (including charge neutralisation or formation of new species) can take place. In some instances, analyte ions are deflected to collide with the mass analyser loosing their electrical charge, which in turn leads to a lower sensitivity (remember that the analyser actually detects the m/z ratio). Effects of Vacuum Systems on Analyte Ions Instruments using an Atmospheric Pressure Ionisation (API) interface will have three or more differentially pumped regions within them to ensure an adequate mean free path and to allow collision and reaction free ion passage through the various stages of the spectrometer. The distance travelled by analyte ions between the ion source and the detector device within the spectrometer will dictate the required mean free path of the instrument. This in turn will dictate the pressure required within the instrument to ensure that the ions will travel via a predictable and controlled route. If the distance (L) between the ion source and the detection device of an instrument were 50cm, a pressure of 10-2 Pa would be required, which can be estimated using the relationship: [1] L P Crawford Scientific 4
6 Mean free path and required pressures for various analyser types are presented in the table below. Table 1. Mean free path and required pressures for various analyser types. [2] Analyser Pressure (torr) Mean free path (L) FTMS < km Magnetic Sector < m TOF < m Quadrupole < cm Ion Trap < cm Rotary Pumps Rotary pumps are pumps that move fluid using the principles of rotation. The vacuum created by the rotation of the pump captures and draws in the liquid. A rotary pump consists of vanes mounted to a rotor that rotates inside of a cavity. This kind of pump operates in a circular motion and displaces a constant amount of liquid with each revolution of the pump shaft. [3] One of the most successful designs uses vanes of variable length and tensioned to maintain contact with the internal walls as the pump rotates. [4] Suction and discharge ports of the pump are preferably on opposite sides of the vanes, which prevents the entrapment of gases. Rotary vane vacuum pumps can be used in a number of applications providing a pressure or vacuum source for laboratory applications and as well as other automation applications. Smaller sizes may have a maximum flow of 3 L/min with pressures ranging from mbar. i Rotary pump Crawford Scientific 5
7 Foreline Pumps Depending upon the design of the spectrometer, foreline pumps (also known roughing pumps) may be employed to assist the high vacuum pumps directly attached to the spectrometer. The function of the foreline pumps is to reduce the pressure within a particular region of the spectrometer to approximately 1 Pa (10-2 torr) prior to the high vacuum pump [5, 6] establishing the required analyser pressure. Foreline pumps are oil-sealed rotary vane pumps in which a piston on an eccentric drive shaft rotates in a compression chamber sealed by spring-loaded vanes, moving gas from the inlet side to the exhaust port. Pumping capacity is limited to around 0.1 Pa (10-3 torr) due to the vapour pressure of the sealing oil and at a speed of 1400 rpm the pump will typically exhibit capacities of between 50 and 150 L/min. When venting an instrument, or if there is a sudden loss of vacuum within the analyser housing, there is a potential risk of the pump sealing oil back streaming into the analyser. To avoid this manufacturers incorporate an anti-suck back valve; [7] however, care should always be taken to follow LC-MS manufacturers instructions on how to correctly vent the instrument. i Crawford Scientific 6
8 Turbomolecular Pumps The high vacuum created in the post skimmer regions of the mass spectrometer may be achieved using a turbomolecular pump. The instrument operating software will normally have a function to monitor and control the speed of the pumping equipment in order that the correct level of vacuum is maintained within the instrument. Sudden ingress of air into the spectrometer can cause significant damage to this type of pump and as such most instruments have safety shutdown mechanisms if the vacuum is compromised. Once again it is important to avoid sudden vent situations by closely following the instrument manufacturers procedures for establishing vacuum (often referred to as pumping down ) and venting the instrument. The pump consists of a system of rotating foils or blades that are angled to compress exiting molecules and progressively draw them down through the stack and out via the vent port. Whilst spinning at around 60,000 rpm these pumps have varying capacities, with L/s being typical. The vapours exiting the turbomolecular pump will normally [8, 9] be entrained directly into the foreline pump. i Turbomolecular Pumps Crawford Scientific 7
