(PHYSICAL CHEMISTRY)

HYPHEMATED TECHNIQUES

Introduction Gas chromatography-mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical techniques. As the name implies, it is actually two techniques that are combined to form a single method of analyzing mixtures of chemicals. Gas chromatography separates the components of a mixture and mass spectroscopy characterizes each of the components individually. By combining the two techniques, an analytical chemist can both qualitatively and quantitatively evaluate a solution containing a number of chemicals. The uses for GC-MS are numerous. They are used extensively in the medical, pharmacological, environmental, and law enforcement fields. This laboratory exercise will look at how environmental chemists evaluate samples containing the pollutants called PAHs. Gas Chromatography In general, chromatography is used to separate mixtures of chemicals into individual components. Once isolated, the components can be evaluated individually. In all chromatography, separation occurs when the sample mixture is introduced (injected) into a mobile phase. In liquid chromatography (LC), the mobile phase is a solvent. In gas chromatography (GC), the mobile phase is an inert gas such as helium. The mobile phase carries the sample mixture through what is referred to as a stationary phase. The stationary phase is a usually chemical that can selectively attract components in a sample mixture. The stationary phase is usually contained in a tube of some sort. This tube is referred to as a column. Columns can be glass or stainless steel of various dimensions. The mixture of compounds in the mobile phase interacts with the stationary phase. Each compound in the mixture interacts at a different rate. Those that interact the fastest will exit (elute from) the column first. Those that interact slowest will exit the column last. By changing characteristics of the mobile phase and the stationary phase, different mixtures of chemicals can be separated. Further refinements to this separation process can be made by changing the temperature of the stationary phase or the pressure of the mobile phase. Our GC has a long, thin column containing a thin interior coating of a solid stationary phase (5% phenyl-, 95% dimethylsiloxane polymer). This 0.25 mm diameter column is referred to as a capillary column. This particular column is used for semivolatile, non-polar organic compounds such as the PAHs we will look at. The compounds must me in an organic solvent. The capillary column is held in an oven that can be programmed to increase the temperature gradually (or in GC terms, ramped). this helps our separation. As the temperature increases, those compounds that have low boiling points elute from the column sooner than those that have higher boiling points. Therefore, there are actually two distinct separating forces, temperature and stationary phase interactions mentioned previously.

As the compounds are separated, they elute from the column and enter a detector. The detector is capable of creating an electronic signal whenever the presence of a compound is detected. The greater the concentration in the sample, the bigger the signal. The signal is then processed by a computer. The time from when the injection is made (time zero) to when elution occurs is referred to as the retention time (RT). While the instrument runs, the computer generates a graph from the signal. (See figure 1). This graph is called a chromatogram. Each of the peaks in the chromatogram represents the signal created when a compound elutes from the GC column into the detector. The x-axis shows the RT, and the y-axis shows the intensity (abundence) of the signal. In Figure 1, there are several peaks labeled with their RTs. Each peak represents an individual compound that was separated from a sample mixture. The peak at 4.97 minutes is from dodecane, the peak at 6.36 minutes is from biphenyl, the peak at 7.64 minutes is from chlorobiphenyl, and the peak at 9.41 minutes is from hexadecanoic acid methyl ester.

Figure 1: Chromatogram generated by a GC. If the GC conditions (oven temperature ramp, column type, etc.) are the same, a given compound will always exit (elute) from the column at nearly the same RT. By knowing the RT for a given compound, we can make some assumptions about the identity of the compound. However, compounds that have similar properties often have the same retention times. Therefore, more information is usually required before an analytic al chemist can make an identification of a compound in a sample containing unknown components. Mass Spectroscopy As the individual compounds elute from the GC column, they enter the electron ionization (mass spec) detector. There, they are bombarded with a stream of electrons causing them to break apart into fragments. These fragments can be large or small pieces of the original molecules.

The fragments are actually charged ions with a certain mass. The mass of the fragment divided by the charge is called the mass to charge ratio (M/Z). Since most fragments have a charge of +1, the M/Z usually represents the molecular weight of the fragment. A group of 4 electromagnets (called a quadrapole, focuses each of the fragments through a slit and into the detector. The quadropoles are programmed by the computer to direct only certain M/Z fragments through the slit. The rest bounce away. The computer has the quadrapoles cycle through different M/Z's one at a time until a range of M/Z's are covered. This occurs many times per second. Each cycle of ranges is referred to as a scan. The computer records a graph for each scan. The x-axis represents the M/Z ratios. The y-axis represents the signal intensity (abundance) for each of the fragments detected during the scan. This graph is referred to as amass spectrum (see Figure 2).

