Tìm hiểu về máy sắc ký ghép nối phổ GCMS

If the ion source is the most important part of the mass spectrometer the ions produced within it wouldn't provide any useful information without the mass analyzer.. These are classified

Gas Chromatography / Mass Spectroscopy

Introduction

Mass Spectroscopy is a powerful analytical technique which can provide a myriad of information about analytes in complex mixtures In principle mass spectroscopy separates ions of a sample based on their mass to charge ratio (m/z) Qualitative as well as quantitative information for both inorganic and organic compounds can be obtained, as well as structural information about a wide variety of molecular species The use of high resolution instruments allows the determination of isotopic ratios of atoms in samples and with specialized instrumentation the structure and composition of solid surfaces can be investigated The first mass spectrometers were developed in the 1920's with the first commercial

instrument becoming available in the 1950's Many significant developments have occurred since that time not only in instrumentation but also in understanding of the fundamental processes occurring within the mass spectrometer Some significant instrumental advances include the development of double focusing instruments, interfacing mass spectrometers to chromatographic separation techniques, the use of tandem mass spectrometer and several new ionization techniques have been developed Fundamental understanding

of the behavior of ions in a magnetic field is the basis for applying mass spectroscopy to the determination

of chemical composition of a sample Additional developments in the understanding of fragmentation of molecular species in the ion source allow the structural analysis and identification of complex molecules Further improvements have included the application of Fourier transform, which greatly improves mass resolution and signal-to-noise ratios Mass spectroscopy has matured into a sophisticated and powerful tool which is widely used in chemical and biochemical analysis

All mass spectrometers have five major components which include an inlet system, ion source, mass analyzer, detector and signal processor The first four components are typically held at high vacuum (10-5 - 10-8 torr) The inlet system's function is to introduce a small amount of sample (typically 1

micromole or less) into the ionization source with a minimal loss of vacuum Typical inlets include batch inlets, direct probe inlets and chromatographic inlets We will be using the latter in this experiment in the form of a gas chromatograph The GC will not only serve as an inlet system but it will also facilitate the separation of our complex mixture before it is introduced to the mass spectrometer

The ion source is perhaps the most important part of the mass spectrometer Ion sources are as varied as the types of samples which can be analyzed by mass spectrometry They can be a simple electron impact ion source, producing ions from the interaction of analyte molecules with energetic electrons in the gas phase, to the more complicated ionization occurring in a MALDI (Matrix Assisted Laser Desorption Ionization) source useful for nonvolatile samples The ion source used is typically mandated by the

physical properties of the sample being analyzed and several types include: electron ionization (EI), chemical ionization (CI), field ionization (FI), field desorption (FD), fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), laser desorption (LD), plasma desorption (PD), thermal

desorption, and matrix assisted laser desorption ionization (MALDI) Understanding the process taking place in the ion source is the key to structure elucidation for qualitative analysis

If the ion source is the most important part of the mass spectrometer the ions produced within it wouldn't provide any useful information without the mass analyzer The main function of the mass analyzer

is to separate ions with different mass to charge ratios (m/z) produced in the ion source Like ion sources, there are a number of mass analyzers which can be used for this purpose These are classified as magnetic sector analyzers, double focusing spectrometers, quadrupole mass filters, ion trap analyzers, time of flight analyzers and Fourier transform instruments Each mass analyzer has its own unique advantages and disadvantages Typically quadrupole mass filters are coupled to chromatographic instruments because of they are typically more rugged, lower priced, and more compact than other mass analyzers Additionally, quadrupole mass filters are capable of scanning a large range of masses in a short time, which is useful for real-time scanning of chromatographic peaks A quadrupole consists of a focusing lens stack and four cylindrical metal rods which act as the electrodes of the mass filter (Figure 1) Ions are accelerated and focused into the space between the rods by the lens stack Opposite rods in a quadrupole are connected electronically, to the positive and negative terminal of a variable dc source Additionally, there is a variable radio frequency ac potential applied, 180 degrees out of phase, to each pair of rods The quadrupole acts as

a mass filter because only ions with a stable trajectory will remain between the rods and pass to the

detector

Figure 1: Schematic representation of a quadrupole mass spectrometer (from Skoog & Leary 1992)

