Quantity errors in HPTLC are often so large

that this excellent analytical tool is disqualified as applicable only for semi quantitative analyses.
On the other hand quantitative HPTLC - in its full instrumentalized version - is not replaceable for very many analytical tasks.

Well done it is as precise as the best HPLC method but more important: the danger of wrong identification is very much smaller than with HPLC. As HPTLC users repeat sampling and separation much more often than HPLC users, the danger of excessive quantitative errors is again much smaller with HPTLC than with HPLC. NOTE that structure identifying highly sensitive spectroscopic detectors are applicable on-line HPTLC even in their micro version. The first direct HPTLC/MS coupling was made by the author in the late sixties but there was not yet a real demand for at these early years.

The reasons, why HPTLC is in quite a good shape with respect to HPLC IF WELL DONE:
HPTLC users have for each sample a freshly unused “column”.
HPTLC users can do multiple simultaneous runs and critically compare data.
Well informed HPTLC users have thousand different and highly specific detection modes available and very many more mobile phases than HPLC users.
HPTLC users can easily multiply the separation power of a plate by multi dimensional separations - two dimensional runs are only a question of proper sampling and the use of proper plates and chambers.
HPTLC is highly sensitive and especially applicable to trace analyses.

As example: drinking water quality analysis MUST be done quantitatively, which is possible by HPTLC for more than hundred toxic traces simultaneously in the ppt-range of concentration (1 ppt = 1/1000 ppb; expressed as weight-concentration; 1 ppt equals 10 to the power of minus 10 weight %)

HPTLC users can easily and directly on-line combine chromatographic techniques with capillary GC, and HPLC/capillary HPLC. They can use HPLC as a quite helpful sample cleaning and sample preparation mode for the final HPTLC analysis.
HPTLC is unbeatable for environmental mass analysis of solid and liquid “dirt” which a HPLC user would never inject into his delicate instruments.
Although circular HPTLC is widely unknown, it is more powerful than linear HPTLC especially for micro preparative chromatography in its “rotation HPTLC mode (Sz. Nyiredy, see HPTLC literature)”.
HPTLC can be used under high pressure (easily up to 3000 kg mechanical forces on a 50 x 50 or 100 x 100 mm circular plate). There is also forced flow linear HPTLC available with promising possibilities for mass screening analyses not only in pharmaceutical research and development.
HPTLC is the only mode applicable in a technically underdeveloped area with missing electrical power and a minimum of space, especially to control product falsification in medicine.
HPTLC can be done in less than 30 seconds for up to 12 samples simultaneously by micro anti circular separation and in a few minutes for up to 80 samples simultaneously. The only problem is then that our to days computers are still too slow for a complete timely quantitation of such a high data stream.

To switch from classical HPTLC with large mobile phase troughs and large classical plates into high pressure circular micro planar chromatography = µ-PLC this is a question of seconds and quite a small investment, we could do this:
- take HPTLC plates
- replace grandfathers trough by a virtual trough in vertical position ( 1mm high, empty
 (this can be seen after clicking a LINK in page µ-PLC of this site).
- use the gas phase with all its analytical power for improving drastically the plate clean up, the sampling,
 the sample focusing to sharp sample bows
- use only one ml mobile phase in a one ml flask contacting the plate center through a wick.
- let separate and dry again completely with a 2 L/min gas flow from a micro pump (costs 10 Euro !)
- make a digital photo, crop it, enter it in µ-PLC integration software (it makes multiintegration and
 reduces this way drastically the plate structure)
- and enjoy a data quality of easily a factor of fife to ten better than ever before. Soon are all details
 in this site.

And why are much too often HPTLC data so badly reproducible, that this analytically most powerful technique has in many laboratories the level of a “semi quantitative” chromatography mode often even not anymore in use ?
 

* The main source of quantity errors in HPTLC standard as well as trace analyses is the plate structure. No existing HPTLC stationary phase is free from structure which kills the analytically possible data quality at any wave length or reduces it by a full factor of ten.

* A next source is the fundamentally non linear calibration function for each substance in any concentration range but the use of the “rule of three” evaluation mode or by the use of data transmission concepts.

* The third source is the quite low separation power of HPTLC resulting in substance overlapping which often causes quantitative side effects falsifying the correct quantity value.

* The fourth source is the still missing or widely underdeveloped planar quantitation techniques, starting with digital photography at any wavelength range but not yet ending with enough accurate and enough precise HPTLC quantitation software.

