Fundamental LC-MS Electrospray Ionisation Instrumentation

8/12/2019 Fundamental LC-MS Electrospray Ionisation Instrumentation

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Mass Spectrometry

Fundamental LC-MS

Electrospray Ionisation Instrumentation
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Aims and Object ives

Aims and Object ives

Aims

Explain the function of the major components of an electrospray Interface

Investigate methods of optimising signals using electrospray Ionisation

Objectives

At the end of this Section you should be able to:

List and describe the most important components of an electrospray Ionisationinterface

Demonstrate an understanding of the principles of optimising instrument responsewhen using electrospray ionisation


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Electrospray source design

Introduction

Electrospray is the dispersion of a liquid into electrically charged droplets, combining thetwo processes of droplet formation and droplet charging. The process of droplet charging

is affected by three main variables:

Eluent flow rate

Liquid surface tension

Electrolyte concentration

If these parameters are not maintained at an optimised minimum level, the electrosprayprocess will become unstable. If any of the variables increases significantly it maybedifficult for the electric field to produce the desired charged aerosol necessary for ionproduction in the API interface. Any effects observed due to a significant increase in anyof the variables may be countered to a certain degree by increasing the capillary voltage(and hence the effective field strength at the capillary tip), but electrical discharge mayoccur, resulting in a decrease in instrument response and an unstable electrospray.

Electrospray capillary design

Standard electrospray capillaries are constructed from stainless steel or a coaxialarrangement of fused silica and stainless steel. If the capillary is of fused silica design,electrical contact is usually made by clamping the capillary in a metal union, however,capillaries with silver or gold deposits upstream from the tip have also been employed forimproved electrical contact.

Conventional Electrospray Capillary (practical upper flow rate limited to 10-20L/min)


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The metal composition used for stainless steel capillaries is of great importance in theoxidative and reductive processes occurring during droplet charging and as suchcontinuity of composition for the metal capillaries should be ensured to give long termrobustness to analytical determinations.

Drying gas

The practical upper limit to eluent flow in pure electrospray is 10-20 L/min dependingupon the solvent composition. Capillary design may be modified to increase the toleranceof the electrospray process to increases in eluent flow rate, liquid surface tension orelectrolyte concentration. One successful approach used in order to increase electrosprayflow rate is the introduction of a nebulising gas via a concentric tube around the capillary(pneumatically assisted ESI).[1]

High-flow electrospray sources (>5-10 L/min.) are normally combined with a supply ofheat within the API source housing to assist the evaporation of solvents. The evaporationof large amounts of solvent is important to ensure that ion evaporation occurs in theoptimum position within the API source to ensure maximum transmission and reduction ofion solvent clusters in the nozzle-skimmer region of the source.

Source heating needs to be optimized for each analytical determination to ensure theproduction of the maximum amount of analyte ions in the source. Practically the use ofheated nitrogen blown into the source housing is normally employed to aid desolvation in


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high flow electrospray source designs. Perkin Elmer (Sciex) (Applied Biosystems, FosterCity, CA), introduced one of the earliest instruments to incorporate pneumatically assistedelectrospray in their IonSpray design. This design used a silica capillary within a stainlesssteel needle that was housed within a concentric PTFE tube. The design of the PTFEtube is such that gas flow rates at the capillary tip are around 200 m/s.

When physically connecting the t-piece or HPLC column to the API interface housing,PEEK (polyetheretherketone) (0.1mm i.d.) tubing is preferred to fused silica tubing due tothe possible adsorption of analyte species to residual silanol species on the inner surfaceof the silica capillary. Most modern instruments employ stainless steel or platinumcapillaries for electrospray to avoid similar problems with fused silica that can adverselyaffect the quantitative response of the instrument. For low flow rate applications micro-electrospray needles are available which consist of either coated or uncoated silicacapillaries with a drawn tip to allow micro-droplet formation. [18] Filters in the needle

assembly help to prevent blocking of the fine capillary tip (


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The distance and potential difference between the tip of the capillary and the samplingplate determine the electric field that creates the electrospray and will influence theperformance of the spray. Optimisation of the sprayer position and capillary voltage areinterrelated and should be optimised empirically together.

Pepperpot (FISONS-Micromass)

Cross Flow (Waters)


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Perhaps the most important practical advance in sprayer positioning has come with theintroduction of orthogonal source design, where the sprayer is positioned orthogonally tothe sampling orifice. This design has several advantages including, reduced down-time ofthe source with decreased coating of the source elements, sampling of fewer chargeddroplets relative to ions and the ability to tolerate higher flow rates.

In orthogonal spray, neutrals and non-volatile materials collide with a plate perpendicularlylocated to the spray axis. Orthogonal design can be combined with a second orthogonalextraction (LCZ or Z-Spray from Waters) or a with an off-axis extraction, such as in theaQa (Thermo-Finnigan) disposition.[5]

Orthogonal designs allow the use of eluent flow rates up to 1ml/min with electrospray andgives the advantage that eluent systems containing non-volatile buffers may be used forextended numbers of samples before source cleaning is required. Orthogonal sourcesare available from several other manufacturers.


