P h a r m a c e u t i c a l t e c h n o l o g yGMP SIzE REDucTION SYSTEMS PROCESS TECHNOLOGIES FOR TOMORROWSM +++ Powder and Particle From millimetre to nanometre – our service range From milligram to tonne: We develop versatile and efficient processing innovative and sPecialised solutions
Principle of mass spectrometry
Principle of LC/MS
II - THE MASS SPECTROMETER:
INSTRUMENT ARCHITECTURES AND MAIN CHARACTERISTICS
Quadrupole, triple quads
Time of Flight
III - LC/MS
The API mass spectrum
IV - PRACTICAL ASPECTS OF USING LC/MS
V - A CLOSER LOOK AT A SINGLE QUADRUPOLE INSTRUMENT:
THE WATERS ZQ
VI - CONCLUSION
HPLC science has always been moving to more specific and moresensitive detection modes. Advances made in LC-MS coupling (newinterfacing techniques, more user friendly systems) make it easier forchromatographers to implement LC/MS.
Waters, the worldwide leader in HPLC, has a long experience ofcombining LC to MS. The acquisition of Micromass has created theopportunity for both companies to reinforce their structures andproduct offerings.
Both Waters and Micromass have a focus on innovative technologiesand high performance products. Now, our new structure is offering toLC-MS users the most complete and homogenous product range forHPLC-MS coupling. Every day, chromatographers make the decision to go for LC-MS, toget better information on their samples, to gain sensitivity andselectivity, or to cope with the need of a higher throughput. When faced with a variety of techniques and instruments, making thecorrect choice of your LC/MS instrument is not an easy task.
We hope that this booklet will assist you in making the correct choiceof an LC/MS instrument.
Waters MarketingBP 608 78056 St-Quentin-en-Yvelines CedexTel. (33) 1 30 48 72 00Fax (33)1 30 48 72 11Internet : www.waters.com
PRINCIPLE OF MASS SPECTROMETRY
The mass spectrometer is an instrument designed to separate gas phase ions according to their m/z (massto charge ratio) value.
The "heart" of the mass spectrometer is the analyser. This element separates the gas phase ions.
The analyser uses electrical or magnetic fields, or combination of both, to move the ions from the regionwhere they are produced, to a detector, where they produce a signal which is amplified. Since the motionand separation of ions is based on electrical or magnetic fields, it is the mass to charge ratio, and not onlythe mass, which is of importance. The analyser is operated under high vacuum, so that the ions can travelto the detector with a sufficient yield.
In addition to the analyser, the mass spectrometer also includes• A vacuum system• Tools to introduce the sample (LC, GC …)• Tools to produce the gas phase ions from the sample molecules• Tools to fragment the ions, in order to obtain structural information, or to get more selective detection• A detection system• Software and computing Quadrupole, Triple quads
Time of Flight
MS/MS is the combination of two or more MS experiments. The aim is either to get structure informationby fragmenting the ions isolated during the first experiment, and/or to achieve better selectivity andsensitivity for quantitative analysis.
MS/MS is done:• either by coupling multiple analysers (of the same or different kind)• or, with an ion trap, by doing the various experiments within the trap PRINCIPLE OF LC/MS
LC/MS is a hyphenated technique, combining the separation powerof HPLC, with the detection power of mass spectrometry. Even with avery sophisticated MS instrument, HPLC is still useful to remove theinterferences from the sample that would impact the ionisation.
Closely related to LC/MS are some other techniques, like flowinjection/MS, CE or CEC/MS, capillary LC or nano LC/MS In all cases, there is the need for an interface that will eliminate thesolvent and generate gas phase ions, then transferred to the optics ofthe mass spectrometer.
Most instruments now atmospheric pressure ionisation (API) techniquewhere solvent elimination and ionisation steps are combined in thesource and take place at atmospheric pressure.
When electron impact ionisation (EI) is the choice, the solventelimination and ionisation steps are separate.
The interface is a particle beam type, which separates the samplefrom the solvent, and allows the introduction of the sample in the form of dry particles into the highvacuum region.
Electron impact is of interest for molecules which do not ionise withAPI technique, or when an electron impact spectrum is necessary,since it provides spectral information independent of the sampleintroduction technique (GC or LC, or direct introduction) andinstrument supplier.
[ M + H] + 221.95 0 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z If we look for the molecular mass of the chloridazon pesticide, we canfind various values in the literature 221.6379: this is the average mass. It is based on the average
221: this is the nominal mass, calculated on the nominal mass of the
most abundant isotopes
221.0278: this is the exact (or monoisotopic) mass, based on
the exact mass of the most abundant natural isotopes
The mass spectrometer measures the exact mass. Looking at the abovemass spectra, the most abundant peak is at 221.95 (top) and 219.81(bottom). These spectra are obtained with positive ionisation (top) andnegative ionisation (bottom). The peaks correspond to the protonatedor deprotonated molecule.
The value is slightly different from the expected 222.0278 and219.0278 because these spectra were obtained with a quadrupoleinstrument, which does not provide sufficient mass resolution andmass accuracy for obtaining the exact mass.
The next smaller peaks correspond to the C13 and Cl37 isotopes.
MASS RESOLUTION, MASS ACCURACY
Mass resolution: represents the ability to separate two adjacentmasses. It measures the "sharpness" of the MS peak.
Mass accuracy: indicates the accuracy of the mass informationprovided by the mass spectrometer.
Mass spectrum of prostaglandin PGF2 α Single Ion method
Double Ion method
Full Width at Half Maximum (FWHM) or at 5% of 2 adjacent ion peaks with a 10% valley max ∆ m = 0.3 Dalton peak width Resolution = m / (FWHM) In that case R= 279 / 0.3 1000 In that case R= 1000 / 1 = 1000
MASS RESOLUTION, MASS ACCURACY
Resolution calculation: the above described methods can be used with various valley and Dm definitions,which are represented on the next figure.
(50 % Max. = FWHM) The mass accuracy is the difference which is observed between thetheoretical mass and the measured mass. Dm accuracy = mreal – mmeasured It is often expressed in parts per million (ppm) ppm = 106 * ∆m accuracy / mmeasuredi.e.: theoretical mass: 1000, measured mass: 999.9 error: 100 ppm Mass accuracy is linked to the resolution. A low resolution instrumentcannot provide a high accuracy 3 different compounds 3 different compounds Same nominal mass 3 different exact masses High resolution, high accuracy A high resolution instrument (time of flight, sector, FTMS…), properlyused with a reference compound provides the mass information withan accuracy better than 5ppm, which is enough to unambiguouslydetermine the elemental composition.
