Resolution
A good chromatographic separation is judged by the resolution between
peaks, that is, adjacent peaks are resolved sufficiently so that accurate
measurement of the peak areas can be obtained.
There should be a baseline separation between the peaks and no coelution or
overlap of the tail of one peak with the leading edge of the next peak.
Considering Gaussian distribution adjacent peaks (A and B) should be
separated by 6σ (3 σA + 3 σB), that is, there would be <0.3% overlap since
±3σ includes 99.7% of a Gaussian distribution.
In practice, resolution (Rs) is assessed by comparing the difference in
retention time for A and B with the half widths of the peaks since 95.5% of a
Gaussian distribution is contained within ±2σ, therefore for a 2.3% overlap
separation should be at least 2σA + 2σB or approx. 4σB, peak width of the
broader peak.
Quantification in chromatography
Data from chromatograms may be used to obtain the relative concentrations
of components in a mixture, provided good resolution is achieved.
Peak area, from integration of the detector signal during elution of a
component, is proportional to the amount of that component in the sample.
Normalising peak areas
The area of each peak is obtained from a series of replicate (5+) injections of
a mixture containing equal (or known) amounts of all the components.
One component is chosen as the reference and the relative responses of the
other components are determined by dividing the peak areas by that of the
reference component.
The detector response factors (DRF) may then be used to calculate corrected
peak areas (Acorrect) for other analyses involving these components and hence
their percentage ratios in the mixture may be determined.
Internal standard (IS) method
A known amount of a known compound called the internal standard is added to the
sample and all calibration samples/solutions. The internal standard must be
chromatographically separated from the analytes. The requirements for the internal
standard are as follows:
must be separated from other compounds in the sample (except with MS
detection),
must have retention time close to the analytes (two or more IS may be
required for multianalyte quantitation),
must behave like the analyte(s) with respect to sample preparation,
extraction, derivatization, and so on,
must be added in a concentration that give preferably equal peak height or
peak area to that of the expected concentration of analyte,
must not be present in the sample,
must be stable (i.e., not reacting with other compounds in the sample,
stationary
phase, or mobile phase), and
must be available with high purity
Since peak area is proportional to the amount of an eluted component and the
detector response factor (DRF)
Analysis of an unknown mixture is achieved by adding an accurately known
amount of internal standard and then carrying out the chromatography. The
concentration of each component calculated using the equation above rearranged to
give
External Standard method
In the external standard method, a calibration curve for the analyte(s) is established
by analyzing calibration solutions containing accurate concentrations of analyte
standard and plotting the detector response as a function of the concentration.
THIN LAYER CHOMATOGRAPHY
Type of planar chromatography
The stationary phase is supported by a flat surface (e.g., a glass plate, a
plastic sheet, paper, etc.).
The mobile phase and analyte pass through the stationary phase by capillary
action and/or gravity.
components of the mixture are separated into different spots depending on
their relative attraction to the stationary phase or nature of solvents that
make up the mobile phase.
The sample is spotted onto the paper/TLC plate and the bottom edge of the
piece of paper/TLC plate is dipped into an appropriate liquid.
The Rf value obtained will be different for different compounds.
Hence, different solvent systems (mobile phases) are scanned to derive
information about a number of components present in the crude sample (the
number of spots on TLC/accounts for the number of components present in
sample).
The TLC plate is visualized either by nondestructive methods such as UV-
visible light/iodine vapors, or by using destructive methods such as spraying
5% H2SO4 in methanol, ninhydrine, etc.
These stationary phases are designated with “F254” (as in silica gel GF254),
meaning that these plates have fluorescent properties when irradiated with
an UV lamp at 254 nm. UV absorbing compounds quench the fluorescence
of the stationary phase appearing as dark spots on the fluorescent plate.
As the mobile phase rises up the TLC plate, the individual components move up at
different rates depending on intermolecular forces between the components with
stationary phase and the components with the mobile phase. These adsorption
strengths of compounds increase with the increasing polarity of functional group
The separation of two analytes on the chromatogram can be expressed as resolution. Hence, the
composition or chemical nature of mobile phase is changed to get the optimum resolution in
which the separation between two components is in the Rf range of 0.2–0.5.
Example of how to separate the following compounds using TLC,
1.
For TLC the solvent system(mobile phase) used is petroleum ether: ethyl acetate, 50:1. The TLC
plate (silica gel) is then observed under UV light 254/365 nm
2.
For TLC the solvent system used (mobile phase) is (petroleum ether: ethylacetate,
1:2). The compounds are observed by immersing the silica gel plate in a
phosphomolybdic acid staining solution and drying it. (The compound cannot be
observed under UV light.)
NB.
The two compounds have different polarity so the solvent system has to be
changed to get Rf value of about 0.2 and good separation. The mobile phase system
(petroleum ether: ethylacetate, 1:2) will be the more polar one.
Other adsorbents are shown in the following table
