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Mass Spectrometry

Fundamental LC-MS

Flow Rates and Flow Splitting

Aims and Objectives

Aims and Objectives

Aims To highlight the flow limitations of various LCMS Atmospheric Pressure Ionisation (API) sources and present possible solutions for use with conventional HPLC pumping systems.

To illustrate the application and benefits of using micro and nano flow in LC/MS.

Introduce the concept of pre and post column flow splitting, and explain the quantitative nature of flow splitting.

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

Explain the flow limitations of various API source types and choose an appropriate solution for coupling with conventional HPLC systems

Discuss the benefits and illustrate the need of using micro and nano flow LC/MS

Describe the instrument adaptations required in order to achieve micro and nano flow HPLC

List and describe the working principles and problems associated with split flow HPLC systems

Explain why it is beneficial working with capillary HPLC systems and choose the situations under which capillary HPLC might be used

Crawford Scientific www.chromacademy.com

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Content Introduction 3 Interface Similarities 4 The ESI Interface 5 The APCI Interface 6 Flow Rates for APCI 7 Flow Rates for ESI 7 Flow Rates for Pneumatically Assisted ESI 8 Micro and Nanoflow ESI 9 Flow Rate Incompatibility –Reasons 11 Flow Rate Incompatibility –Solutions 12 Flow Splitting –Overview 13 Flow Splitting –Adjustable Flow Splitters 15 Flow Splitting –Practicalities 16 MS Detector 17 Columns –Internal Diameter 18 Columns –Low Flow Rates 19 Capillary LC systems –Overview 20 Capillary LC Systems –Practicalities 22 Column Selection 25 References 26


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Introduction Flow rates used in conventional LC separations using non-mass spectrometric detectors may not be compatible with LC-MS systems depending upon the eluent system and the API interface type used.

Generally, the eluent flow rate used in LC-MS experiments will be governed by several factors including:

The API interface type

The eluent system

The vacuum system of the LC-MS instrument

The availability of column and HPLC pumping hardware Perhaps the most limiting factor in the list is the API interface used for a particular application, the two most popular interface types being Electrospray Ionisation (ESI) and Atmospheric Pressure chemical Ionisation (APCI).


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Interface Similarities In general all API interfaces have to be able to accommodate the following processes:

Evaporate liquids into gases.

Ionise neutrals into charged species or transfer charged species in solution to gas phase ions.

Evacuate large amounts of eluent and remove the resulting vapour from the system in order to maintain the required level of vacuum within the mass spectrometer.

The diagram shown below highlights the change of state that applies to all LC-MS

interfaces. Whether the analyte is neutral in the liquid phase ][ )(

O

lM or charged ][ )(

lM it

must make its way to a surface to evaporate into the gas phase. Once in the gas phase a

neutral analyte ][ )(

O

lM must be ionised so that mass analysis can occur. It is the pathway

between the two sides of the diagram that will determine the differences between the types of interface used.

The energy requirements for the thermodynamic change of state are independent of the path taken from one side of the diagram to the other and will be similar for all interface types. It is the way that the energy is applied to the system that will differentiate the interface types. Aerosols are of great importance in LC-MS as they provide the surface area from which the analyte species will evaporate into the gas phase. The higher the surface area, the greater the amount of evaporation and hence the greater the number of analyte species available for sampling into the mass analyser.


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The ESI Interface Electrospray is limited to flow rates below 10 μL/min. Above this the electrolytic droplet charging process that occurs at the capillary tip is inefficient. The size of the initial droplet is critical in determining the surface area available to analyte species for evaporation and the number of charged analyte species resident within each droplet.

The eluent flow rate combined with the potential applied to the capillary will determine the size and charge of the initial droplet, which in turn will determine the sensitivity and reproducibility of the analysis.[1] Modern electrospray (ESI) sources often employ pneumatic assistance to droplet formation in the form of a nebulising gas that is delivered to the capillary tip via a concentric tube around the interface capillary. The fast flowing gas helps to reduce the droplet size at higher flow rates and makes the droplet formation process more reproducible and less prone to small changes in eluent flow rate.