Figure 2: Mass-spectrum generated by an MS. The mass spectrum produced by a given chemical compound is essentially the same every time. Therefore, the mass spectrum is essentially a fingerprint for the molecule. This fingerprint can be used to identify the compound. The mass spectrum in Figure 2 was produced by dodecane. The computer on our GC-MS has a library of spectra that can be used to identify an unknown chemical in the sample mixture. The library compares the mass spectrum from a sample component and compares it to mass spectra in the library. It reports a list of likely identifications along with the statistical probability of the match.

GC-MS When GC is combined with MS, a powerful analytical tool is created. A researcher can take an organic solution, inject it into the instrument, separate the individual components, and identify each of them. Furthermore, the researcher can determine the quantities (concentrations) of each of the components. Figure 3 represents a three-dimensional graph generated when the GC is combined with the MS. Try to visualize how the chromatogram combines with the mass spectrum to produce this image. It is important for you to be able to picture this 3D image and translate it into the previous 2D graphs. (This image is not made from the same compounds in the previous figures.) Note that you can create either a mass spectrum or a chromatogram by making the appropriate cross section of this 3D image. Try to visualize which cross section would produce a spectrum and which would produce a chromatogram.

Figure 3: 3D Depiction of GC-MS output. Interfacing devices

Introduction
Like a good marriage, both gas chromatography and mass spectrometry bring something to their union. GC can separate volatile and semivolatile compounds with great resolution, but it cannot identify them. MS can provide detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separate them. Therefore, it was not surprising that the combination of the two techniques was suggested shortly after the developmentof GC in the mid-1950s. Gas chromatography and mass spectrometry are, in many ways, highly compatible techniques. In both techniques, the sample is in the vapor phase, and both techniques deal with about the same amount of sample (typically less than 1 ng). Unfortunately, there is a major incompatibility between the two techniques: The compound exiting the gas chromatograph is a trace component in the GCs carrier gas at a pressure of about 760 torr, but the mass spectrometer operates at a vacuum of about 106 to l05 torr. This is a difference in pressure of 8 to 9 orders of magnitude, a considerable problem.

How It Works
The Interface The pressure incompatibility problem between GC and MS was solved in several ways. The earliest approach, dating from the late 1950s, simply split a small fraction of the gas chromatographic effluent into the mass spectrometer . Depending on the pumping speed of the mass spectrometer, about 1 to 5% of the GC effluent was split off into the mass spectrometer, venting the remaining 95 to 99% of the analytes into the atmosphere. It was soon recognized that this was not the best way to maintain the high sensitivity of the two techniques, and improved GC-MS interfaces were designed . These interfaces reduced the pressure of the GC effluent from about 760 torr to l06 to 105 torr, but at the same time, theypassed all (or most) of the analyte molecules from the GC into the mass spectrometer. These interfaces were no longer just GC carrier gas splitters, but carrier gas separators; that is, they separated the carrier gas from the organic analytes and actually increased the concentration of the organic compounds in the carrier gas stream. The most important commercial GC carrier gas separator is called the jet separator; s . This device takes advantage of the differences in diffusibility between the carrier gas and the organiccompound. The carrier gas is almost always a small molecule such as helium or hydrogen with a high diffusion coefficient, whereas the organic molecules have much lower diffusion coefficients. In operation, the GC effluent (the carrier gas with the organic analytes) is sprayed through a small nozzle, indicated as , into a partially evacuated chamber (about 102 torr). Because of its high diffusion coefficient, the helium is sprayed over a wide solid angle, whereas the heavier organic molecules are sprayed over a much narrower angle and tend to go straight across the vacuum region. By collecting the middle section of this solid angle with a skimmer and passing it to the mass spectrometer, the higher-molecular-weight organic compounds are separated from the carrier gas, which is removed by the vacuum pump. Most jet separators are made from glass by drawing down a glass capillary, sealing it into a vacuum envelope, and cutting out the middle spacing. It is important that the spray orifice and the skimmer be perfectly aligned.These jet separators work well at the higher carrier gas flow rates used for packed GC columns (10 to 40 mL/min); however, there are certain disadvantages. Packed GC columns are an almost infinite source of small particles upstream of the jet separator. If one of those particles escapes from the column, it can become lodged in the spray orifice and stop (or at least severely reduce) the gas flow out of the GC column and into the mass spectrometer. Part of this problem can be eliminated with a filter between the GC column and the jet separator, but eventually a particle will plug up the orifice. In fact, sometimes it is not a particle at all, but rather tar (mostly pyrolyzed GC stationary phase) that has accumulated in the spray orifice over time. Clearly, these devices require maintenance. Currently, the most common strategy, which is ideally suited for capillary GC columns, is to pass all of the carrier gas flow into the ion source of the mass spectrometer . This works only if the GC gas flow is sufficiently small and the pumping speed of the mass spectrometers vacuum system is sufficiently high to handle the gas flow. For most capillary GC columns, the gas flow is 1 to 2 mL/min,and for most modern mass spectrometers, the pumping speed is at least 300 L/sec. The development offlexible, fused silica capillary columns has made this approach routine. In fact, the only time a jet separatoris now used is for a few applications that require packed or thick stationary phase GC columns(for example, for permanent gas analysis).