THE MASS SPECTROMETER:
In this section, we will cover the mass spectrometers which arecommonly used in LC/MS configurations.
The analysers used in these instruments are quadrupole, ion trap, timeof flight, and combinations such as triple quadrupoles, QTofs.
We will not cover instruments like sectors, FTMS…, which are lesscommonly used in the LC/MS application. Useful information on these instruments can be found onsuppliers web sites,
THE MASS SPECTROMETER:
THE QUADRUPOLE ANALYSER
The quadrupole is the most widely used analyser due to its ease ofuse, mass range covered, good linearity for quantitative work,resolution and quality of mass spectra. All this for a relativelyaccessible price.
The main characteristics are: Working mass range: 10 to 4000 A.M.U.
Resolution: usually operated at a resolution = 1000, but resolution
can be reasonably pushed up to 4000
Mass accuracy: 0.1 to 0.2 A.M.U.
Scan speed: up to 5000 A.M.U per second
rf and -dc dc and Rf voltages rf and +dc The life time of an ion from its formation to detection
is 50 - 100 microsecond.
THE QUADRUPOLE ANALYSER:
HOW IT WOKS
The quadrupole is composed of two pairs of metallic rods. One set of rod is at a positive electrical potential,and the other one at a negative potential. A combination of dc and rf (radio frequency) voltages is appliedon each set .
V(t) = – Vdc – Vrf cosΩt V(t) = Vdc + Vrf cosΩt The positive pair of rods is acting as a high mass filter, the other pair is acting as a low mass filter. Theresolution depends on the dc value in relationship to the rf value. The quads are operated at constantresolution, which means that the rf/dc ratio is maintained constant.
For a given amplitude of the dc and rf voltages, only the ions of a given m/z (mass to charge) ratio willresonate, have a stable trajectory to pass the quadrupole and be detected. Other ions will be de-stabilizedand hit the rods. The performance (i.e. ability to separate two adjacent masses across the applicable range)depends on the quad geometry, on the electronics, on the voltage settings and on the quality of themanufacturing. Increasing the resolution means that fewer ions will reach the detector, and consequentlyimpacts the sensitivity.
The Mathieu stability diagram provides a representation of the ions stability domain. The Q and A axis correspond to the following equations e = charge, m = mass, ro = radius between therods, w = RF frequency, RF = radio frequency voltage, DC = direct current voltage The quadrupole is scanned with A/Q = constant; the resolution depends on the slope of the scan line.
If the continuous voltage DC is switched off, the scan line is the Q axis: We have now a transfer only devicelike the hexapoles or octopoles used to transfer and focus the ions into the mass spectrometer optics
THE QUADRUPOLE ANALYSER:
SCAN AND SIM
The quadrupole can be used in two modes: SIM (single ion monitoring) or Scan. The SIM mode is alsocalled SIR (single ion recording) In SIM mode, the parameters (amplitude of the dc and rf voltages ) are set to observe only a specific mass,or a selection of specific masses. This mode provides the highest sensitivity for users interested in specificions or fragments, since more time can be spent on each mass. That time can be adjusted; it is called thedwell time.
The mass window for observing an ion in SIM mode can be adjusted, in order to compensate small masscalibration shift. This is the span factor.
In Scan mode, the amplitude of the dc and rf voltages are ramped (while keeping a constant rf/dc ratio),to obtain the mass spectrum over the required mass range. The sensitivity is a function of the scanned massrange, san speed, and resolution.
With most LC/MS instrument, it is possible to do positive/negative switching, in order to analyse in the samerun molecules that will ionise in positive and negative modes.
MS/MS WITH TRIPLE QUADRUPOLES
The analyser of a "triple quad" instrument consists in twoquadrupoles, separated by a collision cell. Such a configuration isoften referred as a "tandem in space" instrument.
Precursor ions and product ions are created and analysed in differentphysical spaces. Ions must be moved from "source" to analyser (different physicalregions) where different functions take place.
Collision Induced Dissociation Region A triple quad instrument can be used in various ways, which are represented on the next figure. The firststwo experiments are in fact using the triple quad in a single quadrupole mode MS/MS WITH TRIPLE QUADRUPOLES
The first quadrupole is used to select a first ion (precursor), which isfragmented in the collision cell. This is typically achieved in thecollision cell by accelerating the ions in the presence of a collision gas(argon, helium…). The energy of the collision with the gas can be varied to allowdifferent degrees of fragmentaion. The resulting fragmentsareanalysed by the second quadrupole, used either in SIM or in scanmode Study of mass spectral fragments can provide structural information.
When using a single quadrupole instrument, it is possible to obtainfragmentation by using a technique called in source CID. Thefragmentation takes place before the introduction of the ions into theoptics of the mass spectrometer. This technique is useful if there is nochromatographic interference. With a triple quad system, the firstquadrupole acts as a separation device, reducing the need for aperfect chromatographic separation.
The other use of a triple quad system is quantitation. The first analyser,used in SIM mode, selects the parent ion. The second analyser is alsoused in SIM mode to monitor a specific fragment. Having two analysers increases the selectivity. The ion signal isreduced during the transmission, but the chemical noise, which is amajor limitation for complex samples, is also largely decreased,leading to an improvement of the signal to noise ratio. It is thuspossible to do quantitative analysis on complex samples like serumwith a very short chromatographic separation, and even with noseparation at all. This is the technique of choice for application suchas pharmacology studies.
However, one should keep in mind that, when doing quantitation, thefirst important step is the ionisation, which takes place in thesource.The presence of interfering compounds in the source mightcause unexpected effects, like "ion suppression". Such effects impactthe quantitation, whatever the MS analyser. Using an MS/MS systemmight reduce the problem, but does not eliminate it.
THE ION TRAP ANALYSER
This analyser is also known as the quadrupole ion trap analyser (QIT). It was first used on GC/MSinstruments, then on LC/MS systems.
The principle of the trap is to store the ions in a device consisting of a ring electrode and two end capelectrodes. The ions are stabilized in the trap by applying a RF voltage on the ring electrode. Formaximumefficiency, the ions must be focussed near the centre where the trapping fields are closest to the ideal andthe least distorted - maximizing resolution and sensitivity.This is achieved by introducing a damping gas(99.998% helium) that collisionally cools injected ions, damping down their oscillations until they stabilize.
By ramping the RF voltage, or by applying supplementaryvoltages on the end capelectrodes, or by combinationof both, it is possible to: • destabilise the ions, and eject them progressively from thetrap • keep only one ion of a given m/z value in the trap, andthen eject it to observe it • or keep only one ion in the trap, fragment it by inducingvibrations, and observe thefragments. This is MS/MS.
