LCMSHPLC+MS(ESI)

Purpose HPLCFor analysis of non- volatile analytes & Separation of analytes based on type of column:
e.g relative affinity for stationary phase (column) vs mobile phase.

Purpose MSSeparation of gaseous ions separated based on mass/charge ratio

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 Electro
spray ionization (ESI) is an ionization technique for small amounts of large and/or labile (unstable/
reactive) molecules such as peptides, proteins, organometallics, and polymers.

The most obvious feature of an ESI spectrum is that the ions carry multiple charges which reduces their
mass-to-charge ratio compared to a singly charged species.

Step 1: Analyte in solvent is introduced to the source from liquid chromatography.

Step 2: Analyte solution flows passes through the electrospray needle that has a high potential
difference, approximately 3000V.

Step 3: The strong voltage in the tip charges the molecule in the solvent with the same charge.

Step 4: The charged liquid is repelled from the needle and the droplets burst away from each other into
a fine spray.

Step 5: With the assistance of nitrogen gas pumped into the chamber, solvent evaporation occurs.

Step 6: The droplet shrinks and the charge density in droplet increases until it reaches the point where
the surface tension can no longer sustain the charge. This is called Rayleigh limit -The points at which
the surface charge of the droplet overcomes the surface tension of the droplet leading to ion
evaporation or columbic explosion.

Step 7: The droplet 'explodes‘, creating many smaller, more stable droplets. The new droplets undergo
desolvation and subsequently further Coulomb fissions.
Step 8: This process repeats itself until the solvent is completely evaporated and the droplets have split
up such that each is a single, charged molecule.

Step 9: The naked charged molecules which are free from solvent are detected by Mass Spectrometer.
Peak resolution in LC-MS (liquid chromatography-mass spectrometry) refers to the ability of the LC-MS
system to separate or resolve two adjacent peaks in a chromatogram or mass spectrum. Peak resolution
is typically calculated as the ratio of the distance between the centers of two adjacent peaks to the
average width of the peaks at their half-heights. A higher peak resolution indicates a better separation
between the two adjacent peaks, while a lower peak resolution indicates poor separation.

(Benefits)

One of the advantages of this ionization method is that the molecules remain intact and will not be
broken apart, and very little fragmentation will occur. Hence ESI is a so-called soft ionization technique.

The major advantage of LC-MS is that it is the most robust analytical technique that provides higher
sensitivity and selectivity required to detect an exact molecular weight of a wide range of samples.

It can separate and identify solutes in low concentrations (which are in parts per million- PPM) in a
complex mixture.

(Downside)

Phosphate buffer is not compatible with the LC-MS analysis, which is the most commonly used buffer in
HPLC method development.

The LC-MS has a high maintenance cost as compared with other analytical instruments. LC-MS is an
expensive technique both in terms of capital and analysis costs.

To get around the volatility issue, Liquid chromatography mass spectrometry (LC-MS) has increased in
popularity. One limitation with LC-MS for identification purposes is that the ionization techniques
compatible with LC-MS will produce very little fragmentation, which is useful for determining its
molecular weight, but does not provide any structural information on the molecules.