INTERFACES FOR LC-MS

INTRODUCTION:
- The LC-MS appears to be an obvious choice.
- LC is preferred over GC because of the higher polarity and lower volatility of the sample. Interfaces in LC-MS: 1.Partical beam (PB), 2.Moving belt/wire, 3.Conti. Flow fast atom bombardment (cf-FAB),

4.Direct liquid introduction (DLI),

5.Thermospray (TSP), 6.Atmospheric pressure chemical ionization (APCI) 7. Electrospray (ESP)

1. Direct liquid introduction (DLI):

The DLI interface was developed in order to solve the problems with incapillary evaporation in the capillary inlet.
In a DLI interface, the column effluent is nebulized by the disintegration into small droplets of a liquid jet formed at a small diaphragm (a 2-5m ID pinhole or diaphragm, laser-drilled or electro-etched in 30-100 m thick nickel or stainless-steel plate). After desolvation of the droplets in a desolvation chamber, the analytes can be analysed using solventmediated CI with the LC solvents as the reagent gas.

Advantages: No heat is applied to the interface and it is therefore able to deal with thermally labile materials better than the moving-belt interface. The interface contains no moving parts and is cheap and simple to construct and operate and is inherently more reliable than the moving-belt interface. Both positive- and negative-ion CI spectra can be generated and the interface provides molecular weight information, plus it can also be used for sensitive quantitative and semi-quantitative procedures. Disadvantages: Involatile compounds are not usually ionized with good efficiency. The pinhole is prone to blockage and therefore the system must be kept completely free of solid materials. Only a small proportion of the flow from a conventional HPLC column is able to enter the source of the mass spectrometer and sensitivity is consequently low. Ionization is brought about by CI-like processes and structural information is therefore limited unless a mass spectrometer system capable of MSMS is used.

2. Moving belt/wire:

In a moving-belt interface (MBI), the column effluent is deposited onto an endless moving belt from which the solvent is evaporated by means of gentle heating and efficiently exhausting the solvent vapours. After removal of the solvents, the analyte molecules are (thermally) desorbed from the belt into the ion source and mass analysed.
The MBI for LC.MS was used in a wide variety of applications, including the analysis of drugs and their metabolites, pesticides, steroids, alkaloids, polycyclic, aromatic hydrocarbons and others.

Advantages:

The interface can be used with a wide range of HPLC conditions, flow rates and mobile phases, both normal and reverse phase, particularly if spray deposition is employed.
The analyst does have some choice of the ionization method to be used; EI, CI and FAB are available, subject to certain limitations, and thus both molecular weight and structural information may be obtained from the analyte(s) under investigation. Disadvantages:

An intense chemical background from the material from which the belt was made is often observed in the mass spectra generated by this type of interface unless adequate conditioning is carried out.
The belt is prone to break during operation.

Problems may be encountered in the analysis of thermally labile compounds,

as heat is required for mobile-phase removal and for the transfer of analyte from the belt into the source of the mass spectrometer, and highly involatile compounds which cannot be desorbed from the belt, unless FAB is used for ionization.

 Mobile phases containing high proportions of water often give droplets on the belt rather than an even film and this may produce an erratic TIC trace and irreproducible mass spectra. Memory effects are often experienced, even with the use of a clean-up heater and a wash-bath.

Surface effects can reduce detection limits.

The use of involatile buffers must be avoided in order to prevent build-up of material on the belt and its jamming in the tunnel seals. This type of interface is not easy to construct and therefore reliance has to be

put on commercial manufacturers.

