Summary - Keywords - Understanding µ-PLC.
µ-PLC is named micro, as it allows HPTLC separations on plates of 10 x 10 mm whatever this offers in the analytical practice. But it also works with 100 x 100 mm, 100 x 200 mm, 200 x 200 mm layers based on glass, aluminium, plastic. Its material consumption is minimized. The mobile phase volume used for very small plates is given by micro brushes. A standard µ-PLC run on 100 x 100 mm plates uses one milli liter mobile phase, flowing from 1.5 milli liter micro bottles.
* Truly PLANAR:
It is a truly PLANAR (circular) chromatographic technique, as it uses always the full area of a plate for qualitative and quantitative analyses. Therefore it is simultaneously two dimensional liquid chromatography.
* Not yet used (new ?) Analytical Technique in Chromatography:
Partial overlapping chromatograms offer an unique up to now probably not yet used technique which results in 100 % safe results in case the overlapped chromatograms differ for sure - qualitatively or quantitatively - by a single run. This is in contrast to all standard chromatographic analyses.
* Focussed Sampling
in the range from nano liters to a full milli liter offers a wide range of quantitative µ-PLC from ppt to 99 % concentration. A single sample fully circular separation offers absolute quantitation not available in GC, HPLC, TLC, HPTLC, OPLC, HPPLC. The sampling technique differs completely from all modes used in PLC standard techniques and allows for not yet available concepts. Focussing is mandatory to keep the separation efficiency in PLC at ia high level. Multi focussing may repai systematic sampling errors caused by specific substances showing partial sorption in ‘dirt’ at the sampling place. Multi focussing could enlarge the µPLC separation efficiency by a factor of five. Check for ‘multi focussing’ in the part CONTENT of this site.
* Compare Analysis, Partial Chromatogram Overlapping:
A truly planar AREA chromatography allows for compare separations by partial overlapping of the chromatograms of two samples which are checked if they are equal or if they differ (qualitatively and / or quantitatively)
* 100 % Safe Results by One Single Run ?
The reason is easily to understand if one considers the following: µ-PLC is circular and takes much larger sample volumes at sampling than in standard techniques. This would kill the separation efficiency of PLC as too large sample volumes cause. The sample spots, lines or triangles are focussed to sharp circles or bows. Thus there is enough substance available even in trace runs. The resulting long bows or complete substance circles allow for multiple neighbored chromatograms - we have multi chromatography. The single run is in fact a multiple comparable chromatogram. Thus there is enough safety statistics, much more than by double or triple classical runs.
* The Overlapping Chromatogram Area is Unique
The overlapping chromatogram area is unique in separation analyses. It does not exist in GC, HPLC, OPLC, HPTLC, etc. In this restricted area like a triangle stays the complete physical and chemical situation absolutely equal for the two overlapping chromatograms from to be compared samples. Thus there is no chance for systematic errors which may falsify analytical data of the two samples. There are very many sources for errors in all other and standard chromatographic techniques. Many are widely unknown - click HERE. If the µ-PLC chromatograms however are free from systematic errors AND differ, the sample composition differs, for sure. Time, which is a strong source for chemical and or physical changes in chromatography is top equal for the two samples.
is based on digital photography (10 MPixel digital figures) and the new circular multi integration software of SORBFIL v.2.0
* Multi Integration
is possible in µ-PLC because of the long length of circle lines or bow lines. The quantitation is done by overlapping chromatogram tracks with degree wise position changes and a graphical / mathematical mean of quantitative data. This reduces the worst systematic quantification error in planar chromatography: the plate structure. Thus +-0.5% down to +-0.05 % relative comparability standard deviation is possible in optimally done quantitative analyses.
* Gradient Effects are Strong Sources for Data Falsification in PLC
Therefore it is circular. In µ-PLC we avoid any influence by the sample solvents, the mobile phase vapor along the separation path, the temperature. This is possible by the use of a strong gas flush at room temperature, no any mobile phase chamber and accuracy and precision by an auto optimized constant mobile phase flow strictly out of the plate center with no electronic pump. This Internet-only-book discusses all up-to-now known aspects of circular versus linear PLC.
This all together sounds like µ-PLC needs completely differing instruments
than classical PLC like HPTLC, OPLC, HPPLC ?
