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Gas Chromatography (GC) and Liquid Chromatography (LC)

Gas Chromatography (GC) and Liquid Chromatography (LC)

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246



Mass Spectrometry Basics



Spinach leaf

extract



(



Chlorophyll-b (green)



Sugar

(



(



Xanthophyll (yellow)



Chalk



~~~~~~ (



Carotene (red)



Alumina



-.



LC column



Figure 35.1

A chromatographic column filled in three sections with ground sugar, chalk, and alumina. When a petroleum extract of

spinach leaves is run onto the top of the column, the extract spreads down the column, hut not uniformly; bands of green

chlorophylls stop near the top. yellow xanthophyll further down, and red carotene near the bottom.



mixture to pass through the chromatography column. During its passage through the column

of stationary phase, a mixture gradually separates into its individual components, some passing

through the column of stationary phase faster than others and emerging (eluting) earlier.



Requirements for Chromatographic Apparatus

In principle, a chromatographic instrument is very simple and is little more than an advanced

plumbing system (Figure 35.2a, b). The mixture to be separated is put onto the start (or top) of the

column using an injector. The gas or liquid mobile phase flows from a suitable store (typically a

gas cylinder or bottle of solvent) past the injector and carries the injected mixture through the

chromatographic column, which contains a stationary phase (a liquid for ac and a modified solid

for LC). When the separated components emerge (elute) from the end (or bottom) of the column,

they must be sensed by a detector. Finally, the detector must send an electrical signal, directly or

indirectly, to a readout device or recorder. The time from injection of the mixture to recording the

elution of individual components can be from a few seconds to an hour or more. The resulting

chart from the recorder shows the amounts of separated components plotted against their times of

elution and is called a chromatogram (Figure 35.3a, b).

Many kinds of detectors have been designed, ranging from the widely used, cheap but robust

flame ionization (aC) or ultraviolet absorption type (LC) to the much more exciting and informative,

if much more expensive, mass spectrometer.



Gas Chromatography (GC) and Liquid Chromatography (LC)



247



Sample in



t



Chromatographic

column



(a)



Detector



Recorder



I

I



8Gas supply

cylinder



Chromatogram

(b)



Loop injector Sample in



-,



Detector



Recorder



I



High

pressur

pump



I



8-



Figure 35.2

(a) Schematic layout of a gas chromatograph. A sample is vaporized in the injector, mixed with nitrogen, and carried by

thegas stream through a column, where separation into components occurs. The emerging (eluting) components are detected

and recorded to give a gas chromatogram. Alternatively, a personal computer can be used to acquire and display data. The

injector, column, and the lower part of the detector are placed in an oven. (b) Schematic layout for a liquid chromatograph.

Note the essential similarity to the gas chromatograph shown in Figure 35.2a; however, in detail, the injector, column, and

detector are quite different. No oven is needed.



(a)



Amountl



s.,

lime~



(ij



Amounti



Ttme~



Figure 35.3

Typical (a) gas and (b) liquid chromatograms. The charts show amounts (y-axis) of substance emerging from a column

versus time (x-axis). The time taken (measured at the top of a peak) for a substance to elute is called a retention time.



248



Mass Spectrometry Basics



The Chromatographic Process

The principles behind separation of mixtures by GC or LC are more or less the same, but the

experimental procedures are different. During chromatography, anyone component of a mixture

is partitioned between the mobile and stationary phases such that, at anyone instant, some of the

component is in one phase and the remainder is in the other. In this dynamic equilibrium system,

progress of a component along the chromatographic column is effected by the flow of the mobile

phase in a process of continual exchange between the stationary and mobile phases. The process

can be likened simplistically to the movement of a bus along its route. For some of the time, the

bus moves (molecules in the mobile phase), but, at other times, the bus is stopped (molecules in

the stationary phase). The time taken by a bus to cover its route (retention time for molecules in

chromatography) depends on the number and the length of time of the stops it makes and its speed

between stops.

Similarly, with molecules, their speed of movement through the chromatographic column

depends on the time spent in the mobile phase compared with that in the stationary one and on the

flow rate of the mobile phase.



Gas Chromatographic Phases

For GC, the mobile phase is a gas (usually nitrogen or helium), and the movement of mixture

components along the column will depend on their volatilities; the more volatile a component, the