In practice, most GC-MS interfacing is now done by simply inserting the capillary column directly into the ion source. Fig. 31.2 is a diagram of one such system. The fused silica column runs through a 1/16-in.-diameter tube directly into the ion source. Other gases, such as methane for chemical ionization, are brought into the ion source by a T joint around the capillary column. One of the other two lines into the ion source is used for a thermocouple vacuum gauge tube so that the pressure in the ion source can be roughly measured. The remaining line into the ion source is for the delivery of the mass spectrometer calibration standard, perfluorotributylamine. Most joints are welded together to avoid leaks when this inlet system is thermally cycled or vented. The only removable (Swagelok) fitting is at the junction of the GC column and the far end of the inlet tube (marked with an asterisk in Fig. 31.2). This fitting uses Vespel ferrules. Once the ferrules are on the GC column and it is in the ion source, it is desirable to cut off a few centimeters of the column, if possible. This eliminates the possibility of fine particles partially occluding the end of the column. If the end of the column cannot be placed directly in the ion source, the material in the GC-MS interface becomes important. The interface is held at 250 to 280 C; thus, it should not include a reactive metal (such as copper). In some interfaces, glass-lined stainless steel tubing has been used, even though this tubing is difficult to bend properly. Figure 31.2 A typical GC-MS interface for fused silica capillary GC columns. The end of the GC column enters the ion source of the mass spectrometer

In summary, for capillary GC-MS, the best interface is no interface at all; run the flexible, fused silica GC column directly into the ion source. Using a column that is 25 to 30 m long by 220 to 250 m inner diameter gives an ion source pressure of 106 to 105 torr, a more than acceptable pressure at which to obtain electron impact spectra. This gives a helium or hydrogen GC carrier gas velocity of 25 to 35 cm/sec or a flow of about 1 to 2 mL/min. The GC columns most widely used for GC-MS are those in which the stationary phase has been chemically bonded to the fused silica; DB-5 is a common trade name. Occasionally, there have been problems with the plastic cladding on the outside of the GC column. This cladding is usually hot (typically 250 C) and

under vacuum. Thus, it may decompose, giving background ions in the mass spectrum or weakening the fused silica itself.

What It Does
Gas chromatographic mass spectrometry is the single most important tool for the identification and Gas Chromatography Mass Spectrometry 615 quantitation of volatile and semivolatile organic compounds in complex mixtures. As such, it is very useful for the determination of molecular weights and (sometimes) the elemental compositions of unknown organic compounds in complex mixtures. Among other applications, GC-MS is widely used for the quantitation of pollutants in drinking and wastewater. It is the basis of official EPA methods. It is also used for the quantitation of drugs and their metabolites in blood and urine. Both pharmacological and forensic applications are significant. GC-MS can be used for the identification of unknown organic compounds both by matching spectra with reference spectra and by a priori spectral interpretation. The identification of reaction products by synthetic organic chemists is another routine application, as is the analysis of industrial products for control of their quality. To use GC-MS, the organic compounds must be in solution for injection into the gas chromatograph. The solvent must be volatile and organic (for example, hexane or dichloromethane). Depending on the ionization method, analytical sensitivities of 1 to 100 pg per component are routine. Sample preparation can range from simply dissolving some of the sample in a suitable solvent to extensive cleanup procedures using various forms of liquid chromatography. In addition to the sample preparation time, the instrumental analysis time is usually fixed by the duration of the gas chromatographic run, typically between 20 and 100 min. Data analysis can take another 1 to 20 hr (or more) depending on the level of detail necessary. GC-MS has a few limitations. Only compounds with vapor pressures exceeding about 1010 torr can be analyzed by GC-MS. Many compounds that have lower pressures can be analyzed if they are chemically derivatized (for example, as trimethylsilyl ethers). Determining positional substitution on aromatic rings is often difficult. Certain isomeric compounds cannot be distinguished by mass spectrometry (for example, naphthalene versus azulene), but they can often be separated chromatographically. Quantitative accuracy is controlled by the overall analytical method calibration. Using isotopic internal standards, accuracy of 20% relative standard deviation is typical.