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Cluster Ions

Polar molecules in the gas phase (water, solvent and eluent molecules), tend to formclusters with ions.[23] Cluster ions will appear in the mass spectrum and they are often solarge that they are far outside the detectable mass range of a typical quadrupole (3000-6000 Da):

nOHXOHnX )( 22

+++

Cluster ions may collide with the source elements in the early stages of the spectrometerand give rise to ion bursts which can result in noisy baselines in the Total or Selected IonChromatogram (TIC or SIC).

One commonly applied solution to this problem allows ions to pass into the samplingorifice but excludes water vapour and other neutral species from the entrance to thevacuum system. This is achieved by forcing ions and neutrals in the opposite direction bythe action of an electric field and/or a flow of dried gas.

Cluster formation

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Cluster ions passing into the vacuum region of the source may be de-clustered bycollision with rapidly moving background gas molecules, imparting enough energy tobreak the hydrogen bonds between the ion and the solvating cluster molecules.Increasing the temperature and the potential difference in the first vacuum region of thespectrometer in order to accelerate ions will both assist with ion declustering.

Prevention of Cluster Ion Sampling

A Sciex (Applied Biosystems, Foster City, CA), API source is shown. The region betweenthe interface plate and the sampling orifice plate is continuously flushed with dry nitrogenthat flows into the API source as well as into the vacuum region of the spectrometer.

The gas flow into the source helps to repel water, neutrals and other potentialcontaminants, such as dirt and buffer salts, away from the sampling orifice, increasing theintervals between source maintenance.

Ions in the region of the sampling orifice are driven into the vacuum region by the gas flowcombined with a 600V potential difference between the interface plate and the sampling

plate. The nitrogen employed in this sense is often referred to as a Curtain Gas.[6,7]

Gas curtain

As the nitrogen curtain gas and ions undergo expansion into the nozzle-skimmer region ofthe source, significant cooling occurs. If the source and curtain gas are not heated, thereis a significant possibility of cluster ion formation occurring in this region. Therefore mostmodern sources use both heated drying and curtain gas.

A method preventing cooling effects experienced through expansion and the re-formationof cluster ions uses a heated transfer tube. [25] This device will preheat the mixture of ionsand neutrals prior to expansion using a heated tube of approximately 20 cm long (100 200oC). A further advantage of this type of device is the ability to generate ions (via ionevaporation) from droplets that may be sampled into the nozzle-skimmer region.

An Agilent Technologies (Palo Alto, CA, USA), source is shown which employs an iontransfer tube with metallised ends (also known as a 'dielectric capillary'), that allows theapplication of an accelerating voltage that can be used to promote in-source dissociationof cluster ions.

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Orthogonal source with dielectric capillary (heated transfer tube)


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Source Cleaning

Contamination of the sampling orifice or tube can prove to be detrimental to theperformance of the instrument, in some cases leading to very frequent source cleaningwhen dealing with samples in dirty matrices or when using non-volatile solvents andbuffers.

The layer of contamination inside an orifice or tube will attract a build up of charge thatcan effectively stop the passage of ions, while the flow of neutrals is not affected, thussignificantly decreasing the instrument response.

Cleaning regimes will differ for instruments from different manufacturers but may includesome physical abrasion (aluminium powder is a popular choice) and / or wipe cleaning ofthe sampling cone and other source components followed by sonication in a range ofsolvents matched to the polarity of the contaminants (hexane, acetone and methanol areall popular choices for source cleaning).

Dirty layer formation

The use of curtain gas, off axis spray and/or orthogonal spray can significantly increasethe interval between source cleaning, by directing the deposition of contaminant speciesaway from the sampling plate, orifice or ion transfer tube.

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Ion Optics

Ring Electrode

In the nozzle-skimmer region of the mass spectrometer ions are transported in the free-jetexpansion of gas, some of which will pass through the skimmer into the higher vacuum

region of the mass spectrometer.

Focusing of the ions into a narrow beam is not possible due to the effects of free jetexpansion, which tends to direct the ions and entrained neutrals and gases into a barreltype shockwave, away from the axis of the spectrometer. However, ions may be forced toremain closer to the axis of the beam if a tube or ring lens is placed between the nozzle(sampling plate) and skimmer.[8] A voltage applied to the ring will reduce the spread ofions (but not water vapour or neutrals), away from the axis with a subsequent increase ininstrument sensitivity (mainly due to a reduction in the noise).

Ring electrode operation

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Ion Bridges

In the high vacuum region of the spectrometer after the skimmer lenses, the pressure isstill not low enough to focus ions using traditional ion lenses. In most cases the ions arefocused using a radio frequency (RF) only hexapole or octapole ion bridge, that guidesthe ion beam into the analyzing quadrupole system.