Since everything takes placein the same place, but at adifferent time, that approachis called MS/MS "in time" • repeat the last operation a few times to progressivelyfragment the ions. That is UNDERSTANDING THE TRAP
Understanding the trap: like for a quadrupole, the stability domain of the ions in the trap can be representedwith the Mathieu stability diagram e = charge = z, m = mass ro = radius between the rods RF = radio frequency voltage The ions are stable in the trap if the Q value lies between 0.3 and 0.9 0.3 < Q < 0.9.
This parameter has important consequences when doing fragmentation (in other words MS/MS) in a trap:the fragments which are smaller than about one third of the precursor ion will have a Q value > 0.9 andwill be lost.
By increasing the RF voltage it is possible to extract an ion (m/z is fixed) from the trap. It is also possibleto play with the voltage on the end cap electrodes, which affects the A value. By combining both actions,it is possible to eliminate from the trap the high masses and low masses, and keep in the trap only thedesired ions. The process is more difficult if one wants to select a specific ion, in the presence of an excessof another ion having a close m/z value Charge density in the trap: resolution and performance in an ion trap are dependent upon the
charge density of ions in the trap. If too many ions are present at the same time in the trap, the electrical
fields are distorted. Also, collisions between the ions may occur, leading to unexpected dissociation or
chemical reactions. In this case, the spectra and the quantitation will be impacted.
This is why a short pre-scan is achieved automatically to determine the optimum ion sampling time for eachof the acquisition point, so that enough, but not too many ions will be introduced. The optimum is to havebetween 300 and 1000 ions present in the trap. Consequently the sampling time varies along thechromatographic peak. UNDERSTANDING THE TRAP
Resolution of the ion trap analyser: the resolution which is achievable with an ion trap depends
upon the scan range and scan speed. When scanning over a few hundred Daltons in a fraction of a second,
the typical resolution is similar to the resolution of a quadrupole. However, it is possible to increase the
resolution by scanning at lower speed over a reduced mass range ("zoom scan"). In these conditions the
resolution exceeds 5000 when scanning over a 10 Dalton window, which is sufficient to determine the
number of charges of a multicharged small peptide.
A typical acquisition cycle, for a trap used in MS mode, includes the following automated steps(fromsupplier literature) 1: Prescan: this is to determine the needed injection time : 60 ms 2: Ion injection: about 500 ms. This is the admission of the ions into the trap. The duration depends upon the signal intensity 3: Set the trap parameters for ion isolation, activation…: 80 ms 4: Mass Analysis: about 70 ms Additional steps are needed for MS/MS or MSn operation a Triple Quadrupole and
Data points are evenly Data points are obtained most spaced across the entire peak frequently at the top of the peak an Ion Trap.
The ion trap is more sensitive in scan mode than in SIM mode.
Generally quadrupole instruments used in SIM mode provide an order of magnitude better limit ofquantitation with lower relative standard deviations for quantitative experiments than an ion trap, primarilydue to integration effect (more data points to determine the peak start and end with a quadrupole).
UNDERSTANDING THE TRAP
MS/MS with ion traps:
The principle is to isolate only the ion of interest in the trap , fragment it and then scan the product ions.
Isolation is achieved by increasing the RF voltage to eliminate the low mass ions, then adjusting the voltageon the end cap electrodes to eliminate the high masses (see the Mathieu stability diagram) Fragmentation: to fragment an ion , if is necessary to impart some energy. In the collision cell of a triplequad, or when doing in source fragmentation, this is obtained by applying an acceleration voltage. In anion trap, this is obtained by "shaking" the ion, with an RF voltage. Each ion has it's own resonationfrequency, so the product ions will normally not be fragmented, leading to limited structural information. Inorder to obtain richer spectra, a technique called wide band activation has been developed. It's possibleto build libraries of ion trap spectra, but ion trap spectra are usually different from quadrupole or triple quadspectra.
The figure above compares fragmentation spectra obtained from an ion trap employing two modes offragmentation on the protonated product ion of Labetalol (311 m/z). Top spectrum utilizes the standardmode of activation by isolating the product ion and applying a specific secular frequency. The bottom spectrum was acquired via WideBand Activation™*.
* WideBand Activation™ is a trade mark of Thermo THE TIME OF FLIGHT ANALYSER
This analyser is commonly called the TOF. The TOF is used in single MS systems, with an LC introduction,with a GC introduction, or with MALDI ionisation. In MS/MS configuration, the TOF is associated to aquadrupole (QTof), or to another TOF (TOF-TOF) or to an Ion Trap (QIT/TOF).
Principle of the time of flight analyser: In a Time–Of–Flight (TOF) mass spectrometer, ions
formed in an ion source are extracted and accelerated to a high velocity by an electric field into an analyser
consisting of a long straight ‘drift tube'. The ions pass along the tube until they reach a detector.
After the initial acceleration phase, the velocity reached by an ion is inversely proportional to its mass(strictly, inversely proportional to the square root of its m/z value). Since the distance from the ion origin to the detector is fixed, the time taken for an ion to traverse theanalyser in a straight line is inversely proportional to its velocity and hence proportional to its mass (strictly,proportional to the square root of its m/z value). Thus, each m/z value has its characteristic time–of–flightfrom the source to the detector.
Time of Flight equations: The first step is acceleration through an electric field (E volts). With the
usual nomenclature (m = mass, z = number of charges on an ion, e = the charge on an electron, v = the
final velocity reached on acceleration), the kinetic energy (mv /2) of the ion is given by equation (1).
mv /2 = z.e.E (1)
Equation (2) follows by simple rearrangement.
v = (2z.e.E/m)1/2 (2)
If the distance from the ion source to the detector is d, then the time (t) taken for an ion to traverse the drifttube is given by equation (3).
t = d/v = d/(2z.e.E/m)1/2 = d.[(m/z)/(2e.E)] 1/2 (3)
In equation (3), d is fixed, E is held constant in the instrument and e is a universal constant. Thus, the flighttime of an ion t is directly proportional to the square root of m/z (equation 4).