3. Thermospray (TSP): They are of 2 type: a) Real-TSP ionization

b) Discharge electrode for external ionization and repeller electrode

In a thermospray (TSP) interface, a jet of vapour and small droplets is formed out of a heated vaporizer tube into a low-pressure region. Nebulization takes place as a result of the disruption of the liquid by the expanding vapour that is formed upon evaporation of part of the liquid in the tube. Before the onset of the partial inside-tube evaporation a considerable amount of heat is transferred to the solvent, which assists in the desolvation of the droplets in the low pressure region. By applying efficient pumping directly at the ion source up to 2 ml/min of aqueous solvents can be introduced into the MS vacuum system. The ionization of the analytes takes place by means of ionmolecule reactions and ion evaporation processes. The CI reagent gas can be made either in a conventional way using energetic electrons from a filament or a discharge electrode, or in a process called TSP ionization, where the volatile buffer dissolved in the eluent is involved. In the filament-on mode, high-energy electrons (0.4-1.0 keV) emitted from a heated filament are accelerated into the ion source. In the discharge-on mode, a continuous gas discharge is used to generate electrons. Solvent-mediated CI spectra are obtained in both filament-on and discharge-on mode.

Advantages:

The interface was much easier to use at flow rate of 1ml/min and mobile phases containing high percentages of water .
Can allow unequivocal determination of molecular weight. Can be used to study thermo liable compounds.

High sensitivity.
Disadvantages: The mobile phase used should be volatile. Decomposition of some thermally labile analytes is observed. The interface is not suitable for high-molecular-weight (>1000 Da) analytes. The reproducibility of analytical results is affected by a number of experimental parameters and is sometimes difficult to control.

The formation of adducts may confuse the assignment of molecular weight.

Difficult to interpret the ionic species generated after repeller-induced fragments.

4. Partical beam (PB):

During the desolvation of the droplets, the less volatile analyte molecules coagulate into small particles, typically 50-300nm. The solvent vapour and nebulization gas are separated from the particles by means of molecular-beam technology. The mixture is expanded at a nozzle into a vacuum chamber. The low mass solvent molecules show a greater tendency to diffuse away from the centre of the expansion, while the heavier analyte particles are kept in the core of the vapour jet. The core of the jet is then sampled by a skimmer. The region between the first set of orifices is maintained at between 2 and 10 torr, and that between the second set between 0.1 and 1 torr. In the momentum separator, sufficient pressure reduction is achieved to generate EI and solventindependent CI mass spectra.

Advantages:

A number of thermally labile and relatively involatile compounds which do not yield EI spectra when using more conventional inlet methods do so when introduced via the particle-beam interface.
Disadvantages:

The sensitivity of the particle-beam interface is dependent not only on the specific analyte but also on the experimental conditions employed. Detection limits are invariably higher than are desirable.
Neither extremely volatile or extremely involatile compounds are ideal for investigation using the particle-beam interface. The performance of the particle-beam interface deteriorates as the percentage of water in the HPLC mobile phase increases.

5. Atmospheric pressure chemical ionization (APCI)

Here, the HPLC effluent is passed through a pneumatic nebulizer where the droplets are both generated and desolvated. The spray so formed then passes through a heated region where the vapour is dried. The neutral species thus produced are then passed through a corona discharge the latter occurs when the field at the tip of the electrode is sufficiently high to ionize the gas surrounding it but insufficiently high to cause spark where ionization of the analyte is effected by CI-type processes with the vaporized solvent acting as the reagent gas. The technique is capable of dealing with flow rates between 0.5 - 2 ml/min, so making it directly compatible with 4.6 mm columns, and is much more tolerant to a range of buffers.

Advantages: Applicable to study thermo liable compounds without degradation. It is best applied to compounds with low to moderately high polarities. APCI is a soft ionization technique which usually enables the molecular weight of the analyte under study to be determined. Disadvantages: APCI spectra can contain ions from adducts of the analyte with the HPLC mobile phase or organic modifiers, such as ammonium acetate, that may be present. The presence of ions such as (M + NH4)+ and (M + CH3COO) may hinder interpretation of the spectra obtained. Structural information is not usually available unless cone-voltage fragmentation or MSMS is used. APCI is not able to function effectively at very low flow rates. APCI is not suitable for analytes that are charged in solution.