more time is spent in the gas phase and the faster it will be swept along. This factor is a major

one, but not the only, controlling the rate of movement of a substance through a gas chromatographic

column. The various components of a mixture will have different affinities for the stationary phase,

a nonvolatile liquid over which the mobile gas phase flows. Thus, for two components having the

same volatilities but differing polarities, their rates of passage will be different. A polar component

will tend to stick on a polar stationary phase but not on a nonpolar one, while nonpolar compounds

will be held more strongly by nonpolar stationary phases. The ability of a stationary phase to

separate mixtures in GC depends on its chemical composition. Many types of stationary phase are

used, a few of which are listed in Table 35. I. The choice of stationary phase affects GC operation.

An example of the change in gas chromatographic behavior for two different columns of the same

length is shown in Figure 35.4.

Because volatility is such an important factor in GC, the chromatographic column is contained

in an oven, the temperature of which can be closely and reproducibly controlled. For very volatile



TABLE 35.1

Some Typical Stationary Phases for Use in GC

Approximate

Name



Type



Polarity



(McReynolds Number)*



Nujol



Mineral oil



Nonpolar



11



Silicone SE-30



Elastomer



Nonpolar



41



Grease



Nonpolar



42



Silicone OV-I7



Elastomer



Intermediate



202



Didecyl phthalate



Apiezon L



Pure liquid



Intermediate



235



Carbowax 20M



Polymer



Polar



510



DEGS



Polymer



Very polar



835



*



The McReynolds number gives an approximate indication of polarity on a scale of 0

(nonpolar) to about 1000 (extremely polar).



249



Gas Chromatography (GC) and Liquid Chromatography (LC)



(a)

toluen



o



pyridine



(b)



pyridine



benzaldehyde



Time



toluen



benzaldehyde



/



o



Figure 35.4

Two gas chromatograms showing the effect of polarity of the stationary phase on the separation efficiency for three substances

of increasing polarity: toluene, pyridine, and benzaldehyde. (a) Separation on silicone SE-30, a nonpolar phase, and (b)

separation on elastomer OV-351, a more polar phase. Note the greatly changed absolute and relative retention times; the

more polar pyridine and benzaldehyde are affected most by the move to a more polar stationary phase.



compounds, the oven may be operated at only 30-40'C, but for nonvolatile substances, temperatures

of 200--250"C may be needed. For GC analysis, the column can be operated at one fixed temperature

(isothermally) or over a temperature range (temperature programming).

In general, the longer a chromatographic column, the better will be the separation of mixture

components. In modem gas chromatography, columns are usually made from quartz and tend to

be very long (coiled), often 10--50 m, and narrow (0.1-1.0 mm, internal diameter) - hence their

common name of capillary columns. The stationary phase is coated very thinly on the whole length

of the inside wall of the capillary column. Typically, the mobile gas phase flows over the stationary

phase in the column at a rate of about 1-2 ml/min.



Liquid Chromatographic Phases

For LC, temperature is not as important as in GC because volatility is not important. The columns

are usually metal, and they are operated at or near ambient temperatures, so the temperaturecontrolled oven used for GC is unnecessary. An LC mobile phase is a solvent such as water,

methanol, or acetonitrile, and, if only a single solvent is used for analysis, the chromatography is

said to be isocratic. Alternatively, mixtures of solvents can be employed. In fact, chromatography

may start with one single solvent or mixture of solvents and gradually change to a different mix

of solvents as analysis proceeds (gradient elution).

The stationary phase in LC is a fine granular solid such as silica gel. It can be used as such

(mainly for nonpolar compounds), or the granules can be modified by a surface-bonded coating

that changes (reverses) the polarity of the gel. A very small selection of stationary phases is

listed in Table 35.2.

TABLE 35.2



Some Solid Stationary

Phases for Use in LC

Name



Polarity



Bondapak CI8



Non-polar



Zorbax ODS



Non-polar



Carbowax 400



Intermediate



Micropak CN



Intermediate



Corasil II



Polar



Silica gel Very



Polar



250



Mass Spectrometry Basics



Reversed-phase columns are used to separate polar substances. Although in LC the stationary

phase is a solid, it is necessary to bear in mind that there may be a thin film of liquid (e.g.,

water) held on its surface, and this film will modify the behavior of sample components equilibrating between the mobile and stationary phases. A textbook on LC should be consulted for

deeper discussion on such aspects.

In LC, because the mobile phase is a liquid and the stationary one is a granular solid,

viscosity limitations rule out the simple use of the long capillary columns found in Gc. Short

