Separation of chemical compounds is carried out by passing the mobile phase, containing the mixture of the components, through the stationary phase, which consists of a column packed with solid particles.
The cause for retention is physical and chemical forces acting between the solute and the two phases, on the chromatographic column. The reason for retention is the difference in the magnitude of forces; this results in the resolution and hence separation of the individual solutes. The separation of compounds occurs by distribution of solutes between the two phases.
11.2.1 Chromatography Classifi cation
Chromatography can be classifi ed according to mechanism of separation as: adsorption chromatography, partition chromatography, ion ex- change chromatography, size exclusion chromatography and affi nity chroma- tography. In HPLC, separation is mainly governed by adsorption and partition chromatography. In adsorption chromatography, separation is based on the difference between the adsorption affi nities of the sample components on the surface of an active site, whereas in partition chromatography separa- tion is mainly based on the difference between the solubility of sample components in the stationary phase and the mobile phase.
There are two modes of analysis depending on the operation techniques viz. isocratic and gradient. Isocratic analysis is the procedure in which the composition of the mobile phase remains constant during the elution process. In gradient elution, the composition of the mobile phase changes continuously or stepwise during the elution process. HPLC can also be classifi ed according to special techniques, such as reverse phase (RP) and normal phase chromatography. Reverse phase is an elution procedure used in liquid chromatography where the mobile phase is signifi cantly more polar than the stationary phase. On the other hand, in the normal phase
procedure, the stationary phase is more polar than the mobile phase. Li- pophilic substances like oils, fats and lipids are separated by normal phase chromatography. Commonly used mobile solvents are n-hexane, heptane, chloroform, and alcohols. Most biomedical substances are separated by reverse phase chromatography using aqueous mixture with methanol, ac- etonitrile and additives (buffers, ion-pairs).
11.2.2 Important Factors that Infl uence HPLC Separation
HPLC separation is infl uenced by dead volume, capacity factor, theoretical plate count and selectivity:
Dead volume
• (V0) is the volume at which an un-retained com- ponent elutes.
Capacity factor
• (K’) is a measurement of the retention time of a sample molecule, relative to column dead volume. It changes with variations in mobile phase composition, col- umn surface chemistry or operating temperature. Capacity factor is calculated as follows:
K' = V1 – V0
V0
Ei
V1 = Retention volume of peak 1 Theoretical plate count
• (N) is a measure of column effi ciency in terms of band-spreading of a peak. The smaller the band- spread, the higher the number of theoretical plates, which indicates good column and system performances.
Resolution
• (Rs) is the distance between the peak centres of two component peaks divided by the average base of the peaks, as follows:
RS = V2 – V1
√W1 + W2
W1 = width of peak 1 W2 = width of peak 2 Selectivity
• (α) is the relative retention of two peaks in a chro- matogram.
α = K'2
K'1 = V2 – V0 V1 – V0 K1 and K
2 = capacity factors for retention volume of peak 1 and peak 2 respectively.
Capacity factor (K’), selectivity (α) and column effi ciency (N) are three fundamental parameters that infl uence the resolution of a chromato- graphic separation, as follows:
Rs = 1
4
(
α – 1α)
√⎯N(
1 + KK' ')
11.2.3 Main Components of HPLC
An HPLC system contains the following components:
Reservoir
a) . This is meant for the mobile solvents. Acetonitrile, methanol, heptane, isopropanol and cyclohexane are the organic modifi ers most commonly used. Trifl uoroacetic acid, heptafl uorobutyric acid, phosphor- ic acid and triethylamine phosphate are ion-pairing reagents for better chromatographic results. All tubing and fi ttings should be chemically inert. Solvent must be fi ltered through a 0.45-μm fi lter unit.
Degasser
b) . In analytical operations, the mobile phase should be free of air bubbles. For this purpose, a degasser is used.
Pumps
c) . These are devices that deliver the mobile solvent at a controlled fl ow rate to the separation system. HPLC uses reciprocating pumps: a pump with a single or multiple chambers, from which the mobile phase is displaced by reciprocating pistons or diaphragms. Binary gradients are cre- ated by the selected mixing of two solvents, on a single-headed two-pump system. Accurate gradient is maintained by microprocessor control.
Injector/autosampler.
d) This device introduces a liquid sample into the mobile phase or onto the chromatographic bed. An autosampler can perform repeated functions without operator attendance, and thus is a labor-saving device.
Column
e) . Silica and modifi ed silica columns are available for various ap- plications. Examples are octyl (C8), octadecyl (C18), phenyl (C6H5), and cyno (CN) columns.
Guard column.
f) This is used to protect the main column.
Detectors
g) . No universal detector is available for all molecules. However, according to the characteristic of the molecules investigated, various detectors are used (Table 1).
Fraction collector
h) . This device collects the fractions containing the mol- ecules of interest during the chromatographic run.
Records.
i) A computer is used for chromatographic data acquisition.
Table 1: Characteristics of various HPLC detectors
Detector Application Advantages and limitations
Electrochemical Responds to substances that are oxidizable or reducible
Commercially available.
Non-specifi c. High LOD
Detector Application Advantages and limitations
Fluorescence Detects trace-level analytes such as afl atoxins, carbamates and polycyclic aromatic hydrocarbons
Very specifi c. Low LOD.
Not everything fl uoresces Infrared Works for all molecules Many solvents are infrared-active Mass
spectrometry
Analyte identifi cation Ability to ionize analyte. Low LOD Photodiode
array
Works for wavelengths 190-800 nm
High LOD
Refractive index Works for nearly all molecules Temperature sensitive. High LOD Scattering Uniform response Non-specifi c. LOD, 5 ng per 25
mL. Interference from solvent Ultraviolet
and visible
Works for molecules with chromo- phores and for complex samples
Non-specifi c. All molecules that absorb UV and visible light can be detected.
LOD, level of detection
11.2.4 HPLC Classifi cation
HPLC may be characterized depending on column diameter, which is the governing factor for fl ow rate from microscale to industrial scale chromatography. Column internal diameter (i.d.) defi nes the sample load and fl ow rates (Figure 1).
N-NJO