Application
Environmental monitoring and cleanup GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. There are some compounds for which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of environmental samples, including many major classes of pesticides, it is very sensitive and effective.

Criminal forensics GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of fire debris using GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire debris analysis. GCMS/MS is especially useful here as samples often contain very complex matrices and results, used in court, need to be highly accurate. Law enforcement GC-MS is increasingly used for detection of illegal narcotics, and may eventually supplant drug-sniffing dogs.[1] It is also commonly used in forensic toxicology to find drugs and/or poisons in biological specimens of suspects, victims, or the deceased. Sports anti-doping analysis GC-MS is the main tool used in sports anti-doping laboratories to test athletes' urine samples for prohibited performance-enhancing drugs, for example anabolic steroids Security A postSeptember 11 development, explosive detection systems have become a part of all US airports. These systems run on a host of technologies, many of them based on GC-MS. There are only three manufacturers certified by the FAA to provide these systems,one of which is Thermo Detection (formerly Thermedics), which produces the EGIS, a GC-MS-based line of explosives detectors. The other two manufacturers are Barringer Technologies, now owned by Smith's Detection Systems, and Ion Track Instruments, part of General Electric Infrastructure Security Systems. Food, beverage and perfume analysis Foods and beverages contain numerous aromatic compounds, some naturally present in the raw materials and some forming during processing. GC-MS is extensively used for the analysis of these compounds which include esters, fatty acids, alcohols, aldehydes, terpenes etc. It is also used to detect and measure contaminants from spoilage or adulteration which may be harmful and which is often controlled by governmental agencies, for example pesticides. Astrochemistry Several GC-MS have left earth. Two were brought to Mars by the Viking program.Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS.[15] The Huygens probeof the Cassini-Huygens mission landed one GC-MS on Saturn's largest moon, TitanThe material in the comet 67P/ChuryumovGerasimenko will be analysed by the Rosetta mission with a chiral GC-MS in 2014. Medicine Dozens of congenital metabolic diseases also known as Inborn error of metabolism are now detectable by newborn screening tests, especially the testing using gas chromatographymass spectrometry. GC-MS can determine compounds in urine even in minor concentration. These compounds are normally not present but appear in individuals suffering with metabolic disorders. This is increasingly becoming a common

way to diagnose IEM for earlier diagnosis and institution of treatment eventually leading to a better outcome. It is now possible to test a newborn for over 100 genetic metabolic disorders by a urine test at birth based on GC-MS. In combination with isotopic labeling of metabolic compounds, the GC-MS is used for determining metabolic activity. Most applications are based on the use of 13C as the labeling and the measurement of 13C-12C ratios with an isotope ratio mass spectrometer (IRMS); an MS with a detector designed to measure a few select ions and return values as ratios. Radiochromatography: Radiochromatography is based on paper or thin layer chromatography though it could also be used in ion-exchange column or gas chromatography as well as in electrophoresis. It uses a radiotracer as cation or anion or radiolabelled organic compound where separated cation, anion or a compound is identified on the basis of characteristic radiation emitted by the radionuclide. Thus, any chromatographic separation can be carried out using radiotracers. However, no developer/chromogenic reagent or detector is used to study the movement of initial spot except nuclear counting equipment or autoradiography. In this way distribution of activity is measured which increases sensitivity of the measurement by several fold. Radiochromatography has played an important role in the discovery of some trans- plutonium elements by Glen T. Seaborg. The identity of each element of 5f series was established beyond any doubt by the sequence of their appearance analogous to the sequence of the corresponding 4f elements. There are many instances of the use of radiochromatography for analytical purposes. typical example of complete separation of the alkali metal ions Na+, K+, Rb+, and Cs+ by using respective tracer solutions.