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The APCI Interface In APCI the eluent is introduced into the interface using a capillary of similar design to the ESI source. However, no potential is applied to the capillary but instead the liquid emerges from the capillary surrounded by a flow of inert, heated, nebulising gas into a heated region. The combination of nebulising gas and heat forms an aerosol that begins to rapidly evaporate. A pin is placed within or just beyond the heated region that has a high potential applied to it and produces an electrical discharge that ionises solvent molecules as shown. Gas phase analyte molecules are subsequently charged by a series of gas phase reactions with the ‘plasma’ of charged solvent molecules.

Liquid flow rate is important here for two main reasons. Firstly, the eluent flow into the heated region will determine the rate of evaporation needed to ensure that all species are in the gas phase as they encounter the discharge pin. Secondly, stable droplet formation is required in terms of droplet size to ensure a stable plasma of ionised solvent molecules exists around the discharge pin. This will ensure the same discharge conditions affect all analyte molecules within the sample. Essentially APCI is a high flow rate technique optimising at flow rates of over 1.0 mL/min, above 0.4 mL/min an unstable analyte signal is usually observed due to the instability of the gas plasma caused by non-reproducible discharge processes. At high flow rates (2-4 mL/min), the heating and drying gas should be adjusted to ensure that all species are in the gas phase as they encounter the discharge region, allowing maximum ion generation.[2,3,4]

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Flow Rates for APCI APCI is a technique that uses high flow-rates (in general 1.0 mL/min or above). The diagram shown below illustrates the relative intensity of the protonated [M+H]+ ion of penicillin G for APCI applications over the flow rate range of 0.1 - 2.0 mL/min.[5] The mobile phase was 50:50 acetonitrile:0.1% formic acid (aq).

Penicillin G ESI/APCI relative intensity versus flow rate The signal intensity increases as the flow rate is raised corresponding to the formation of a stable and reproducible solvent gas plasma that is effective in ionising the analyte molecules present in the discharge region of the source. The actual value of the optimal flow rate for a particular application will depend upon the analyte, the nature of the eluent system and the instrumentation used. To this effect, flow rate must be optimised when analysing compounds not previously studied using LC-MS. Flow Rates for ESI Electrospray is limited to flow rates below 10 μL/min, above this the electrolytic droplet charging process that occurs at the capillary tip is inefficient. As larger droplets are formed at higher flow rates the surface area available for evaporation is decreased. This increases the distance between the charged species at the surface of the droplet leading to inefficient surface charging. It is this parameter that determines the position within the interface housing that the initial coulombic fissions will occur. Large droplets will take longer to reach the point of fission and as such the process of ion production in the interface may not be optimal.

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Penicillin G ESI/APCI relative intensity versus flow rate As can be seen from the diagram the electrospray penicillin G signal optimises at flow rates lower than 0.05 mL/min. The ideal in terms of capillary tip droplet charging is to have a small drop that forms over a relatively long period of time. This will ensure that the droplet contains many charged species and the surface spacing of the charges will cause the initial coloumbic fissions at a point within the source that will allow optimal sampling of gas phase analyte ions. Flow Rates for Pneumatically Assisted ESI The use of high pressure, high flow rate nitrogen from a concentric tube at the capillary tip means that this Pneumatically Assisted ESI optimises at slightly higher flow rates than the non-assisted technique. In general pneumatically assisted ESI optimises at flow rates of around 0.2 mL/min, but flow rates of up to 1.0 mL/min can be endured by the source with a moderate reduction in sensitivity. The diagram below presents a sensitivity reduction of around 40% for Penicillin G [M+H]+ when the eluent flow rate is increased from 0.2 to 1.0 mL/min, this significant reduction can be tolerated in some cases.

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Penicillin G ESI/APCI relative intensity versus flow rate Pneumatically assisted ESI allows the use of conventional pumping equipment with ESI sources and results in the formation of small efficiently charged droplets at conventional LC flow rates. Micro and Nanoflow ESI For applications where sample amounts are limited or for high sensitivity work, micro- and nano-flow ESI interface devices are available. Flow rates of between 100 and 10,000 nL/min are usual with these devices and the samples may be loaded directly into the needle, infused from pressurised vials or introduced via packed silica capillaries when using capillary LC systems. Sensitivities in the range of fempto-moles on column have been reported.[6,7,8]

Capillary HPLC columns (typical flow rates 100 to 10,000nL/min)

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Table 1. Column diameter versus flow rate.