t = (m/z) 1/2 x a constant (4)
Equation (4) shows that an ion of m/z 100 will take twice as long to reach the detector as an ion of m/z 25: In order to increase the resolution, the ion trajectory is bent by an electronic mirror, the reflectron. Whengoing through the reflectron, the dispersion of ions of the same m/z value is minimized, leading to a greatimprovement of resolution THE TIME OF FLIGHT ANALYSER
Characteristics of the time of flight analyser:
Mass range: there is no upper theoretical mass limitation; all ions
can be made to proceed from source to detector and the the upper
mass limit exceeds 500 kDa. In practice, there is a mass limitation, in
that it becomes increasingly difficult to discriminate between times of
arrival at the detector as the m/z value becomes large. Another
limitation is that very large molecules are difficult to ionise. Using an
ionisation technique which produces multiply charged ions, like
electrospray ionisation, extends the working range of the TOF
Resolution: with a TOF instrument, it is possible to obtain
10000 FWHM resolution
Mass accuracy: better than 5 ppm, using a reference mass; that
allows unambiguous formula determination of small organic
MALDI TOF, OA-TOF
Two different sample introduction/ionisation techniques are used with time of flight analysers: MALDI andorthogonal acceleration MALDI (Matrix Assisted Laser Desorption Ionisation): this is an off linetechnique. A laser beam hits the samples deposited in a matrix on atarget plate. Samples are volatilised and ionised. The systemmeasures the time of flight between the laser pulse and the detection.
MALDI TOF spectrum of a mix of Angiotensin I, Angiotensin II,Bradykinin, and Fibrinopeptide A (20 femtomoles of each peptideloaded). Each peak corresponds to a peptide.
oa-TOF: this is for in line coupling. This configuration is used forLC/MS with API ionisation, and for GC/MS. The ions are transferredfrom the source to the analyser through a transfer optics. The "pusher"accelerates the ions to the same level of energy, and gives the startsignal for timing the flight.
Electrospray TOF spectrum of Lysozyme. Withelectrospray ionisation, multiply charged ions areobtained. The peaks corresponds to various levelsof charge.
The QTof is a hybrid MS/MS instrument combining a quadrupole with a Tof analyser. Thiscombination provides the benefits of in space MS/MS (selectivity, flexibility for collisionexperiments) with the advantages of the Tof (sensitivity in scan mode, fast scan, accurate mass,resolution). This is an ideal combination for sophisticated applications.
The detector is the device which detects the ions separated by the analyser.
3 different types of detector are used with the analysers described in the previous pages: Electron multipliers, dynolyte photomultiplier, microchannel plates A conversion dynode is used toconvert either negative orpositive ions into electrons.
These electrons are amplifiedby a cascade effect in a hornshape device, to produce acurrent. This device, also calledchanneltron, is widely used in quadrupole and ion trap instruments.
Ions exiting the quadrupole are converted to elec-trons by a conversion dynode.These electrons strike a phosphor which when excited, emit pho-tons. The photons strike a photocathode at the front of the photomultiplier to produce electronsand the signal is amplified by the photomultiplier.
The photomultiplier is sealed in glass and heldunder vacuum. This prevents contamination andallows the detector to maintain its performance fora considerably longer period than conventional electron multipliers.
Most TOF spectrometers employ multichannelplate (mcp) detectors which have a time response< 1 ns and a high sensitivity (single ion signal >50 mV). The large and plane detection area ofmcp's results in a large acceptance volume of thespectrometer system. Only few mcp channels outof thousands are affected by the detection of a sin-gle ion i.e. it is possible to detect many ions at thesame time which is important for laser ionisationwhere hundreds of ions can be created within afew nanoseconds.
INTERFACING HPLC AND MS
Interfacing a HPLC system with a mass spectrom-eter is not trivial.
The difficulty is to transform a solute into a gas phaseion. The challenge is to get rid of the solvent whilemaintaining adequate vacuum level in the mass spec-trometer, and to generate the gas phase ions. Since the early seventies, a number of approach-es have been used. LC/MS became really popu-lar with the introduction of of the thermospray inter-face and the particle beam interface. The next bigimprovement was the introduction of the electro-spray and APCI techniques. The thermosprayinterface is no longer available on the market, butthe particle beam is still available from Watersbecause it it the only method to provide electron GET REPRO RIGHTS
Actually, the large majority of applications are done with electrospray and APCI ionisation. New techniqueslike APPI (atmospheric photo ionisation) are appearing, but are not yet largely used.
In the next pages, we will cover the principle and application of atmospheric pressure ionisation modes.
We will not discuss older techniques, which are now mainly of historical or theoretical interest.
Electrospray and APCI are both API (atmospheric pressure ionisation) techniques. Ionisation takes place atatmospheric pressure and both are considered to be soft ionisation method. The mass spectrum providesmainly the molecular weight information, unless fragmentation techniques are used. The possiblefragmentation techniques are in source CID (collision induced dissociation), CID in the collision cell of atandem type instrument, fragmentation in an ion trap. This is very different from the spectra obtained withEI (electron impact ionisation).
negative APCI (A), fragmentation (B), ionisation (C).
ELECTROSPRAY IONISATION (ESI)
Description: the HPLC line is connected to the electrospray probe, which consists of a metallic capillary
surrounded with a nitrogen flow. A voltage is applied between the probe tip and the sampling cone. In most
instruments, the voltage is applied on the capillary, while the sampling cone is held at low voltage. First step
is to create a spray. At very low
flow rate ( a few µl/mn), the
difference in potential is
Atmospheric Pressure Region sufficient to create the spray. Athigher flow rate, a nitrogen flow is necessary to maintain a stable spray.
ESI Needle 3-8 kV The API sources include a heating device, in order to speed up solvent evaporation.
(Disintegration) A mandatory condition to work with electrospray is that the compound of interest must be Droplet After Reduction ionized in solution.
If it is not compatible with theHPLC conditions (I.e. in case ofnormal phase chromatography), it is possible to use post column addition to get appropriate conditions.
In the electrical field, at the tip of the capillary, the surface of the droplets containing the ionized compoundwill get charged, either positively or negatively, depending on the voltage polarity . Due to the solventevaporation, the size of the droplet reduces, and, consequently, the density of charges at the droplet surfaceincreases. The repulsion forces between the charges increase until there is an explosion of the droplet. Thisprocess repeats until analyte ions evaporate from the droplet.
Multiply charged ions can be obtained depending on the chemical structure of the analyte. This is why ESIis the technique of choice for analyzing proteins and other biopolymers on quadrupole or ion trapanalyzers.
Typical ions produced by electrospray ionisation:
[M+H]+ protonated molecule[M+Na] +, [M+K] + … adducts[M+CH3CN+H] + protonated, + solvent adducts [M-H] - deprotonated molecule[M+HCOO -] -, … adducts ELECTROSPRAY IONISATION (ESI) (2)
Flow rate: the best sensitivity is achieved at low flow rate. Working at 1 ml/mn or even higher is
technically possible, but may cause a reduction in the signal to noise ratio.
