Two-dimensional chromatography

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. (For instance, a C18 reversed-phase chromatography column may be followed by a phenyl column.) Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents. Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. (For instance, a C18 reversed-phase chromatography column may be followed by a phenyl column.) Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents. Modern two-dimensional chromatographic techniques are based on the results of the early developments of Paper chromatography and Thin-layer chromatography which involved liquid mobile phases and solid stationary phases. These techniques would later generate modern Gas chromatography and Liquid chromatography analysis. Different combinations of one dimensional GC and LC produced the analytical chromatographic technique that is known as two-dimensional chromatography. The earliest form of 2D-chromatography came in the form of a multi-step TLC separation in which a thin sheet of cellulose is used first with one solvent in one direction, then, after the paper has been dried, another solvent is run in a direction at right angles to the first. This methodology first appeared in the literature with a 1944 publication by A. J. P. Martin and coworkers detailing an efficient method for separating amino acids- “...but the two-dimensional chromatogram is especially convenient, in that it shows at a glance information that can be gained otherwise only as the result of numerous experiments” (Biochem J., 1944, 38, 224). Two-dimensional separations can be carried out in gas chromatography or liquid chromatography. Various different coupling strategies have been developed to 'resample' from the first column into the second. Some important hardware for two-dimensional separations are Deans' switch and Modulator, which selectively transfer the first dimension eluent to second dimension column The chief advantage of two-dimensional techniques is that they offer a large increase in peak capacity, without requiring extremely efficient separations in either column. (For instance, if the first column offers a peak capacity (k1)of 100 for a 10-minute separation, and the second column offers a peak capacity of 5 (k2) in a 5-second separation, then the combined peak capacity may approach k1 × k2=500, with the total separation time still ~ 10 minutes). 2D separations have been applied to the analysis of gasoline and other petroleum mixtures, and more recently to protein mixtures. Tandem mass spectrometry (Tandem MS or MS/MS) uses two mass analyzers in sequence to separate more complex mixtures of analytes. The advantage of tandem MS is that it can be much faster than other two-dimensional methods, with times ranging from milliseconds to seconds. Because there is no dilution with solvents in MS, there is less probability of interference, so tandem MS can be more sensitive and have a higher signal-to-noise ratio compared to other two-dimensional methods. The main disadvantage associated with tandem MS is the high cost of the instrumentation needed. Prices can range from $500,000 to over $1 million. Many form of tandem MS involve a mass selection step and a fragmentation step. The first mass analyzer can be programmed to only pass molecules of a specific mass-to-charge ratio. Then the second mass analyzer can fragment the molecule to determine its identity. This can be especially useful for separating molecules of the same mass (i.e. proteins of the same mass or molecular isomers). Different types of mass analyzers can be coupled to achieve varying effects. One example would be a TOF-Quadrople system. Ions can be sequentially fragmented and/or analyzed in a quadrupole as they leave the TOF in order of increasing m/z. Another prevalent tandem mass spectrometer is the quardupole-quadrupole-quadrupole (Q-Q-Q) analyzer. The first quadrupole separates by mass, collisions take place in the second quadruple, and the fragments are separated by mass in the third quadrupole. Tandem MS has been gaining popularity and relevance as analytical techniques are becoming more and more precise. This is an active area of research by many analytical chemists around the world, including David E. Clemmer of Indiana University, who is in the forefront of mass spectrometer instrumentation. Gas chromatography-mass spectrometry (GC-MS) is a two-dimensional chromatography technique that combines the separation technique of gas chromatography with the identification technique of mass spectrometry. GC-MS is the single most important analytical tool for the analysis of volatile and semi-volatile organic compounds in complex mixtures. It works by first injecting the sample into the GC inlet where it is vaporized and pushed through a column by a carrier gas, typically helium. The analytes in the sample are separated based upon their interaction with the coating of the column, or the stationary phase, and the carrier gas, or the mobile phase. The compounds eluted from the column are converted into ions via electron impact (EI) or chemical ionization (CI) before traveling through the mass analyzer. The mass analyzer serves to separate the ions on a mass-to-charge basis. Popular choices perform the same function but differ in the way that they accomplish the separation. The analyzers typically used with GC-MS are the time-of-flight mass analyzer and the quadrupole mass analyzer. After leaving the mass analyzer, the analytes reach the detector and produce a signal that is read by a computer and used to create a gas chromatogram and mass spectrum. Sometimes GC-MS utilizes two gas chromatographers in particularly complex samples to obtain considerable separation power and be able to unambiguously assign the specific species to the appropriate peaks in a technique known as GCxGC-(MS). Ultimately, GC-MS is a technique utilized in many analytical laboratories and is a very effective and adaptable analytical tool. Liquid chromatography-mass spectrometry (LC/MS) couples high resolution chromatographic separation with MS detection.  As the system adopts the high separation of HPLC, analytes which are in the liquid mobile phase are often ionized by various soft ionization methods including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), which attains the gas phase ionization required for the coupling with MS. These ionization methods allow the analysis of a wider range of biological molecules, including those with larger masses, thermally unstable or nonvolatile compounds where GC-MS is typically incapable of analyzing.