Analytical Chemistry - Gas Chromatography / Mass Spectrometry

GAS CHROMATOGRAPHY - MASS SPECTROMETRY

What is gas chromatography (GC)?	Chromatography using a GC-MS (video)	GC-MS Analysis of pharmaceutical drugs in blood samples using Deconvolution
Gas Chromatography Detectors	Gas Chromatography – Quantitative Analysis	Gas chromatography sample introduction techniques
Headspace Analysis - Gas Chromatography - Analysis of Ethanol in Blood (Forensic Chemistry)	How does a mass-spectrometer work?	Interpreting Mass Spectra / GC–MS Data Analysis
Troubleshooting and maintenance of GC Systems	Understanding Columns in Gas Chromatography

 

Gas chromatography (GC) is a separation technique based on the repeated partition or adsorption, between a mobile phase and a stationary phase, of components to be separated. The mobile phase is always a gas known as the carrier gas. The stationary phase can be either a solid or a liquid.

Traditionally, the scope of gas chromatography has been for the separation of the following:

Complex mixtures
Components closely related chemically and physically
Mixtures consisting of a wide breadth of compounds

 

Mass spectrometry is the analytical technique that provides the most structural information for the least amount of analyte material. It provides qualitative and quantitative information about the atomic and molecular composition of inorganic and organic materials and their chemical structures. As an analytical technique it possesses distinct advantages:

Increased sensitivity over most other analytical techniques because the analyzer, as a mass-charge filter, reduces background interference.
Excellent specificity from characteristic fragmentation patterns to identify unknowns or confirm the presence of suspected compounds. closely related chemically and physically
Information about molecular weight
Information about the isotopic abundance of elements

 

The combined analytical technique GC-MS became - since the eighties - the most powerful method for the analysis of organic compounds such as drugs, pesticides, pollutants and their metabolites in clinical, forensic and food chemical determinations. It is also used in doping control as well as in environmental and occupational toxicology.

 

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References

1. D. Harvey,  “Modern Analytical Chemistry”, McGraw-Hill Companies Inc., (2000)
   

2.  K. Pfleger et al. “Mass Spectral and GC Data of Drugs, Poisons, Pesticides, Pollutants and their Metabolites”, 2nd Edition, VCH, 1992E. C. McGoran, J. Chem. Educ., 68, 19-23 (1991)
3. H.M. McNair, J.M. Miller, “Basic Gas Chromatography”, John Wiley &Sons, (1997)

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GC-MS Analysis of pharmaceutical drugs in blood samples using Deconvolution

For over 50 years, mass spectrometers have been used as gas chromatographic (GC) detectors. During this time, the capability and reliability of the mass spectrometer (MS), and the reproducibility of analytical data, has increased tremendously.

A modern GC-MS system is capable of performing an analysis on a complex matrix of 25 compounds in less than 30 minutes. Both the qualitative and quantitative information is provided to the analyst in a short period of time. The GC-MS data system is used to process each GC peak found during the analysis and to compare the mass spectrum from each GC peak to a mass spectrum from a library of standards stored in a database.

A GC-MS analysis can fail, however, when acquired spectra are “contaminated” with mass spectral peaks that arise from co-eluting compounds and ionization chamber contaminants. These peaks can pose a serious problem for automated identification methods where they can cause identifications to be missed by reducing the spectrum comparison factor below some pre-set identification threshold. In addition, the presence of such peaks in a spectrum adds to the risk of making false identifications.

Since the inception of GC-MS, there has been a continuing interest in extracting “pure” component spectra from complex chromatograms. A deconvolution software – AMDIS (Automatic Mass Spectral  Deconvolution and Identification System) in our case - is used to automatically find any of a set of target compounds in a gas chromatography / mass spectrometry (GC-MS) spectrum. The basic steps are the following:

AMDIS first deconvolutes the GC-MS data file to find all of the separate components
Each of these components is then compared against a library (or libraries) of target compounds
The match factor between the target spectrum and the deconvoluted component spectrum is then reported
If this match factor is above a user set value (for example 80%), then there is positive identification.