Eluent pH: the mobile phase should have a pH such that the analytes will be ionized. An acidic mobile
phase is suitable for the analysis of basic compounds, using positive ESI, while a basic pH will be chosen
for analyzing acidic molecules. However, some exceptions exist to this general rule: positive ESI of basic
compounds with a high pH mobile phase has been published.
Buffers: volatile buffers
are to preferred for routine
use. Operating the instru-
ment with non-volatile buffers such as phosphate is technically possible, but the salt deposit in the 10 12 14 16 18 20 source will have to be XTerra™ MS C18, 2.1 x 150 mm, 5 µm XTerra™ MS C18, 2.1 x 150 mm, 5 µm ES positive ion mode, TIC removed periodically. The ES positive ion mode, TIC Gradient elution: Acetonitrile/ 10 mM Aqueous ammonium Gradient elution: Acetonitrile/Aqueous 0.04% TFA bicarbonate pH adjusted with ammonium hydroxide buffer, or acid or baseused to adjust/control thepH should be as low as possible. If not, competition between analyte and electrolyte ions for conversion togas-phase ions decreases the analyte response.This can be explained as follows: if a species is in large excess,it will cover the droplet sur-face and prevent other ions 1. Surface competition
to access the surface, and thus to evaporate. A species in large excess will also catch all charges available and prevent the ionisation of other 2. Charge competition
molecules present at much OAc– + AH+ → HOAc + A Ion pairing agents (sodium octanesulfonates….) : these molecules have surfactant properties. The presenceof surfactants in the mobile phase impacts the spray formation and droplet evaporation. There is also asurface competition mechanism phenomenon.
Matrix effects: when the sample contains high concentration of salts, or an excess of another analyte
that can ionise in the operating condition, there might be a competition effect in the ionisation. This is called
"ion suppression". The chromatographic separation must be developed to remove this effect, at least when
doing quantitative analysis.
CHEMICAL IONISATION (APCI)
Corona Discharge Needle Description:the HPLC line is is connected to the APCI probe which consists normally of a glass capillarysurrounded with a nitrogen flow used for mobilization. Part of the APCI probe, or close to the probe tip area heating device and an additional gas flow, to instantaneously volatilize the solvent and sample. Close to the probe, there is a metallic needle, which is at potential of a few kilovolts. This is the "coronadischarge electrode". The "corona effect" term describes the partial discharge around a conductor placedat a high potential. This leads to ionisation and electrical breakdown of the atmosphere immediatelysurrounding the conductor. This effect is known as corona discharge. St. Elmo's fire is an example of anaturally occurring corona. In the case of an APCI source, the atmosphere surrounding the corona electrodeconsists mainly in the vapour generated from the HPLC eluent, nitrogen, and the analyte molecules.
The eluent vapours are ionised by the corona effect, and react chemically with the analyte molecules in thegas phase.
The above figure shows the solvent molecules (S) being protonated by the corona (SH+), then reacting withthe analyte molecule (M) to give the protonated form MH+.
The following conditions are required for APCI to work:• the analyte must be volatile and thermally stable• the mobile phase must be suitable for gas phase acid-base reactions - for working in positive mode, the proton affinity of the analyte must be higher than the proton affinity of the eluent (in other words, the analyte can catch a proton from the protonated solvent)SH+ + M _ S + MH+ - for working in negative mode, the gas phase acidity of the analyte must be lower than the gas phase acidity of the eluent (in other words, the analyte can give a proton to the deprotonated solvent)[S – H] - + M _ S + [M – H] – Thermodynamic values can be obtained from the internet (i.e. the NIST site http://webbook.nist.gov/chemistry/)or from handbooks of chemistry.
Solvent adducts or radical cations (M . + ) can be observed ATMOSPHERIC PRESSURE
CHEMICAL IONISATION (APCI) (2)
Flow rate: APCI is usually used at higher flow rate than ESI.
Optimum conditions are at a few hundreds µl/mn
Eluent: the mobile phase must be suitable for ionisation. If it is not
the case, a small amount of modifier can be added, in order to
generate the solvent gas phase ions.
Buffers: buffers must be volatile.
Atmospheric pressure photo ionisation is a newly introducedtechnique. It was presented at ASMS 2000 by Andries Bruins fromthe University of Groningen, Netherlands. The principle is to usephotons to ionise gas phase molecules. The source is a modified APCI source, with the corona electrodereplaced by an UV lamp Ionisation mechanism: the ionisation can be obtained directly,
or through a dopant (I.e acetone, or toluene)
D+ + M _MH+ + D[-H] Applicability: APPI is said to allow the ionisation of compounds
which cannot be ionised with APCI or ESI, to be compatible with flow
rates down to 100 µl/mn, and to be quantitative. Obviously, it is too
early and there is not enough published work to estimate the range of
applications. Is it of interest for a few specific cases, or will it be a
significant complement to the traditional API techniques?
General rules can be given to chose between ESI, APCI, or EI ESI is preferred for compounds which are ionic or very polar or thermo labile, or with masses higher than1000… APCI is preferred for compounds which are not very polar If we take the example of pesticides, we can rule out that:polar pesticides will be in ESI, less polar in APCI, volatiles in APCI, non polar better done with GC/EI…. But practical consideration are also important: changing from ESI to APCI cannot be done automaticallyduring the separation. The instrument itself have an influence: paraquat and diquat are reported to workonly in ESI on some instruments but also in APCI on other systems*. Depending on the supplier, someinstruments might show better performance in APCI, while other ones will perform better in ESI.
So, when it comes to practice, and when the choice is not obvious, the best approach for determining themost suitable ionisation method is still to try to inject in various conditions, or to refer to existing work.
API SOURCE DESIGN
All MS suppliers are making a continuous effort to improve the design of the API sources, particularly asthe source is a key element of the LC/MS instrument. A good source is essential for: - clean MS spectra- sensitive detection- robustness and reliable operation- easy use and maintenance An API source always comprises - the probe (ESI or APCI)- the corona electrode (for APCI operation)- Gas flows for mobilization, evaporation and desolvatation- sampling cone- transfer optics to the MS analyser Trends in source design:
Looking back at the source design of various suppliers, we observe that, only a few years ago (1997-1998)all were using an axial design. The probe was in the axis of the optics. To limit the risk of plugging thesampling cone orifice with non volatiles from the eluent or from the sample, various approach were used. Among the most efficient were the the "pepper pot" counter electrode from Micromass, and the "curtaingas" from PE-Sciex "Pepper pot" design: the ions have to travel through the channels of the metallic counter electrode, whichis heated. The counter electrode protects the samplingcone orifice and helps in ion desolvatation Counter electrode Hexapole Ion Bridge "Curtain gas" design: a flow of heated gas protectsthe orifice plate and helps in the desolvatation API SOURCE DESIGN
Ion desolvatation is also an important aspect of source design. Some suppliers use a heated metallised
transfer capillary.This approach was already available in the early electrospray source designs
(Whitehouse, 1985) . Another approach is to use heated gas flows, which eliminates the risk of plugging
the capillary, or of sample adsorption and degradation on the capillary walls.
