Liquid Chromatogaphy Mass Spectrometry (LCMS)

Take Home Point:

Separation and measurement of unknown volatile and non-volatile components  

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What It Provides:

Chromatogram (intensity versus retention time) showing the separation of volatile and non-volatile components within a sample.  The intensity is proportional to the amount of each component within the sample allowing the technique to be quantitative with proper calibration.   Thus, it can provide a list of the organic compounds present in a mixture and their amount.      

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Brief Description:

 

Separation of Volatile and Non-Volatile Mixtures

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LCMS is a subset of HPLC that uses MS for detection.  The HPLC portion of the instrument separates the mixture components while the MS portion identifies the separated species by producing a mass spectrum of each and comparing it against a library of known compounds.  Concentrations of the detected species can also be determined by comparing against appropriate standards.   In addition to analyzing volatile/semi-volatile species, it can also analyze non-volatile compounds which GCMS can't.  LCMS is a much better technique for looking at unknowns than conventional HPLC.  Samples can be liquids or solids (must be dissolved first in a solvent).  

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In LCMS the liquid sample is injected into a flow of pressurized liquid (i.e. the mobile phase) and proceeds down a column packed with an appropriate adsorbent (i.e. the stationary phase).  As the sample passes through the column, the various compounds within the sample 'partition' between the mobile and stationary phases.  Those compounds having greater preference for the mobile phase will travel through the column faster than those with a greater affinity for the stationary phase allowing their separation.  The analytes exit the column and enter into an Atmospheric Pressure Ion Source (API) where they are ionized and/or desolvated (i.e. the solvent/eluent is separated from the analyte).  There are three common APIs.  

 

1.  Electrospray Ionization (ESI):  In this API the analyte is already an ion prior to entering the API by controlling the pH of the eluent.  The eluent/analyte mixture flows into an inlet capillary with a voltage applied between it and the sampling cone leading into the MS.  This voltage drop coupled with a nebulizing gas results in the eluent/analyte forming droplets from the inlet capillary that are accelerated towards the sampling cone.  (Note some sources claim that ionization occurs because of this voltage drop rather than from the eluent pH in the HPLC portion of the instrument.)  A heated drying gas (typically N2) flows in a counter direction desolvating the eluent from the analyte ions.  The analyte ions then enter the MS.   ESI is particularly good for large biomolecules, samples that are ions in solutions and ones that contain heteroatoms.  

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2.  Atmospheric Pressure Chemical Ionization (APCI):  Here the eluent/analyte mixture flows into an inlet capillary that has no charge applied.  It exits the capillary and is subjected to a N2 nebulizing gas which forms it into an aerosol.  A heated dissociation gas (N2) is used to desolvate the eluent from the analyte.  A Corona Electrode Pin with a high potential then ionizes the eluent molecules which can transfer charge to the analyte molecules.  The analyte ions are extracted into the sampling cone of the MS.  APCI is good for small polar to non-polar molecules and ones that contain heteroatoms.   

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3.  Atmospheric Pressure Photo Ionization (APPI):  APPI is less commonly used and is considered complementary to ESI and APCI.  The setup is similar to that of APCI except that ionization occurs via UV photons emitted from a discharge lamp.  APPI is good for non-polar compounds that can't be ionized by ESI or APCI.   

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All three APIs are considered 'soft ionization' techniques as little if any fragmentation occurs (i.e. only psuedo molecular ions are observed).  Note that many commercial LCMS systems are equipped with both ESI and APCI that can be utilized simultaneously. 

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Following the API, the analyte ions are extracted into the MS for mass separation.  Like the ASIs, multiple MS systems are commonly used for LCMS.  The most common are as follows:

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1.  Quadrupole:  Ion separation is achieved by varying the electric fields between 4 parallel equidistant rods (poles) as the ions traverse them.  This MS is inexpensive and reproducible but has low mass resolution.  

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2.  Time-of-Flight:  Ions enter a field free tube where ions are separated as their speed down the tube is controlled by their mass and charge.  This MS is very sensitive and has high mass resolution.

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3.  Ion Trap:  Ions of a chosen mass to charge ratio are 'trapped' in a cell by using applied DC and RF fields.  Inert gas can be added to facilitate fragmentation to obtain structural information.  This MS is also very sensitive.

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4.  MS/MS:  This set-up combines two (or more) mass spectrometers (most commonly quadrupoles)  with a collision cell in between for fragmenting ions.  This fragmentation set-up provides structural information allowing better identification of unknown samples or confirmation of larger molecules.  

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Following the MS, the separated ions are counted via a detector (e.g. Electron Multiplier, Dynode, Photodiode, Multi-Channel Plate).   

The time it takes for a compound to traverse the system is termed the 'Retention Time'.      

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