Application Flow rate range Typical column I.D.

Micro LC 10 – 100 μL/min 1 mm

Capillary LC 1 – 10 μL/min 300 μm

Nano LC 0.1 – 1 μL/min 75 μm

The advantages of nano-ESI in comparison to the high-flow rate techniques can be rationalized based on the fundamentals of the ESI process. A lower solution flow rate is known to reduce the size of the charged droplets initially produced in the spraying process. The smaller initial droplets require fewer droplet fission events and less solvent evaporation prior to ion release into the gas phase. As a consequence, a larger portion of the analyte molecules present in the primary droplets is made available for the MS analysis. The alternative to capillary or micro-scale pumping devices is the use of flow splitting, where only a portion of the eluent is sampled by the MS. The needles used can be drawn glass, silica or stainless steel with internal diameters ranging from 5-20 μm for microflow or 1-5 μm for nanoflow work. The potential is applied to these fine capillaries either through the use of metallic coating or using liquid junctions.[9,10]

Nanospray source configured to use conventional capillary columns ended with coated fused-silica tips where ion formation takes place.


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LCMS chip Flow Rate Incompatibility –Reasons For successful mass filtering and analysis the spectrometer system must be under high vacuum. The amounts of vapour produced by spraying conventional LC eluents at traditional flow rates would compromise the MS vacuum system and so there is a fundamental incompatibility between the separation and detection techniques. In order to solve this incompatibility and to avoid the loss of analyte within the LC-MS interface, considerable analyte enrichment has to occur i.e. the analyte must be preferentially transferred to the mass spectrometer relative to the mobile phase constituents that should be removed within the interface.[7] The maximum amount of LC eluent that can be introduced into a vacuum system without a substantial increase in background pressure is limited by the pumping capacity of the vacuum system. Modern spectrometer systems use differentially pumped ion sources whose effective pumping speeds are around 0.3 to 0.7 m3/s. It can be shown that a typical LC eluent at a flow rate of 1mL/min. results in a gas flow of between 0.3 and 2.1 Pa m3/s. depending upon the molecular masses of the solvents used.

Important:

Different manufactures have produced different LCMS chips (in general

no bigger than a normal credit card), they are able to perform not only the

HPLC separation but also the analyte ionisation with optimum results.


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Assuming a pressure of 10-3 Pa is required in the ion source housing, this would mean a maximum flow rate of around 1.8 μL/min of water could be introduced without compromising the level of vacuum within the instrument —about five hundred times less than a conventional 1mL/min. 4.6 mm i.d. LC column flow rate! Flow Rate Incompatibility –Solutions Several solutions to this vapour–vacuum incompatibility are available:

Remove a significant part of the eluent vapour relative to the analyte ions prior to introduction into the MS high vacuum region (i.e. analyte enrichment interface designs such as a mechanical transfer system, molecular beam system or jet separator).

Split and divert part of the column effluent prior to introduction into the LC-MS interface.

Miniaturize the LC column in terms of internal diameter and thus achieve much lower eluent flow rates.

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Mechanical separation devices are widely used as an analyte enrichment tool and to remove a significant portion of the non-ionised eluent vapour, as are devices such as orthogonally sprayed sources. Splitting of the eluent flow and the use of micro-bore and capillary LC systems are both commonly employed techniques and will be discussed further.[11] Flow Splitting –Overview A split is used to reduce the flow of the LC eluent to the MS interface. Typically the splitter will be positioned post analytical column, prior to the MS interface. Post column tees may also be used in the system to introduce reagents to modify the analyte charge state or promote volatilisation of the eluent system.[12]

Post-column splitting Splitting the eluent flow has the added advantage of increasing the time interval between source cleaning to remove deposits of electrolyte and matrix residues. When dealing with eluent systems containing involatile buffers or with dirty sample matrices (i.e. biologically derived samples or environmental samples etc.) the source may need cleaning on a frequent basis –affecting the instrument productivity. The inclusion of a splitter allows the source to remain contaminant free over extended periods of time as there is physically less contaminating material entering the source during each injection.

