7
INSTRUMENTAL CHROMATOGRAPHY
7.1 Introduction
There are two forms of chromatography, very widely used in analytical laboratories, which rely on
electronic control of the process and detection of the species. Both are column-based:

gas chromatography - where the mobile phase is a gas, and the separation is based on polarity
attraction to the stationary phase and volatility attraction to the mobile phase

high performance liquid chromatography – where the mobile phase is a liquid, and separation is
based on competition between the polarity of the two phases
7.2 Gas Chromatography
In gas chromatography (GC), components are carried through a heated column containing the
stationary phase by an inert gas, which is the mobile phase.
Mobile phase
The mobile phase, often called the carrier gas, is most often nitrogen or helium. It has no chemical
attraction for the components of the mixture being separated; it competes for the compounds by heat –
the more volatile (lower boiling) compounds will be attracted to the mobile phase. This method of
attraction gives GLC its one important limitation: only volatile compounds can be analysed, since
travel through the medium is in the gas phase.
Stationary phase
The stationary phase can be a solid (gas-solid chromatography, GSC) or more commonly, a liquid
(gas-liquid chromatography, GLC). In the latter case, the liquid is high-boiling (> 400ºC) and is either
coated on a inert powder material (a packed column), known as a support or on the inside walls of the
column (a capillary column). The stationary phase competes for the analytes by polarity(GLC) or
adsorption (GSC).
Stationary
phase
Polarity
Compounds in
sample
Mobile
phase
Heat
Instrument configuration
The basic layout of a gas chromatography instrument is shown in Figure 7.1.
Oven
Carrier
gas
reserve
Flow
rate
control
FIGURE 7.1 Typical gas chromatograph
Injection
port
Column
Readout
Detector
7. Instrumental Chromatography
CARRIER GAS
Carrier gases are supplied in compressed form in bottles, and the flow rate is then controlled by a tap
mechanism on the instrument. Flow rates are typically 1-10 mL/minute. the faster the flow rate, the
faster the components move through the column.
INJECTION PORTS
Sample sizes in GC are very small: typically 0.1 to 10 uL in solution. This is done manually or by a
robotic controller with a calibrated microsyringe, through a rubber septum into a heated area at the
head of the column. The septum provides a seal so that the sample doesn’t vaporise back out the
injection port!
Manual injection is prone to errors because of the small volumes and likelihood of trapping air
bubbles. Duplicate injection s are essential, and the use of internal standards even better 9see
Applications)
The injection port must be heated to immediately vaporise all of the sample, typically using
temperatures of at least 50ºC above the boiling point of the least volatile component of the mixture. If
the sample was not vaporised upon injection, the carrier gas would selectively evaporate the more
volatile components, leaving the others behind. So when you are injecting a sample into a GC, be
careful not to put your fingers on the metal port; otherwise you’ll be rewarded with the smell of
burning flesh!!
COLUMNS
In this subject, we will only deal with packed columns: these are not as efficient as capillary columns,
but have other advantages. A typical packed column is made of stainless steel, and is 2-3 metres long.
Its internal diameter is 3-5 mm. The column is coiled so that the oven it is placed into is as small as
possible.
Packed columns for GLC need the liquid stationary phase to be physically or chemically bonded
to an inert support material. The solid support generally consists of small, uniform spherical particles
with good strength and high surface area. The first, and still most widely used, supports are silicates
prepared from diatomaceous earth, which consists of the skeletons of thousands of single-celled plants
that inhabited ancient lakes and seas.
Desirable properties for the liquid phase in a GLC column are:

low volatility (b.p. at least 100ºC above maximum operating temperature)