9 Diffusion Pumps An alternative to the turbomolecular pump for maintaining high vacuum in LC-MS systems is the Oil-Filled Diffusion pump. Heated oil rises up the diffusion pump chimney, jets out through circular openings at various levels and condenses on contact with the cool walls, trapping gases from the mass spectrometer. The oil then runs down the cool walls exhausting entrained gases through the pump outlet and towards the foreline pump. The boiler will normally operate between 280 and 300 o C and the vapour exiting the pump will often have supersonic velocities (>750 m/s) (formatting) and will have temperatures around 100 o C. The pumps can have capacities in the same order as turbomolecular pumps ( L/s), which is important for evacuating the high vacuum regions within the instrument. Diffusion pumps operate by boiling a fluid, often a hydrocarbon oil (like polyethylene oil), and forcing the dense vapour stream through central jets angled downward to give a conical curtain of vapour. Gas molecules from the chamber that randomly enter the curtain are pushed toward the boiler by momentum transfer from the more massive fluid molecules. Water cooled diffusion pumps have coils through which cooling water [10, 11, 12] circulates during operation. i Oil-Filled Diffusion pump Crawford Scientific 8
10 Vacuum and Flow Rate Incompatibility We have already studied some of the concepts associated with the inherent incompatibility between the amount of vapour produced by introducing 1mL/min. of a typical chromatographic eluent into the mass spectrometer and the levels of vacuum that could be attained. The vapour pressure created by the vaporised eluent and the level of vacuum attainable with affordable pumping equipment differs by approximately two to three orders of magnitude. Several solutions to this fundamental incompatibility have been suggested: Miniaturise the LC column. Split part of the column effluent. Increase the pumping capacity. Remove a significant part of eluent. i In order to significantly increase the pumping capacity of LC-MS vacuum systems, cryogenic pumps would have to be used. This adds a great deal of complexity to the instrument is more maintenance heavy and greatly increases the costs of the instrument. Most manufacturers prefer the alternative approach of preferentially removing the eluent vapour relative to the analyte species and the principles behind these techniques will be studied in more detail. Crawford Scientific 9
11 Establishing Vacuum and Transmission Levels of vacuum within LC-MS instruments are usually established in stages, the level of vacuum increasing with the distance away from the atmospheric pressure interface. Because the vacuum is established in this way, relatively uncomplicated (and inexpensive) pumping equipment may be used and transmission of the analyte from the interface to the mass analyser can be made more efficient via the use of larger sampling orifices. Further, there are advantages in having a relatively high background pressure in the regions of the spectrometer immediately after the interface that allow the degree of fragmentation of analytes to be controlled as well as increasing the effectiveness of ion focussing components. The transfer efficiency of the interface may be calculated using the relationship. [1] Y Q Q MS LC 100 Where Q LC is the amount of analyte eluting from the LC column and Q MS the amount of analyte introduced to the mass spectrometer. Crawford Scientific 10
12 The Sampling Orifice Plate In the first vacuum region of the spectrometer, after the atmospheric pressure interface, a background pressure of Pa is usually established. This first vacuum region (Nozzle-Skimmer region) is separated from the interface via a transfer capillary or orifice plate (nozzle). The nozzle performs a split by sampling only a small portion of the nebulised eluent via a fixed diameter orifice through which the vapours are drawn by a combination of electrostatic attraction of charged species and the pressure difference between the atmospheric pressure interface and the vacuum of the nozzle-skimmer region. The split ratio at the nozzle is usually in the ratio of 1:100. The transmission of the interface will depend upon the size of the orifice in the nozzle. Also the vacuum system in any region of the spectrometer must be capable of removing the gas that passes through the orifice between the two vacuum regions as well as any gas that may naturally leak into the vacuum region. Arpino et al, [13] showed that as long as the pressure difference between the two regions does not exceed one order of magnitude, the area of the orifice may be as large as 1cm 2. In most modern spectrometers the pressure difference between the atmospheric pressure interface and the nozzle-skimmer region is approximately three orders of magnitude ( Atm) and therefore the size of the sampling orifice is considerably reduced. The pressure of the nozzle-skimmer region and the orifice area are balanced to allow maximum transmission of the analyte species whilst using pumping equipment that is affordable, low maintenance and straightforward to operate. i Crawford Scientific 11