Flow splitting and split ratio

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Splitting is primarily used with 2.1 and 4.6 mm i.d. columns to keep the flow rate to below 200 μL/min with pneumatically assisted ESI applications. When operating in APCI it is generally not necessary to use a splitting device as flow rates of up 4 mL/min can be tolerated by the source. The magnitude of the split is dependent upon the column i.d., the design of the interface and the sensitivity requirements of the analytical application. Table 2. HPLC column characteristics.[5]

Column I.D Flow rate Volume. Injection volume

4.6mm 1 mL/min 4.1 mL 100 L

2.0mm 0.2 mL/min 19 L 19 L

1.0mm 47 L/min 4.7 L 4.7 L

320m 4.9 L/min 485 nL 485 nL

50m 120 nL/min 12 nL 12 nL

Post-column addition

Important:

With the advent of new HPLC columns that can separate ionic species,

post-column addition is becoming less relevant.


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Flow Splitting –Adjustable Flow Splitters Pre-column splitting is used for micro, capillary, and nano HPLC applications, where the flow from the pump is split from analytical flow rates down to microliter or nanoliter flows. It is important to note that even though the split ratio created by the splitter knob will remain constant, the split ratio will change when a HPLC column is installed. This is due to the added resistance on the low flow rate channel from the HPLC column. This added resistance must be factored in to make sure the fluid resistor selected for the flow splitter provides the correct split ratio.

Micro-splitter Post-column splitting is less used than pre-column splitting. As in pre-column flow splitting, any significant additional pressure (resistance) downstream from the splitter may affect the split ratio. Post-column devices also contribute to chromatographic dispersion so care must be given to connecting tubing and fittings, especially at low flow rates.

Micro-flow system


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Flow Splitting –Practicalities To achieve the correct flow rate or flow volume to the MS, the internal diameter or length of the split PEEK tubing is adjusted. Reducing the internal diameter or lengthening the split tubing will give rise to higher MS capillary flow rates / volumes. It is also possible to purchase adjustable flow spitting devices which are entirely adjustable or switch between pre-programmed split ratios such 10:1, 50:1, 100:1 etc.

Zero Dead Volume (ZDV) Union The magnitude of the split may be determined by measuring the volume of eluent from the split line over a set period of time. This can then be used with the LC flow rate to determine the flow rate into the MS interface. It should be noted that this approach is only valid if the split line and the MS transfer line are constructed from the same material with the same internal diameter. For more accurate work, the same volume measurement procedure may be used with the interface housing removed and the eluent volume from the capillary tip measured over a set time period. This helps to avoid problems that may be encountered with inaccurate LC flow rates or blocked t-pieces and avoids the need to use the same tubing for both the split line and the MS transfer line.

Volume meassurement


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MS Detector Most common detectors (UV, Fluorescence etc.) used within the analytical laboratory are concentration dependant, that is, the detector response is directly proportional to the concentration of analyte in the LC eluent contained within the measurement cell of the detector.

Flow rate independent detector

In theory, the mass spectrometer is a mass-flow sensitive instrument, which means that its response is dependent upon the instantaneous analyte concentration multiplied by the flow rate of the eluent. As long as the interface flow rate and split ratio remain constant (between successive samples or between samples and standards), the difference between concentration and mass flow dependant detectors is not noticed as the response of the instrument is directly proportional to concentration in each case.

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The MS detector is mass flow dependent

It would be expected that signals from experiments with and without split flow would vary, if the detector were mass flow dependent. The split signal should be much lower than the signal due to the non-split experiment as both the flow rate and analyte mass have reduced. However in practice this is not always observed and the signal may actually increase in magnitude if an eluent split is applied to the analytical system. This may be explained by the fact that at low flow rates, droplet formation at the capillary tip takes longer, the charging process is much more efficient and greater numbers of gas phase ions are produced –giving an enhanced signal. Columns –Internal Diameter The flow rate incompatibility can be solved through the use of LC columns with reduced internal diameters as these columns generally have flow rates that can be directly introduced into both APCI and pneumatically assisted ESI sources.[8] The use of micro-bore columns is assumed to be advantageous in terms of the absolute detection limits in LC-MS when eluent splitting is necessary. However, as the mass detection limit increases when using smaller internal diameter columns (these columns have limited load ability), there is no real gain in concentration detection limit.

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Important:

The instrument signal R(t) can be described by the equation:

MSFtCdt

dmtR )()(

Where is the analyte response factor, C(t)is the analyte concentration profile, FMS is the flow rate to the MS.