In our case, a blood sample was analyzed by GC-MS and AMDIS for compounds of pharmacological interest. Several compounds were identified (see video below) amongst them: venlafaxine (antidepressant, Effexor), diazepam (anti-anxiety, Valium, Diastat) and levomepromazine (neuroleptic, Nosinan, Nozinan, Levoprome).

The match factor for a positive identification was set to above 80%. The actual analysis time was 25 min (GC-MS) and 5 min (deconvolution process using AMDIS and several spectra libraries such as NIST, AAFSDRUG, SWGDRUG, WILEY, MPW2007).

 

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Relevant Posts - Relevant Videos

Interpreting Mass Spectra / GC–MS Data Analysis

What is gas chromatography (GC)?

How does a mass-spectrometer work?

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References

1. D. Harvey,  “Modern Analytical Chemistry”, McGraw-Hill Companies Inc., 2000
2. R.D. Brown, “Introduction to Chemical Analysis”, McGraw-Hill Companies Inc, 1982
3. R. G. Dromey et al., Anal. Chem., 48, 1368-1375, (1976)

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Key Terms

gc-ms, gc-ms analysis of drugs, mass spectrometry, gas chromatography, deconvolution,

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Headspace Analysis - Gas Chromatography - Analysis of Ethanol in Blood (Forensic Chemistry)

Headspace techniques are employed in conjunction with gas chromatography (GC) analysis for certain type of samples. The GC can handle any gaseous sample and any liquid sample that can be vaporized completely and instantaneously before it goes to a proper column for separation. Unfortunately there are many liquid samples (biological, environmental) that cannot be directly injected into the GC. For such samples headspace analysis can be used.

The principle underlying GC headspace analysis is that in a sealed vial at constant temperature equilibrium is established between volatile components of a liquid or solid sample in the vial and the gas phase above it – the “headspace” (Fig. I1). After allowing due time for equilibration (normally 15 min.) a portion of the headspace – ambient volume above a sample matrix where the volatile compounds exist in gaseous form at predictable levels - may be withdrawn via a rubber septum using a gas-tight syringe and injected into the GC column.

Fig. I1: GC headspace vial

Headspace analysis is useful for situations where:

• The analyte of interest is volatile at temperatures below 290 ^∘C

• The sample matrix is a solid, liquid, paste that is not easy to inject into a GC inlet

• Sample preparation to allow easy liquid injection is difficult 

The following video demonstrates the headspace sampler of a GC-FID system:

  Advantages of Headspace Analysis

Headspace analysis provides several advantages over normal injections:

• Simpler sample preparation

• Directly analyze a wide range of sample matrices such as liquids, solids and pastes

• Columns last longer, with less maintenance. The headspace volume above the sample matrix is more clean than the matrix. By injecting fewer contaminants the analytical column lasts longer.

• High precision

• Solvent peak is smaller or nonexistent compared to traditional liquid injection GC techniques.

An internal standard may be added prior to the heating process, and quantitative analyses may be performed after constructing a calibration graph.

This technique is widely used in the analysis of ethanol in blood (blood-alcohol in driving under influence (DUI) and driving whil and other volatile substances in biological samples and in the pharmaceutical industry for measuring solvent residues in tablets, amongst other applications. A gas chromatogram of an in-house reference material containing ethanol, methanol and n-propanol obtained by using headspace analysis is shown in Fig. I2.

Fig. I2: A gas chromatogram of an in-house reference material containing ethanol, methanol and n-propanol obtained by using headspace analysis is shown above. The first peak is acetaldehyde (retention time: 0.886 min), the second peak is ethanol (retention time: 1.407 min) and the last peak is isopropanol the internal standard (retention time: 3.070 min)

Chromatography using a GC-MS (video)

Chromatography is the separation of a mixture of compounds into individual components.

There are three major steps involved with separating and identifying components of a mixture using a GC-MS. They are:

Injecting a sample into the GC inlet

Separating the sample into individual components

Detecting the compounds present in the sample. This is done in the detector. In the video shown below, the detector is a mass spectrometer.

Chromatography using a GC or GC-MS has been described in previous posts:

Gas chromatography

The Injection System in GasChromatography – Sample Introduction

Understanding Columns in GasChromatography

Gas Chromatography Detectors

How does a mass-spectrometer work?