Axial source design with heated transfercapillary (first versions of the Finnigan LCQ).
Similar design is still used on variousinstruments.
Design evolution: designs have changed,
to give better protection of the sample orifice
(gain in robustness), for introducing more ions
into the optics (gain in sensitivity), for better and
smoother desolvatation (gain in sensitivity,
cleaner spectra, less thermal decomposition).
Those improvement are found on the MKII Zspray source, which is used in various versions on the Waters-Micromass LC/MS instruments. More than 2000 of these sources are in operation in the field.
MKII source design:
The probe is perpendicular to the sampling cone, which is protected by a "cone gas" flow. The secondextraction cone is also perpendicular to the ion beam. This so-called "ZSpray" geometry protects veryefficiently the optics against non-volatile material and non-ionised molecules. Consequently, cone orificescan be larger than in previous designs. The combination of larger orifices and noise reduction largely compensates for transmission losses due to theorthogonal geometry, giving a large gain insensitivity. Efficient desolvatation is provided by a heatednitrogen flow close to the probe and by thecone gas. The source block is also heated, forfinal desolvatation.
For easy maintenance, an isolation valve, isinstalled, so that it is possible to disassemblethe sampling cone for cleaning without havingto vent the system. A glass window givesthe operator the possibility to observe thespray and the interior of the source duringoperation.
THE API MASS SPECTRUM
Both APCI and ESI are soft ionisationtechniques. Thus, the MS spectra obtained with Parent Ion Region API ionisation will consist mainly of the"molecular" ions, unless fragmentation Positive ion mode, ESI, 2 ul /min, 50% techniques are applied.
Looking at the sulfamethazine spectrum obtained by infusion of the sample, we observe the protonated molecule, and a few small ions.
These small ions might be fragments, or more A closer look at the [M + H] + region shows the peak and isotopes. The acquisition is made in continuous (profile) mode and indicates a resolution of about1000 (FWHM). The isotope pattern often provides useful information, since the well known rule of MSspectra interpretation can be applied to API spectra (nitrogen rule, double bond equivalent value). It is alsopossible to use the isotopic pattern to determine the number of carbon, chlorine, bromine…atoms Adducts/clusters: the UPAC nomenclature is as follows:
"Adduct Ion - An ion formed by interaction of two species, usually an ion and a molecule, and often withinthe ion source, to form an ion containing all the constituent atoms of one species as well as an additionalatom or atoms. Cluster Ion - An ion formed by the combination of two or more molecules of a chemical species, often inassociation with a second species. For example, [(H2O)nH]+ is a cluster ion. " Adducts and cationised or anionised molecules are often observed with API techniques. Those ions can be formed "accidentally", due for instance to the presence of sodium in the mobile phase or intentionally, for more specific or sensitive detection Adduct formed with acetonitrile. By switching on the "cone gas", the desolvatation i With "C one gas " improved, and the solventadduct disappears. Without " Cone gas " The formation of adducts or clusters should be controlled when doing quantitative analysis, since the compound ofinterest should be in a well defined and stable form. THE API MASS SPECTRUM
Negative ESI mass spectrum for glucose, with chloride adducts.
The gradient was 80% acetonitrile-water to 50%acetonitrile-water, both containing 0.001M LiCl.
Chloride was added to form the sugar-chloride adduct, providing sensitive and selective detec-tion. The characteristic Cl35/Cl37 isotope pat-tern makes carbohydrate identification easy.
Multicharged spectrum: with electrospray ionisation, multiply charged ions are obtained for proteins,
peptides oligonucleotides, and in general, for any molecule which presents multiple ionisation sites. Various level
of charge are present simultaneously, giving a spectrum with multiple peaks. The mass of the molecule can be
obtained by calculation from the multiply charged spectrum. This can be done manually for small peptides, but
de-convolution software options, available from instrument suppliers, are very useful for large molecules.
Each of the peaks of this mass spectrum representsa different charge level. The lower masses corre-spond to the larger number of charges.
Deconvolution can be made by a mathematicalapproach (resolution of a system of equations)using the Transform software, or by a statisticalapproach, using the Waters/Micromass MaxEntsoftware.
with Waters/Micromass MaxEnt with Waters/Micromass Transform THE API MASS SPECTRUM
In source fragmentation: this is is obtained by accelerating the ions in the source, in a region where thepressure is in the millibar range. The acceleration is produced by applying a voltage (cone voltage) betweenthe sampling cone and the next extraction lens.The ions collide with residual gas molecule, gain energy andfragment.
Increasing fragmentation withincreasing cone voltage.
Standard 550 pg/ul , ES , FS positive , CV variables
The fragmentation obtained by in source fragmentation is useful for confirming a peak identity, getting structure information, improving selectivity, building 100 120 140 160 180 200 220 240 260 280 300 320 340 PRACTICAL ASPECTS OF USING LC/MS:
When planning LC/MS equipment, it is necessary to make sure of the availability of a nitrogen supply.
Possible options are nitrogen generators, liquid nitrogen and nitrogen cylinders.
An important point is that the gas must not contain organic impurities that would be visible on the massspectra and could completely contaminate the mass spectrometer.
Severe contamination of the instrument due to an inappropriate gas installation are rather common. Themain causes are the soldering flux (N2 tubing should be preferentially stainless steel, connected withSwagelock type connectors), and not maintained N2 filtration/purification devices.
Using compressed air in place of nitrogen is sometimes mentioned. Despite that we are not aware of anyaccident, there are potential safety issues, due to the presence of solvent vapours, high temperatures andhigh voltages in the source. In addition, oxygen presence might be visible on the mass spectra. The figure below shows that the use oflow purity nitrogen can lead to the formation of superoxyde adducts.
Thus, replacing nitrogen by air must be avoided.