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Generally, low flow capillary type columns will only be used with electrospray and pneumatically assisted electrospray applications as these flow rates are too low to establish a stable corona discharge in APCI. Table 3. Tipical flow rates used with narrow and micro-bore LC columns.

Column i.d. Typical flow

Compatibility

Pneumatically assisted ESI

APCI

4.6 mm 1.0 mL/min Yes (10:1) Yes



1.0 mm 40 - 50 L/min Yes No

Capillary < 10 L/min Yes No

Using LC Capillary columns, sensitivity increases dramatically, whereas solvent use and disposal decrease. The main advantages include: high efficiency, reproducibility and easy coupling to the MS detector.

HPLC capillary columns

Hopfgartner demonstrated that when using columns with differing internal diameters, the signals yielded by the detector were approximately equal given that the split ratio was adjusted to allow the same eluent flow rate into the interface.[13] Columns –Low Flow Rates As the LC-MS is a flow rate limited detection system, the column internal diameter should be chosen to best suit the interface optimum flow rate range. In this way lower absolute detection limits may be achieved. This phenomenon was investigated by Henion who demonstrated on-column detection limits of 500pg methylparathion on a 4.6mm i.d. column in combination with a split ratio of 991 and of 30 pg for phenothiazine derivatives on a 0.5mm i.d., fused silica, packed LC capillary column without a split.[14,15] For low flow rate applications (below 0.1 mL/min) specialist pumping equipment is required to maintain flow rate, reduce gradient dwell times and ensure low system dead volumes. A useful approximation when converting flow rates from 4.6mm i.d. at 1.0 mL/min. to smaller i.d. columns is: Flow = (new column i.d. / 4.6)2

For example Flow = (0.3 / 4.6)2 = 4.25 L/min. This relationship may be used when estimating the flow rates required for an equivalent separation using reduced internal diameter columns. Capillary LC systems –Overview


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Specialist capillary LC systems have the advantage that the eluent flow rate is suitable for direct introduction into an ESI or Nano-flow ESI source.[16] All systems are characterised by extremely low dead volumes to reduce band broadening and reduce solvent gradient delay volumes. Without a reduction in dead volume any advantage in sensitivity gained will be nullified by the reduction in signal height (due to increased band broadening).

Capillary LC systems are divided into two broad categories:

1. Split flow systems: the eluent that is delivered from the pumping system is split prior to sample introduction (usually based on reciprocating pumps).

2. Direct delivery systems: the eluent volume is directly supplied by the pumping system without any eluent splitting (usually based on syringe type pumps).


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Capillary LC Systems –Practicalities Flow splitting is based on relative backpressures in two flow paths, a sample path and a path to waste designed with a fixed volume restriction. Problems have previously been reported when the backpressure ratio changes due to blocking of the LC column. Similarly, the direct delivery systems need to be extremely well designed in order to overcome problems when forming gradients, eluent components may have to be delivered at low fractions of the total flow (for example 50 nL/min out of a total flow rate of 5 μL/min. Mixing of eluent components in low flow rate systems can be a particular challenge and manufacturers take fundamentally different approaches to the problem. The analytical column is expensive and can be damaged by particulate material depositing at the head of the column, as well as by attack of the eluent on the packing itself. Eluents with pH outside the 2 to 7 range may dissolve the silica support of the column. To lengthen the useful column life Filters, Guard columns and Precolumns can be used. Generally there are two different methods for forming gradients at low and high pressures, see below.

Definitions:

Filters: A major cause of column deterioration and damage is the buildup of particulate and

chemical contamination at the head of the column. This can lead to increased back

pressure and anomalous chromatographic results. Columns normally contain stainless steel

inlet and outlet filters or frits to retain the column packing. The pore size of the frit must be

smaller than the particle diameter of the packing, e.g., a 2 μm frit for 5 μm packing.

Guard columns: A guard column is inserted between the injector and analytical

column to protect the latter from damage or loss of efficiency due to the presence of

particulate matter or strongly adsorbed impurities from analytical samples.

Precolumns: Precolumns are short segments of tubing packed roughly with similar

material to that used in the column. Their purpose is to saturate the mobile phase

with silica so that the silica or bonded silica analytical column packing is not

dissolved during use.


