Gas chromatography
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Gas
chromatography
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A
gas chromatograph with a headspace sampler
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Acronym
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GC
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Classification
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Gas chromatography (GC), is a common type of chromatography
used in analytical chemistry for separating and analyzing compounds that can be vaporized
without decomposition. Typical uses of GC include testing the purity of a
particular substance, or separating the different components of a mixture (the
relative amounts of such components can also be determined). In some
situations, GC may help in identifying a compound. In preparative
chromatography, GC can be used to prepare pure
compounds from a mixture.[1][2]
In gas chromatography, the mobile
phase (or "moving phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen.
The stationary phase is a microscopic layer of liquid or polymer
on an inert solid
support, inside a piece of glass or metal tubing called a column (a homage to the fractionating column used in distillation). The instrument used to perform gas
chromatography is called a gas chromatograph (or "aerograph",
"gas separator").
The gaseous compounds being analyzed
interact with the walls of the column, which is coated with a stationary phase.
This causes each compound to elute
at a different time, known as the retention time of the compound. The
comparison of retention times is what gives GC its analytical usefulness.
Gas chromatography is in principle
similar to column chromatography (as well as other forms of chromatography, such as HPLC, TLC), but has several notable differences. Firstly, the process
of separating the compounds in a mixture is carried out between a liquid
stationary phase and a gas mobile phase, whereas in column chromatography the
stationary phase is a solid and the mobile phase is a liquid. (Hence the full
name of the procedure is "Gas–liquid chromatography", referring to
the mobile and stationary phases, respectively.) Secondly, the column through
which the gas phase passes is located in an oven where the temperature of the
gas can be controlled, whereas column chromatography (typically) has no such
temperature control. Thirdly, the concentration of a compound in the gas phase
is solely a function of the vapor pressure
of the gas.[1]
Gas chromatography is also similar
to fractional distillation, since both processes separate the components of a mixture
primarily based on boiling point (or vapor pressure) differences. However, fractional
distillation is typically used to separate components of a mixture on a large
scale, whereas GC can be used on a much smaller scale (i.e. microscale).[1]
Gas chromatography is also sometimes
known as vapor-phase chromatography (VPC), or gas–liquid partition
chromatography (GLPC). These alternative names, as well as their respective
abbreviations, are frequently used in scientific literature. Strictly speaking,
GLPC is the most correct terminology, and is thus preferred by many authors.[1]
History
Chromatography dates to 1903 in the work of the Russian scientist, Mikhail
Semenovich Tswett. German
graduate student Fritz Prior developed
solid state gas chromatography in 1947. Archer
John Porter Martin, who was awarded the Nobel Prize
for his work in developing liquid–liquid (1941) and paper (1944)
chromatography, laid the foundation for the development of gas chromatography
and he later produced liquid-gas chromatography (1950). Erika Cremer
laid the groundwork, and oversaw much of Prior's work.
GC
analysis
A gas chromatograph is a
chemical analysis instrument for separating chemicals in a complex sample. A
gas chromatograph uses a flow-through narrow tube known as the column,
through which different chemical constituents of a sample pass in a gas stream
(carrier gas, mobile phase) at different rates depending on their
various chemical and physical properties and their interaction with a specific
column filling, called the stationary phase.
As the chemicals exit the end of the column, they are detected and identified
electronically. The function of the stationary phase in the column is to
separate different components, causing each one to exit the column at a
different time (retention time). Other parameters that can be used to
alter the order or time of retention are the carrier gas flow rate, column
length and the temperature.
In a GC analysis, a known volume of
gaseous or liquid analyte is injected into the "entrance" (head) of the
column, usually using a microsyringe
(or, solid phase microextraction fibers, or a gas source switching system). As
the carrier gas sweeps the analyte molecules through the column, this motion is
inhibited by the adsorption of the analyte molecules
either onto the column walls or onto packing materials in the column. The rate
at which the molecules progress along the column depends on the strength of adsorption,
which in turn depends on the type of molecule and on the stationary phase
materials. Since each type of molecule has a different rate of progression, the
various components of the analyte mixture are separated as they progress along
the column and reach the end of the column at different times (retention time).
