Hyphenated techniques
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Analytical chemistry involves separating, identifying, and quantifying components of matter. Hyphenated techniques combine two analytical methods, such as gas chromatography coupled with mass spectrometry (GC-MS). GC-MS separates chemical mixtures using gas chromatography and then identifies components using mass spectrometry. Other common hyphenated techniques include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-infrared spectroscopy (GC-IR). These coupled techniques provide enhanced sensitivity and accuracy for analyzing organic compounds, pollutants, drugs, proteins, and more.
Hyphenated techniques
- 2. Analytical Chemistry • Analytical chemistry is the study of the separation, identification, and quantification of matter for the purpose of chemical characterization of matter. • Analytical chemistry answers the two important questions... 1. What is it..?? 2. How much is it..?? • Qualitative analysis gives an indication of the identity of the chemical species in the sample. • Quantitative analysis determines the amount of certain components in the substance. 1. Conventional/ Classical methods 2. Instrumental methods • The separation of components is often performed prior to analysis. • The separation of materials is accomplished using- – Chromatography, – Electrophoresis, – Field flow fractionation methods.
- 3. • To select an appropriate method of analysis, following factors must be taken into account- a. Nature of information which is sought b. Size of sample available c. Proportion of the constituents to be determined d. Purpose for which analytical data is required
- 4. Qualitative Analysis • A qualitative analysis determines the presence or absence of a particular compound, but not the mass or concentration. • By definition, qualitative analyses do not measure quantity. – Color Tests – Microcrystal Tests – Flame Tests – pH Tests
- 5. Hyphenated Techniques • A Hyphenated technique is combination or coupling of two different analytical techniques with the help of proper interface. • Hirschfield (1980) introduced the term "hyphenation" to refer to the on-line combination of a separation technique and one or more spectroscopic detection techniques. • Chromatography - Produces pure or nearly pure fractions of chemical components in a mixture. Spectroscopy – Produces selective information for identification using standards or library spectra. • The hyphenated technique is developed from the coupling of a separation technique and an online spectroscopic detection technology. • The number of existing techniques has been combined to expand the utility. • The direct conjugation of chromatographic technique with spectroscopic examination of separated fraction constitutes several powerful analytical techniques.
- 6. Hyphenation • The hyphenation does not always have to be between two techniques; the coupling of separation or detection techniques • Recently, more than two techniques coupled together to form a more powerful integrated system have revolutionized the trace element analysis industry. • Also called double hybrid e.g. LC-PDA-MS; LC-MS-MS; LC-NMR-MS instruments have become available and have been applied to pharmaceutical problem solving. • Online coupling with solid phase extraction (SPE), solid phase micro extraction or large volume injection can be incorporated to build in a more powerful integrated system e.g. SPE-LC-MS.
- 7. •Hyphenated techniques ranges from the combination of- 1. separation-separation, 2. separation-identification & 3. Identification-identification techniques.
- 9. Advantages of Hyphenated Techniques 1. Fast and accurate analysis 2. Higher degree of automation 3. Higher sample throughput 4. Better reproducibility 5. Reduction of contamination due to its closed System 6. Separation and quantification achieved at same time.
- 10. List of Hyphenated Techniques 1. GC-MS 2. LC-MS 3. LC-NMR 4. EC-MS 5. CE-MS 6. GC-IR 7. LC-MS-MS 8. LC-ESI-MALDI-TOF 9. GC-MS-MS 10. GC-NMR 11. GC-AES 12. ICP-MS 13. ICP-AAS 14. ICP-OES
- 11. Hyphenated GC • Simple detector (such as thermal-conductivity detection (TCD), flame- ionization detection (FID) and electron-capture detector (ECD), etc.) for a GC system. • Based on different detection mechanism, information-rich detectors can be mainly classified as- 1. detection based on molecular mass spectrometry, 2. detection based on molecular spectroscopy such as Fourier-Transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, and 3. detection based on atomic spectroscopy (elemental analysis) by coupling with such as inductively-coupled plasma (ICP)-MS, atomic absorption spectroscopy (AAS) and atomic emission spectroscopy (AES), respectively. • In addition to these hyphenations mentioned above and which are mounted after a gas chromatograph, it can also include automated online sample preparation systems before a GC system such as- 1. Static headspace (HS), 2. Dynamic headspace, 3. Large volume injection (LVI) and 4. Solid-Phase Micro Extraction (SPME).
- 12. GC-MS • A GC-MS instrument is composed of following two major building blocks: i. a gas chromatograph and ii. a mass spectrometer. • GC-MS separates chemical mixtures into individual components (using a gas chromatograph) and identifies / quantifies the components at a molecular level (using a MS detector). • It is one of the most accurate and efficient tools for analyzing volatile organic samples.
- 13. GC Components: •Carrier Gas, N2 or He, 1-2 mL/min •Injector •Oven •Column •Detector
- 15. In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.