columns of 10-25 em and 2-4 mm internal diameter are more usual in LC. It is difficult to

force the mobile solvent phase through such columns, and high-pressure pumps must be used

to get a reasonable flow rate - hence the name high-pressure liquid chromatography (HPLC).

More recently, very narrow LC columns have become available, but these nanocolumns must

be operated at very high pressure to force the mobile liquid phase through them. Typically, a

liquid phase flow of some I to 2 mllmin at a pressure of 20 to 200 bar (300 to 3000 lb/in-)

is used. Very often the high-pressure part of the terminology is omitted, as in LC/MS rather

than HPLCIMS



Injectors

For GC, the injector is most frequently a small heated space attached to the start of the column.

A sample of the mixture to be analyzed is injected into this space by use of a syringe, which pierces

a rubber septum. The injector needs to be hot enough to immediately vaporize the sample, which

is then swept onto the head of the column by the mobile gas phase. Generally, the injector is kept

at a temperature 50°C higher than is the column oven. Variants on this principle are in use, in

particular the split/splitless injector. This injector can be used in a splitless mode, in which the

entire injected sample goes onto the column, or in a split mode, in which only part of the sample

goes onto the column, the remainder vented to atmosphere. For other less usual forms of injector,

a specialist book on GC should be consulted.

For liquid chromatography, a sample of the mixture solution is injected through a loop injector

which allows a quantity of the solution to be placed in a small tubular loop at atmospheric pressure,

By manipulating a valve, the high-pressure flow of solvent to the column is diverted through the

loop, carrying the sample with it (Figure 35.5).



Detectors

Specialized detectors and inlet systems for GC/MS and LC/MS are described in Chapters 36

and 37, respectively.

The effluent from the end of a GC column is usually nitrogen or helium and contain a very

small proportion of organics as they emerge (elute) from the column. The most widely used detector

is one in which the eluate is burned continuously in a flame after admixing it with hydrogen and

air (flame ionization detector). Ions and electrons formed when emerging organics burn in the flame

are monitored electronically, and the resulting electric current is used to drive a chart recorder

(Figure 35.6).

In LC, the most common means for monitoring the eluant is to pass it through a cell

connected into an ultraviolet spectrometer. As substances elute from the column, their ultraviolet absorption is measured and recorded. Alternatively, the refractive index of the eluant is

monitored since it varies from the value for a pure solvent when it contains organics from the

column.



251



Gas Chromatography (GC) and Liquid Chromatography (LC)



Sample in (atmospheric pressure)



t



{oVerflOW



Injector block

Sample loop -f------i~



4.1-+--+--+-- Rotatable center

block



t



~



Solvent flow To column

(pressurized)



Figure 35.5

A typical loop injector showing the sampling position with pressurized solvent flowing through one loop onto the column

and the sample solution placed in the other loop at atmospheric pressure. Rotation of the loop carrier through 180' puts

the sample into the liquid flow at high pressure with only momentary change in pressure in the system.



~--+--



Cylindrical electrode



'"-==c===~==1

Electrical

current ~I!\)-E-+--+-- Flame



t

t



Outer screening can



Hydrogen~ _ _--r----r_--,



t



Column effluent



Figure 35.6

Schematic diagram of a flame ionization detector. Ions and electrons formed in the flame provide an electrically conducting

path between the flame at earth potential and an insulated cylindrical metal electrode at high potential surrounding the

flame; the flow of current is monitored, amplified, and passed to the recording system.



Uses of GC and LC

As a rule of thumb, one can say that the efficiency of separation of mixtures and the simplicity of

operating and maintaining apparatus are much greater for GC than for LC. Hence, other things

being equal, GC is most often the technique of first choice and can be used with a very wide variety

of compound types. However, for nonvolatile or thermally labile substances like peptides, proteins,

nucleotides, sugars, carbohydrates, and many organometallics, GC may be ruled out completely



252



Mass Spectrometry Basics



and LC comes into its own. Apart from availability of instrumentation, in choosing between GC

and LC, it is necessary to consider the nature of the sample to be analyzed. Whereas GC cannot

be used with nonvolatile or thermally labile compounds, LC can be used for almost all the types

of substance routinely analyzed by Gc. The major reasons for not simply using LC for all types

of compound lie mostly in the far greater achievable resolution of mixtures into their components