13 Nozzle Skimmer Region Analyte Enrichment The nozzle skimmer region of the instrument functions as an expansion chamber (molecular beam system) to perform analyte enrichment. The background pressure in the region immediately after the skimmer cone (around 1 Pa) would be compromised if the all eluent vapour is allowed to pass. There is a necessity to separate the analyte species from the remaining eluent vapour to allow good transmission but to reduce vapour pressure. When the eluent vapour is sampled through the nozzle into the low-pressure region, higher mass particles show a lower momentum perpendicular to the axis of expansion than lower mass particles. Therefore the lower mass particles diffuse to a greater extent from the core of the expansion and enrichment of heavier species (which in LC/MS are generally the analyte molecules) will occur. In LC-MS the analyte molecules generally have a much higher mass than the eluent constituents, and the nozzle skimmer system ultimately results in a gas-phase enrichment of the analyte molecules. i A vapour flow of around 3 L/min can be introduced from an atmospheric pressure interface into the nozzle-skimmer region maintained at 10 3 Pa by means of a mechanical pump operating with an effective pumping speed of 300 L/min. In principle this is enough capacity to allow the introduction of eluent at 1 ml/min. into the mass spectrometer interface. However, in practice most LC-MS instruments would have a second stage of pumping in the ion-optic region. Crawford Scientific 12
14 Vacuum Systems In atmospheric pressure interfaces, droplet desolvation is rarely complete. A much larger fraction of liquid than is intended enters the nozzle skimmer region (usually in the form of ion clusters). Solvent clusters are preferentially sampled through the skimmer orifice into the post skimmer region, due to their low momentum. On contact with hot metal surface, solvent clusters may decompose (into analyte ions and eluent molecules) giving rise to increases in background gas pressure high enough to compromise the vacuum level. The use of the second pumping stage allows the removal of vapour created by the evaporating solvent clusters prior to the mass analyser region of the spectrometer. Manufacturers usually use this second expansion chamber to house ion optics used to pre-focus the analyte beam. A second pumping stage, used to form what is more commonly referred to as a momentum separator, also allows the use of slightly larger sampling orifices within the nozzle and subsequent skimmer plates, which is favourable with respect to analyte transfer efficiency (Y). i Resolution compromised through ion cluster sampling Improved resolution due to reduction in ion cluster by use of second vacuum pumping stage High molecular weight analyte cluster ions Crawford Scientific 13
15 Molecular Beam Theory in the Nozzle Skimmer Region When vapour species pass through the nozzle their motion is approximately directed into straight streamlines. The velocity of the vapour borne species increases dramatically, the difference in speeds between the various species narrows and rapid cooling occurs. This phenomenon is known as free jet expansion into a vacuum with the highest intensity of vapour species situated on and around the axis of the nozzle. As the vapour species move appreciable distances away from the nozzle, the gas molecules move in a non-directed fashion, the lighter molecules that deviate most from the nozzle axis will be pumped away. The transition from directed to random motion will involve multiple collisions with vapour species and background gas molecules resulting in shock-waves that scatter the ions and neutrals within the plasma of vaporised species to approximately the geometry shown (known as the barrel shock wave). The region inside the barrel shock wave is called the zone of silence where molecules will move with equal speed and in the same direction, undergoing strong and rapid cooling. [14] The zone of silence terminates in a shock wave known as the Mach Disk where molecules undergo more frequent collisions and may completely lose the directed flow and travel perpendicular to the nozzle axis. The location of the Mach disk relative to the nozzle and skimmer components is of vital importance in manufacturing LC-MS interfaces and will affect transmission of neutral species and clusters. Crawford Scientific 14