- 16. Gas Chromatography-Mass Spectrometry (GC-MS)
- 17. Gas Chromatography-Mass Spectrometry (GC-MS) Example •To analyze a urine sample for tetrahydrocannabinol, (THC) the principal psychoactive component, the organic compounds are extracted from urine, purified, concentrated and injected into the GC-MS. •THC appears as a GC peak, and gives a molecular ion at 314, its molecular weight. Mass spectrum of Tetrahydro- cannabinol (THC)
- 18. Problem Bank President Who robbed the bank? Biologist What protein was isolated?
- 19. Gather Evidence Police Officer 1. Interview witnesses 2. Dust for fingerprints Mass Spectrometrist 1. Interview biologist who isolated the protein 2. Cleave protein to obtain peptide mixture 3. Analyze peptide mixture by MS to obtain peptide molecular masses! enzym e
- 20. Database Search Police Officer Height: 5’7” Weight: 160 lbs Gender: male Age: 35-40 Fingerprints Mass Spectrometrist Approx. molecular weight: 30,000 Origin: bovine liver Peptide mass list from MS analysis: 975.4832, 1112.5368, 632.3147, 803.4134, 764.3892 DATABASE OF KNOWN FELLOWS PEPTIDE MASS DATABASE OF KNOWN PROTEINS search search
- 21. Database Search Results Police Officer Identifies the robber Mr. XYZ Mass Spectrometrist Identifies the protein e.g. bovine carbonic anhydrase
- 22. Working of GC-MS • The separation occurs in the gas chromatographic column (such as capillary) when vaporized analytes are carried through by the inert heated mobile phase (so-called carrier gas such as helium). • The analytes spend different time (called retention time) to come out of (elute from) the GC column due to their different adsorption on the stationary phase (of a packed column in gas-solid chromatography). • As the eluted substances emerge from the column opening, they flow further into the MS through an interface. • This is followed by ionization, mass-analysis and detection of mass-to-charge ratios of ions generated from each analyte by the mass spectrometer. • The combination of the gas chromatograph and mass spectrometer in a GC-MS, allows a much accurate chemical identification than either technique used separately.
- 23. Application 1. Quantitation of pollutants in drinking and waste water. 2. Quantitation of drug in metabolites and urine is done for the pharmacological and forensic use. 3. Identification of unknown organic compounds in hazardous waste dumps and reaction products by synthetic organic chemistry. 4. Used for drug analysis, pesticide and herbicide detection.
- 24. LC-MS • Liquid chromatography-mass spectrometry is the technique which performs separation by liquid chromatography and mass analysis with the help of the mass spectrometry. • Electrospray needle is used as bridge to connect the liquid chromatography with that of the mass. • LC-MS is mainly separated into the three parts- 1. Chromatography – In liquid chromatography separation is performed which is detected with the help of Photo diode Array. – These separated components then transferred to the interface. 2. Interface – In interface the liquid is volatilized and transferred to the MS. 3. Spectrometry – With the help of various ionization techniques the compound is ionized and then it is analyzed by mass analyzer. – Various mass analyzers are used viz. Quadrupoles, quadrupole ion traps, time-to-flight (TOF), time-to-flight reflection (TOFR), and ion cyclotron resonance (ICR) mass analyzers.
- 25. Applications 1. LC-MS used to detect compounds from polyaromatic (non-polar) to peptide and proteins. 2. LC-MS used for compounds identification and purity. 3. Used for determination of pesticides, herbicides & organic pollutant for environmental monitoring. 4. Proteome analysis is done by this technique.
- 26. GC-IR • GC-IR technique is hyphenation of gas chromatography and Infrared spectroscopy. • This technique is very sensitive, very expensive, sample recovery is also possible because IR is non-destructive technique. • In this technique the GC does the separation part where as IR perform the function of identification. • Effluent from GC is directly forwarded into the heated pipe of IR at atmospheric pressure. • Infrared red spectroscopy identifies the compound by identifying the functional groups.
- 27. Applications 1. Pharmaceutical, Industrial 2. DNA Analysis of blood samples, other fluids.
- 28. GC-AES/ GC-OES • This technique is combination of gas chromatography with atomic emission spectroscopy. • Atomic emission spectroscopy is one of the elemental analysis techniques. • GC performs the separation of the components and with the help of AES the elemental identification of the components is performed. • Elemental composition of every peak separated by GC is determined. i. GC effluent is directly introduced into the Quartz atomization furnace. ii. Analytes are first atomized using either ICP or microwave irradiation (high temperatures), where the atoms are transferred to electronically excited state. iii. Then these electrons are return to the lower energy levels at that time photons are emitted at certain wavelengths that are characteristic of the particular element. iv. In both the techniques sample is in gas phase so the techniques are complementary to each other.