6
WHAT IS CHROMATOGRAPHY?
6.1 Introduction
Chromatography dates back to the turn of the century when a Russian botanist found that he could
separate a various plant pigments, such as chlorophylls, by pouring a solution of the complex extracted
mixture through a glass tube, holding finely powdered calcium carbonate. The various components of
the mixture took different times to pass through the tube.
Chromatography refers to separation of mixtures by making use of differences in the physical
properties of the substances in the mixture. There are a great range of chromatographic techniques,
but all have two common factors:

a stationary phase – the components interact by surface adsorption or solubility,

a mobile phase – this carries the mixture through the stationary phase.
All chromatographic separations are based upon differences in the extent to which components of a
mixture are attracted to the mobile and stationary phases, as shown in Figure 6.1.
Stationary phase (SP)
direction of flow of mobile phase (MP)
FIGURE 6.1 Representation of the separation process
CLASS EXERCISE 6.1
In Figure 6.1, which substance is most attracted to (a) the stationary phase and (b) the
mobile phase?
6.2 Some General Principles
Stationary Phases
Stationary phases are so-named because they don‟t move! This means they are either solid – in
powder form – or liquid which is bonded to a solid surface. For successful separation, the components
of the mixture must be capable of interacting physically or chemically with the stationary phase. This
will occur through some chemical or physical interaction, such as polarity („like dissolves like‟).
6. What Is Chromatography?
Mobile Phases
Mobile phases move, which means they must be either liquid or gaseous. The components of the
mixture can only move along the chromatography path when in the mobile phase. If the compound is
so strongly attracted to the stationary phase, and not to the mobile phase, it will never move. Thus, a
component which is strongly attracted to the stationary phase will move much more slowly than one
which is very soluble in the mobile phase.
The relative attraction of a component between the two phases is often determined by the polarity of
each of the substances. The polarities of the components of the mixture are, of course, fixed; to
achieve separation the polarities of the mobile and/or stationary phases must be varied.
The mobile phase in most cases can be infinitely varied, liquid phases being chosen from any
suitable solvent or solvent mixture. The easiest method by which a poor separation of a mixture can
be improved is by changing the polarity of the mobile phase.
The simplest measure of the polarity of potential mobile phases is the dielectric constant for
each liquid (as given in Table 6.1), the less polar solvents having a lower dielectric constant. The
dielectric constant of a mixture of solvents can be approximated, using the proportion of each solvent
in the mixture.
TABLE 6.1 Dielectric constants of pure liquids at 20ºC
Liquid
Cyclohexane
D.E.
2.02
Liquid
Toluene
D.E.
2.44
Trichloromethane
4.81
Ethyl ethanoate
6.02
Ethanoic acid
6.15
1-Butanol
17.8
Ethanol
24.3
Water
80.4
CLASS EXERCISE 6.2
Arrange the following solvents in order of increasing polarity:
cyclohexane
toluene
ethanol
water
trichloromethane
ethanoic acid
ethyl ethanoate
1-butanol
ethanol/water (1:1)
ethanol/water (4:1)
ethanoic acid/
1-butanol/water (7:2:1)
So, the movement of a particular compound through the chromatography path is determined by its
attraction to both phases. If the stationary phase has a stronger attraction, then the compound will
move slowly, because it will not spend as much time in the mobile phase, as a compound that is more
attracted to the mobile phase. The term given to the movement of a compound in a chromatography
path is elution, and generally refers to the compound reaching the end of the path.
Sci Inst Analysis (Spectro/Chrom)
6.2
6. What Is Chromatography?
CLASS EXERCISE 6.3
Predict the order of elution of the following mixture from a non-polar stationary phase, with
ethanol as mobile phase:

sodium benzoate (contains ionic groups)

caffeine (contains polar covalent bonds)