by GC, the generally greater sensitivity of GC detectors (nanogram amounts can be analyzed easily),

and the easier operation of GC instruments.



Conclusion

Mixtures passed through special columns (chromatography) in the gas phase (GC) or liquid phase

(LC) can be separated into their individual components and analyzed qualitatively and/or quantitatively. Both GC and LC analyzers can be directly coupled to mass spectrometers, a powerful

combination that can simultaneously separate and identify components of mixtures.



Chapter



Gas Chromatography/Mass

Spectrometry (GC/MS)

Introduction

As its name implies, this important analytical technique combines two separate procedures: gas

chromatography (GC) and mass spectrometry (MS). Both individual techniques are quite old. OC

developed as a means of separating volatile mixtures into their component substances and provided

a big step forward in the analysis of mixtures. The method is described fully in Chapter 35, but it

can be summarized as follows (Figure 36.1): by passing a mixture in a gas stream (the gas phase)

through a long capillary column, the inside walls of which are thinly coated with a liquid (the

liquid phase), the components of the mixture become separated and emerge (elute) one after another

from the end of the column. In a simple OC instrument, the emerging components are either burnt

in a flame for detection (the popular flame ionization detector) or passed to atmosphere after

traversing some other kind of detector. The detected components are recorded as peaks on a chart

(the gas chromatogram). The area of a peak correlates with the amount of a component, and the

time taken to pass through the column (the retention time, i.e., the time to the peak maximum)

gives some information on the possible identity of the component. However, the identification is

seldom absolutely certain and is often either vague or impossible to determine.

In complete contrast to a GC apparatus, a mass spectrometer is generally not useful for dealing

with mixtures. If a single substance is put into a mass spectrometer, its mass spectrum (Figure 36.2)

can be obtained with a variety of ionization methods that are described in Chapters I through 5.

Once the spectrum is obtained, it is often possible to make a positive identification of the substance

or to confirm its molecular structure. Clearly, if a mixture of substances were put into the MS, the

resulting mass spectrum would be a summation of the spectra of all the components (Figure 36.3).

This spectrum would be extremely complex, and it would be impossible to identify positively the

various components. (Note that some MS instruments can deal with mixtures, and these are

described in Chapters 20 through 23, which deal with hybrid MSIMS systems, and Chapters 33

and 34, which deal with linked scanning.)

Thus, there is one instrument (GC) that is highly efficient for separating mixtures into their

components but is not good on identification, and another instrument (MS) that is efficient at

identifying single substances but is not good with mixtures. It is not surprising to find that early

efforts were made to combine the two instruments into one system (OCIMS) capable of separating,

positively identifying, and quantifying complex mixtures, provided these could be vaporized. Com-



253



254



Mass Spectrometry Basics

Mixture (AB,C,D) Separated components

Sample in

out

Detector Signal



Injector



Electronics



GC Column



Amountl



Gas chromatogram



Figure 36.1

Schematic diagram showing the injection of a mixture of four substances (A, B, C, D) onto a GC column, followed by

their separation into individual components, their detection, and the display (gas chromatogram) of the separated materials

emerging at different times from the column.



Mass spectrometer

I

I



Sample (A

in



If-+



Electronics



I

Ionization



Ion

separation



\

Ion

detection



Abundancej

of Ions



I u1.



.I.

mlz~



Mass spectrum of A



Figure 36.2

Schematic diagram of a mass spectrometer. After insertion of a sample (A), it is ionized, the ions are separated according

to mlz value, and the numbers of ions (abundances) at each m/z value are plotted against m/z to give the mass spectrum

of A. By studying the mass spectrum, A can be identified.



bining GC and MS was not without its problems (see below), but modem GCfMS is now a routinely

used methodology in many areas, ranging from interplanetary probes to examination of environmental dust samples to determine dioxin levels. Further, the addition of GC to MS does not simply

give a sum of the two alone; the information provided by combined GCfMS yields information that

could not be extracted from either technique alone, an aspect that is discussed below.



Connection between GC and MS

As described above, the mobile phase carrying mixture components along a gas chromatographic

column is a gas, usually nitrogen or helium. This gas flows at or near atmospheric pressure at a rate

generally about 0.5 to 3.0 ml/min and eventually flows out of the end of the capillary column into

