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The qualitative errors in HPTLC are less critical as in HPLC

although the planar chromatographic separation power is much smaller than available for modern HPLC. The latter offers a total peak separation capacity of around 50 (baseline separated peaks - however check ‘multi focussing -> here ). Standard HPTLC reaches a total peak separation capacity of about 15.
Thus spot overlapping exists most often.

About the “MAIN ERRORs” in all PLC’s you can read here - click this LINK

TLC_image

Lets have a look onto the planar chromatogram left: It shows the beauty of a visualization by fluorescence. It shows the power of spectral information. But in case of wider ranging concentrations one will immediately reach limits especially when these spot lines even only partially overlap.
There is of course full spectral signal power available. There is time to enhance even very weak peak signals which is not available with elution chromatography as in HPLC or with forced flow HPTLC.

This page concentrates on a classical systematic error in HPTLC:
the falsified quality value “Rf”. By the Rf value HPTLC  users express qualitative results.
 
The linear Rf value for substance “x” = s / f                                       See figure 1 below.

f = length of the mobile phase path developed from start to front.
s = the path length the substance x was moved until the HPTLC run stopped.

The main reason to concentrate here in all details on the Rf value error is the fact, that its correction allows to use HPTLC-k-values which are comparable with HPLC-k-values. If the mobile and stationary phases in HPLC are exactly equal in HPTLC, the HPLC-k-value corresponds with the HPTLC-k-value. Although both differ by value it becomes evident that the two liquid chromatography techniques can support each other qualitatively. Due to the very limited separation efficiency of both if compared to modern micro gas chromatography a both sides support of these sister techniques is good to avoid serious systematic qualitative errors. As the life combination of HPLC with HPTLC is a next and powerful bridge to gap error trouble, an equal “language” for both techniques is mandatory. For this the Rf value fails.

For qualitative HPTLC and qualitative HPLC isocratic separation techniques are preferable. In this case qualitative HPLC data are time or volume based. The best quality value is k = residence time in the stationary phase over the residence time in the mobile phase: k = ts / tm .

It is not immediately visible, that HPTLC data are also time based. But this is shown in the figures below.

Especially helpful in HPTLC is the comparison and the simultaneous use of linear, circular and anti circular HPTLC. Circular techniques are not available in HPLC. NOTE that HPLC is blind to detect substances which chemisorb or very strongly adsorb in the stationary phase, that is for substances with HPLC-k-values of over 1000. Especially circular HPTLC with enlarged separation power for substances with higher
HPTLC-k-values can help HPLC users. Substances with very large HPLC-k-values remain often fixed in the HPLC column, that is: remain invisible. HPTLC allows to make substances with extreme large k-values visible Whilst a HPTLC information about substances with extreme k-values changes HPLC quantitation techniques towards quantitative inner standard modes, anti circular HPTLC data may help to avoid HPLC errors hidden under peaks with very small k-values.
Circular HPTLC techniques are important for HPLC users .
NOTE:  µ-PLC IS CIRCULAR. A complete top level HPTLC working place needs quite some table space, lots of mobile phase volume - far above what modern micro - HPLC needs today and it costs a fortune. In using µ-PLC instead of instrumentalized HPTLC all is available for a HPLC ./. Planar Chromatography combination but at one to two orders of magnitude smaller in space, mobile phase consumption and investment.

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Figure 1:

The main error source for a false
Rf value = s / f. Reason: the mobile phase compresses already sorbed mobile phase traces in the layer which results in a wrong upper limit of the mobile Phase front “mP”. The more volatile the mobile phase or the more saturated the linear HPTLC chamber gas volume is, the more falsified is the visible position of the mP front line. What can help: adding a test substance with NO any sorption power in the stationary phase. Optimal is a sprayed line of such a substance solution exactly positioned onto the starting line for the samples. Text error: lokking = looking

Figure 2:

HPTLC can be understood as an elution chromatography procedure based on time: residence time in the mobile phase = tm and residence time in BOTH phases = tms.

If the mobile phase would be evaporated at the “end line” E, the mobile phase would still move. The substances “1” and “2” would continue to move. But the relation of the mobile front path and the substance moving path length remain constant despite the fact, that the mobile phase speed decreases from second to second in case we have standard HPTLC, that is no forced flow HPTLC.

