Archive for the ‘Kromatografi’ Category
If you’ve had your laboratory run low-level polyaromatic hydrocarbons (PAHs) or other low level analyses, chances are you have heard of Gas Chromatography/Mass Spectroscopy- Selective Ion Monitoring (GC/MS-SIM). Over the years clients have asked us “What’s the difference between GC/MS-Full Scan and GC/MS-SIM?” To address this question we must start with the basics. (For our example we will be talking about a standard quadrupole mass spectrometer using electron ionization.)
GC/MS is an instrumental analytical technique comprised of a gas chromatograph and a mass spectrometer. In general, the GC is used to separate complex chemical mixtures into individual components. Once separated, the chemicals can be identified and quantified by the mass spectrometer.
Before analysis can occur a sample must be prepared, usually by extracting the analytes of interest into a liquid solvent phase. This extract is then injected into the GC where it is swept onto a separation column by an inert carrier gas such as hydrogen or helium. The analytes in the mixture are carried through the column by the carrier gas where they are separated from one another by their interaction between the coating (stationary phase) on the inside wall of the column and the carrier gas. Each analyte interacts with the stationary phase at different rates. Those that react very little move through the column quickly and will exit into the mass spectrometer before those analytes having longer interaction and retention times.
When the individual analytes exit the GC column they enter the ionization area (ion source) of the MS. Here they are bombarded with electrons which form ionized fragments of the analyte. These ionized fragments are then accelerated into the quadrapole via a series of lenses and separated based on their mass to charge ratio. This separation is accomplished by applying alternating RF frequency and DC voltage to diagonally opposite ends of the quadrapole, which in turn allows a specific mass fragment to pass through the quadrapole filter. From here the fragments enter the mass detector (electron multiplier) and are recorded. The MS computer graphs a mass spectrum scan showing the abundance of each ionized mass fragment.
A GC/MS system in Full Scan mode will monitor a range of masses know as mass to charge ratio (abbreviated m/z). A typical mass scan range will cover from 35-500 m/z four times per second and will detect compound fragments within that range over a set time period. Laboratories have extensive computer libraries containing mass-spectra of many different compounds to compare to the unknown analyte spectrum. The Full Scan mode is quite useful when identifying unknown compounds in a sample and providing confirmation of results from GC using other types of detectors.
Operation of a GC/MS in SIM mode allows for detection of specific analytes with increased sensitivity relative to full scan mode. In SIM mode the MS gathers data for masses of interest rather than looking for all masses over a wide range. Because the instrument is set to look for only masses of interest it can be specific for a particular analyte of interest. Typically two to four ions are monitored per compound and the ratios of those ions will be unique to the analyte of interest. In order to increase sensitivity, the mass scan rate and dwell times (the time spent looking at each mass) are adjusted.
When properly setup and calibrated, GC/MS-SIM can increase sensitivity by a factor of 10 to 100 times that of GC/MS-Full Scan. Because unwanted ions are being filtered, the selectivity is greatly enhanced providing an additional tool to eliminate difficult matrix interferences.
The ability of the mass spectrometer to identify unknowns in the full scan mode and quantitiate know target analytes in the SIM mode, makes it one of the most powerful tools available for trace level quantitative analysis in the lab today.
source : ALS Environmental
The invention of gas chromatography by James and Martin was evoked by their work on the synthesis of fatty acids in plants. To aid in their research, a method was needed to separate the fatty acids extracted from plant tissue and to quantitatively estimate the different fatty acids present. As a consequence, the technique suggested by Martin and Synge in 1941 (26) (GC) was developed into a practical separation procedure. Subsequently, the synthetic pathways for the different fatty acids were examined using 13C and 3H markers. Thus, having established a technique to separate the fatty acids, those that were radioactive needed to be identified and the relative activity of each peak compared and to do this successfully, a proportional radioactive detector was required. James and Piper described a radioactivity detector 1961-3 [27,28] suitable for this purpose is still in use today, although the detector has been fabricated in various different forms by a number of different manufacturers. A diagram of the radioactivity detector based on the device of James and Piper is shown in figure 46.
There are two basic forms of the radioactivity detector, one that measures 13C only and the other that measures both 13C and 3H. In both systems the carrier gas used must be helium or argon and the column eluent is fed through a furnace packed with copper oxide to oxidize all the solutes to carbon dioxide and water.
- LC-MS relies on drying away all the solvent. It therefore follows that you cannot use solvents with non-volatile components. Phosphate buffers and salts are bad. They will crystalize out as the solvent is dried away, and fill the ion source with solid gunk. Ion exchange chromatography is therefore unlikely to work. But you can try changing non-volatile salts to volatile ones, such as ammonium acetate.
- All the solvent must be dried away, so the less solvent there is, the better. Consider reducing your flow rate. Electrospray mass spec signals depend on concentration, not total amount of stuff squirted into source per minute, so reducing flow rate will also increase sensitivity!
- You are no longer dependant on perfect separation of peaks, and good retention time, for the identification of your chemical of interest. But the machines are more expensive and in demand. Consider sacrificing a little separation for improved throughput. Most LC-MS people use very short columns, 100mm maximum, often less.
What matters to retention time is how fast the solvent flows linearly along the column. You can calculate a flow rate for a narrower column very easily.
The cross-sectional area of the column is πr2
The cross-section of a 4.6 mm column is about five times that of a 2.1 mm column.
Therefore you should use a flow of about one fifth, i.e. 200 µl.min-1 instead of 1000µl.min-1
Note that 200 µl.min-1 is absolutely ideal for most mass specs. Buy 2mm columns instead of 4.6mm where ever possible!
Written by : Dr. Lionel Hill (JIC, UK)