1. 6.4 COLUMNAR & PLANAR CONFIGURATIONS
In GC, stationary phase is always packed = columnar (! Meaning contained) in a column in order to contain mobile phase, a gas.
A similar configuration is common in LC, but since mobile phase is a liquid, it is also possible to use another configuration.
one in which stationary phase is spread on a flat, planar surface. This configuration is called either paper chromatography (PC) or thin-layer chromatography (TLC).
In TLC, stationary phase is coated on a supporting planar surface, which can be glass, plastic, or metal.

2. Many of stationary phases used in columnar LC can also be used in TLC,
Background for comparison between LC and TLC
Peaks Compared to Bands
Band, peak, and zone have been used synonymously, but as commonly used, all three terms describe distribution of analyte molecules in space (concentration profile), but band represents this distribution while analyte is still in system, while peak refers to a distribution of analyte that has eluted from system. Zone is more general & includes both bands & peaks;

3. Zone is used in those cases when we do not wish to be more specific
Zone is used in those cases when we do not wish to be more specific. This means that zones in TLC and PC are called bands and those in column LC and in GC are called peaks.
Figure 6.10, which shows the difference between bands and peaks in a separation of two analytes, A and B by (a) TLC and (b) column LC.
The chromatogram in (a) is obtained by densitometering plate shown above it, and in ( b ) is from a conventional online detector.
The partition coefficient for A, KA, is greater than that for B, KB. Consequently, B migrates faster than A, and as shown in Figure 6.10a, B has moved farther down bed than A;

4. in Figure 6. 10b, B is shown eluting from bed before A
in Figure 6.10b, B is shown eluting from bed before A. Chromatograms of the two situations show the zones in reverse order and width.
Thus, zone broadening does not depend on length L of bed, as suggested by definition of column dispersivity.

7. Retardation Factors
Another difference between column techniques & planar techniques in LC is the way that retardation factor is expressed.
Figure 6.11a shows that migration distances are measured from analyte starting spot.
Clearly, both Rf and R , can be easily calculated from their respective chromatograms, making them very useful measures for describing chromatographic results.
Recall also (Table 2.6) that k = (1 ~ R )/R and that k is proportional to distribution constant Kc, the basic thermodynamic variable in chromatographic theory.

8. So, a comparison of HPLC and TLC for systems with equivalent stationary and mobile phases can also be made by comparing their respective k values, each calculated with the appropriate retardation factor, R or Rf .
It is noted that there are several reasons why the planar techniques could have retardation factors different from the column methods.
For example, many times the flow is not controlled in TLC, and consequently it is not constant.

9. The column LC peaks in Figure 6
The column LC peaks in Figure 6.12 are in reverse order and reverse width compared to the TLC bands.
There are some operational differences that cause differences between HPLC and TLC. Even if the exact same stationary phase is coated on a TLC plate and packed in a column, the TLC material usually contains an additional binder to hold the stationary phase on the plate.
This binder will most likely slightly alter the characteristics of stationary phase and result in differences between the R and R,- values.
And, finally, in normal TLC procedures the stationary bed is usually dry when chromatographic elution process is begun, whereas it is usually wet with mobile phase in column processes.
Difference may also contribute to differences between retention parameters.

11. 11.1 Paper Chromatography
filter paper such as Whatman # 1 are common for PC chromatography, but chromatographic grades are also available.
formats vary widely from large sheets to cylinders to circular sheets (Figure 11.1).
large sheets, necessary for good separations (Figure l l . l a) , must be developed by a descending flow of mobile phase since solvents will not flow the required distance against gravity.
smaller sheets ( Figure 11.1 b) develop by ascending flow and need to be supported at top or formed into stable cylinders.
also popular is format as circular sheet of filter paper (Figure 1l.ld) is placed over a Petri dish with a wick cut out of it for solvent transfer.

12. Figure 11 .l. Developing chambers for planc chromatography: (0) descending-used with PC;
( b ) ascending-used with TLC and PC; ( c ) sandwich-used with TLC; ( d ) horizontal-used
with paper as shown, but also adaptable for HPTLC

13. because non-dried paper contains a significant amount of adsorbed water, PC can be classified as LC method & separation mechanism as absorption (partition).
other liquids can be applied to change its characteristics. For example, silicone oils, petroleum jelly, paraffin oil, & rubber latex are used as non-polar phases.
special papers are also commercially available that contain adsorbents or ion exchange resins or are specially treated (e.g., acetylated) or are made of other fibers (e.g., glass, nylon).
paper chromatography still finds some use at precollege level and for elementary demonstrations.