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High-pressure mixing gradient elution chromatographic instrumentation

High-pressure mixing gradient:

The flow from two high pressure pumps is directed into a high pressure-mixing chamber. The first pump reservoir contains a weak eluent while the second contains the stronger eluent. The different eluents are mixed in the chamber before the mobile phase flows through the injection valve and then onto the analytical column. The total flow from the two pumps remains constant, and controlling the relative pump rates forms the gradient. The gradient commences with a high flow of the weak eluent pump and a low flow of the strong eluent pump.


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Low-pressure mixing gradient elution chromatographic instrumentation

Low-pressure mixing gradient:

At low pressure the gradients are formed from the combinations of the different solvents by metering a defined amount from the various eluent reservoirs into the pump. The composition in the low pressure mixing chamber is controlled by timed proportioning valves, only one of which is open at given time. The gradient is formed by the relative time that the valves are open; at the start of the chromatographic run, the valve connected to the weak eluent is open the longest time. As the gradient progresses, the valve attached to the strong eluent is open for longer and longer times, while conversely the weak eluent valve is open shorter times. Often the gradients are formed using just two of the valves, allowing the others to be used for cleaning solutions.


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Column Selection Column selection is not a simple process. The best approach is to search for work published on a separation that is the same as, or similar to the one that needs to be performed. Knowledge of the physicochemical properties of the sample, combined with a systematic trial and error approach, is probably the method used most often in column selection. The choice of LC system, analytical column and the flow rate employed is very much governed by both the application and the interface type being used. Where flow rates of 1.0 mL/min. or above are needed (due to limited LC equipment availability), the use of reduced dimension LC columns below 1 mm i.d. may be problematical due to the excessive backpressures created. Conversely, when the need to operate at flow rates of 0.1 mL/min or below is important, 4.6 mm i.d. columns cannot be used due to excessive retention and band broadening of the analyte. When a high column loading of sample is required, a larger i.d. column needs to be chosen. The flow diagram shown should assist in obtaining the correct LC configuration.

Column selection


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References 1. J. Abian. “The Coupling of Gas and Liquid Chromatography with Mass Spectrometry.” J. Mass Spectrom. 34, 15-168 (1999) 2. www.waters.com 3. R. D. Voyksner in Electrospray Inization Mass Sectrometry. R. B Cole. John Wiley and Sons,Inc 1997. 4. A. L. Burlingame, S. C. Hall, D. A. Maltby, J. P. Salzmann, C. J. Hughes, T. Hutton McKenna, S. A. Jarvis, A. E. Ashcroft, and E. Kapp. Micromass Application Note. 223, (1996) 5. R. D. Voyksner in Electrospray Inization Mass Sectrometry. R. B Cole. John Wiley and Sons,Inc 1997. 6. A. L. Burlingame, S. C. Hall, D. A. Maltby, J. P. Salzmann, C. J. Hughes, T. Hutton McKenna, S. A. Jarvis, A. E. Ashcroft, and E. Kapp. Micromass Application Note. 223, (1996) 7. I. C. Mylchreest, M. E. Hail and J. R. Herron. United States Patent 5, 157, 260, October 20, 1992. 8. J. Abian, A. J. Oosterkamp and E. Gelpï. Journal of Mass Spectrometry. J. Mass Spectrom. 34, 244-254 (1999) 9. www.dionex.com 10. Pat Sandra, Gerd Vanhoenacker, Fréderic Lynen, Liu Li and Marc Schelfaut. “Considerations on Column Selection and Operating Conditions for LC–MS.” Agilent Technologies Dec 2001. 11. Michael Pickering. “An Over view of Post-column Derivatisation Methods from a Pharmaceutical Applications Perspective.” Pharma Outsourcing 2005, 1-5 12. K. B. Tomer, M. A. Moseley, L. J. Deterding and C. E. Parker. Mass Spectrom. Rev. 13, 431 (1994). 13. G. Hopfgartner, K. Bean, J. D. Henion, and R. A. Henry. J. Chromatogr. 647, (1993), 51. 14. J. D. Henion and G. A. Maylin. Biomed. Mass Spectrom. 7, (1980), 115. 15. J. D. Henion. Anal. Chem. 50, (1978), 1687. 16. DIONEX. LCMS Tools.

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