Figure 40 : One shot analysis, the most important power of µ-PLC. Still causing critical discussions because a majority of analytically working colleagues CANNOT believe -
what they see. The authors statement, that this result you see above does not need any repetition, as long as to be compared samples run partially overlapped and show DIFFERENCES in circle position (or color) and signal strength, then we already have a 100% safe analytical result. The compared samples differ for sure.
We analysts know about the not avoidable uncertainty in standard analytical techniques. We know of the MUST using repetition statistics in quantitative analysis and many believe blindly in error free qualitative analysis.
Check the following: In sector 5 of figure 40 above you see a chromatogram of two substances. This sector belongs to an authentic product. The producer declared this product as correctly produced under complete control and packed into a commercial product with all regulated guarantees. Let us name this product A for “authentic”. In sector 3 above you see the chromatogram of a product about which a market partner says: this is just the same authentic product like A seen in sector 5. The salesman guarantees: it is not only comparable, it IS equal.
You take it. May be first as a test sample. You have now two samples in your hand. Let us name the second sample as Q for “questionable”. You prepare for a COMPARE analysis. The sample Q you treat exactly the same way as sample A. As analyst you know how to prepare absolutely equal sample solutions - by procedure, tools, materials, concentration. You use the same type of top clean bottles, solvents and what so ever. Preparing samples exactly equal is what analysts know since more than hundred years. Learned from generation to generation. Otherwise analytical chemistry would be useless.
Figure 40 shows the circular chromatogram of a further sample in sector 6. One substance bow only is detectable. Between sector 5 and sector 6 is also an overlapped chromatogram part but no sector lines drawn and no number given. It may be that the detectable substance in 6 equals chemically the main substance in sector 5. The substance in 6 differs absolutely sure from the substances in 5 at least by concentration. There is also a chromatogram seen in sector 1. It is a polarity test used to check for correct PLC run conditions. One should know, that in figure 40 three further samples have been chromatographed besides the run test sample 1 . Somebody told that all three samples are equal.
But lets concentrate only on the compare analysis of sample A versus Q. We look at the overlap sector 4 and on sector 3 and 5.
It is in sector 4 where we find the special analytical power of µ-PLC in case samples show detectable differences.
In sector 4 µ-PLC offers a special analytical condition which the author never has realized in ANY other analytical procedure. Definitively not in any modes of gas and liquid chromatography including those with the highest separation efficiency or with MS/MS on line coupling.
* This is the special analytical condition:
In sector 4 chemistry and physics are exactly equal for all substances and substance concentrations which overlap because there is a PLC layer area in which a part of sample A runs together with a part of sample Q. There is no time effect nor any gradient which could differ for the A- and the Q- substances in the overlap area. We simply see: the two separated substances in A and in Q may be chemically equal but definitively the relative and absolute concentrations differ. Thus Q is NOT A.
Concentrations are not only cost factors but in medicine too much or too few of a specific substance may be crucial.
A is not Q, definitively, for sure. And by 100 % guarantee.
Important: We do NOT need any calibration nor any spectroscopy. Any repetition of this compare analysis is wasting time and money in case you could prepare the two samples A and Q correctly. Also important: there are already lots of qualitative and quantitative repetitions seen: the substance signals are long enough parts of circles. Just look along the circular lines. There are qualitative repetitions. Look from the plate center to the phase front: there are quantitative repetitions.
The only remaining risk is: if you got and took the wrong samples. If A is in fact not A or Q is not Q or some other organizational disaster happened.
Let us assume, we made no sample taking - preparing and giving error - in the minute both samples are in the circular chromatographic system, no further compare uncertainty exists in the overlap sector.
Summary to figure 40:
We see no top separation efficiency. We see no complex substance mixtures. The only detection mode used was UV 254 on a Silicagel F254 100x100 mm aluminium based HPTLC plate from MERCK. Only a simple mobile phase mix = hexane : methylen chloride (10 : 90; v:v ) was used in one single run. There is nothing special but the top analytical power of micro circular planar chromatography is used and documented in one single photo. It works for compare analysis of two samples A and Q.
If the compare chromatogram area, where the two samples overlap partially, show DIFERENCES (by any mode of detection) and ONLY then, we realize:
In this compare area is no difference of chemistry, no difference of physics, no time dependent effects. If we detect differences, then sample A differs from sample Q with 100% safety. There is no need to use any identification technique, nor any calibration.
This is the power of circular PLANAR chromatography because we use the technique PLANAR in an overlap area. We only have to guarantee for absolutely correct taking, preparing and giving the to be compared samples, must focus a way that we reach a partial overlapping and must use µ-PLC the way described here in detail. Although there is no classical statistics for multiple repetition necessary we should know, that there is plenty of comparability available. In case quantitative data are wanted we use the circular PLC multi integration.
Circular PLC is simultaneous multi chromatography.
It may look that the NOT EQUAL results are less important. The opposite is true. It is the most important analytical question. If two samples differ qualitatively and are representative material of series or mass production, quite something NOW must be done. If two commercial products of the same name differ qualitatively, than there is action necessary, because there may be quite some application danger or risk. Quantitatively there are always some differences. Those differences however have to be limited to the level of the quantitative uncertainty. As regulated TLC/HPTLC easily works with the not acceptable +-5% relative standard deviation level and in case only N=2..3 repetitions are made for the main substance in an important product, the quantitative uncertainty today reaches 15 %, roughly. Click on uncertainty.