carotene (contains only C-C and C-H bonds)
Choosing the phases
Most frequently, separation will be required of chemically similar substances, such as the components
in petrol which are all hydrocarbons. To separate these similar components requires that they be
attracted to the stationary phase; if they weren‟t, then they would travel through the path at the same
rate.
General Rule for choice of phases (where polarity is the attraction mechanism)
Stationary phase – match the polarity of the stationary phase to that of the analytes
Mobile phase – use a mobile phase that is different in polarity to the stationary phase (but not
too different, otherwise the analytes won’t shift at all)
EXAMPLE 6.1
What combination of phases would be suitable to separate a mixture of hydrocarbons?
Given the analytes are hydrocarbons, this means their polarity is very low, so a non-polar
stationary phase would be suitable. The mobile phase can’t be too pol ar (eg water),
otherwise, the hydrocarbons would never go into it. Therefore, something of medium -low
polarity, such as ethyl ethanoate would be suitable.
CLASS EXERCISE 6.4
Choose a combination of phases that would satisfactorily separate the following mixtures:
(a) DDT and other chlorinated hydrocarbons (non-polar compounds).
Non-polar
compounds require a non-polar stationary phase for any interaction, and therefore, a
mobile phase as polar as possible.
(b) A mixture of polar sugar compounds. The highly polar sugar compounds would need a
polar stationary phase, and a mobile phase of limited polarity.
Stationary phase
Mobile phase
(a)
(b)
Sci Inst Analysis (Spectro/Chrom)
6.3
6. What Is Chromatography?
It should be noted that these generalisations cannot be applied to all techniques, because there are
limitations on the variety of either phase available. For example, in thin layer chromatography, only
relatively polar stationary phases are available, while in gas chromatography the mobile phase is a
chemically non-interacting gas.
6.3 Variations in chromatographic systems
Chromatographic methods may be classified according to a variety of schemes because of the number
of possible variations in the way in which separation can be achieved.
1. By physical state of mobile phase – the most common mobile phase used in chromatography is
liquid; but the technique of gas chromatography is a very useful quantitative tool in organic
laboratories.
2. By mechanism of interaction between stationary phase and solute – adsorption refers to the
physical interaction between a solute and a solid stationary phase; partition refers to the case where
both phases are liquids and the solute will be dissolved in each, depending on the relative polarities of
it and the two liquid phases (i.e. the micro-scale equivalent of an extraction in a separatory funnel);
ion-exchange involves transfer of ions from solute to stationary phase and vice versa.
3. By movement of compounds through system – development chromatography produces a result
where the solute components remain in the chromatography path for analysis; elution chromatography
has the separated components move out of the system where they are analysed
4. By geometry of system – column chromatography has the stationary phase packed into a column, or
bonded into its walls, and the stationary phase is pumped or filters through the column; planar
chromatography has the stationary phase spread out in a thin, flat layer, and the mobile phase passes
across it by capillary action (upward travel) or by gravity (downward travel).
During this subject, two different methods will be examined: gas (GC) and high performance liquid
(HPLC). Table 6.2 shows how they fit into the categories above. You may have seen other forms of
chromatography, such as thin layer, in other subjects.
TABLE 6.2 Classification of HPLC and GLC
Technique
GC
HPLC
By Mobile Phase
gas
liquid
By Stationary Phase
partition or
adsorption
By Means of Movement
By Geometry
elution
column
partition or
ion-exchange
elution
column
6.4 Chromatographic Analysis
Whether the technique relies on elution or development will determine how the separated component
bands are detected and analysed. Because both techniques examined in the next chapter are elution
types, that is all we will look at in this subject.
Elution, where the compounds are washed out the end of the column, requires the “collection”
of the eluate and detection of the compound bands either visually or instrumentally. The band will
appear as a peak, where the number of molecules of the compound forming the band reaching the end
of the column will steadily increase to a maximum and then decrease. Figure 6.2 shows the
chromatogram obtained from the elution of two different compounds, X and Y.
Sci Inst Analysis (Spectro/Chrom)
6.4
6. What Is Chromatography?
Detector response
X
Y
0
10
20
30
40
Time (s)
FIGURE 6.2 Elution chromatogram
Qualitative analysis
If everything stays the same, a given compound will travel at the same rate through a chromatography
system each time it is run, regardless of whether it is in a mixture or by itself. This is an important
qualitative aspect of chromatography, in that an unknown solute from a mixture may be identified due
to its similar behaviour to a known compound.
The retention time (R t), used in column chromatography as an identification measure is the
time taken for a compound to pass through the system.
CLASS EXERCISE 6.5
Determine the retention times for compounds X and Y in Figure 6.2.
X
Y
If you want to identify a given peak in a chromatogram, you could compare its retention time to that of
a series of standards, run under the same conditions.
CLASS EXERCISE 6.6
Determine the possible identities of compounds X and Y in Figure 6.2, given the R t data
below.
Compound
Methanol
Ethanol
1-Propanol
2-Propanol
Ret. time (s)
5
10
14
15
Compound
1-Butanol
2-Methyl-1-propanol
1-Pentanol
1-Hexanol
Ret. time (s)
20
21
25
30
X
Y
Sci Inst Analysis (Spectro/Chrom)
6.5
6. What Is Chromatography?
Where identification is made difficult by the fact that a number of possibilities have similar retention
times, as you found for compound X, then a method known as spiking is used. This involves adding a
small amount of the possible pure substance to a portion of the sample, and re-running the “spiked”
sample. One of two things will occur:

one of the peaks will get larger (see Figure 6.3(a)) – this means that the spiking compound is
causing that the peak in the sample

another peak will appear (see Figure 6.3 (b)) – the spiking compound is not in the sample
(a)
Detector response
X
Y
0
5
10
15
20
25
30
35
40
Time (s)
(b)
Detector response
X
Y
0
5
10
15
20
25
30
35
40
Time (s)
FIGURE 6.3 Outcomes of spiking (a) peak X increases relative to peak Y (b) another peak appears
Quantitative analysis
The techniques of gas- (GC) and high performance liquid (HPLC) chromatography are instrumentbased, and use electronic detectors which measure the amount of a solute emerging from the end of a
column. A peak is obtained as each compound emerges, from which information can be obtained as to
the total amount of each component. The area under the peak is proportional to the amount of
substance. The peak height for normal „triangular” peaks is a reasonable alternative.
To determine how much substance produces a given size peak, calibration standards of known
concentration are run, and the peak area or height, plotted against concentration. The volume of
sample and standard must be exactly the same, which can be a difficulty because in GC and HPLC,
microlitre volumes are involved.
Sci Inst Analysis (Spectro/Chrom)
6.6
6. What Is Chromatography?
Measurement of peak area has been made much simple in recent times through the advent of, first,
mechanical integrators and now electronic ones. These allow an accurate determination of peak area.
Without these, peak height is the better option.
CLASS EXERCISE 6.7
Calibration standards for compound X in Figure 6.2 have been measured. determine the
sample concentration, using peak height in mm.
Standard X conc. (mg/L)
100
250
500
Peak height (mm)
20
46
95
6.5 Improving the separation
Chromatographic separations should be made as efficient as possible, which means getting all the
compounds out as quickly as possible, whilst still maintaining separation. The separation of peaks is
known as resolution.
Apart from saving time, a quicker separation also improves the peak shape. The longer a
compound stays in the chromatographic path, the more its molecules will spread out from each other,
making it more dilute and consequently difficult to detect, and also risking overlap with another
compound's band. This is known as band broadening.
The reason for band broadening is simple. For any given compound travelling through the
medium, some molecules will travel faster than the average, and some slower. Since, distance is
proportional to time, the longer the time spent in the system, the greater the difference in the distances
travelled by the fast and slow molecules.
Reducing or eliminating slow separations can be done in a number of ways, the most common
being a change in one or both of the phases, or in GC, an increase in temperature.
Sci Inst Analysis (Spectro/Chrom)
6.7
6. What Is Chromatography?
What You Need To Be Able To Do
 define relevant terms
 describe the basic process of chromatography
 describe the general nature of stationary and mobile phases
 explain how polarity of solvents (mixed and pure) can be estimated
 predict the order of elution of a given mixture from a particular stationary phase
 choose a combination of phases (in terms of their polarities) that would satisfactorily separate a
given mixture
 describe the chromatographic techniques studied in terms of the various methods of classification
 draw a representation of the results obtained from elution and development chromatography
 describe how qualitative and quantitative analysis can be performed by chromatography
Terms And Definitions
Match the term with the definition.
1.
3.
5.
7.
stationary phase
elution
resolution
band spreading
2.
4.
6.
mobile phase
retention time
spiking
A.
B.
C.
D.
E.
F.
G.
broadening of peaks caused by long retention times
when the compound reaches the end of the system
adding a small amount of pure substance to help identify a peak
the liquid or gas that moves the compounds through the system
the usually solid material that the analytes are attracted to, causing separation
the separation of two peaks
the time taken to pass through the system
Review Questions
1.
What three factors are common to all chromatographic techniques?
2.
Describe the passage of two different compounds in a mixture through a chromatography path.
3. Describe the passage of two different molecules of the SAME compound through a
chromatography path.
4.
Describe how (a) planar and (b) column chromatography could be used to identify a component
in a mixture.
5.
Explain why retention factor is used for identification in planar chromatography rather than
simply the distance travelled by the solute.
6.
(a) Using the retention times in Table 6.2, identify the peaks in the chromatogram below.
Sci Inst Analysis (Spectro/Chrom)
6.8
6. What Is Chromatography?
Detector response
A
B
0
5
10
15
20
25
30
35
40
Time (s)
(b) Given the following calibration data for peak A, determine the concentration in the sample.
Standard A conc. (%v/v)
0.25
1
2
Sci Inst Analysis (Spectro/Chrom)
Peak height (mm)
10
38
77
6.9