thermal stability

chemical inertness

suitable polarity characteristics to separate the mixture
Table 7.1 lists some of the common stationary phases.
TABLE 7.1 Common stationary phases for GLC
Name *
OV-1
Max. Temp (ºC)
350ºC
Polarity
non-polar
DNP
150ºC
medium
Carbowax 20M
250ºC
polar
Applications
hydrocarbons
alkanones
essential oils, alkanols
* the different companies making stationary phases may have other names for equivalent materials
DETECTORS
The detector is responsible for measuring the gas stream as it emerges from the column, and
responding rapidly to microgram amounts of the solutes. Other desirable properties of a detector are
linear response, stability and uniform response to a wide variety of chemical species. Table 7.2
describes the two most common GC detectors.
Sci Inst Analysis (Spectro/Chrom)
7.2
7. Instrumental Chromatography
TABLE 7.2 Common GC detectors
Name
Thermal
conductivity
Mechanism of detection
Measures heat conduction of gas
stream relative to carrier gas
Features
Simple, cheap, responds to all species,
relatively insensitive
Flame
ionisation
Gas stream passes through flame;
combustible species produce ions
which are measured
Requires separate hydrogen and air lines
for flame, very sensitive to species that
burn well (eg hydrocarbons), less
sensitive to oxidised species (eg alkanoic
acids), insensitive to water, CO2
Improving Separation Efficiency
The stationary phase polarity should be similar to that of the substances being separated. The only
influence that the gaseous mobile phase in GC has on the components of the mixture is heat. To
achieve the most efficient separation (good resolution of peaks in the shortest time possible), the most
appropriate column in terms of polarity is chosen, and then the oven temperature is adjusted to
improve the separation.
CLASS EXERCISE 7.1
Complete the following sentences.
The more volatile a compound is, the GREATER/LESS the time it will spend in the mobile
phase, and the SHORTER/LONGER its retention time will be.
The higher temperature in the oven, the GREATER/LESS the time all compounds will spend
in the mobile phase, REDUCING/INCREASING retention times.
CLASS EXERCISE 7.2
A mixture of alkanes – pentane, hexane and heptane – are to be analysed by GLC.
(a) From the columns in Table 7.1, choose the most suitable.
(b) Predict the retention order for the compounds.
(c) The three chromatograms below are recorded at different temperatures. Comment on
the differences and choose the optimum temperature.
60ºC
90ºC
0
3
6
9
12
15
0
2
4
6
8
10
Time (mins)
Time (mins)
125ºC
0
2
4
6
8
10
Time (mins)
Sci Inst Analysis (Spectro/Chrom)
7.3
7. Instrumental Chromatography
Applications
Trace analysis of organic analytes in a mixture is a difficult process, but gas chromatography enables
many complex mixtures (containing chemically very similar components) to be determined accurately
and rapidly. Both qualitative and quantitative work can be carried out on instruments which are
generally robust, reliable and simple to operate. However, remember that only volatile compounds
can be analysed.
The methods of retention time comparison or spiking described in Chapter 6 are used for
identification in GLC. Quantitative analysis is carried out by using a series of standards to prepare a
calibration graph much in the same way as spectrophotometric analyses, but in this case, it is detector
response as measured by peak area or height that is plotted on the vertical axis.
However, as mentioned above, injection volumes are very hard to keep constant because of the
small measures involved. An internal standard is used in a similar way to that in flame photometry. A
constant volume of a compound chemically similar to the analytes, but not present in the sample, is
added to all solutions. The analytical measure for the calibration graph becomes the ratio of the peak
areas of the analyte to internal standard.
7.3 High Performance Liquid Chromatography
GLC, until recently, has been the dominant form of analytical chromatography. It has, of course, the
limitation of being only suitable for volatile analytes. Liquid chromatography – where the mobile
phase is liquid – has been available since almost the very earliest days of the field, but has been
plagued by poor separation efficiency: to achieve a reasonable separation generally took at least two
hours!
The long separation times were primarily the result of very small flow rates of mobile phase,
caused by very large particle size in the column packing. Any attempts at pumping the mobile phase
through reduced the efficiency of separation even further. However, recent developments in
producing much small packing material have meant that the technique known as high performance
liquid chromatography (HPLC) has become as important as GLC.
Mobile phase
The mobile phase is a liquid, frequently a mixture of pure solvents or containing a dissolved species
to change the overall characteristics. It uses polarity attraction for the analytes to aid separation.
Stationary phase
Packed and capillary columns also exist in HPLC, though the former are far more common. As in
GLC, the liquid stationary phase is coated physically or chemically onto the support material or the
column walls. Like the mobile phase, the stationary phase attracts the analytes by polarity.
Stationary
phase
Polarity
Compounds in
sample
Polarity
Mobile
phase
Instrument configuration
A schematic diagram of a typical HPLC instrument is shown in Figure 7.2.
Mobile
phase
reservoir
Pump
Guard
column
Injection
port
Readout
Main
Column
Detector
FIGURE 9.11 Typical HPLC instrument
Sci Inst Analysis (Spectro/Chrom)
7.4
7. Instrumental Chromatography
MOBILE PHASE STORAGE
The mobile phase in HPLC is kept in reservoirs of 0.5-1 L capacity. The reservoir outlets generally
include a filter to remove suspended particles, and a degassing device, such as a sparger (in which the
dissolved gases are carried out of solution by a stream of bubbles of an inert, insoluble gas).
Nothing wrecks a HPLC system – both the pump and the column - quicker than suspended
solids, so it is absolutely essential to filter the mobile phase immediately before use.
PUMPS
A reliable pump was one of the things standing in the way of progress in liquid chromatography. The
pressures needed to push the mobile phase through the packed column are 10-100 times atmospheric
pressure!
INJECTION PORTS
The simple injection ports in GC instruments are unsuitable in HPLC because of the high pressures
encountered. You couldn’t possibly force a syringe into a liquid being forced through at such high
pressures. And even if you could, the flow would come spurting out the injection port.
The most widely used method is known as a sampling loop, where the sample is placed into a
non-pressurised chamber, and then a measured aliquot automatically transferred to the top of the
column. The advantage of such a system is that the volume of sample is highly reproducible, since a
large excess is placed into the sampling loop, and the mechanical system takes the microlitre amount
of sample. This means the internal standards are not necessary in HPLC.
COLUMNS
HPLC columns are usually constructed from stainless steel tubing, because of the pressure
requirements. Typically the main column will only be 30 cm in length. A guard column is placed in
front of the main column: it is a miniature version (1 cm) of the main column, designed to catch any
remaining solid material before it could damage the expensive main column.
Stationary phases were originally physically bonded to support material in the column, but
they suffered the problem of slowly dissolving in the mobile phase. For this reason, most modern
instruments use chemically-bonded packings, where covalent bonds are involved.
Normal-phase HPLC stationary phases are polar, while reversed-phase means non-polar. Ion
exchange columns for anions and cations also exist.
CLASS EXERCISE 7.3
Choose a column type to suit the following analyses.
Chloride and nitrate in river water
Sugars in fruit juice
Caffeine (non-polar) in tea
DETECTORS
The most widely used detectors for HPLC are based on conventional laboratory instruments:

UV/visible absorption

refractive index

conductivity
Like GC detectors, they need to respond rapidly to microgram amounts of analyte, and must operate
with very small volumes. The measurement cells must be flow-through to allow continuous
measurement.
Sci Inst Analysis (Spectro/Chrom)
7.5
7. Instrumental Chromatography
Improving Separation Efficiency
As with all forms of chromatography, the stationary phase polarity should match that of the analytes.
Therefore, column choice in HPLC is relatively simple. However, choice of mobile phase is not so
easy. Its polarity cannot be so different that the compounds will not dissolve at all, and therefore
never move. However, if it is too similar, then the stationary phase will lose the battle to attract the
analytes, and no separation will occur.
CLASS EXERCISE 7.4
The chromatograms below were recorded using a non-polar column and the mobile phase
shown. Suggest how each separation could be made more efficient by changing the mobile
phase. Use the table of dielectric constants in Chapter 6 to help you select a different mobile
phase. Stick to pure solvents.
(a)
0
(b)
ethanol
3
6
9
trichloromethane
12
15
0
2
4
6
8
10
Time (mins)
Time (mins)
Applications
Not bound by the limitation of analyte volatility, HPLC is essentially capable of separating and
detecting all classes of compounds at trace levels: chlorinated hydrocarbon pesticides, vitamins,
sugars, amino acids, ions (cations or anions), the list of possibilities is endless. It finds substantial
usage in the pharmaceutical industry where drugs of various chemical nature are qualitatively and
quantitatively analysed.
Qualitative and quantitative analysis is the same as for GC, except that internal standards are not
necessary. Table 7.3 compares the basic aspects of both forms of instrumental chromatography.
TABLE 7.3 Comparison of instrumental chromatography methods
Characteristic
Classification
GC
Column, elution
HPLC
Column, elution
Mobile phase
Inert gas
Liquid
Stationary phase
Liquid coated on a powder or on the walls
of the column; can be a powdered solid
also
Chemically bonded to silica
particles
Separation
mechanism
Compounds are attracted between polarity
of stationary phase and the heat of the
mobile phase
Polarity attraction to both phases
Introduction of
sample
By syringe injection
By syringe injection
Detection
By electronic measurement
By electronic measurement
Type of analytes
Volatile compounds only
All analytes possible
Sci Inst Analysis (Spectro/Chrom)
7.6
7. Instrumental Chromatography
CLASS EXERCISE 7.5
Determine which technique – GC or HPLC – would be better for the following analyses.
Identification of volatile components of lavender oil
Water content in ethanol
Sugars in fruit juice
Aspirin in tablets
What You Need To Be Able To Do
 for each technique studied:
- draw a schematic diagram of the components of the instrument
- briefly describe aspects of instrument components
- describe some common applications
- describe how separations can be improved
Terms And Definitions
Match the term with the definition.
1.
3.
carrier gas
reversed-phase
2.
4.
packed column
normal-phase
A.
B.
C.
D.
polar HPLC column
GC or HPLC column filled with fine particles coated with stationary phase
mobile phase in GC
non-polar HPLC column
Review Questions
1.
Explain why gas chromatography is limited to volatile components.
2.
Why would increasing the temperature of a column oven speed up the elution of all
components?
3.
Why do compounds of similar polarity elute from a GLC column on the basis of their
boiling points?
4.
A hydrocarbon mixture is suspected to consist of the alkanes (b.p. in ºC): heptane (98),
octane (126), 2,3,4-trimethylpentane (113), 3-ethylhexane (119) and 2,2,3,3-tetramethylbutane
(106). Gas chromatographic analysis shows five peaks as expected.
(i) Predict the order of elution.
(ii) Describe how you would confirm that this prediction is correct.
(iii) What type of stationary phase would be used in this analysis? Give an example.
(iv) Choose a internal standard for this analysis.
(v) The retention times are relatively long. What could be done to improve them? What
problem could occur?
5.
What GC column would suitable for the separation of the components of lavender oil?
6.
What advantages does the high pressure pumping of mobile phase give to HPLC?
7.
A mixture of pharmaceuticals is analysed by HPLC, using a reversed-phase column and
propanone as mobile phase. The separation does not work, since all compounds elute very
quickly in one peak. What could be done to improve the separation?
Sci Inst Analysis (Spectro/Chrom)
7.7