A detector is used to monitor the outlet stream from the column; thus, the time
at which each component reaches the outlet and the amount of that component can
be determined. Generally, substances are identified (qualitatively) by the
order in which they emerge (elute) from the column and by the retention time of
the analyte in the column.
Physical
components
Diagram of a gas chromatograph.
Autosamplers
The autosampler provides the means
to introduce a sample automatically into the inlets. Manual insertion of the
sample is possible but is no longer common. Automatic insertion provides better
reproducibility and time-optimization.
Different kinds of autosamplers
exist. Autosamplers can be classified in relation to sample capacity
(auto-injectors vs. autosamplers, where auto-injectors can work a small number
of samples), to robotic technologies (XYZ robot vs. rotating robot – the most
common), or to analysis:
- Liquid
- Static head-space by syringe technology
- Dynamic head-space by transfer-line technology
- Solid phase microextraction
(SPME)
Traditionally autosampler
manufacturers are different from GC manufacturers and currently no GC
manufacturer offers a complete range of autosamplers. Historically, the
countries most active in autosampler technology development are the United
States, Italy, Switzerland, and the United Kingdom.
Inlets
The column inlet (or injector)
provides the means to introduce a sample into a continuous flow of carrier gas.
The inlet is a piece of hardware attached to the column head.
Common inlet types are:
- S/SL (split/splitless) injector; a sample is introduced
into a heated small chamber via a syringe through a septum – the heat
facilitates volatilization of the sample and sample matrix. The carrier gas then
either sweeps the entirety (splitless mode) or a portion (split mode) of
the sample into the column. In split mode, a part of the sample/carrier
gas mixture in the injection chamber is exhausted through the split vent.
Split injection is preferred when working with samples with high analyte concentrations
(>0.1%) whereas splitless injection is best suited for trace analysis
with low amounts of analytes (<0.01%). In splitless mode the split
valve opens after a pre-set amount of time to purge heavier elements that
would otherwise contaminate the system. This pre-set (splitless) time
should be optimized, the shorter time (e.g., 0.2 min) ensures less tailing
but loss in response, the longer time (2 min) increases tailing but also
signal.
- On-column inlet; the sample is here introduced directly
into the column in its entirety without heat, or at a temperature below
the boiling point of the solvent. The low temperature condenses the sample
into a narrow zone. The column and inlet can then be heated, releasing the
sample into the gas phase. This ensures the lowest possible temperature
for chromatography and keeps samples from decomposing above their boiling
point.
- PTV injector; Temperature-programmed sample
introduction was first described by Vogt in 1979.[citation needed]
Originally Vogt developed the technique as a method for the introduction
of large sample volumes (up to 250 µL) in capillary GC. Vogt introduced
the sample into the liner at a controlled injection rate. The temperature
of the liner was chosen slightly below the boiling point of the solvent.
The low-boiling solvent was continuously evaporated and vented through the
split line. Based on this technique, Poy developed the programmed
temperature vaporising injector; PTV. By introducing the sample at a low
initial liner temperature many of the disadvantages of the classic hot
injection techniques could be circumvented.[citation needed]
- Gas source inlet or gas switching valve; gaseous
samples in collection bottles are connected to what is most commonly a
six-port switching valve. The carrier gas flow is not interrupted while a
sample can be expanded into a previously evacuated sample loop. Upon
switching, the contents of the sample loop are inserted into the carrier
gas stream.
- P/T (Purge-and-Trap) system; An inert gas is bubbled
through an aqueous sample causing insoluble volatile chemicals to be
purged from the matrix. The volatiles are 'trapped' on an absorbent column
(known as a trap or concentrator) at ambient temperature. The trap is then
heated and the volatiles are directed into the carrier gas stream. Samples
requiring preconcentration or purification can be introduced via such a
system, usually hooked up to the S/SL port.
The choice of carrier gas (mobile
phase) is important. Hydrogen has a range of flow rates that are comparable to
helium in efficiency. However, helium may be more efficient and provide the
best separation if flow rates are optimized. Helium is non-flammable and works
with a greater number of detectors and older instruments. Therefore, helium is
the most common carrier gas used. However, the price of helium has gone up
considerably over recent years, causing an increasing number of chromatographers
to switch to hydrogen gas. Historical use, rather than rational consideration,
may contribute to the continued preferential use of helium.
