Benchtop GCMS Introduction         

Dr. Lhost G.J.J.

1. Introduction to GCMS

Gas chromatography  (GC) being a very good method for separating components of a mixture and mass spectrometry ( MS) a powerful technique for identifying components in a mixture, combination of the two techniques (GCMS) may be considered as a natural marriage.  GC and MS deal with samples in the gas phase and can handle very small quantities of material.  A basic introduction will be provided here to explain how GC separate ans MS identify compounds.   Gas chromatography relies on the fact that different compounds dissolve to different extents in a particular liquid.  In the experimental practice a long thin tube with such a liquid adhering to the inner wall , which is heated and has a gas such as helium flowing through it may be used.  As chromatographers call it, the tube is called a column or a capillary column and when a mixture is introduced into the hot column, a component that does not dissolve in the liquid would be vaporized by the heat and carried straight though the capillary column at the same speed as the helium gas which is called the carrier gas.  A compound of the mixture that dissolves in the liquid called the stationary phase and has less interactions with the gas phase would remain near the start of the column and move through it with difficulty.   Consequently, different compounds are separated within the column because they move through it at different rates, depending of the partition between the stationary phase and the mobile carrier gas.

The experimental situation is depicted in figura 1.

                   

       Fig. 1   Separation of different molecule species in a capillary gas chromatographic column

The compounds separated by GC are passed for identification into a mass spectrometer.  The eluted molecules from the capillary column are introduced in the ion source of the mass spectrometer where neutral molecules are converted into charged particles.  The gaseous charged particles move in electric and magnetic fields in a way that is dependent on their masses.  Determining the molecular mass of a compound does not allow an unambiguous identification because acetic acic and urea have the same mass of 60 daltons to the nearest whole number.  The molecular ions may be submitted to fragmentation processes characteristic of the substance and usually sufficient to identify the compouind.   In the ionization process molecules are converted into ions which are caused to undergo fragmentation reactions.  A record of all the ions produced in this process is a mass spectrum.  In gas chromatography (GC) alone the time taken for a compound to pass through the column is called the retention time. which is the sole criterium for identifying the substance.  In GC/MS analysis a second identifying parameter is available which is the mass of the compound.  With this instrumental combination , the technique rarely fails to identify compounds under experimental investigation.

    

                   Fig. 2   Schematic illustration of a GC/MS system

GC/MS mass spectrometers have all some pecularities in common:

a)  an interface between gas chromatograph and mass spectrometer

b)  an ionization chamber (ion source)

c) an analyzer where the ions are separated according to their mass

d) A detector with various amplification systems

e) A computer acquiring and processing the data

Some commercial modern GCMS systems are illustrated with some JEOL Scientific Instruments  serving advanced technology..                                                                                                                                                                                                                                                                                                                                                                              

 

 

JEOL QUADRUPOLE GC/MS

EASY MAINTENANCE

1. Remove front flange

2. Remove ion source block (slide to the left side)

3. Remove the Chamber (remove a pin)

4. Remove the lens block ( Turn counter clockwise)

5.  Cleaning operation

 

Fig. 3  Quadrupole and GC/Time of flight mass spectrometer 

 New generation of Instruments for GC/TOFMS Analysis

1. Achieves stable, high-sensitivity spectrum analysis.

2. High-resolution measurement conditions are always possible.

3. Fast GC analysis which improves productivity.

4. Variety of optional attachments improves analytic capabilities.

 

2.  Practical aspects of Gas Chromatography (GC)

Figure 4 illustrates  a gas chromatograph coupled to a quadrupole mass spectrometer.

                          Fig. 4  Main parts of a Jeol quadrupole GCMS

A sample of 0.1 to 5 L containing a mixture of compounds is injected manually or automatically onto the column via an injection port.  An oven maintains the column at a certain temperature and a carrier gas under pressure causes the sample to flow through the column where a separation of compounds of different polarities occurs.   A lot of factors are governing the degree of separation of the compounds such as

1.  Nature of the carrier gas and  flow rate settings

2. Type of column and nature of the stationary phase

3. Column and injector temperature

The column may be kept at a defined isothermal temperature or its temperature may be changed at a programmed linear rate.