14. 11.2 Thin-layer Chromatography
difference between PC and early TLC is stationary phase (SP), usually silica gel coated on carriers such as glass, aluminum, or plastic.
because SP was polar, chromatographic mode was classified as normal phase.
TLC plates are placed in small glass chambers ( Figure 1 l . l b), and developed by an ascending mobile phase (MP).
still, for many scientists, main use of TLC is for qualitative analysis and screening, and it is performed in a fashion that could be described as quick and dirty.
TLC procedure have been instrumented with less labor, as better reproducibility and quantitation, and can be handled with modern data systems.
manual TLC,
instrumental TLC

15. Manual TLC
steps in TLC include the following:
Sample application
Development (name used for actual running of the chromatographic process)
Visualization or detection of the separated analytes
Quantitation
sample should occupy as small an area as possible on bed.
sample solution can be applied as spots or bands at one end of bed using micropipet or microsyringe.
in neither case should application of sample disturb the bed, so that for soft TLC plates, sampling device cannot touch the surface.
harder layers on commercial plates are preferred for this reason.

16. sample spots should be dried before development, so solvent is usually chosen for its volatility as well as its ability to dissolve sample.
solvent does not need to be mobile phase (MP) or have any relationship to MP, unlike situation in column LC.
to keep the spots small, sample can be applied repetitively to same area, allowing previous application to dry before reapplication.
in general, placement of spots must be far enough from end of bed to prevent them from dipping into solvent reservoir (1-2 cm).

17. Figure 11.8. Composition of TLC plate with two different stationary phases.

18. prepared plate is developed in a closed, presaturated chamber usually made of glass and large enough to accommodate the conventional 20x20-cm plates.
when development is completed and the plate dried, the analyte bands are located unless they are colored. If the analytes fluoresce, they can be located under a UV lamp. If not, the use of a phosphor in the TLC plate may make it possible to locate them as nonfluorescing (quenching) spots on phosphorescing plate.
for qualitative identification, data obtained by planar techniques. alternatively, the distances migrated can be measured relative to a standard run on same plate, which usually is more reproducible.
colors caused by selective visualizing reagents can also be used for identification.

19. Two methods are commonly used.
the first method involves a visual estimate of the quantity of analyte by comparing its spot size with sizes of standards that bracket it.
the semiquantitaive result is reported as being between two of standards and provides an accuracy of about 25%.
somewhat greater accuracy can be achieved if spot sizes are actually measured and plotted.
alternatively, spots can be scraped off TLC plate and extracted to remove analytes, which can then be quantitated by UV-Vis spectrophotometry

20. Stationary Phase
most popular thin layer is silica gel, and it is estimated that about 90% of TLC separations are performed on conventional TLC silica plates.
silica gel used in TLC has same properties as that used in LSC in columns.
silica has a heterogeneous surface and many very active silanol groups. It picks up water from the atmosphere very readily and will preferentially adsorb the most polar component in a mobile-phase mixture.
calcium sulfate (gypsum) and compounds related to polyvinyl alcohol (PVA) are most common.
PVA plates are very stable and will withstand rather rough handling.

22. Mobile Phase
liquid mobile phase is a mixture of liquids chosen by consulting literature and optimized by trial and error.
for normal-phase systems on silica gel, a nonpolar liquid is modified with a more polar one, and small amounts of a third component such as acetic acid are often added to deactivate the plate slightly and decrease tailing.
amount of water is critical in determining the activity of the plate, the chosen mobile phase is simply a mixture that works, and a typical example is a mixture of butanol-acetic acid-water.
for reversed-phase systems, a polar mixture like those used in column LC are satisfactory, and combinations of water and acetonitrile or methanol are common.

23. Plate: silica gel plate; 20 x 20 cm
Sample: 13 Sulfonamides. 1 mg/ml in
(1) Silica gel development to 14 cmdissolved in Acetone/Methanol (9O:lO)
Development: 2-Dimensional
(1) Toluene/aceonitrile (8020)
(2) (Silica gel layer): Ethyl acetate/MeOH/
NH4OH (85:15:0.6)
Figure Two-dimensional separation of 13 sulfonamides on Multi-K plate.