Figure 3:

One must very carefully look onto the positions of a start line, a mobile front line and where and how the substance “x” has been sampled.
Figure 3 shows Rfdirect versus Rf lin and                                    Rflinear values
and where the f and s data must be measured. NOTE: In order to let the substance “x” start exactly at the center of the circular run the sample solution is injected into the layer just together with the mobile phase. In circular runs the phase flow volume is constant .
 Text error: Direkt = direct

Figure 4:

In anti-circular HPTLC there is a quite complex Rf formula necessary to express LINEAR Rf data. Thus Rf linear - the second line formula under Rf direct = (s / f)ac looks complicated. But NOTE: If an anti-circular run is done twice, the resulting Rf linear values are LINEAR and equal with Rf direct = (s / f)ac.
Important is now the right part of figure 4: Here one sees the correlation of Rf values and chromatography related  HPTLC-k-values. Whilst the linear (standard) HPTLC is only “good” in k ranges from 0.1 to 5, the circular technique helps to get data between  0.1 and 500, and the anti-circular technique is powerful in the
k-range 0.01 to 5. Remember: HPTLC-k-values are NOT numerically equal to HPLC-k-values.

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Figure 5:

From figure 2 it follows that Rf = tm/tms. As discussed on GC and HPLC pages the residence time in the mobile phase is equal for all substances and thus has no chromatography information impact. The non adjusted (raw) tms value is only useful for chromatography until we know the value of tm, as tms = ts + tm and only ts is chromatography related. Thus the Rf data as tm / tms are really not a qualified value, just a not to easy measurable “number”. Therefore figure 5 is IMPORTANT :      kHPTLC = 1/Rf - 1

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Figure 6:

This figure prepares the comparison of the HPTLC related k value with the HPLC related k-value. In isocratic runs the partition coefficient (large) K is fundamentally equal in PLC and HPLC. Reason: the substance nicotine in a HPLC column packed with silicagel and in the HPTLC layer consisting of exactly the same silicagel “does not know” if confronted with the mobile phase methanol. For nicotine the HPLC column stationary phase is a round rod and in HPTLC it is a flat open rod - or layer. Thus the base for the relation of HPLC-k and HPTLC-k values is the EQUAL value of the partition coefficient K. Look onto the “triangel” comparison model given in fig. 6.

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Figure 7:

The “triangle” comparison model is used again comparing now HPLC-k data with HPTLC k-data.
It is simple to understand :
The analyst works with TWO compounds, (this is always a good idea: chromatography  separates and thus needs as a minumum two substances). As the relation of ka to kb in HPTLC is exactly equal the relation of ka to kb in HPLC, the HPTLC-k-value can be used as HPLC-k-value by an inter method standard substance. This is comparable to the use of the inner standard technique in any mode of quantitative chromatography either GC, HPLC or HPTLC.

Figure 8:

Now we can help HPTLC by HPLC or HPLC by HPTLC in case of equal phase compositions. But still there is a critical point to be considered: Qualitative HPTLC data can easily be falsified by the gas phase effect in classical chambers. The gas phase is absent in all HPLC runs and is present in all HPTLC runs besides in forced flow techniques. Thus the strong effect of the gas phase MUST be brought under control. How this becomes possible is intensively discussed in a series on ‘HPTLC errors’ published (and continued at the time of this site production) in Journal of Planar Chromatography - Modern TLC (JPC) - issue 102 (2005), Vol 18, 118-126 and 50-55.

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Figure 9:

HPPLC - high pressure planar liquid chromatography - can solve special ‘large k’ problems. The linear single column HPLC cannot. Special problems in liquid chromatography arise with substances of large k-values. If k is larger than 100, the elution time is larger than 100 times the dead time. Those substances elute - if ever - from a column in large dilution. Therefore their detectability is poor. In addition the analysis time is very long, mobile phase consumption is high. Circular high pressure planar chromatography is an answer. ASK us using interchromforum@t-online.de .

In summary:
Rf values are poor quality numbers in any HPTLC application as they are most often systematically false and fundamentally have nothing to do with chromatography.
But correct values of the path length a substance has moved on the plate and the path length the ‘acting mobile phase’ has moved is not the distance between the visible front and the sample start line.
In fact there is only a real sharp START line if the sampled spots or lines have been focussed after sampling and prior separation. The real position of the front line can be found by a colored or UV active test substance with NO any residence time in the stationary phase. The values must be measured by a local precision of at least accurate to 0.1 mm, which is possible using either top scanners or top image processors.
Now a simple online computer program expresses the Rf value as  k = 1/Rf - 1. As k is correlated with the partition coefficient K, it is possible to compare k-HPTLC values with k-HPLC values using an inter method standard substance and warn the HPLC-only  user of the critical ranges for k larger 100 and k smaller 0.1.

For both groups of chromatographers a critical look onto the right part of figure 4 above may be helpful.
One should have such fundamental drawings on the LC - laboratory wall.

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