Detectors
The most commonly used detectors are
the flame
ionization detector (FID) and the thermal
conductivity detector (TCD). Both are sensitive to a wide
range of components, and both work over a wide range of concentrations. While
TCDs are essentially universal and can be used to detect any component other
than the carrier gas (as long as their thermal conductivities are different
from that of the carrier gas, at detector temperature), FIDs are sensitive
primarily to hydrocarbons, and are more sensitive to them than TCD. However, a
FID cannot detect water. Both detectors are also quite robust. Since TCD is
non-destructive, it can be operated in-series before a FID (destructive), thus
providing complementary detection of the same analytes.[3]
Other detectors are sensitive only
to specific types of substances, or work well only in narrower ranges of
concentrations. They include:
- Thermal Conductivity detector (TCD), this common
detector relies on the thermal conductivity of matter passing around a
tungsten -rhenium filament with a current traveling through it.[4]
In this set up helium or nitrogen serve as the carrier gas because of
their relatively high thermal conductivity which keep the filament cool
and maintain uniform resistivity and electrical efficiency of the
filament.[5][4]
However, when analyte molecules elute from the column, mixed with carrier
gas, the thermal conductivity decreases and this causes a detector
response.[5]
The response is due to the decreased thermal conductivity causing an
increase in filament temperature and resistivity resulting in flucuations
in voltage.[4]
Detector sensitivity is proportional to filament current while it's
inversely proportional to the immediate environmental temperature of that
detector as well as flow rate of the carrier gas.[4]
- Flame Ionization detector (FID), in this common
detector electrodes are placed adjacent to a flame fueled by hydrogen /
air near the exit of the column, and when carbon containing compounds exit
the column they are pyrolyzed by the flame.[5][4]
This detector works only for organic / hydrocarbon containing compounds
due to the ability of the carbons to form cations and electrons upon
pyrolysis which generates a current between the electrodes.[5][4]
The increase in current is translated and appears as a peak in a
chromatogram. FIDs have low detection limits (a few picograms per second,
but they are unable to generate ions from carbonyl containing carbons.[4]
FID compatible carrier gasses include nitrogen, helium, and argon.[5][4]
- Catalytic combustion
detector (CCD), which measures combustible hydrocarbons and hydrogen.
- Discharge ionization detector (DID), which uses a high-voltage electric discharge to
produce ions.
- Dry electrolytic conductivity detector (DELCD), which
uses an air phase and high temperature (v. Coulsen) to measure chlorinated
compounds.
- Electron capture detector (ECD), which uses a
radioactive beta particle (electron) source to measure the degree of electron
capture. ECD are used for the detection of molecules containing
electronegative / withdrawing elements and functional groups like
halogens, carbonyl, nitriles, nitro groups, and organometalics.[5][4]
In this type of detector either nitrogen or 5% methane in argon is used as
the mobile phase carrier gas.[5][4]
The carrier gas passes between two electrodes placed at the end of the
column, and adjacent to the anode (negative electrode) resides a
radioactive foil such as 63Ni.[5][4]
The radioactive foil emits a beta particle (electron) which collides with
and ionizes the carrier gas to generate more ions resulting in a current.[5][4]
When analyte molecules with electronegative / withdrawing elements or
functional groups electrons are captured which results in a decrease in
current generating a detector response.[5][4]
- Flame photometric detector (FPD),which uses a
photomultiplier tube to detect spectral lines of the compounds as they are
burned in a flame. Compounds eluting off the column are carried into a
hydrogen fueled flame which excites specific elements in the molecules,
and the excited elements (P,S, Halogens, Some Metals) emit light of
specific characteristic wavelengths.[5]
The emitted light is filtered and detected by a photomultiplier tube.[5][4]
In particular, phosphorus emission is around 510-536nm and sulfur emission
os at 394nm.[5][4]
- Atomic Emission Detector (AED), a sample eluting from a
column enters a chamber which is energized by microwaves that induce a
plasma.[5]
The plasma causes the analyte sample to decompose and certain elements
generate an atomic emission spectra.[5]
The atomic emission spectra is defracted by a difraction gradient and
detected by a series of photomultiplier tubes.[5]
- Hall electrolytic conductivity detector (ElCD)
- Helium ionization detector
(HID)
- Nitrogen–phosphorus detector
(NPD), a form a thermionic detector where nitrogen and phosphorus alter
the work function on a specially coated bead and a resulting current is
measured.