2: Scan AP- M32+test06 Mass chromatograms Negative Ion Mode (M + 32)- = 890.8 m/z Superoxide adduct (O • -
Proper exhaust must be also available, to avoid the dispersion of solvent vapours (from the source) and oil Mass chromatograms vapours (from the vacuum Positive Ion Mode (M + H)+ = 859.8 m/z pump) into the laboratoryenvironment.
The lab temperature should be reasonably stable, to avoid mass drifts.
For long term successful opera- tion, LC/MS needs a clean environment. This includes the lab environment, the quality of solvent and buffers,the quality of the columns, and sample preparation.
Laboratory environment: the lab must be ACAP (as clean as possible). Contaminations, which are not visiblewith a HPLC/UV system, may be a real headache with LC/MS! Traditional causes are solvent vapours, (i.e:DMSO), oil vapours (from not properly installed vacuum pumps). The HPLC system can also be a source ofcontamination. Traces from buffers, solvents or sample from previous runs are common. Traditional ones aresurfactants (from ion pairing agents, TFA, THF, TEA….).
PRACTICAL ASPECTS OF USING LC/MS:
Instrument maintenance: proper maintenance of the LC/MS system is mandatory for successful longtermoperation. This concerns both the HPLC part, and the MS.
Routine maintenance of the LC/MS consists mainly in cleaning the source. This includess the
probe,the source enclosure, the sampling cone and eventually the transfer optics. When the source becomes
dirty the quality of the signal degrades. This is due to contaminant ions and to a degradation of the ions
transmission. Even without a visible blockage of lens orifices, a dirty surface affects the voltages and impacts
Source enclosure after operation with phosphate buffer. The instrument is still operational, due to the cone gaswhich protects the sampling cone orifice (left photograph). However the phosphate deposit needs to be removed.
After turning the isolation valve, the cone can be disassembled and cleaned without venting the system.
Cleaning of the source parts located after the sampling cone is sometimes needed. The cleaning frequencydepends upon the system usage, nature of the samples, and source design. This operation can be made bythe user, but is more or or less difficult to do, depending on source design. In any case, the system needsto be vented.
The Waters ZQ source, disassembled formaintenance. All parts are self aligning.
Vacuum system: the roughing pump (s) oil
need to be replaced every few months. There is no
user maintenance on the turbomolecular pumps.
Detector: if the detection system is an electron
multiplier, it will need a replacement after about
Maintenance contracts: every supplier proposes maintenance contracts. The content of the contract
is very variable. Opting for a preventive maintenance (like for a car) is the approach to minimize instrument
PRACTICAL ASPECTS OF USING LC/MS:
Using a mass detector brings some constraints upon the eluent selection. Theseconstraint are different from what is to be considered with other detectors. Forexample, the eluent UV absorbance, which is of high importance with UV detection isirrelevant with MS detection.
Eluent selection criteria:
Ionisation: the eluent must be suitable for the ionisation. Thus the eluent must be
selected depending on the Ionisation mode (ESI or APCI, positive or negative mode)
and the analyte (pKa, gas phase acidity).
Eluent molecular weight: it is not possible to analyse compounds which MW is
lower than the one of the eluent (or eluent additives
Volatility: for routine operation, it's easier to use volatile buffers.
Acids: HCl, H2SO4, methane sulfonic acid… might damage the instrument and
should not be used. They must be replaced by volatile organic acids (TFA, formic,
Adduct formation: ions (Na, NH4, acetate …) from the eluent will trend to form
adducts. In the case of phosphate, multiple adducts are observed, which produce
complicated mass spectra. The formation of adduct is usually not a reason for avoiding
an eluent, and at the opposite, adduct formation might be forced for analytical
Ion pairing reagents, surfactants: impact the spray formation, the droplet
evaporation, and compete in term of ion formation
Buffer concentration: must be kept as low as possible (mM range). If the buffer
concentration is too high, ion suppression occurs.
Common eluents for LC/MS: Methanol/water, acetonitrile/water (methanol
usually gives a better sensitivity than acetonitrile) pH modifiers: formic, acetic acids,
TFA, NH4, TEA, DEA, carbonates, ammonium formate, ammonium acetate, ammonium
carbonates, ammonium phosphate (non volatile)…
HPLC column: the column must give a good separation without using high
concentration of buffers, nor ion pairing reagents. The bonding must be stable, so that
the column will not "bleed". Special MS versions of columns are available from
ACLOSER VIEW AT A SINGLE QUADRUPOLE
INSTRUMENT: THE WATERS ZQ
Instrument history: this instrument was introducedin theyear 2000 replacing the Micromass Platformand Waters ZMD family of single quadrupoles. Itbenefits from the experience gained with previousgenerations, but most of the components havebeen replaced with the latest technology, with thegoal to reduce instrument size and gain inperformance.
The instrument imbeds a syringe pump for infusionexperiments and for calibration.
A switching valve is included, which can be, forexample, used to discard the injection peak for asample containing a high salt concentration.
Waters ZQ components: main sub assemblies are:
the sourcethe opticsthe detectorthe RF generatorthe voltage suppliesthe vacuum systemthe electronics the software The source: it is a redesigned MKII type source,
with the probe positioned vertically Having the
probe placed vertically allows shorter connection
to the cell of a detector (UV) placed on the top of
the mass spectrometer. Optimised connections
minimise peak broadening, and help in gaining
It also saves some lab or bench space.
Gas flows and source temperature are controlledfrom the software, to allow full documentation ofexperimental conditions.
ACLOSER VIEW AT THE WATERS ZQ:
Depending on the flow rate the probe positioncan be adjusted to get the maximum sensitivity.
If needed it is possible to lock the position of theprobe.
The ion optics, consist of the extraction cone and lens (part of the source block), the transfer hexapole,
the quadrupole, and the detector
The quadrupole is made of molybdenum rods,which are proven to provide excellent stability.
For easier instrument manufacturing, and in theunlikely case of a cleaning need, all parts areself aligning and self connecting.
On this photograph, the quadrupole has beenremoved. The detector (dynolyte photo-multiplier) is placed perpendicular to the ionbeam, leading to an improved signal to noiseratio, by reducing the noise from the neutrals.
Also visible on the photo are the top of the turbopump and a vacuum gauge.
ACLOSER VIEW AT THE WATERS ZQ:
The vacuum system consist of an Edward roughing pump, and in an Edwards split flow turbo pump.
The pumping capacity is largely dimensioned, allowing very fast return to operation after venting theinstrument.
BOC Edwards EXT200/200Hi190 l/s N2 at main inlet port160 l/s N2 at side inlet portAir CooledOnly one pump and one controller The electronics consist of a PC board and in an embedded PC
Using an embedded PC presents many advantages. The main one isfast signal processing. The ZQ is able to scan at 5000Dalton/second, which is a real advantage for LC/MS applications.