A GC-MS analytical instrument equipped with an autoinjector is shown in the following video. A sample is automatically injected into the GC inlet and separation and identification of its constituents takes place. 

 https://www.youtube.com/watch?v=R4sITWA8xrc

Gas Chromatography – Quantitative Analysis

Gas Cromatography - Quantitative Analysis

The basic theory for quantitation of sample components involves the measurement of peak heights or peak areas. For peaks that are well resolved, both peak height and area are proportional to the concentration.

Three different calibration methods, can be used in quantitative analysis:

1. The external standard
2. The internal standard
3. The standard addition method

For gas chromatography, the most commonly used quantitative methods are the internal standard and standard addition method.

An internal standard is a standard whose identity is different from the analyte’s, that is added to all samples and standards (calibrants) containing the analyte.

Since the analyte and internal standard in any sample or standard receive the same treatment, the ratio of their signals will be unaffected by any lack of reproducibility in the procedure. With this method, an equal amount of an internal standard (IS) is added to both the sample and calibrator solutions. The IS selected should be chemically similar to the analyte and have a similar retention time and similar derivatization. It is also important to ensure that the IS is stable and does not interfere with any of the sample components. The IS should be added before any preparation of the sample so that extraction efficiency can be evaluated. Quantitation is achieved by using ratios of peak areas of the component to the internal standard:

 

Conc-sample = [( Area_IScalibrator) / ( Area_ISsample)] x [Area_sample / Area_calibrator] * (Conc_calibrator)

Standard addition: If only a few samples are to be chromatographed, it is possible to employ the method of standard addition(s). The chromatogram of the unknown is recorded. Then a known amount of the analyte(s) is added, and the chromatogram is repeated using the same reagents, instrument parameters, and procedures. From the increase in the peak area (or peak height), the original concentration can be computed by interpolation. The detector response must be a linear function of analyte concentration and yield no signal (other than background) at zero concentration of the analyte. Sufficient time must elapse between addition of the standard and actual analysis to allow equilibrium of added standard with any matrix interferant.

If an instrumental reading (area or height), Rx, is obtained from a sample of unknown concen-tration x and a reading R1 is obtained from the sample to which a known concentration C of analyte has been added, then x can be calculated from the relation.

x /( x + C)  =  R_x / R₁

 

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Relevant Posts

What is Gas Chromatography?

Understanding Columns in Gas Chromatography

Gas Chromatography Sample Introduction Techniques

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References

1. D. Harvey,  “Modern Analytical Chemistry”, McGraw-Hill Companies Inc., 2000
2. “Gas Chromatography”, J. Willett, John Wiley &Sons, 1987
3. "High Resolution Gas Chromatography”, K.J. Hyver, P. Sandra, 3rd Edition, 1989

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Key Terms

gradient elution, gas chromatography, mobile phase, external standard, internal standard in gas chromatography, standard addition method in gc, quantitation of sample components in gc,

 

Troubleshooting and maintenance of GC Systems

Troubleshooting a GC chromatograph can be made easier, if one learns to recognize the symptoms produced by an instrument malfunction, column and detector problems, leaks. Many symptoms appear as unusual peaks as shown below:

Symptom: No peaks (Fig. 1) 

Fig. 1: No peaks appear after the injection of the sample

Possible Cause: Main power off, fuse burned out

Action: Plug in the instrument, replace fuse

Possible Cause: Detector off

Action: Turn detector on and adjust its sensitivity

Possible Cause: No carrier gas flow

Action: Check the carrier gas lines and correct the problem i.e. replace empty gas cylinders, possible obstructed or broken gas lines

Possible Cause: Injector temperature too low. The sample is not vaporized

Action: Increase injector temperature.

Possible Cause: Injector septum is leaking

Action: Replace the septum.

Possible Cause: The syringe used for the injection of sample is plugged up.

Action: Clean the syringe or replace it if it is damaged.

Possible Cause: The FID flame is out.

Action: Check the FID flame. Check if water vapor condenses on mirror. Light up the flame if needed.

Possible Cause: Column connections are loose.