2.1  Types of carrier gases

Helium is most employed for capillary columns , nitrogen has been often used with packed columns but has a high viscosity and when used with narrow-bore capillary columns the elution of the compounds would be slowed down.  Moreover traces of oxygen are to be eliminated and compared to helium , nitrogen may produce interferences in the lower mass range since nitrogen ha a mass of 28 daltons and helium a mass of 4.   Hydrogen is the best gas to use with capillary columns , it is oxygen free, is inexpensive and has a low viscosity producing fast separations.  Leaks must be avoided because of the formation of explosives mixtures with air.  The use of hydrogen in GC/MS is not frequent.

2.2  Injection

Samples are introduced into the analytical instrument in a vapor state and vaporizarion can take place during or after injection of the sample.  Liquid samples may be introduced in the gas chromatograph using a microliter syringe and solid samples are to be dissolved before injection.  The use of a hot injector system is the easiest method of vaporization.  The critical step in gas chromatography is the introduction of the sample where reaction of the components of the sample may occur.

Samples may be injected using : splitless injection, 

                                                             split injection, 

                                                             temperature-programmed injection 

                                                             on-column sample injection procedures.

 

2.2.1  Splitless Injection 

    For this type of injection used for dilute solutions, the top of the column is overloaded with the solvent and for this reason the column in the GC oven is maintained at a low temperature during injection so that the top of the column is 10-20C  below the boiling point of the solvent. so that sample components are condensed in a narrow area.  Subsequently, the separation is carried out by temperature programming and consequently this instrumental method is not recommended for volatile compounds or for isothermal analysis.  In the splitless mode, the split valve is closed when injection occurs and septum purge valve is set so that a small flow is maintained.  After 30 to 60 sec the split valve is opened and the exceeding solvent not focused and "cold trapped" is eliminated from the injector.  The solvent is selected to have a boiling point more or less 20C below that of the most volatile compound of interest for the analysis.  One of the drawbacks of splitless injection in directly coupled capillary column is that large amounts of solvent enter the ion source leading to the possibility of source contamination and a decrease in the mass spectrometer performance.

 

2.2.2 Split Injection

When the split injection technique is choosen, only a part of the sample is delivered to the capillary column.  The sample is injected through a septum into the carrier gas stream, vaporized in the vaporizer tube and mixed with the carrier gas.  The split valve is open and adjusted to a split flow of for example 40 ml/min and the septum purge valve is open to allow a small flow (0,5 mL/min) but can be closed in order to prevent sample loss.  Only a small proportion of the sample reaches the column and the split injection is consequently only suitable for concentrated solutions and mixtures and not for trace analysis.  In this technique overloading of the capillary column is prevented. 

2.2.3  Temperature-Programmed Injection

The liquid sample is injected into a "cold" injector and after introduction of the sample the injector is submitted to a temperature-programmed heating in which the split is programmed by a so called Event Programme .  A focussing at the top of the column can be produced depending on the column temperature.  The advantage of the procedure is that the solvent can be removed by a time-controlled split before the actual separation takes place.  The method is suitable for thermolabile compounds and for components which may form artifacts at high injector temperatures. such as oxazepam.  The split/splitless injector leads to artifact formation, but this can be largely prevented by a temperature-programmable injector.

2.2.4  On-Column Injector

The on-column injection port is similar to the slit/splitless injector and can have a septum purge.  The syringe has a long needle (stainless steel or fused-silica) which allows the whole sample to be placed directly into a cool capillary column via a special valve or septum.  The needle of the syringe enters the capillary column by 1 cm during injection which should be smooth and rapid.  As on-column injection is a splitless method, only low-concentration sample can be injected.  On-column injection is particularly suited for quantitative work and analysis of polar and thermally unstable compounds.