16 Practical Implication of the Skimmer Position In practice pumps and nozzle orifice diameters are chosen to give highest transfer efficiency with reasonably priced and low maintenance pumping systems. In most modern LC-MS systems the skimmer entrance is positioned so that the Mach disk is in front of the sampling orifice. This approach has several advantages: Intermolecular collisions in the Mach disk and the associated shock waves leads to an increase in the gas temperature, which aids with the dissociation of ion-solvent clusters. Applying a voltage to the skimmer allows ions to be sampled in preference to neutral species that are off-axially diverted in the Mach disk. Altering the skimmer voltage can give rise to highly accelerated ions that undergo higher energy collisions causing fragmentation of the analyte ions to gain analyte structural information. i To aid in the transmission of ions through the nozzle-skimmer region relative to neutral species, manufacturers often include a ring electrode or transfer capillary to keep ions focused onto the nozzle axis and stop them from diffusing from the expansion core. Crawford Scientific 15
17 Vacuum System Troubleshooting & Maintenance The performance of a mass spectrometer will be severely impaired by the lack of a good vacuum in the ion transfer region of the mass analyser. As the vacuum deteriorates it will become insufficient to maintain the instrument in the operating mode. Before suspecting a leak, the following points should be noted: Foreline pump maintenance High vacuum pumps switch off if an over temperature is detected Turbomolecular pumps switch off if full speed is not reached within a set time following start up Finally, an excessive analyser pressure results in general loss of performance. This is indicated by loss of resolution and an increase in background noise. Foreline (rotary) vane pump High vacuum pumps (turbomolecular and/or diffusion pumps) will not operate if the forline pump has failed. If the foreline pump is not maintained, the oil may become so contaminated that the optimum pumping is no longer possible. Initially, gas ballasting may clean the oil. If the oil has become discoloured then it should be changed according to the pump manufacturers maintenance manual. Crawford Scientific 16
18 High vacuum pumps switch off if an over temperature is detected On most instruments turbomolecular or diffusion pumps switch off if an over temperature is detected. This could be due to: Poor foreline vacuum Failure of the water supply (if pumps are water cooled) Leak in the mass analyser and/or ion transfer regions. Turbomolecular pumps switch off if full speed is not reached. Turbomolecular pumps switch off if full speed is not reached within a set time following start up. This could be due to a leak or raised ambient temperature. Crawford Scientific 17
19 Vacuum leaks If a leak is suspected, the following points may help to locate it: Leaks very rarely develop on an instrument that has been fully operational. Suspect components that have recently been disturbed Leaks on flanges can sometimes be cured by further gentle tightening of the flange bolts or by replacing the seal When re-fitting flanges pay particular attention to the o-rings and/or gaskets. Any cuts or scratches may cause a leak. They should be clean and free from foreign matter A hair across a viton o-ring is sufficient to prevent the system pumping down Source components that operate at, or slightly above atmospheric pressure are not susceptible to vacuum leaks Vacuum Gauges The Pirani gauge is a roughing pressure vacuum gauge. It uses the thermal conductivity of gases to measure pressure. Useful range: torr. Pirani gauges (roughing, low vacuum gauges) do not require routine maintenance. Penning gauges or Ion gauges (high vacuum gauges) may be cleaned or repaired. You should refer to the manufacturers literature for specific instructions. Crawford Scientific 18
20 Foreline Pumps Gas Ballasting Gas ballasting serves two important purposes: [15] When rotary pumps are used to pump away solvent vapours, the solvent can become dissolved in the oil causing an increase in backing line pressure. Gas ballasting is a means of purging the oil to remove dissolved contaminants Oil mist expelled from the rotary pump should be trapped in an oil mist filter. This oil is then returned to the pump during gas ballasting Gas ballasting should be performed routinely on a weekly basis for approximately 30 minutes. If the instrument is used heavily in APCI or high flow ESI modes, then more frequent gas ballasting is recommended. Pump elements Crawford Scientific 19
21 i Important: Consult your pump manufacturers information for the specific ballasting instructions It is normal for the rotary pump to make more noise when the gas ballast valve is open Failure to gas ballast the rotary pump regularly will lead to shortened oil lifetime which in turn may shorten the rotary pump lifetime Gas ballasting should NOT be performed during operation of the instrument Crawford Scientific 20
