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“ GAS CHROMATOGRAPHY/ MASS SPECTROMETRY/ MASS SPECTROMETRY ” sumandeep vidyapeeth university Sisal Shah.. Final Year B-Pharm.. 1 Guide Mrs. Aarti Zanwar

What is GC?:

What is GC? GC is a separation technique Sample is usually a complex mixture, that is separated into constituent components. Why? usually to quantify some or all components e.g. Pharmaceuticals, Environmental pollutants, etc Occasionally as a qualitative tool 2 Sisal Shah

Key components of GC :

Key components of GC Hardware to introduce the sample Technique to separate the sample into components Hardware to detect the individual components. Data processing to process this information. Sisal Shah 3

Basic Block Diagram!:

Basic Block Diagram! Sisal Shah 4

Separation Process:

Separation Process Sample is introduced into system via hot vaporising injector. Typically 1ul is injected. Flow of “Carrier Gas” moves vaporised sample (i.e. gas) onto column Column is coated with wax type material with varying affinity for components of interest Components are separated in the column based on this affinity. Individual analytes are detected as they emerge from the end of the column through the Detector. Sisal Shah 5

Example Chromatogram (Capillary):

Example Chromatogram (Capillary) Time  Inject Point Detector Response Sisal Shah 6

GC Step by Step :

GC Step by Step Carrier Gas Injector Column Capillary Stationary Phase Detectors Mass Spectrometer Sisal Shah 7

Carrier Gas:

Carrier Gas Inert Helium Choice dictated by detector, cost, availability Pressure regulated for constant inlet pressure Flow controlled for constant flow rate Chromatographic grade gases (high purity) Sisal Shah 8


Columns Material of Construction Metal (1957) Glass (1959) Fused Silica (1979) Aluminium Clad (1984) Inert Metal (1990) Sisal Shah 9

Stationary Phases:

Stationary Phases Choice of phase determines selectivity Like dissolves like Use polar phases for polar components Use non-polar phases for non-polar components Sisal Shah 10

Choosing a Column:

Choosing a Column Internal Diameter Film Thickness Length Phase Column Capacity The Maximum amount that can be injected without significant peak distortion Column capacity increases with :- film thickness temperature internal diameter stationary phase selectivity If exceeded, results in :- peak broadening asymmetry leading Sisal Shah 11


Detectors Flame Ionization Detector Photo Ionization Detector Electron Capture Detector Infrared Detector Helium Ionization Detector Sisal Shah 12


Applications Purity of compounds Atropine sulphate tablets Presence of foreign substances Cimetidine Assay or drugs Antazoline HCL Sisal Shah 13

Introduction of Mass Spectrometry:

Introduction of Mass Spectrometry Sisal Shah 14 Mass spectrometry (MS) is based on the production of ions, which are subsequently separated or filtered according to their mass-to-charge ( m/z ) ratio and detected. The mass to charge ratio (m/ z) is used to describe ions observed in mass spectrometry.

Instrumentation of MS:

Instrumentation of MS Sisal Shah 15


Application Detection of molecular mass Structure elucidation Detection of impurity Drug metabolism study Cis -trans difference Determine bond dissociation energy Sisal Shah 16


GC MS COMBINATION Combination of GC-MS provides extremely powerful tool because it permits direct and effectively continuous and correlation of chromatographic and mass spectroscopic properties . The separation and identification of the components of complex natural and synthetic mixtures are achieved more quickly than any other technique with less sample . Molecules then undergo electron /chemical ionisation Ions are then analysed according to their mass to charge ratio Ions are detected by electron multiplier which produces a signal proportional to ions detected Sisal Shah 17


GC/MS Sisal Shah 18


GC/MS PROCESS Sisal Shah 19

Basic GCMS Theory(2):

Basic GCMS Theory(2) Electron multiplier passes the ion current signal to system electronics Signal is amplified Result is digitised Results can be further processed and displayed Sisal Shah 20

Interpretation of Mass Spectra(1):

Interpretation of Mass Spectra(1) Sisal Shah 21


GC/MS/MS Gas chromatography coupled to tandem mass spectrometry ( gc /ms/ms) is used to separate the organic compounds in the mixture. Ms/ms is a highly selective mass spectrometric technique, target analytes are detected regardless of the sample matrix. Gc /ms/ms is ideal for fire debris analysis and has been used for the analysis of target compounds in various matrices such as complex environmental samples, pesticides in foods, drug metabolites, drug residues in biological fluids and other forensic trace evidence. Sisal Shah 22

GC/MS/MS (continued..):

Gc/ms/ms is a more powerful analytical technique that provides a method for detecting target compounds in complex matrices at trace levels . This method is designed to provide a better analysis for weak, highly weathered or older samples. It is particularly well suited for the detection of gasoline and can be used on any sample. GC/MS/MS (continued..) Sisal Shah 23


WORKING Sisal Shah 24 MS/MS operates by selecting target ions of specific mass Separates the ions from selected parent ions by collision with helium molecules. GC is used for seperation of components. GC/MS/MS provides identification in cases where GC/MS spectra of compounds are difficult to interpret.

