2. Principal Chemical and Analytical Methods Used in Reverse Engineering
2.4 Chromatography
2.4.1 Introduction
Chromatography is basically a technique of separation. This separation is either physi- cal or chemical. In chromatography the two immiscible phases are brought together: one phase is stationary, and the other is mobile. A sample introduced into a mobile phase is carried along through a column containing a distributed stationary phase. Components in the sample undergo repeated interactions (partitions) between the mobile phase and the stationary phase.
Every chromatographic separation is based on differences in the rates of migration of the sample components through the column. The different sample components spend differ- ent times in the stationary phase depending on its molecular characteristics, whereas the time spent in the mobile phase is identical for all components. The mobile phase, which alone affects transport through the column, can be a gas, a liquid, or a supercritical fluid.
Classification of the chromatographic process is based on the types of mobile and station- ary phases used.
250 200 150 100 Temperature (°C)
50 0 –50 250
200 150 100
(a) (b)
0
–50 0
0.15 0.45 0.75 8.75
8.25 7.75 7.25
Log G´(Pa)
6.75 6.256.00
Tan δ
1.05 1.35
Temperature (°C)
FIGURE 2.17
(a) Storage modulus versus temperature plot. (b) Tan δ versus temperature plot for hydrogenated nitrile rubber at 10, 20, 30, 40, 50 phr of carbon black loading.
Various types of chromatography include:
• Gas chromatography
• Liquid chromatography
• Bonded-phase chromatography
• Ion-pair chromatography
If a solid with a large active surface is employed as the stationary phase and a gas as the mobile phase, the chromatography principle is called gas-adsorption chromatography or gas-solid chromatography (GSC). With a liquid mobile phase, the method is liquid- adsorption chromatography or liquid-solid chromatography (LSC). When the separation involves predominantly partitioning between two immiscible liquid phases, the process is called liquid-liquid chromatography (LLC). Two other liquid chromatographic methods differ somewhat in their modes of action. In ion-exchange chromatography (IEC), ionic components of the samples are separated by selective exchange with counter-ions of the stationary phase. The use of exclusion packing as the stationary phase brings about a clas- sification of molecules based largely on molecular geometry and size. Exclusion chroma- tography (EC) is referred to as gel-permeation chromatography by polymer chemists and as gel filtration by biochemists.
When a solid is used as the stationary phase, one generally speaks of adsorption chro- matography; if a liquid is coated on an inactive solid support, it is called partition chroma- tography. No clear differentiation can be made between the two, especially in adsorption chromatography where precoated adsorbents are frequently used and there is a continu- ous transition from pure adsorption to a more or less distinct partition.
Gas chromatographic methods are appropriate for the separation of volatile substances.
Many substances that are nonvolatile at ordinary pressures can be converted simply and quantitatively into volatile derivatives that can be separated by gas chromatography.
Liquid chromatographic methods are utilized mainly for the separation of substances, which decompose on vaporization.
The chromatographic behavior of a solute can be described in numerous ways like reten- tion volume, VR (or corresponding retention time, tR), and the partition ratio k.
2.4.1.1 Retention Behavior
Retention behavior reflects the distribution of a solute between the mobile and stationary phases. The volume of mobile phase necessary to convey a solute from the point of injec- tion, through the column, and to the detector is defined as the retention volume VR. It may be obtained directly from the corresponding retention time tR on the chromatogram by multiplying the latter by volumetric flow rate Fc, expressed as the volume of the mobile phase per unit time:
VR = tR Fc (2.41)
2.4.1.2 Partition Ratio
The partition ratio (capacity ratio) k relates the equilibrium distribution of the sample within the column to the thermodynamic properties of the column and to the tempera- ture. For a given set of operating parameters, k measures the time spent by the sample in
the stationary phase relative to the time spent in the mobile phase. It is defined as the ratio of moles of a solute in the stationary phase to the moles in the mobile phase.
A suitable chromatography column may be selected on the basis of three criteria:
1. The attainable resolution 2. The speed of analysis
3. The load capacity of the column 2.4.1.3 Resolution
The resolution R of two sample bands is defined in terms of the distance between the two peak maxima, expressed as the difference in the two retention times, tR2 and tR1, and the arithmetic mean of the two bandwidths, W1 and W2, and is expressed as
R t t
W W
R R
=2( – ) +
2
1 2
1 (2.42)
In chromatography one should go for optimum resolution (i.e., the peaks should be sepa- rated from each other as far as necessary).
2.4.1.4 Speed of Analysis
A separation is optimum if it completes in the shortest time. High resolution is undesirable because it can only be achieved at the expense of analysis time. For every analysis there is an optimum system of stationary and mobile phases with an optimum temperature which offers the greatest selectivity for the desired separation and thus provides a large relative retention. The column properties and packing exert considerable influence on the analysis time. However, a certain length is necessary to attain the required plate number.
It is important to remember that the plates do not really exist; they are imaginary and are used to help in conceptualizing the working principle of the column. Plates also indicate column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height—the height equivalent to a theoretical plate (HETP) (the smaller the better).
If the length of the column is L, then the HETP is
HETP = L/N (2.43)
The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution:
N t
w
=5.55R2
1/22 (2.44)
where N is the number of theoretical plates, tR is the time between sample injection and an analyte peak reaching a detector at the end of the column which is termed as the retention time, and w1/2 is the peak width at half-height. Since the plate number is inversely propor- tional to the square of the particle size, smaller particles should always be used.
For a given separation, the required plate number can be obtained with a long column packed with larger particles as well as with a shorter one containing smaller particles.
However, the speed of analysis is always greater with shorter columns packed with smaller particles. Another advantage of small particles is in the higher detection sensitiv- ity they permit, which results from the sample components migrating as sharper zones, hence reaching the detector less diluted and giving rise to higher peaks.
The temperature affects the analysis time only indirectly. The viscosity of the mobile phase decreases with rising temperature, resulting in increasing eluent velocities for a constant pressure drop. Since the rate of diffusion also increases with rising temperature, sharper peaks are obtained at higher temperatures.
The chromatographic techniques principally used in the rubber industry are as follows:
1. Thin-layer chromatography (TLC)
2. Liquid-solid column chromatography (LSC) 3. Gas-liquid chromatography (GLC)
4. High-performance liquid chromatography (HPLC)
These techniques may be variously used as analytical tools to establish the complexity of mixtures and purity of samples, and as preparative tools for separating mixtures into individual components. The selection of a particular technique is to some extent a matter of experience.