By simultaneously adjusting the ac and dc potential applied to the rods it is possible to change the m/z of an ion which will have a stable trajectory Therefore, by rapidly adjusting the ac and dc potentials it is possible

to rapidly scan a range of masses and continually collect a complete mass spectrum of the column eluent The use of a hyphenated GC-MS system is powerful for qualitative as well as quantitative determinations

of complex mixtures

Once the ions have been separated by the mass analyzer they must be collected and the beam of ions converted into an electrical signal which can be recorded, stored and/or displayed for analysis The most common type of detector is the electron multiplier Other detectors include the Faraday cup,

scintillation detectors and photographic plates Similar to a photomultiplier tube in optical instruments, the electron multiplier ejects a cascade of electrons from a dynode held at a fixed voltage Through a series of dynodes, each at a successively higher voltage, the current caused by analyte ions is amplified before being recorded and measured Another electron multiplier, and the type used in this instrument, is a continuous dynode type This detector works on the same principle as a discrete dynode electron multiplier, however it

is constructed of a single trumpet shaped device which acts to collect and amplify the signal from

impacting ions The last component of most mass spectrometers is the signal processor In most modern instruments analog signals from the detector are converted to digital output which is collected and stored by

a computer The computer can also be used to control instrumental parameters of both the GC and MS as well as for data acquisition and manipulation Through the use of software programs various analysis

modes and data work up can be performed depending on the analysis being carried out

Procedure:

Part 1: Tutorial

The first part of this experiment will introduce you to the Shimadzu QP-5000 Gas Chromatograph / Mass Spectrometer and the CLASS-5000 software (Pay attention to the steps which introduce how to use the library to analyze the mass spectrum and how to prepare a table to automatically id peaks in the unknown)

-Follow the procedure outlined in the Shimadzu GCMS-QP5000 (ver 2) “Quick Guide” User’s Manual, Section 2 Perform all tutorial steps as outlined for Scan mode analysis This will prepare the instrument for the analysis which will be performed later

-Skip Step 4 in the CLASS-5000 tutorial (p 2-9,2-10)

-Resume the Tutorial at the bottom of page 2-11 by opening the Real Time Analysis from the Main menu Continue following the Tutorial through page 2-19 where the MC Table is created

-Resume the Tutorial at Step 9 (Similarity Search using Chromatogram in Browser, p 2-48 through 2-50) This will be useful in qualitative identification of the components in your unknown sample

-*Note: Section 3 in the GCMS-QP5000 “Quick Guide” has a list of commonly used abbreviations and glossary which you may find useful when referencing the manual and while reading GC/MS literature

Part 2: Qualitative Sample Analysis

Reminder before starting your scans each day run an auto tune At the end of the day set the oven temperature to 200 degrees for overnight standby

Part A:

This section will allow you to try out the basic operation of the instrument in Id-ing and unknown sample You will be given a vial containing a hydrocarbon in pentane Follow the instructions below under essential instrument instructions to run a chromatogram and then use the software to ID the unknown from its Mass Spectrum

Part B

You will be given a sample that contains a standard mixture of six aromatic hydrocarbons and a diluted gasoline mixture in a solvent of methylene chloride for analysis Prepare an analysis plan to quantify the amount of two of the components in the gasoline unknown (Do not use the same method you used in the GC lab or HPLC lab.) You will be using the systems’ software to qualitatively identify the components in the standard mixture and then see if those components are present in the gasoline

Essential Instrument instructions

-First, be sure that you have performed the Auto-tune procedure as outlined on p 2-2 of the “Quick Guide” user’s manual The instrument must be tuned each day before analysis is carried out (If you have already set-up and saved the tuning method you can simply Load the previous tuning method from the File menu.)