6. Conti. Flow fast atom bombardment (cf-FAB)

In a continuous-flow fast-atom bombardment (Cf-FAB) interface, typically a 5-15 l/min liquid stream, mixed with 5% glycerol (or) thioglycerol (or) nitrobenzyl alcohol as FAB matrix, flows through a narrow-bore fused-silica capillary towards either a stainless-steel frit or a (goldplated) FAB target. At the target or frit, a uniform liquid film is formed due to a subtle balance between solvent evaporation and solvent delivery. Ions are generated by bombardment of the liquid film by fast atoms or ions, common to FAB.
The fast atom like Ar or Xe is used for bombarding the sample and ions are sputtered out of the solution and enters into MS.

Advantages:

Can study thermally labile materials, since the only heat applied to the probe tip is that required to prevent freezing of the mobile phase as it evaporates.
Due to low flow rates used with microbore, it produce good sensitivity small amounts of sample.

The interface is simple in design and relatively easy to construct.

Mobile phases with a high percentage of water may be used. Disadvantages: The presence of the matrix can cause chromatographic problems if added to the mobile phase before the column, especially if this is of small diameter. The low flow rates that are used require an increased concentration of matrix to be present in the mobile phase to ensure an appropriate amount reaches the probe tip. If conventional HPLC columns are used, with splitting of the eluate to provide the necessary flow rate, an overall decrease in sensitivity usually results.

7. Electrospray (ESP):

 ESI nebulization involves a variety of electrochemical processes at the needle

and at the counter electrode. The ESI interface can be considered as a electrochemical cell, in which part of the ion transport takes place through the gas phase.
In positive-ion mode, an enrichment of positive electrolyte ions occurs at the solution meniscus as the result of an electrophoretic charge separation. Electrospray spectra are produced by passing a liquid stream through a metal capillary maintained at high voltage (typically 34 kV for the production of positive ions; slightly less, and of opposite polarity, for the production of negative ions). The liquid meniscus is pulled into a cone which emits a fine mist of droplets with an excess positive charge. Charge balance is attained by electrochemical oxidation at the capillary tip and reduction at the counter electrode. This high voltage disperses the liquid stream, forming a mist of highly charged droplets that undergo desolvation during their passage across the source of the mass spectrometer. As the size of the droplet reduces, a point is reached (within 100 s) at which the repulsive forces between charges on the surface of the droplets are sufficient to overcome the cohesive forces of surface tension.

Advantages:

Ionization occurs directly from solution and consequently allows ionic and thermally labile compounds to be studied.
Allows to study of molecules with higher molecular weights Disadvantages: Electrospray is not applicable to non-polar or low-polarity compounds. The mass spectrum produced from an analyte, in terms of the m/z range of the ions observed and their relative intensities, depends upon a number of factors and spectra obtained using different experimental conditions may therefore differ considerably in appearance. Suppression effects may be observed and the direct analysis of mixtures is not always possible. Electrospray is a soft-ionization method producing intact molecular species and structural information is not usually available.

APPLICATIONS OF LC-MS:

-In Environmental,
a) Pesticides: Eg: Triazine derivatives, Chlorophenols, Phenoxy alkanoic acids, Sulfonyl urea, etc., b) Polycyclic Aromatic compounds,

c) Organometallic compounds: Eg: Lead in gasoline, arsenic compounds, di- & tributyltin chlorides in marin paints, etc.,
-In Biomedical, a) Amino acids, nucleosides (in human urine) and nucleotides (in plants tissues) , saccharides, peptides & protiens, Steroids, etc., b) Pharmaceutical: Eg: Antibiotics; Detromethorphan and Metaprolol enantiomers in plasma, diphenhydramine, doxylamine & carbinoxamine, Sulphanamides in samples. c) Drug metabolites: Rantidine, Betamethasone, Doxylamine and Almitrine in body fluids. - In Natural products: Lipids, Alkaloids, Fatty acids.