- Infrared detector (IRD)
- Mass spectrometer (MS) – also called (GC-MS) highly effective and
sensitive, even in a small quantity of sample.
- Photo-ionization detector
(PID)
- Pulsed discharge ionization detector (PDD)
- Thermionic ionization detector (TID)
Some gas chromatographs are connected
to a mass spectrometer which acts as the detector. The combination is known as GC-MS. Some GC-MS are connected to an NMR spectrometer
which acts as a backup detector. This combination is known as GC-MS-NMR. Some GC-MS-NMR are connected to an infrared
spectrophotometer which acts as a backup detector.
This combination is known as GC-MS-NMR-IR. It must, however, be stressed this
is very rare as most analyses needed can be concluded via purely GC-MS.[citation needed]
Methods
This image above shows the interior
of a GeoStrata Technologies Eclipse Gas Chromatograph that runs continuously in
three minute cycles. Two valves are used to switch the test gas into the sample
loop. After filling the sample loop with test gas, the valves are switched
again applying carrier gas pressure to the sample loop and forcing the sample
through the Column for separation.
The method is the collection
of conditions in which the GC operates for a given analysis. Method
development is the process of determining what conditions are adequate
and/or ideal for the analysis required.
Conditions which can be varied to
accommodate a required analysis include inlet temperature, detector
temperature, column temperature and temperature program, carrier gas and
carrier gas flow rates, the column's stationary phase, diameter and length,
inlet type and flow rates, sample size and injection technique. Depending on
the detector(s) (see below) installed on the GC, there may be a number of
detector conditions that can also be varied. Some GCs also include valves which
can change the route of sample and carrier flow. The timing of the opening and
closing of these valves can be important to method development.
Carrier
gas selection and flow rates
Typical carrier gases include helium, nitrogen,
argon,
hydrogen
and air.
Which gas to use is usually determined by the detector being used, for example,
a DID requires helium as the carrier gas. When analyzing gas
samples, however, the carrier is sometimes selected based on the sample's
matrix, for example, when analyzing a mixture in argon, an argon carrier is
preferred, because the argon in the sample does not show up on the
chromatogram. Safety and availability can also influence carrier selection, for
example, hydrogen is flammable, and high-purity helium can be difficult to
obtain in some areas of the world. (See: Helium—occurrence and
production.) As a result of helium becoming
more scarce, hydrogen is often being substituted for helium as a carrier gas in
several applications.
The purity of the carrier gas is
also frequently determined by the detector, though the level of sensitivity
needed can also play a significant role. Typically, purities of 99.995% or
higher are used. The most common purity grades required by modern instruments
for the majority of sensitivities are 5.0 grades, or 99.999% pure meaning that
there is a total of 10ppm of impurities in the carrier gas that could affect
the results. The highest purity grades in common use are 6.0 grades, but the
need for detection at very low levels in some forensic and environmental
applications has driven the need for carrier gases at 7.0 grade purity and
these are now commercially available. Trade names for typical purities include
"Zero Grade," "Ultra-High Purity (UHP) Grade," "4.5
Grade" and "5.0 Grade."
The carrier gas linear velocity
affects the analysis in the same way that temperature does (see above). The
higher the linear velocity the faster the analysis, but the lower the
separation between analytes. Selecting the linear velocity is therefore the
same compromise between the level of separation and length of analysis as
selecting the column temperature. The linear velocity will be implemented by
means of the carrier gas flow rate, with regards to the inner diameter of the
column.
With GCs made before the 1990s,
carrier flow rate was controlled indirectly by controlling the carrier inlet
pressure, or "column head pressure." The actual flow rate was
measured at the outlet of the column or the detector with an electronic flow
meter, or a bubble flow meter, and could be an involved, time consuming, and
frustrating process. The pressure setting was not able to be varied during the
run, and thus the flow was essentially constant during the analysis. The
relation between flow rate and inlet pressure is calculated with Poiseuille's
equation for compressible fluids.