the ion source of the mass spectrometer. The ion sources in GefMS systems normally operate at

about 10-5 mbar for electron ionization to about 10-3 mbar for chemical ionization. This large pressure



Gas Chromatography/Mass Spectrometry (GC/MS)



255



(a)



Figure 36.3

By way of illustration. very simple spectra for



fOUT substances (A, B, C, D) are shown: (a) separately and (b) mixed in unequal

proportions. The mixture spectrum is virtually impossible to decode if A, B, C, 0 are not known beforehand to be present.



change between the end of the chromatographic column and the inside of the ion source causes the

gas to expand and reach a flow equivalent to several liters per minute. Therefore, large pumps are

required to remove the excess gas while maintaining the vacuum inside the source near the level

optimumfor ionization. In modem GCIMS installations, the use of capillary chromatographic columns

and high-speed pumps means that the end of the column can be literally right inside the ion source.

In older GCIMS instruments, gas flow rates were much greater, and it was necessary to have an

interface between the end of the column and the ion source. This interface or separator removed much

of the GC carrier gas without also removing the eluting mixture components, which traveled into the

ion source. The jet separator was a popular form that can still be found occasionally.



Recording Mass Spectra

As each mixture component elutes and appears in the ion source, it is normally ionized either by

an electron beam (see Chapter 3, "Electron Ionization") or by a reagent gas (see Chapter 1,

"Chemical Ionization"), and the resulting ions are analyzed by the mass spectrometer to give a

mass spectrum (Figure 36.4).

For capillary GC, separated mixture components elute in a short time interval, often lasting only

a few seconds. Thus, the amount of anyone component in the ion source is not constant as its mass

spectrum is being obtained. Rather, it starts at zero, rises rapidly to a maximum, and drops rapidly

back to zero. If this passage through the ion source is faster than the mass spectrometer's ability to

scan the spectrum, then a true spectrum will not be found because the start and end of the scan will

show less compound than at the middle of the scan. This changing concentration of eluting component

results in a distorted mass spectrum that might not be recognizable (Figure 36.5). The answer to this

problem is to scan the spectrum so fast that, in effect, the concentration of the eluting component

scarcely changes during the time needed to acquire a spectrum.

For a quadrupole mass spectrometer, this high rate of scanning is not difficult because it requires

only simple changes in some electrical VOltages, and these changes can be made electronically at

very high speed, which is why quadrupoles are popular in GCIMS combinations. In the early days

of magnetic-sector mass spectrometers, the required scanning speed was not possible because of

serious hysteresis effects in the magnets. With modem magnet technology, scanning can be done



Mass Spectrometry Basics



256



A



Separated

components

B



MS



C

Sample in

(mixture A, B, C, 0)



D



Mass spectra of individual

components



Figure 36.4

In a GC/MS combination, passage of the separated components (A, B, C. D) successively into the mass spectrometer yields

their individual spectra.



(i)



{iii



!



!



OCLJ ..'.

Figure 36.5

Slow scanning (i) of the mass spectrum over a GC peak for substance A gives spectrum (a), but rapid scanning (ii) gives

spectrum (b), which is much closer to the true spectrum (c).



at high speed with insignificant hysteresis, and magnetic-sector instruments can now compete with

quadrupoles, While ultimate scan speed for a magnetic instrument is not as good as the quadrupole's,

the former does have the advantage of providing greater mass resolution at higher mass. However,

in GCfMS, where the highest analyzed mass is likely to be less than about 600 Da, this advantage

of the magnetic instrument is of little consequence.

As described above, the concentration of an eluting component in the ion source goes from

zero to zero through a maximum. Where should the scan be taken? Usually, the greater the

amount of a substance in an ion source, the better will be the resulting mass spectrum (within

reason!). This situation suggests that the best time for a scan will be near the maximum,

concentration (the top of a GC peak) and that the instrument operator must watch the developing

chromatogram continually, trying to judge the best moment to measure a spectrum. Since a gas

chromatogram can routinely take 20 to 50 min to obtain, such a watching brief is labor intensive

(and deadly dull l). The simple answer is to set the mass spectrometer scanning continuously.

Therefore, as the mixture is injected onto the chromatographic column, repetitive scanning is

instituted over a preset mass range (e.g., 50 to 500 mass units) at a preset interval (e.g., every



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