2.2.5  Moving Needle Injector

The simple and ingenious design of this injector first described by Ros and improved by van den Berg is represented herunder.:

The injector consists of a glass tube with an adjustable leak at the top, a septum port for introduction of the sample solution, a carrier gas inlet and a column outlet. The leak is adjusted such that the gas flow is split with 20 mL/min leaving via the leak and 1 mL/min flowing though the column.  Inside the glass tube is a moveable glass needle with a small iron rod attached at the upper end allowing the needle to be moved with an external magnet. In the sample loding mode 1 L of the sample solution is trasferred to the needle tip and the solvent, which should be reasonably volatile, is blown off in the 20 mL/min gas stream.  After evaporation of the solvent, the needle is moved rapidly so that the tip enters the hot injector block surrounding the column entrance.  At the injection point space is restricted reducing dead volume and giving a very high carrier gas velocity on the sample surface and ensuring rapid sample transfer to the column.  This injection system is well suited for high temperatures isothermal operation.  Since involatile components and contaminants are left on the needle tip surface, care must be taken for regular cleaning to avoid deterioration of column performance. by for example entrance of solid dirt particles into the column.

2.2.6  Headspace Technique

Gas chromatography (GC) combined with headspace techniques (HS) and recognized as headspace gas chtomatography (HSGC), is important in the investigation of volatile components in samples from clinical chemistry, biochemistry, food chemistry and in environmental analysis.  HSGC is an indirect analysis method for the determination of volatile compounds in liquid or solid samples.  In a static  headspace , the sample is in a closed static system at equilibrium as shown herunder.

After the equilibrium is reached, the analyte is removed directly from the vapor space above the sample and transferred to the gas chromatograph. Equilibrium must be reached for reproducible measurements and equilibrium is influenced by the conditioning time and temperature.  The vials are closed with polytetrafluoroethylene (PTFE) or aluminium-coated silicone septum. The septum itself is protected from the overpressure of the interior space by an aluminium cap.  A blank determination with an empty HS vial is carried out to detect whether any constituent of the materials of the septum or vial are being emitted.

 

 

 

 

2.2.7  Injection Port Care and Syringe Needles

Injection Port : The metal port  should be cleaned with methanol and dried before re-using

 Inserts:  The liner or fused silica insert found in several injectors should be regularly replaced before a deterioration in performance is observed.  Contaminated inserts mya be left in a chromic acid bath ( concentrated H2SO4 + potassium dichromate) until clean, washed with distilled water and methanol before air-drying and re-installation.  

Syringe needles:  A bevelled syringe needle is better than a pointed syringe needle.

Boiling point of common solvents as an aid for GC oven and injector temperature setting (see table herunder)

  Solvents and reagents Boiling Point at 760 mm Hg
Diethyl ether 35C
n-Pentane 36C
Dichloromethane 40C
Carbon disulphide 46C
Chloroform 61C
THF 66C
n-Hexane 69 C
Ethyl acetate 77C
Acetonitrile 82C
n-Heptane 98C
Toluene 111C
Pyridine 116C
n-Octane 126C
MSTFA 131C
BSTFA 146C
DMF 153C
MTBSTFA 169C

3.  Columns

In GC-MS the minimization of the release of stationary phase called bleeding due to heating giving background in the mass spectrometer is an important factor in selecting a column.

3.1 Types of columns

3.1.1  Packed columns

Glass tube of dimension 2m by 0.18 cm internal diameter packed with stationary phase coated diatomeous earth particles.  These columns are easy to pack and install and can cope with larhe amounts of sample or dirty samples.  A molecular separator for example of the Ryhage type is needed before the high vacuum of the mass spectrometer ion source because of the large carrier gas flow rate of 30 ml/min.    No more used with new instruments but sometimes still in use.

3.1.2  Wide -bore capillary column

These are fused-silica tubes of 5m by 0.53 mm coated on internal surface with a stationary phase which can be chemically-bonded to the internal surface of the column and for this reason these columns bleed less than packed columns.  The carrier gas flow rate is more or less 10 ml/min and a molecular separator is also needed although certain mass spectrometer have pumps able to cope with such flow rates.