Action: Check for leaks. Use a leak detector. If needed tighten column connections

Possible Cause: Oven temperature too cold. Possibly, the sample condenses in the column

Action: Increase oven temperature

 

Symptom: Retention times or areas are not reproducible (Fig. 2)

Possible Cause: Septum is leaking

Action: Replace the septum if it is damaged. If there is a premature septum failure (less than 200 injections) check also if:

The syringe needle is not straight

The syringe is not installed correctly

The septum retainer nut is too tight

Possible Cause: Syringe is dirty or damaged

Action: Replace the syringe if it is damaged. Clean the syringe with an appropriate solvent if it is dirty.

Possible Cause: Sample is not stable under the conditions of the analysis

Action: Check the sample stabiblity. Some samples change with heat or U.V. light. In case the sample is photosensitive use amber sample vials.

Possible Cause: Sample volume is too low or too high

Action: Check the sample vials. If the sample vials are not filled correctly, evaporation or contamination may affect the analysis. The sample level should be approximately half the volume of the vial.

 

Fig. 2: Retention times or areas are not reproducible

Symptom: Poor sensitivity with normal retention time

 

Fig. 3: Poor sensitivity with normal retention times

 

 Possible Cause: Insufficient sample size

Action: Increase sample size. Check syringe needle for plugging.

Possible Cause: Poor sample injection technique

Action: Check if the proper injection technique is used.

Possible Cause: High attenuation

Action: Reduce attenuation.

Possible Cause: FID response low

Action: Optimize the flow rates of H₂ and air. Use N₂ for make-up gas

Possible Cause: Thermal conductivity response low

Action: Use higher filament current.

Possible Cause: Syringe or septum leaking when injecting

Action: Replace syringe and/or septum

Symptom: Poor sensitivity with increased retention time and broadening of the peak

Possible Cause: Carrier gas flow rate too low.

Action: Increase carrier gas flow.

Possible Cause: Septum is leaking.

Action: Replace septum.

Symptom: Irregular baseline drift (isothermal)

  

Fig.4: Irregular baseline drift when operating isothermally

Possible Cause: The instrument location is not according to the manufacturer’s specifications .

Action: Instrument should not be placed directly under heater or air conditioner or any other place where it is subject to excessive drafts and ambient temperature changes.

Possible Cause: Column packing bleeding.

Action: Let column to stabilize as described by the manufacturer.

Possible Cause: Carrier gas leaking.

Action: Locate leaks and correct.

Possible Cause: Carrier gas regulators do not work properly.

Action: Check carrier gas regulators and flow controllers.

Possible Cause: Poor air or H₂ regulation (FID detectors)

Action: Check regulators and flow controllers.

Possible Cause: Detector is contaminated

Action: Clean detector according to manufacturer’s specifications. Raise temperature and bake out detector overnight.

Possible Cause: Detector filaments are defective (TCD detector only)

Action: Change the filaments.

Symptom: Contamination or ghost peaks

Possible Cause: Vial cap septum is dissolving in solvent

Action: Check the vial septum. If it is not resistant enough to the solvent you are using try a more resistant type.

Possible Cause: Injection port septum is giving off volatiles

Action: Make several blank runs with a small piece of aluminum foil backing the inlet septum. If the contamination peaks disappear, they were probably due to the septum. Use another type of septum that is stable to the required injector temperatures.

Possible Cause: Column is contaminated

Action: Examine the first 10 cm of the capillary column for contamination holding a light behind it. If possible cut the contaminated part of the column. Replace or clean the inlet liner.

Possible Cause: The sample is not stable

Action: Store the sample in a protected environment and use amber sample vials.

Symptom: Sinusoidal baseline drift

 

Fig.5: Sinusoidal baseline drift

Possible Cause: Oven temperature defective

Action: Replace the oven temperature sensing probe .

Possible Cause: Oven temperature control was set to a low value

Set the oven temperature control to a higher value.

Possible Cause: Carrier gas flow regulator defective

Replace the carrier gas flow regulator. Set to a higher pressure in order to stabilize the gas flow.

Possible Cause: Carrier gas cylinder pressure too low to allow regulator to work properly                                                                                                                   

Replace the carrier gas cylinders.