There are quite some further error sources which are in discussion in a series of publication in the only international PLC journal “JPC, Journal of Planar Chromatography - Modern TLC”;
Springer; ISSN 0933-4173.
Contact the Editorial Assistant under eufeps@axelero.hu

Even if this tail belongs only to a sample solvent peak of no analytical interest in quantitation, the green peak value remains falsified, as the “tail line” is the real base line for the “green” substance. In figure 2 both red lines - the perpendicular separation line and the prolongation of a false positioned base line - falsify the peak area value of the green substance peak. These quantitation problems can be reduced drastically by an optimized selection of stationary phases in GC and HPLC, by the programming of the stationary phase in HPLC instead of a mobile phase programming and if the chromatographer observes the limits of working ranges for his separation system, the programming limits, the detector- and the detection limits.

Completely different is the baseline problem in PLC / HPTLC.
It is much more critical and needs a fundamental correction of to days practice:

The base line in PLC with plate layers made of granular materials causes a base line structure, which falsifies drastically all quantitative PLC data. Up to now (year 2005) nearly all users of quantitative PLC accept a mathematically false - statistically even illegal - mode of structure correction: this is statistical smoothing. The structure of PL- chromatograms have however a stable signal which overlays the chromatogram signal precisely. It is therefore a MUST to treat the base line - or base plane - structure signal as a systematic error which has nothing to do with chromatography and which must be subtracted from the total signal. Thus we need TWO runs: to get the structure signal prior a chromatogram and later to get the total signal “structure plus chromatogram” signal when separated. Both signals are taken at differing time. Therefore the quantitation steps and the signal data must remain absolutely reproducible (light intensity, wave length, signal strength and position precision) during the two correlated runs. Even the line voltage and the line frequency must remain constant or kept constant by qualified scanner hardware. The raw signals must be stored, at best in computer memory. After the empty plate structure signals and the PL- chromatogram signals are taken the difference “substance signal plus structure minus structure” is calculated and only NOW the structure free signal can be integrated. Software helps to subtract the structure signal precisely. As a result quantitative PLC data become this way at least one order of magnitude better reproducible and correct.

There is a second procedure to reduce the structure caused errors in PLC: this is the mean of multiple circular quantitations at changed angle position, see figure 3.

Figure 3: Circular PLC, 7 substances (1...7). St = substances which are not moving. They have k- values of 50 and higher, are invisible in HPLC (remain on the column packing) and change there stationary phase characteristics. The scans are linear - see figure 4 - and repeated at changing angles. 9 to 16 repeated scans at differing angles are added together and divided by 9 or 16.

The sum divided by 9 or 16 results in a chromatogram with strongly reduced plate structure - see the lowest line-

Figure 4 illustrates the multiple linear scan of one circular chromatogram of only one sample. The quantitative signals of each scan are summarized. Each scan is taken only at one fixed angle, than the plate is rotated to a next angle - in figure 4 the 9 positions differ by 10 degree each.
It is understandable from figure 4 that we get double data, as for instance the outermost green circle is scanned twice: at the beginning of the scan and at its end. This happens which each substance circle.
The figure looks like the scan track is rotating but in fact the plate rotates, not the scanner. We used CAMAG´s linear scanner for the experiments. Each single scan is shown in figure 3, upper chromatogram part. The scatter in the multiple chromatogram lines result from the plate structure.
NOTE: the lower chromatogram line is NOT the result of smoothing, but is the mean and mathematically OK.

Figure 5:
A sample containing very low concentrations of two substances have been chromatographed - see the two peaks left with one large spike right of the first peak. The plate was scanned in UV at the highest available signal sensitivity. No integration program could localize and quantitize the two peaks because of the very strong plate structure signals.
What is not well visible in figure 5 is the fact that TWO signal lines lay over each other. Only slight deviations of the empty plate structure signal are seen in the left chromatogram side overlayed by the chromatogram signal.
However under the two peaks there is the structure baseline visible. One sees the left and right of the two peaks.

No any mode of chromatogram improvement could help to get any analytical signal qualified for an integration. We chromatographed such a small amount of substances that we clearly remained a factor of ten below the detectability limit.

Figure 6:
The complete raw data - about thousand binary values - respresenting the scan signal of the empty plate precisely at the later position of the double trace chromatogram are stored in the lab computer. The complete raw data set of the scan taking the two nearly not visible traces are stored as well. The difference of all 1000 signal data pairs is calculated and saved as raw signal “chromatogram minus structure”. This data set is now integrated and resulted in well reproducible values (+- 2.6 % rel. standard deviation) for the two traces. The repeatability was “only” +- 2.6 %, because the base line quality was still not good enough - see the “base line” right of gthe two peaks. Later technical checks showed the following problem: the scanner moved the plate linearly by stepper motor action. Each step was about 100 micrometer long but the frequency of the stepper motor electronics was not kept constant enough. It was line frequency controlled and the line frequency is NOT precise enough during the short time a chromatogram is taken.