3.1.3  Wall-coated open tubular (WCOT) capillary columns

Length of fused-silica capillary columns is often 25 m with an internal diameter of 0.2 or 0.3 mm. and according to the application lengths of 10 or 50 m also are used.  Since the fused silica is very brittle a coating of heat-resistant polyimide plastic provides support and preserves flexibility.  The columns ends must be cleanly cut and all the connections must be made with care to avoid contamination from the polyimide coating and dead volume giving rise to spaces in the connections leading to band broadening and loss of resolution.  The choice of capillary columns is governed by a number of factors:

Liquid phase: Stationary phases covalently bonded to the inner column surface provide greater stability so that the column tolerates high temperatures ,can be washed with solvents and gives low bleeding.  The general rule in phase selection is:

separate apolar compounds on apolar liquid phases

separate polar compounds on polar phases

The table herunder gives some common stationary phases together with chemical details

Phase Similar to Structure Max temp Polarity
OV-1 SE-30, BP-1, DB-1, CP-Sil5 100 % dimethyl silicone 325C non
OV-101 SP-2100 100 % dimethyl silicone (fluid) 325 C non
OV-17 HP-17 50 % phenyl 50 % methylsilicone 325 C medium
OV-225   cyanopropylmethyl-

phenylmethylsilicone

250 C high
CW-20M HP-20M, DB-Wax polyethylene glycol 20000 220 C high
SE-52 DB-5, BP-5 5 % phenylsilicone 95% methyl silicone 325 C low
SE-54   1 % vinyl silicone 5 % phenyl silicone 94 % methyl silicone 325 C low
FFAP SP-1000, OV-351 polyethylene glycol - 2-nitroterephtalic acid 250 C high
Dexsil 500   carboranemethyl silicone 450 C non
Chirasil-Val   silicone with chiral center 220 C high

OV-1 and OV-101 stationary phases of low polarity, stable to high temperatures tend to separate comopunds according to their boiling points and are frequently used in gas chromatography.  The phenylsilicone more polar phases such as OV-17 show stronger retention of aromatic compounds and are used for the separation of TMS and TFA derivatives of steoids, sugars and drugs in the biomedical field.   Cyanosilicone phases such as OV-225 used in the analysis of fatty acid methyl esters (FAME) show strong cis-trans selectivity with trans before cis elution.   Carbowax 20M, sensitive to water and oxygen exposure, used for separation of essential oil components (hydrocarbons and terpenes) not at high temteratures provides excellent results.  SE-52 and SE-54 are frequently used to separate polycyclic aromatic hydrocarbons (PAH).   FFAP was developed for the separation of free fatty acids.   Dexsil , a carboranemethyl silicone was developed for applications with high temperature operation.  Chirasil-Val, used for the resolution of D- and L-amino acid derivatives and chiral drugs, is a silicone-based phase with an in-built chiral centre allowing the separation of enantiomers . 

Film thickness

The thickness of the stationary phase film is 0.3-0.5 m.  Sample capacity increases with film thickness and thicker films and/or longer columns are more suitable for lower molecular weight volatile compounds.  Thin film columns (0.1-0.2 m) are used for high boiling compounds eluting near the upper temperature limit of the column.

Length of the column

The selected column should be as short as possible taking into the analysis requirements.  Analysis time with a 50 m column will take twice as long as with a 25 m column and will not dedliver twice the resolving power.which is proportional to the square root of the length.  When more than 50 compounds have to be separated a 50 m column is required.  A 12 m column is frequently adequate for a lot of separations.

Bore of the column

The narrower the bore of a column, the greater the resolving power but there is a danger of column overloading.  Column bores of 0.2 or 0.3 mm inside diameter are common but  0.1 mm inside diameter columns of very high resolving power are also available.  For these small bore columns, higher carrier gas pressures are required.

