High Performance Liquid Chromatography and Gas Chromatography Coupling With Mass Spectrometry
April 30, 2009
Although chromatography is useful for separating complex mixtures, many non-routine constituents of a sample can not be readily identified or characterized from the chromatogram alone. While mass spectrometry (MS) is capable of identifying enumerable compounds, discerning them from a complex mixture can be difficult or impossible. Direct physical coupling of high performance liquid chromatography (HPLC) and gas chromatography (GC) to MS allows each instrument to compensate for the others weakness. Efforts to couple MS and chromatography began with GC, which was successfully coupled with MS in 1955 when a homemade gas chromatography column was linked to a time-of-flight mass spectrometer, thus allowing the MS to analyze the effluent as it exited the GC column.1 For this GC/MS, depicted in Figure 1, a small portion, about 0.3%, was directed into the MS before entering the thermal conductivity cell.1 The mass spectrum of effluent material was displayed on an oscilloscope at 2000 scans per minute and recorded using a Polaroid camera.2
The primary issue facing developers in the endeavor to create an interface between the two instruments was pressure. GC columns at the time were packed and ran at atmospheric pressure, while MS requires pressures of 10-6 to 10-5 torr.3 Several interfaces have been developed, such as pressure reduction systems and jet separators.4 Jet separators can been utilized to separate the carrier gas from the sample using variation is the diffusibility of the carrier gas and the sample.3
The advent of fused silica capillary GC columns eliminated the need for complex interfaces for most applications of GC/MS, as it solved the issues of pressure reduction and flow rate. Unlike the first GC/MS, in which only a fraction of the analyte was diverted to the MS, the majority of today’s instruments allow for all of the analyte to be sent to directly to the MS for analysis, as depicted in Figure 2.3 The sample can be ionized directly in the gas phase either by electron ionization (EI) or chemical ionization (CI). During EI, the sample is bombarded with electrons as it enters the MS, forming free radicals that can be separated on the basis of their mass to charge ratio (m/z) by a strong magnetic field. During EI, a constituent of the solvent serves as a reaction intermediate that leads to the formation of radical species.10
The development of an interface for HPLC and MS did not turn out to be so serendipitous. HPLC flow rates are many times more than an MS can accommodate, operate at atmospheric pressure, and is generally employed for the analysis of compounds to thermally and kinetically fragile for typical GC temperatures and pressures.7 Direct ionization of compounds for MS requires that they be gaseous, a phase at which most analytes necessitating HPLC decompose. Numerous interfaces have been developed including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), moving belt interfaces, and the thermospray technique.11 Moving belt interfaces, depicted in Figure 3, deposit HPLC effluent onto a belt, which mechanically transports it through a series of vacuums, to evaporate the solvent, and eventually carry the analyte to the ion source of the MS.11 Thermospray involves direct vaporization of sample as it exits the HPLC column and enters the source.9 Moving belt interfaces are generally inaccurate, though, and thermospray comprises samples that, due to their sensitivity, may not have survived vaporization anyway.
Altering the sample, through vaporization or evaporation is clearly not ideal, as it reduces the advantageous range of applications of HPLC/MS and compromises its quantitative power. APCI and ESI represent interfaces that form analyte ions directly from the solvent.9 During ESI, depicted in Figure 4, effluent exits the column and enters an electrospray needle, from which it is sprayed as a fine mist called an aerosol. A strong electrical field, beginning with the needle, spans the region through which the aerosol travels. This causes a concentration of positive or negative solvent ions in the aerosol particles. As the solvent evaporate, Columbic forces increase, increasing the rate of solvent dissociation. Analyte ions can then be separated by the MS.8 During this process, radical derivatives of analyte are not being formed by this process, as they are during EI or CI. Instead, the compounds of interested are introduced as ionized species, in addition to come components of the solvent being ionized, into the solvent before chromatographic separation occurs. The electrical field of the ion source acts upon the solvent ions, concentrating positive or negative charge aerosol as it exists he electrospray needle.10
During APCI, depicted in Figure 5, effluent is mixed with a nebulizing gas, such as N2, as it exits the column, is vaporized, and then emitted as an aerosol. A corona needle introduces electrons into the aerosol as it transverses the ion source, which initiates a series of gas phase chemical reactions that lead to formation of cationic and anionic analyte species. Like ESI, radical analyte derivatives are not being formed. The introduction of elections leads to the formation or positive and negative ions, such as H+ or OH– from water, which associate with the analyte and impart upon it a charge.10
Despite the more complex nature of the HPLC/MS interface, it does have some advantages over GC/MS, including the potential analysis of a much broader category of compounds, including proteins and other biological polymers. However, HPLC/MS comes with the additional expense of large volumes of waste solvent, a decrease in resolution and sample senestivity, and an increase in retention times. While HPLC/MS requires a general minimum of 100ng or analyte of interest per injection, GC/MS requires between 0.1 and 100ng of anylate per injection. If GC/MS is feasible, it is preferred over HPLC/MS because of its cheaper costs, shorter analysis time, and better resolution.10
1. Gohlke, R., McLafferty, F., Early gas chromatography/mass spectrometry,
Journal of the American Society for Mass Spectrometry
2. Gohlke, R. S. Anal. Chem. 1959, 31,535-541.
3. Hites. R, Gas Chromatography Gas Spectroscopy, Chapter 31, acquired from http://www.prenhall.com/settle/chapters/ch31.pdf on April 25, 2009
4. J. T. Watson and K. Biemann, Analytical Chemistry, 37 (1965), 844–51.
5. R. Ryhage, Analytical Chemistry, 36 (1964), 759–64.
6. T. E. Jensen and others, Analytical Chemistry, 54 (1982), 2388–90.
7. Vestel, M., October 19, 1984, High-performance liquid chromatography-mass spectrometry, Science, p275(7) Vol. V226 ISSN: 0036-8075
8. Pramanik, B., Ganguly, A., Gross M., 2002, Applied electrospray mass spectrometry
9. University of Bristol Life Science Mass Spectrometry Facility, aquired from http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html on April 25, 2009
10. Harris, D., 2006, Quantitative chemical analysis
11. Giddings, J., Keller, R., Cazes, J., 1998, Advances in chromatography