Tandem Mass Spectrometry

What is tandem mass spectrometry? Tandem mass spectrometry (MS/MS) is perhaps the most significant advance in newborn screening in the past 30 years. A tandem mass spectrometer is a specialized instrument that detects molecules by measuring their weight (mass). Mass spectrometers measure weight electronically and display results in the form of a mass spectrum. A mass spectrum is a graph that shows each specific molecule by weight (mass) and how much of each molecule is present. A simple newborn blood specimen contains thousands of molecules ranging from small molecules such as salt to large proteins such as hemoglobin. When alcohol is added to the dried blood spot, several hundred molecules are pulled out and can be studied. If this entire mixture is studied directly, a complex graph is produced and it is difficult to tell biochemically important molecules from other less important molecules that have the same weight. To make the graph easier to understand, the biochemically important molecules must be separated from all other molecules. Before MS/MS technology, these molecules could only be separated by physical methods (gas and liquid chromatography) that take between 10 to 30 minutes each. A tandem mass spectrometer can accomplish this task accurately in approximately two minutes. How does a mass spectrometer work? A mass spectrometer works similar to how a person might sort a pocketful of change. Each coin has a unique weight and size (quarters weigh more and are larger in size than dimes). After sorting by weight and size, the quantity of each type of coin can be counted quickly. Tandem mass spectrometers can take the sorting even further. For example, consider the new quarters that feature specific state designs on the back. Each state quarter has a similar weight and size but has a slightly different structure. A

tandem mass spectrometer can quickly sort biochemically important molecules of similar weight in the same manner that you can sort how many of the newer Pennsylvania, Massachusetts, or Georgia quarters you have. Why is MS/MS the best method for newborn screening? Tandem mass spectrometry measures many different molecules in a single test. Older testing methods require the use of several tests to look at different types of molecules making them time-consuming and expensive. With tandem mass spectrometry, the results are available quickly and are also more accurate. For example, MS/MS testing for PKU studies the levels of two chemicals: phenylalanine (phe) and tyrosine. While most babies with PKU will have an elevated level of phe, virtually all babies with this disorder will have a high level of phe compared to tyrosine. This is important because older testing methods look only at phe and may miss some babies with PKU. MS/MS testing is also more accurate than the study of phe alone because there are several reasons other than PKU that a child may have an elevated blood level of phe. These include prematurity, special diets, liver disease, and differences in blood collection. The phenylalanine to tyrosine comparison is generally not high in these instances so MS/MS testing reduces the number of falsely "abnormal" PKU results. Tandem mass spectrometry is also superior to other testing methods because the study of multiple chemicals at a time allows us to accurately pinpoint which specific disorder a child may have. This technology is particularly helpful in the identification of specific disorders that share common chemical elevations. For example, MCAD deficiency and MADD both present with high levels of a chemical called octanoylcarnitine (C8). If we look at C8 alone and it is high, we cannot tell which of the two disorders a child might have. But, if we look at C8 in combination with other chemicals the picture becomes clearer. For example, individuals with MADD also show an unusual level of decanoylcarnitine relative to C8 and higher levels of long and short chain acylcarnitines. These findings are not seen in MCAD deficiency and distinguishes the two conditions. Finally, there is an obvious cost benefit to running both amino acids and acylcarnitines in a single profile. Currently, PerkinElmer Genetics monitors more than 60 chemical and chemical relationships in a single test. These markers provide indicators for disease, quality assurance, and potential contaminants that may interfere with other studies. The markers also provide information regarding hyperalimentation, method of collection, and drug treatment. Tandem Mass Spectrometry measures these indicators or markers in a typical two-minute run. The analyses conducted by PerkinElmer Genetics produce results that can be used by qualified physicians in the diagnosis of certain disorders. While evidence of such conditions will be detected in the vast majority of affected individuals, due to genetic variability, it may not be detected in all.

Appications of MS-MS Tandem mass spectrometry (MS/MS) has become a key technology in the fields of biochemical genetics and newborn screening. The development of electrospray ionisation (ESI) and associated automation of sample handling and data manipulation have allowed the introduction of expanded newborn screening for disorders which feature accumulation of acylcarnitines and certain amino acids in a number of programs worldwide. In addition, the technique has proven valuable in several areas of biochemical genetics including quantification of carnitine and acylcarnitines, in vitro studies of metabolic pathways (in particular beta-oxidation), and diagnosis of peroxisomal and lysosomal disorders.

REFERENCES R.P.W.Scott,TandemTechniques,Wiley IndiaPvt.Ltd. Reprint 2009. www.gmu.edu/depts/SRIF/tutorial/gcd/gc-ms2.htm www.intechopen.com/download/pdf/32813 en.wikipedia.org/wiki/Gas_chromatographymass_spectrometry www.piercenet.com Protein Methods Library en.wikipedia.org/wiki/Tandem_mass_spectromet www.spincotech.com/chy/Applications/Radio%20Chromatography.pdf ry