Flow rate

The flow rate of carrier gas can be controlled by injecting a small amount of an inert gas such as methane on to the column and by measuring the time for the signal to appear in the mass spectrometer.  This time is generally 1.5 min for a 25 m x0.2 mm i.d. column.   The linear velocity V is

where L is the column length and tg gas retention time

The volumetric flow rate F in ml/min is given by   

where r is the column radius

 

Capillary columns flow rates are set normally at 0.5-2.0 ml/min and actual flow rate can be measured with a bubble flow meter connected to the end of the column.

Compounds Retention

Compounds of different polarities move at different rates along the column and the retention time (tR) is a property of a specific compound and may be considered as an aid in its identification.  Since the experimental operating conditons are changing with time (modification of column properties with time) retention times may be expressed relative to an internal standard and this is known as the relative retention index RRI.

Relative retention index (RRI)

where tRc is the retention of the compound

   and   tRs is the retention time of the standard

 

Carrier gas pressure and flow

Carrier gas pressure can be regulated by pressure or flow.  The carrier gas viscosity increases when the GC oven temperature increases and the flow rate would decrease if pressure regulation only were employed.  When using spli/splitless injection, the use of a flow controller and pressure regulator connected in parallel will provide constant pressure on the column during injection and then a constant flow through the column even during temperature programming.  Pressure control is also important with an on-column injector.

Retention gap

A retention gap is a length of deactivated tubing placed between the injection port and the start of the capillary column and 1 m is recommended for every l os sample.  It serves as a guard column and can help to prevent peak splitting and distortion often caused by differences in the boiling points and polarities of the solvents and the solutes and their interaction with the stationary phase.

Column care

Cutting:  Columns should be cut with a diamond knife to ensure smooth edges and to avoid contamination from polyimide and increase in dead volume.

Carrier gas:  The carrier gas air content should be as low as possible because certain polar phases ( Carbowax 20 M) are very sensitive to the presence of oxygen as well as non polar phases such as SE-30 at elevated temperatures.

Rinsing of the column : In order to effect regeneration of cross-linked chemically bonded columns solvent wash is possible as illustrated herunder

A wash sequence in the opposite direction to the original carrier gas flow for an OV-1 column would be 5 ml of water, 5 ml of methanol, 5 ml of dichloromethane and finally 5 ml of hexane.

 

 

 

 

 

4. Gas Chromatography-Mass Spectrometry Interfacing

With the introduction of open capillary columns for GC and  GC-MS, an analyte enrichment interface is no longer required because the flow rate of such a column is compatible with the vacuum system of a benchtop GC-MS instrument.   Two types of GC-MS coupling are now in use: the direct coupling and the open split coupling.

Direct coupling : Efficient pumps can cope with the reduced amount of carrier gas emanating from capillary columns and the helim flow rates of 0.5 to 2.0 ml/min are handled by modern pumps.  In priciple the best interface is no interface at all, just a flexible GC column introduced directly into the ion source.  The main advantages of such a coupling are::

Simplicity,  no interface dead volume degrading the chromatographic resolution, no sample loss because transfer efficiency is 100 % providing good analytical sensitivity.   Moreover, the molecules only come into contact with the stationaty face until they reach the ion source and there are no other glass or metal surfaces on which molecules might decompose or adsorb.  The drawbacks are: a risk of source detuning and contamination due to the solvent pulse, to flow-rate changes during temperature programming and sample contaminants.  While in direct coupling the vacuum system must be switched off for changing the GC column and this operation is not required with the open split coupling.

Open split coupling

The open-split interface allow the column outlet to remain at atmospheric pressure.  The design of an open-split interface is shown herunder:

The term open is used because the GC column ends in an open system at atmospheric pressure and the term split is used because the gas flow has two different outlets, one being the ion source of the mass spectrometer.  The tube of small diameter leading to the mass spectrometer acts as a restrictor allowing a constant flow of about 1 ml/min into the ion source.  If the flow from the column is smaller than 1 ml/min , gas in helium make - up gas controlled by a valve is supplemented.  When the flow from the column exceeds 1 ml/min , the excess flow escapes though gas out at atmospheric pressure.  The device allows maximum chromatographic performance under conditions used in normal GC practice.