Many modern GCs, however,
electronically measure the flow rate, and electronically control the carrier
gas pressure to set the flow rate. Consequently, carrier pressures and flow
rates can be adjusted during the run, creating pressure/flow programs similar
to temperature programs.
Stationary
compound selection
The polarity
of the solute is crucial for the choice of stationary compound, which in an
optimal case would have a similar polarity as the solute. Common stationary
phases in open tubular columns are cyanopropylphenyl dimethyl polysiloxane,
carbowax polyethyleneglycol, biscyanopropyl cyanopropylphenyl polysiloxane and
diphenyl dimethyl polysiloxane. For packed columns more options are available.[4]
Inlet
types and flow rates
The choice of inlet type and
injection technique depends on if the sample is in liquid, gas, adsorbed, or
solid form, and on whether a solvent matrix is present that has to be
vaporized. Dissolved samples can be introduced directly onto the column via a
COC injector, if the conditions are well known; if a solvent matrix has to be
vaporized and partially removed, a S/SL injector is used (most common injection
technique); gaseous samples (e.g., air cylinders) are usually injected using a
gas switching valve system; adsorbed samples (e.g., on adsorbent tubes) are
introduced using either an external (on-line or off-line) desorption apparatus
such as a purge-and-trap system, or are desorbed in the injector (SPME
applications).
Sample
size and injection technique
Sample
injection
The rule of ten in gas
chromatography
The real chromatographic analysis
starts with the introduction of the sample onto the column. The development of
capillary gas chromatography resulted in many practical problems with the
injection technique. The technique of on-column injection, often used with
packed columns, is usually not possible with capillary columns. The injection
system in the capillary gas chromatograph should fulfil the following two
requirements:
- The amount injected should not overload the column.
- The width of the injected plug should be small compared
to the spreading due to the chromatographic process. Failure to comply with
this requirement will reduce the separation capability of the column. As a
general rule, the volume injected, Vinj, and the volume of the
detector cell, Vdet, should be about 1/10 of the volume
occupied by the portion of sample containing the molecules of interest
(analytes) when they exit the column.
Some general requirements which a
good injection technique should fulfill are:
- It should be possible to obtain the column’s optimum
separation efficiency.
- It should allow accurate and reproducible injections of
small amounts of representative samples.
- It should induce no change in sample composition. It
should not exhibit discrimination based on differences in boiling point,
polarity, concentration or thermal/catalytic stability.
- It should be applicable for trace analysis as well as
for undiluted samples.
Column
selection
The choice of column depends on the
sample and the active measured. The main chemical attribute regarded when
choosing a column is the polarity
of the mixture, but functional groups can play a large part in column selection. The polarity of
the sample must closely match the polarity of the column stationary phase to
increase resolution and separation while reducing run time. The separation and
run time also depends on the film thickness (of the stationary phase), the
column diameter and the column length.
Column
temperature and temperature program
A gas chromatography oven, open to
show a capillary column
The column(s) in a GC are contained
in an oven, the temperature of which is precisely controlled electronically.
(When discussing the "temperature of the column," an analyst is
technically referring to the temperature of the column oven. The distinction,
however, is not important and will not subsequently be made in this article.)
The rate at which a sample passes
through the column is directly proportional to the temperature of the column.
The higher the column temperature, the faster the sample moves through the
column. However, the faster a sample moves through the column, the less it
interacts with the stationary phase, and the less the analytes are separated.
In general, the column temperature
is selected to compromise between the length of the analysis and the level of
separation.
A method which holds the column at
the same temperature for the entire analysis is called "isothermal."
Most methods, however, increase the column temperature during the analysis, the
initial temperature, rate of temperature increase (the temperature
"ramp"), and final temperature are called the "temperature
program."
A temperature program allows
analytes that elute early in the analysis to separate adequately, while
shortening the time it takes for late-eluting analytes to pass through the
column.
Data
reduction and analysis
Qualitative
analysis
Generally chromatographic data is
presented as a graph of detector response (y-axis) against retention time
(x-axis), which is called a chromatogram. This provides a spectrum of peaks for
a sample representing the analytes present in a sample eluting from the column at different
times. Retention time can be used to identify analytes if the method conditions
are constant. Also, the pattern of peaks will be constant for a sample under
constant conditions and can identify complex mixtures of analytes. In most
modern applications however the GC is connected to a mass spectrometer or similar detector that is capable of identifying the
analytes represented by the peaks.