22 Foreline Pumps Exhaust Filters Foreline pumps can contaminate the working environment by expelling oil or chemicals that have contaminated the pump oil. When vacuum pumps are used in instruments such as mass spectrometers, all the residual organic chemicals (some of them harmful) analyzed by the mass spectrometer are trapped in the vacuum pump oil. Eventually these contaminants are expelled out of the oil. A two stage vacuum pump filtering system is highly recommended to keep high air quality levels in the instrumentation laboratory. The first stage filter consists of an oil mist filter to trap the heavy oils exiting the pump. [16, 17, 18] The trapped oil is then returned to the rotary pump during gas ballasting. The second stage is a charcoal trap, this trap is especially designed to retain volatile organics. Two Stage Vacuum Pump Exhaust System Purifies vacuum pump exhaust gases Provides for a safe laboratory environment Crawford Scientific 21
23 i Oil mist eliminator. Oil mist eliminator Change the odor element monthly or whenever the pump emits an oily odor. Change the mist element every time the rotary pump oil is changed. i Charcoal Filter. Used to trap organic chemicals Crawford Scientific 22
24 Foreline Pumps Rotary Pump Oil The rotary pump oil should be maintained at the correct level at all times. Check the oil level weekly and top up as necessary. It is important to monitor the condition of the oil regularly. [19] Replace the oil when it has changed to a noticeable dark colour, or routinely at 4-month intervals. Change the rotary pump oil as follows: Gas ballast for 30 minutes. Vent and shut down the instrument (see manufacturers procedure) It is much easier to drain the oil while the pump is still warm Drain the oil through the drain hole. Flush the pump, then replace the drain plug and refill with the correct grade oil to the correct level. Gas ballast for 30 minutes. Eliminate back streaming hydrocarbons from your rotary vane pump by using a trap. Just place the trap between your roughing pump and the vacuum chamber or the high vacuum pump. Important: Anti-suckback valves in conjunction with oil traps are used to prevent backsteaming of oil vapours to the high pressure pump. [20] Crawford Scientific 23
25 High Vacuum Pumps Turbomolecular pumps are usually sealed units and no user repair or maintenance is [21, 22] possible. If the unit fails it is generally replaced with a new one. Oil diffusion pumps require very little maintenance. The oil level should not drop unless a vacuum accident occurs and this is unlikely on modern, self-protected instruments. The most likely failure of an oil diffusion pump is the heating plate. This can usually be replaced by the user, consult the instrument manual for specific information. High vacuum pumps are often water cooled and will switch off if the water supply fails. Remember, high vacuum pumps will not function if the rotary (foreline) pump has failed Turbomolecular pump Oil diffusion pump Crawford Scientific 24
26 References 1. W. M. A. Neissen, in Liquid Chromatography-Mass Spectrometry. 2nd edition. Marcel Dekker Inc. NY. USA R. Willoughby. E. Sheehan and S. Miltrovich in A Global View of LC/MS. 1st edition, (Appendix D), Global View Publishing, PA, USA, David R. Staley, Rolland D. Giampa, Alan W. Hayman. Engine Oil System with Variable Displacement Pump U.S. Patent No US July 20, Rotary Vane Pumps Features and Benefits. Varian Inc. Dec Dual Stage Rotary Vane Pumps. Varian Inc. Vacuum Technologies. Pp 1-8. Dec Pump Systems. Dionex Application Note. Pp 1-12 March Bornemann Alfred H. Antisuckback device for rotary piston pumps European patent application number Nov 27, Turbomolecular Pumping Systems. Adixen by Alcatel Vacuum Technology. Pp P.H. Mokler, A. Bardonner and U. Kopf. New Turbomolecular Pump with Central Opening for Free Axial Access. 10. R. F. COE, M.Sc., Grad.Inst.P., and L. RIIJDIFORMD,. Sc., Ph.D., A.Inst.P., Physics Department. The final vacua of oil diffusion pumps JOURNAL OF SCIENTIFIC INSTRUMENTS. Pp Issue 6 (June 1955) 11. DIP Oil Diffusion Pumps. LEYBOLD VACUUM PRODUCTS AND REFERENCE BOOK 2001/ G. LIEBMASND,. Phil., F.Inst. P. An Uncooled All-Metal Oil Diffusion Pump Pp Vol 25 June P. J. Arpino, G. Guiochon, P. Krein, and G. Devant. J. Chromatogr. 185, (1979), R. Campargue. J. Phys. Chem. 88, (1984), Robert E. Poole. Rotary Vane Pump With Ballast Port US patent number May 2, John J. Manura. Vacuum Pump Exhaust Filters The Mass Spec Source Summer Pp Filters for Vacuum Pumps. Graver Technologies application note GTX VACUUM PUMP FILTER KITS. Scientific Instrument Services application note. Pp Heinz P. Bloch. Centrifugal Pump and Lubricating Application A Best Technology Update. Proceedings of the Twenty-Second International Pump Users Symposium Pp Harold Shapiro. Trap for preventing diffusion pump backstreaming. US patent application number Dec 31, Turbomolecular Pumping Systems. Alcatel application note. Pp C F Weston. DEVELOPMENTS IN HIGH-VACUUM PUMPS Phys Techno1 Vol 15. Pp Printed in Northern Ireland. Crawford Scientific 25
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