Yes. Most simple ones, easy to construct them by yourself. See the figures. Click HERE.
What means truly planar ?
Classical TLC as well as HPTLC and OPLC use the linear Thin-Layer Chromatography concept for several samples simultaneously in locally isolated singular tracks.
Planar separation means to separate simultaneously samples in the complete stationary phase AREA on tracks, which are touching each other or even in a partially overlapped mode for best possible qualitative comparison. Whilst especially modern OPLC takes care for locally isolated sample tracks (for good practical reasons) planar separations take care to keep the local position of a substance free from systematic changes and if necessary even partially overlapped. For this we have serious reasons. Especially compare analyses are fundamentally supported by partial overlapping.
Linear PLC with isolated tracks show quite strong substance position changes just - but not only - for mechanical reasons. This may be a non uniform layer thickness or a special non uniform layer structure. Mechanical effects have nothing to do with chemistry and are unknown to HPLC- or GC chromatographers. Even the top qualified ready made HPTLC plates show some flow gradients due to the mechanical production directions. Nearly all plates have edge effects. More serious are other non uniformity effects in the area like temperature and changing vapor phase composition in contact with the stationary phase.
Truly planar would be a separation condition where the separated substance lines partially overlap each other. The simple reason for this are locally strictly equal physics (temperature, flow speed), chemistry (vapor phase composition in contact with the stationary layer) and time in the overlapped area. Thus the local isolation of tracks in classical PLC is not a good concept. It may cause systematic errors of several kinds.
True planarity and partial overlapping are next to a truly planar acting vapor phase the most important key conditions for an analytically most powerful chromatography technique. This will be discussed in all details in this SITE.
Let me add a nearly all analysts shocking - at least simply not yet believed - statement already here: A single µ-PLC analysis with overlapped areas of to be compared samples is ready for a clear decision making if it shows differences clearly detectable. There is no statistics necessary, that means: no any repeated analysis as long as the samples have been given error free. One single run (N=1 !) delivers a 100 % save result. The samples differ. Court case stable.
The author will in all details discuss the AREA based PLC in the sense of planar separations. He knows very well, that a truly planar technique is not regulated and certified except in the rare cases of accepted two dimensional SPOT PLC. Unfortunately spots do not allow for planar compare techniques and are not the very best for quantitation. But “regulated & certified” is known as the most effective blockage of analytical progress and has nothing to do with accuracy and precision. It just helps to keep certain interested groups happy at an enough low level of a repeatability standard deviation and helps for method comparability, not for accuracy. Note: accurate data are perfectly comparable but it does not work the other way round.
The PLANAR argument however is just too important even if the author swims against the flow, because:
* It will make understandable, why linear spot TLC on isolated tracks is the weakest mode of PLC.
* It will make clear, that linear PLC with sprayed sample lines is much better than the spot technique but gives away its only theoretical advantages in case the tracks remain isolated from each other.
* It will make clear why the circular mode of PLC is far more easy and powerful in nearly all practical aspects beginning with sample application and not ending with its quantitation by a new concept of photo integration.
By the way: µ-PLC offers such a wealth of analytical power that even without partial overlapping we can easily end up with quantitative analytical data and a statistics of
+- 0.05 % comparability standard deviation at N = 4 comparisons.
(NOTE: comparisons, not only repetitions. This means: in this SITE there may be something special which is named “Multi Integration”. This reduces drastically the main error source of classical planar chromatography quantitation - the plate structure.
However: +- 0.05 % as an up to now best measure of accuracy is not in easy reach but +- 0.5 % should be standard by photo based multi integration in comparison mode.)
A next less believable statement: the new micro planar liquid chromatography µ-PLC has the widest range of sample volume capacity: from nanoliters to easily a full milli liter and no kill over of the separation power. If one gram sample allows for specific detection in the femtogram range - well, lets calculate this trace detectability level later.