Quantitative
analysis
The area under a peak is
proportional to the amount of analyte present in the chromatogram. By
calculating the area of the peak using the mathematical function of
integration, the concentration of an analyte in the original sample can be
determined. Concentration can be calculated using a calibration curve created by finding the response for a series of
concentrations of analyte, or by determining the relative response factor of an analyte. The relative response factor is the expected
ratio of an analyte to an internal standard (or external standard)
and is calculated by finding the response of a known amount of analyte and a
constant amount of internal standard (a chemical added to the sample at a
constant concentration, with a distinct retention time to the analyte).
In most modern GC-MS systems, computer software is used to draw and integrate peaks, and match MS
spectra to library spectra.
Applications
In general, substances that vaporize
below ca. 300 °C (and therefore are stable up to that temperature) can be
measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance
can be measured, but it is often required that the sample must be measured in
comparison to a sample containing the pure, suspected substance known as a reference standard.
Various temperature programs can be
used to make the readings more meaningful; for example to differentiate between
substances that behave similarly during the GC process.
Professionals working with GC
analyze the content of a chemical product, for example in assuring the quality
of products in the chemical industry; or measuring toxic substances in soil,
air or water. GC is very accurate if used properly and can measure picomoles
of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
In practical courses at colleges,
students sometimes get acquainted to the GC by studying the contents of Lavender
oil or measuring the ethylene that is secreted by Nicotiana
benthamiana plants after artificially injuring
their leaves. These GC analyses hydrocarbons (C2-C40+). In a typical
experiment, a packed column is used to separate the light gases, which are then
detected with a TCD. The hydrocarbons
are separated using a capillary column and detected with an FID. A complication with light gas analyses that include H2
is that He, which is the most common and most sensitive inert carrier
(sensitivity is proportional to molecular mass) has an almost identical thermal
conductivity to hydrogen (it is the difference in thermal conductivity between
two separate filaments in a Wheatstone Bridge type arrangement that shows when
a component has been eluted). For this reason, dual TCD instruments are used
with a separate channel for hydrogen that uses nitrogen as a carrier are
common. Argon is often used when analysing gas phase chemistry reactions such
as F-T synthesis so that a single carrier gas can be used rather than 2
separate ones. The sensitivity is less but this is a tradeoff for simplicity in
the gas supply.
GCs
in popular culture
Movies, books and TV shows tend to
misrepresent the capabilities of gas chromatography and the work done with
these instruments.
In the U.S. TV show CSI, for example, GCs are used to
rapidly identify unknown samples. For example, an analyst may say fifteen
minutes after receiving the sample: "This is gasoline
bought at a Chevron station in the past two weeks."
In fact, a typical GC analysis takes
much more time; sometimes a single sample must be run more than an hour
according to the chosen program; and even more time is needed to "heat
out" the column so it is free from the first sample and can be used for
the next. Equally, several runs are needed to confirm the results of a study –
a GC analysis of a single sample may simply yield a result per chance (see statistical
significance).
Also, GC does not positively
identify most samples; and not all substances in a sample will necessarily be
detected. All a GC truly tells you is at which relative time a component eluted
from the column and that the detector was sensitive to it. To make results
meaningful, analysts need to know which components at which concentrations are
to be expected; and even then a small amount of a substance can hide itself
behind a substance having both a higher concentration and the same relative
elution time. Last but not least it is often needed to check the results of the
sample against a GC analysis of a reference sample containing only the
suspected substance.
A GC-MS can remove much of this ambiguity,
since the mass spectrometer will identify the component's molecular weight. But this
still takes time and skill to do properly.
Similarly, most GC analyses are not push-button
operations. You cannot simply drop a sample vial into an auto-sampler's tray,
push a button and have a computer tell you everything you need to know about
the sample. The operating program must be carefully chosen according to the
expected sample composition.
A push-button operation can exist
for running similar samples repeatedly, such as in a chemical production
environment or for comparing 20 samples from the same experiment to calculate
the mean content of the same substance. However, for the kind of investigative
work portrayed in books, movies and TV shows this is clearly not the case.