Ion bridges work on a similar principle to a quadrupole mass analyser but operate withradio frequency potential only. The quadrupole DC component is removed, and theapplication of a radio frequency potential allows ions of all m/z values to pass from oneregion of the spectrometer to another.[9]

The multipole ion bridge has the advantage of slightly focusing the ion beam, thereforeincreasing transmission as well as allowing the removal of a significant amount of neutralspecies which are not held within the multipole ion bridge, so increasing signal to noiseratio.

Ion bridges operation

Collision Induced Dissociation

Classical API spectra tend to show very little fragmentation due to the 'soft' nature of theionisation processes. That is, during the formation of ions, the analyte molecules do notreceive enough energy to break the intra-molecular bonds.

However, fragmentation may easily be induced in one of the higher-pressure regions andstructural information can be gathered.

Acceleration of ions between the sampling orifice and the skimmer, or between theskimmer and the RF-only multipole results in collisions of ions with the background gas.This process is known by various different names depending upon the instrument

manufacturer and includes 'in-source collision induced dissociation (CID)', 'nozzle-skimmer fragmentation', 'cone-voltage fragmentation', etc.

By increasing the potential difference between the skimmer and the quadrupole V(S-Q)

or between the nozzle and skimmer V(N-S)), the energy imparted to the analytemolecule through increasing frequency and energy of collisions can be enough to causeintra-molecular bonds to be broken and for fragmentation to occur.[10,11] In the case oflarger molecules, such as peptides and proteins, the excess energy can often beabsorbed in several vibrational modes and high potential differences are required tofragment these kind of molecules.

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CID working principle

The major advantage of this technique for the production of spectra containing a greater

amount of structural information is its simplicity. Only one voltage needs to be adjustedand there is no need for switching or adjustment of collision gases and no retuning of ionoptics.

It is possible to test the degree of CID using probes.[11]

In negative ion mode the degree of CID can be estimated using the drug naproxen thatnormally shows the [M-H]-ion as the only at m/z 229. With small nozzle-skimmer potentialdifferences the naproxen molecule readily loses CO2 giving rise to a base peak at m/z185.

In the positive ion mode the [M+H]+ion of dibutyl phthalate at m/z 279 can be used as atest ion. Fragmentation to m/z 149 takes place readily even under mild CID conditions,making declustering of ions impossible with these labile sample ions.

Mass spectrum of naproxen (used to estimate the degree of CID in negative ion mode)

Fragmentationi

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Ion Declustering

Collision Induced Dissociation can also be used to improve the baseline noise andincrease the signal-to-noise ratio in LC-MS experiments.[9,12]When ions pass through thesampling orifice into the vacuum region the background density of neutral gas ions fallsrapidly. If ions moving in a low-density gas are accelerated by the nozzle-skimmer region

mild-CID may be affected, which will be enough to cleave the hydrogen bonds inside theion-water or ion-neutral gas clusters. Further, heating of the ion clusters, which occursduring collisions, will also aid desolvation of the cluster ions.

Moderate acceleration of clusters is effective and widely used to decluster ions, leading toa reduction in baseline noise and the numbers to cluster ions detected. A disadvantage ofthis type of approach is the moderate scattering of the ion beam that is associated withion-neutral gas collisions that may lead to a small reduction in the numbers of ionspassing through the skimmer element and into the mass analyser (i.e. reducedspectrometer transmission).

Ion declustering process by using CID

References

1. A. P. Bruins, T. R. Covey and J. D. Henion, Anal. Chem. 59, (1987), 2642.2. M. R. Emmett and R. M. Caprioli. J. Am. Soc. Mass Spectrom. 5, (1994), 605.3. M. S. Wilm and M. Mann. Anal. Chem. 68, (1996), 1.4. A. P. Bruins, T. R. Covey and J. D. Henions. Anal. Chem. 59, (1987), 2642.5. K. Hiraoka. Rapid Commun. Mass Spectrom. 6, (1992), 463.6. J. Abian. The Coupling of Gas and Liquid Chromatographywith Mass Spectrometry. J. Mass Spectrom. 34, (1999), 157 168.7. C. K. Meng and J. B. Fenn. Org. Mass. Spectrom. 26, (1991), 542.23. C. M. Whitehouse, R. N. Dreyer, M. Yashamita, J. B. Fenn. Anal. Chem. 57, (1985),675.

8. I. C. Mylchreest, M. E. Hail and J. R. Herron. United States Patent 5, 157, 260,October 20, 1992.9. M. E. Hail and I. C. Mylchreest. Presented at the 41st ASTM Conference on MassSpectrometry and Allied Topics. May 31 Jun 1, 1993, San Francisco, CA, 745.10. R. D. Smith, J. A. Loo, C. J. Barinaga, C. G. Edmonds and H. R. Hudspeth. J. Am.Soc. Mass Spectrom. 1, (1990), 53.11. R. D. Smith and C. J. Barinaga. Rapid Commun. Mass Spectrom. 4, (1990), 54.12. A. P. Bruins in Electrospray Ionisation Mass Spectrometry R. B. Cole [ed.] JohnWiley and Sons Int. 1997, 132-133.

Declustering

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Fundamental LC-MS Electrospray Ionisation Instrumentation

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