Fast scanning allows the acquisition of more data points per second,giving thus better peak shape, better integration and betterreproducibility.
Other advantage is the possibility to do multiplexing:
Using the MUX source, up to four HPLC lines can be connected to the same Waters ZQ instrument for very
high throughput applications, or for method development
Photographs: The ZQ
equipped with the MUX source
and close up view of the 4 ESI nebulizers. A rotor puts each nebulizer
successively in line with the extraction cone. The signal is deconvolut-
ed by the software, so that 4 independent data channels are acquired
ACLOSER VIEW AT THE WATERS ZQ
The software: the Waters ZQ is controlled either by MassLynx software, or by Millennium software.
MassLynx is the software developed by Micromass, a division of Waters Corporation, for the control of allMicromass instruments. MassLynx users benefit from a complete series of application managers, designedto fulfil specific needs, like Open Access to the instrument, screening of libraries from combinatorialchemistry, MS or UV triggered automated purification, protein applications…Millennium is the Waters chromatography software. This software is used by more than 35000 users in theworld, either for control and data acquisition from a single chromatograph, or in large networkconfigurations. To fulfil Millennium users needs, Waters has implemented the control of the ZQ massspectrometer into Millennium. Millennium users can easily benefit of MS detection by simply complementingtheir LC with a ZQ mass detector.
Software aspects: we will review only what is linked to the ZQ mass spectrometer. Information on
HPLC methods (system control, signal acquisition and processing, reporting…) is available from Waters
sales and support organisation.
The tune pages: these pages allow a direct control of the ZQ, for instrument fine tuning and
In the tune page, you can adjust the temperatures,gas flows, cone voltages, resolution, achieve themass calibration of the instrument… A real time display provides the visualisation ofany modification. Acquisition can also be startedfrom the tune page.
It is also possible to control a syringe pump forinfusing the sample or calibration solution.
The tune page parameters are saved in a methodwhich can be used for analysing sample series.
The diagnostic page provides a direct informationon instrument "vital" functions.
For example, the rotation speed of the turbo pumpis monitored. A vacuum leak will translate into anabnormal speed. Various voltages are alsomonitored.
SIGNAL ACQUISITION MODES
The Waters ZQ offers a large flexibility for choosing the signal acquisition parameters.
The raw signal from the photomultiplier is acquired in the form of data pairs (mass : intensity). The maximumresolution is 16 acquisition points per a.m.u. That value can be adjusted in the advanced parameters of theinstrument method.
The information can be stored in this way. This is the continuous
acquisition mode. To reduce data file size, it is also possible to
compute immediately the signal, to provide a bar (or stick)
representation of the signal. The bar height is proportional to the
abundance of the ion. This is the centroid mode, which gives to
much smaller files. It is possible to convert a spectrum from continuous
to centroid, but a signal acquired in centroid cannot be converted into
1 A.M.U, i.e. 16acquisition points.
A third acquisition mode is the MCA mode (Multi Channel
Acquisition). This mode is adopted when infusing a sample into the
MS with a syringe pump. The software accumulates the mass spectra
acquired over a user defined time period. This is a way to enhance
the quality of the signal.
SIM mode: In SIM mode [single ion monitoring or recording (SIR)],
the instrument observes a specific mass, or a series of masses. The
time spent to observe each ion is called the dwell time, and the
observation window the "span". These parameters can be adjusted
individually for each ion.
Scan mode: the mass range scan window, scan time, are the
parameters to be defined
Positive/Negative mode: each of the above listed modes can
be performed in positive or in negative polarity. Switching polarity is
very fast and can be made during a single acquisition.
Acquisition function: an acquisition function is a line in the
method, which sets the acquisition mode (continuous or centroid, SIM
or Scan), and various other parameters (cone voltage, ESI or APCI
voltages….). A method can combine up to 32 functions
ACLOSER VIEW AT THE WATERS ZQ
The MS instrument method: in this method, the user defines the acquisition parameters of the mass
spectrometer. This can be compared to the method which is developed for using a traditional HPLC detector.
The MS instrument method will be one of the elements of the global LC/MS method used to run sample andgenerate results.
The MS instrument method consist of a series of lines, each line corresponding to a specific function (task)of the mass spectrometer. The ZQ method can combine up to 32 functions, allowing complete flexibility.
For example:Function 1: from 0 to 17 minutes, acquisition in positive ESI, scan from 100 to 350 amu in 0.8 seconds,Function 2: from 0 to 17.5 minutes, acquisition in negative ESI, scan from 100 to 350 amu in 0.8 secondsFunction 3: from 0 to 17 minutes, acquisition in positive ESI, SIR (same as SIM) on mass 226.2 and 309.1,dwell time 0.2 second, cone voltage at 25 voltsFunction 4: from 6 to 8 minutes, acquisition in negative ESI, SIR on mass 275.9, dwell time 0.2 second,cone voltage at 25 voltsFunction 5: from 6 to 9 minutes, acquisition in negative ESI, SIR on mass 307.1, dwell time 0.2 second,cone voltage at 25 volts The instrument will go through all these functions and will generate 5 chromatograms simultaneously. Thetwo scan function will provide a global view of the sample, while the 3 SIR function will be used to observeand quantitate targeted compounds.
The application field of LC/MS is extremely large and is covered by a wide range of instruments andtechniques.
Looking globally at the users, it is possible distinguish three groups, depending on how they use LC/MS • Users for which the main useful information from the mass spectrometer is the mass information (molecular weight or fragments). The quantitative aspect is of no or little importance. Typically, these users wish to: - monitor or confirm an organic chemistry synthesis,- or to trigger a fraction collector when the expected compound elute from the column or to check if a peak on a chromatogram is a metabolite or degradation product of a known parentcompound - or to get molecular weight and structure information from their compound • Users for which the main interest is getting a very selective and sensitive detection. These users are targeting specific molecules. The quantitative aspect is important, but the mass information is of secondaryimportance.
• Users targeting specific molecules, wanting the quantification and the confirmation of the identity. The molecular weight, and the presence of a few specific fragments which the expected abundance are asimportant as the sensitivity and selectivity.
Waters/Micromass has developed instruments and software to address those various needs. Our MSspecialists will be pleased to help you in selecting the instrument which is the best adapted to your need.
Waters European Marketing 78056 St-Quentin-Yvelines Cedex Tel. (33) 1 30 48 72 00 - Fax (33)1 30 48 72 11 Internet : www.waters.com
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