* It will make understandable why arguments - like “we never will change from linear to circular” are as qualified as the historically wrong statement “we never will remove the systematical PLC error caused by the plate structure”. (Who really believes the plate structure swings in noise frequencies ?). The structure problem in PLC is normally 10 times stronger than the detection noise level - the latter to be cured by signal polishment. Thus PLC could easily be a factor of up to ten better than today. (Really regulated for pharma analyses ? +-5% at N=2 means roughly a 15 % uncertainty range)
Well poor enough precision and accuracy makes some parts of life easier and the simple formulas which translate data sharpness into knowledge and economy are widely
This SITE discusses of course classical techniques as well. So we will see spots, linear TLC analysis and non structure corrected quantitative data including the under those conditions unavoidable calibration lines. But the author will use fifty years own experiences in PLC and discussions with more than 5000 chromatography colleagues from more than 55 countries about ways and steps to solve problems for reaching correct analytical information as good as necessary, as cheap as possible and for his/heir best personal success. Because analysts carry a high burden of responsibility. She/he has to work hard to get qualified data based on materials for decision makers. IF THESE DATA ARE SYSTEMATICALLY WRONG, the decision made is wrong at least as the size of the error, for sure. And this may be very much more expensive than HPTLC analysis even if done with the utmost of the latest instrumental equipment.
About the author you will find relevant details under AUTHOR (click onto blue underlined words).
Date of publication : April 2, 2009.
Last updating : March. 1, 2018.
This book is about four years old. It got many thousand clicks from chromatographers in > 60 countries.
All figures in this book are copyright of IfC (Institute f. Chromatography Bad Duerkheim, Germany) and the ones in Hypercard mode are based on a close cooperation with Ute Hezel-ZEISS, Germany; J. Blome, Germany; H. Halpaap-MERCK, Germany; D. Jaenchen-CAMAG, Switzerland; J. Ripphahn-MERCK, Germany.
The author wants to thank these chromatographer colleagues for the great cooperation lasting over many years since the time when we together invented HPTLC in Bad Duerkheim (whatever else you can read about the true start of HPTLC in other publications).
All colored figures in this Internet book are also copyright of IfC.
Only the µ-PLC equipment with no mobile phase chamber allowed to realize the first time THREE PHASES CHROMATOGRAPHY. The stationary phase is in contact with a liquid and a gaseous mobile phase but not necessarily all over the run time. This third phase allows a wide range of freedom to modify separation selectivity prior to or during the separation run. It also showes the importance of a complete control of the sample solvent traces prior to a chromatographic run and makes understandable, why solvent tanks in gas phase contact to the stationary phase are sources for systematically falsified mobile front positions, reason for beta-fronts and reason for poor analytical reproducibility in classical TLC / HPTLC modes of analysis. For more to the THREE PHASES CHROMATOGRAPHY click HERE
Responsible for this Internet-ONLY-book:
Prof. Dr. Rudolf E. Kaiser; Fax: +0049 6322 989751; e-mail: firstname.lastname@example.org
µ-PLC was developed in close cooperation with Dr. Olga Kaiser, Bad Duerkheim
I owe heir a great debt of gratitude
NOTE: There is a new critical paper about methods (over-) regulation in the by now only English SITE www.interchromforum.com , which discusses sources, ident-methods and error reduction procedures in analytical chromatography [GC, CGC, GC*GC, HPLC, HPLC*HPLC, TLC, HPTLC]. Click onto PAPER 2010. The paper is a ppt-pdf type and needs the latest Google Chrome, firefox, or safari and pdf reading software like Adobe reader later than version 9.0. Loading time is normally below 1 minute.
Quite some additional information about µ-PLC and further application possibilities including sub trace analysis by on-plate mass sampling - is given - however only in German language - in www.institut-f-chromatographie.de .
A 4-minutes-Video is available, necessarily done under very weak light conditions because of heat problems during the separation. It shows all steps from the plate cleaning up to the result of a pharma products compare analysis. This video is found
in institut-f-chromatography or click onto this LINK below:
NOTE: It works best under Google Chrome as browser for an easier access to necessary plug-ins. http://www.institut-f-chromatographie.de/html/mu_plc-video.html
Most easy however - no plug-ins are needed, any screen size available:
Go to YOUTUBE. The video is immediately on your screen (if you are Youtube user) by this input: Micro-Planar-Chromatography1 (Note: 1 at the end)
Youtube author: rudolfkaiserduew1
Or just use this LINK:
Although there was stress onto the chromatogram by the video-light (non uniformly surface heat, the final chromatogram showed: authentic product A differs by 100 % sureness from the questionable product Q. To see the chromatogram click HERE and go to the end of the page - green (UV) foto.