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Sisal Shah 25

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After separation by gas chromatography , the ms/ms operates by first selecting the target ion(s) of choice at a specific mass during the first stage of ms/ms, which separates the ions from the chemical background or matrix . These selected precursor ions or parent ions are then induced to further dissociate by collision with helium molecules. The resultant unique product ion spectrum provides confirmation of the target analyte . This increased selectivity of ms/ms also results in an enhancement of the signal to noise ; thus somewhat lower limits of detection are achieved. Gc/ms/ms provides unequivocal identification in cases where gc/ms spectra of compounds are difficult to interpret. Sisal Shah 26

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Thus, even if the matrix contains another compound with the same mass as the parent ion for the analyte of interest , it is extremely unlikely that the interfering ion would yield the same daughter ion spectra as the analyte , thus gc /ms/ms is more specific for an ignitable liquid. In order to overcome the pyrolysis product interference and improve detection levels, ms/ms can be utilized as the method of detection. As gasoline is one of the more common distillates used by arsonists, the identification of gasoline in fire debris samples is important. The parent ions and daughter ions are isolated and the ms/ms chromatograms for a variety of hydrocarbon distillates are obtained and subsequently compared to ignitable liquid standards, run under identical conditions. Sisal Shah 27


SAMPLE PREPARATION Metals, strong acid or bases, salts, oligomeric and polymeric material. Those compounds will damage the column . Suitable solvents are methylene chloride, methanol, hexane, cyclohexane, and octane . Use 2ml vials with plastic insert . Fill up to at least 10 ul of the insert . Take your sample out from the tray if you want it back, otherwise it will be thrown away. Sisal Shah 28


Applications A MULTIPLEX GC-MS / MS TECHNIQUE FOR THE SENSITIVE AND QUANTITATIVE SINGLE-RUN ANALYSIS OF ACIDIC PHYTOHORMONES AND RELATED COMPOUNDS, AND ITS APPLICATION TO ARABIDOPSIS THALIANA A highly sensitive and accurate multiplex gas chromatography–tandem mass spectrometry (gc-ms/ ms) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid . Sisal Shah 29

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2. TESTING FOR GHB IN HAIR BY GC/MS/MS AFTER A SINGLE EXPOSURE. APPLICATION TO DOCUMENT SEXUAL ASSAULT Gamma- hydroxybutyric acid , is naturally present within mammal species. Properties of neurotransmitter or neuromodulator are generally given to this substance. Ghb is therapeutically used as an anesthetic , but can be used for criminal offenses (date-rape drug). It appears that the window of detection of ghb is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. In order to document single exposure, we investigated the use of hair. Hair was collected one month after the allegated event in order to sample the corresponding period after regular growing . This study demonstrates that a single exposure to ghb in a case of sexual assault can be documented by hair analysis when collected about one month after the crime. Sisal Shah 30

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3. DETERMINATION OF ESTROGENS IN SLUDGE AND SEDIMENTS BY LIQUID EXTRACTION AND GC/MS/MS Two methods have been developed that enable the determination of estrogens in digested and activated sludge from domestic sewage treatment plants ( stps ) and in freshwater sediments. The method for sludge analysis consists of solvent extraction; a gel permeation chromatography ( gpc ) cleanup step 4. COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY WITH MASS SPECTROMETRIC DETECTION (GC × GC/MS) APPLIED TO THE ANALYSIS OF PETROLEUM Sisal Shah 31

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5. HIGHLY SENSITIVE GC/MS/MS METHOD FOR QUANTITATION OF AMINO AND NONAMINO ORGANIC ACIDS GC/MS/MS targeted metabolite profiling method enabling absolute quantification of all detected metabolites . The method is based on methyl chloroformate derivatization and quantification spiking samples with metabolite standards separately derivatized with reagents. Sisal Shah 32

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6. Using GC-MS/MS for Superior Sensitivity, Specificity and Precision in Free Testosterone Analysis Highly efficient for the analysis of complex mixtures, GC-MS/MS could not address the requirement of the industry for the analysis of larger non-volatile molecules. Sisal Shah 33


OTHER APPLICATIONS Pesticides in fruits/ veges /marine biota PCBs Nitro-PAHs in air particulates Organo -tins Melamine in milk and baby formula Drug Screens PBDE Flame Retardants Anabolic Steroids in urine/blood Sisal Shah 34


DISADVANTAGES It's expensive to buy a GC/MS/MS machine. The tubes you use for each sample are expensive. You need a trained operator. GC/MS is very, very accurate. If you smoke weed, being tested on an extremely accurate machine is a disadvantage. Sisal Shah 35


REFERENCES Paul W. Steinwedel H: " Einneuesmassens pektrometerohne magnetfeld ":1953; vol8 (7): 448–450 R. E. " Quadrupole ion trap mass spectrometry: a view at the turn of the century’Int . J. Mass spectrom .’; march 2000; vol 200 (1–3): 285–312. Schwartzjae C. Michael W. Senko and John E. P. Syka :"a Two-dimensional quadrupole ion trap mass spectrometer".J . Am. Soc. Mass. Spectrom .; 2000; vol 13 (6): 659–669 Pavia, Donald L., Gary M. Lampman , George S. Kritz , Randall G. Engel: Introduction to Organic Laboratory Techniques (4th Ed.);2006; Thomson Brooks/Cole. pp. 797–817. Harris, Daniel C.: "24. Gas Chromatography". Quantitative chemical analysis (Chapter) (Fifth ed.);1999; pp. 675–712. Sisal Shah 36

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Sisal Shah 37 Any queries….????

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