-Once the Autotune has been performed open the Real Time Analysis and from the File menu, Load the method file “Gas.met”

-Using the Setup Method menu, browse through the method parameter screens and record important chromatograph and mass spectral parameters for this analysis

-From the Single Sample menu fill in the pertinent information in the Sample Registration window which opens Pick a single file name which will be used for all your samples and incremented for subsequent injections (Be sure to note the Data path to which your data will be saved)

-Click on the “Send Parameters” button (this will send the method parameters to the instrument for data analysis)

-Select the Option menu and select CC/MS Monitor… from the choices shown This will open a monitor window which shows the state of various chromatographic parameters throughout the run -Place the aromatic mixture in the first position of the auto injector and ensure that the solvent rinse vial contains enough solvent to rinse the auto injector syringe between sample injections

-Once all the items in the “Status” window are highlighted green the GC and MS are ready for analysis -Press the Start button on the auto injector and the first sample will be injected and analyzed

-After the chromatogram has developed determine the appropriate ‘Solvent Cut’ and modify that parameter in the method file by selecting Setup Method and then Mass Spectrometer… from the Real Time Analysis menu Set the Solvent Cut time to just after the solvent elutes (The solvent cut time turns the mass spectrometer off while the solvent elutes from the column This preserves the ion source and detector while the solvent elutes from the column.)

-Click on “Send Parameters” again to initialize the instrument Once the Status box indicates the instrument is ready, press the Start button on the auto injector and make a second injection of the standard aromatic mixture

-As this sample is being analyzed reprocess the data from the first injection you made

Do this by opening the Post Run Analysis from the CLASS-5000 Main menu and then open the Browser

Load the data file for your first injection and qualitative identify each peak in the chromatogram by first performing a background subtraction and then a library search

(As outlined in the Tutorial; Step 9 (p 2-48 to 2-50)

Make a note of the M/Z for the ‘Base Peak’ in the Mass Spectrum of each compound eluted in the mixture, you will use this information when analyzing the unknown

-Once the chromatograph of the second injection of the standard mixture has fully developed you are ready to inject the unknown sample Place the diluted gasoline sample into the first position of the auto sampler and click the “Send Parameters” button in the Real Time Analysis window

-Once the analysis is complete open the data file for the gasoline sample in the Browser window in order

to reprocess this data You should notice the complexity of this chromatogram compared to that of your standard mixture

-In order to selective identify the Aromatic composition of the gasoline sample you will want to use the Multiple Ion Chromatograph option for displaying the results This will allow you to view the chromatograph for selected M/Z ratios only

-Select Display from the menu and then MIC (for Multiple Ion Chromatogram)

-Now set the parameters for the ions you wish to view Select Setup Table from the main menu and then MIC Table… Input the M/Z range for the six aromatic hydrocarbons you identified in your standard mixture and then click “OK” Your MIC should appear on the screen with the aromatic components more clearly identifiable than in the TIC (Total Ion Chromatogram)

-Qualitative Identify each peak based on retention time and the results of a background corrected library search (As outlined in the Tutorial; Step 9 (p 2-48 to 2-50)

Part 3: Quantitative Sample Analysis

Present your analysis plan to the TA/Instructor They will assist you in further setting up the instrument

to perform this analysis

Results and Data analysis

- Print out the TIC of the standard aromatic mixture as well as a peak report and the mass spectrum for each peak The chromatogram should have printed out when you first analyzed the sample You can print the peak report from the Post Run Analysis, Browser window Open the data file for your aromatic mixture and then select Peak Detect and Peak Report from the Browser’s menu, then select Report! from the Peak Report menu and click the “Report” button The mass spectrum for each peak can be printed from the Setup Table and Spectrum Process Table in the Browser’s menu Make sure that each peak is present in the table (they are either listed by scan number or retention time) and then select Report and Mass Spectrum In the Mass Spectrum Report window be sure the Output size is set

to 1/3 and then click the “Report” button

- Print out the TIC and MIC of the diluted gasoline sample and a peak report for the MIC

Based on the mass spectra you obtained for each aromatic compound in the standard mixture suggest structures for the major ion peaks observed and possible fragmentation pathways which resulted in those ions Why is there more significant fragmentation for some compounds than others? What is the origin of the m/z=51 peak found in all the spectra?

Discuss differences in the appearance of the TIC and MIC of the gasoline sample How is the MIC more useful for qualitative as well as quantitative analysis of this sample?

Report the qualitative results of your analysis for this gasoline sample citing how you identified each peak

Report the quantitative results for the two aromatic compounds you determined including the precision

of the standardization technique used Given that the intensity and energy of the electrons emitted by the ion source can be highly variable with time (i.e within the same chromatographic run ionization efficiency can increase and decrease) suggest the optimal standardization technique which would account for this fluctuation (hint, think of the other possible quantation It could be the method you used.)