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How to use this INTERNET book:
Contents is linked to pictograms showing hardware, technical details, tools, procedures and results in qualitative and quantitative planar chromatography - the latest one and the established one. There are TWO pictogram sites: one which contains µ-PLC and a second which is concentrated on TLC / HPTLC.
You can reach the pictogram pages through the bar switches on left: µ-PLC pictograms and TLC HPTLC pictograms. The pictogram pages are concentrated and organized by blue and violet numbers and a few shortened “ident” words. But when having done the 1 to 40 clicks for µ-PLC or 1 to 64 clicks for PLC / HPTLC you get the main content of this book: figures and drawings. A figure is worth thousand words and the language is no longer “broken English” as the readable book part (sorry, please excuse, my German is much better).
The click onto one of the pictograms brings you to the enlarged figure with detailed figure captions - often more exhaustive than standard for captions. These exhaustive captions should offer you complete specific information. Thus this book is mainly figures plus maxi captions.
Through the pictogram pages you reach any figure, but you can also move from figure to figure one by one forward and backward . There are three click icons on top of each figure page: <-- backward, forward -->, and up to the figure summary page of pictograms.
Not everything in an INTERNET book can be treated through figures with extensive captions. Therefore the topics “Multi Integration”, “µ-PLC helps HPTLC”, “Trace Analysis by µ-PLC”, “Makingt a µ-PLC Instrument” and “PLC literature” have their own switch bars as seen on the left side here. In case the switch bars disappeared, move with the scroll bar on the right page side down to the bottom of the page. At the bottom you will find a complete navigation board which in all cases contains HOME, this page “Introduction” or all of the other pages mentioned above.
Immediate comparison of details is often helpful, therefore you will find many LINKs here named as clickword. All of them are BLUE and underlined like this click here.
Clicking on a blue underlined word will bring you to a SITE place which should answer a question, identify a word or give arguments for a statement. To return to the place from where you left by clicking is easy by a click on a backwards oriented arrow like <--- or a next clickword. Pages or places you already have visited show the formerly used clickword in RED. There are blue page numbers which are NOT underlined. These are not click words.
You need some parts of this SITE on paper ? Well: this depends on your computer hardware and your printer, but printing on paper should work, normally, as the SITE software used in this INTERNET book is one of the globally best. At least at writing this book it was on top of the SITE techniques and software.
Some links point to the non commercial www.interchromforum.com, It contains error warnings and information how to detect, reduce or bypass systematic analytical errors. Unfortunately all colleagues I invited to cooperate and publish a series on error detection and the way out or around told me their thank you very much and that they do not want to loose friends and commercial partners. But somehow we should be aware, that there are unbelievably powerful and nevertheless very economical analytical ways to get correct analytical answers to solve critical problems. Only in case one is willing to look round, remain flexible and is free in analysis that is: not bound by regulations. Errors are everywhere. Poor accuracy and precision is sooner or later VERY expensive. Beautiful agreements on regulation and clever looking “grey one knob boxes” are standard. But beauty is nothing without brain
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To the history of TLC:
Victor G. Berezkin and Tadeus H. Dzido discussed the discovery of thin-layer chromatography by N.A.Izmailov and M.S.Shraiber (1938) , and their contribution to the development of this mode of chromatography in JPC 21, Number 6, December 2009 (399 - 403)
Joseph Sherma and Gertrud Morlock reported in JPC 21, Number 6, December 2009
(471 - 477) about 115 mile stones of the Chronology of Thin-Layer Chromatography focussing on Instrumental Progress. This report gives also a Chronology of HPTLC Symposia, which started with the First International Symposium on Instrumentalized High Performance Thin-Layer Chromatography - 18-21 May 1980. in Bad Duerkheim, Germany. This Symposium was organized by Institute f. Chromatography Bad Duerkheim
25 Symposia followed, the last mentioned in this Chronology was on 11-13 June, Helsinki, Finland, numbered as 20th Symposium, HPTLC 2008. The numbering is not too correct.
To the history of HPTLC:
As mentioned in this electronic book under HOME, HPTLC was developed in Bad Duerkheim but there was up to now no real historical background published. As with the start of TLC in 1938 - caused by technical accident - HPTLC started also because of an technical accident in 1975.
L. V. Andreev, a Biochemist from Moscow, presented a new plate material to R.E.Kaiser at an Analytical Symposium in Slovakia. The new plate in the size of a microscope glass slide (2.5 x 7.5 cm) was covered by a 100 µm thin layer of binder free sub 5 µm silica particles. On return from the conference Kaiser asked his friend F. Eisenbeiss-MERCK-
Darmstadt to reproduce such a plate which was done within a day. Eisenbeiss made the silica layer a factor of two thicker than the ones made by Andreev by soft sedimentation of a sharp silica gel cut. The plate was presented by Eisenbeiss together with Kaiser in front of a 24 GC chromatographers course at IfC in a life experiment. We announced a world record separation. We could take this risk as we had two plates at hand. The one showed ten minutes prior to the record session “it will work”.
Kaiser sampled a micro spot of a ten compounds color mix, inserted the small plate into a small glass cylinder with some benzene as mobile phase and got the sharp complete separation of ten spots in 13 seconds on a 7.5 cm long run witnessed by the 24 colleagues. The plate exists still today. Some months later 24 highly interested TLC experts met at the first “IfC course on high speed TLC”. The author had a contract with experts from Merck not to use mechanically instable microscope glass plates but exclusively the just developed new Nanoplates from MERCK. Perfect surface, dense fine particle layer plus binder. In preparing the course technically a few days prior to the arrival of the TLC experts it became clear, that we will have a full range disaster. The new plates had been uselessly slow. Kaiser developed immediately circular micro separation instruments with fine capillaries for the mobile phase transfer into a 5 x 5 cm small Nano plates layer. The 100 x 100 mm size had to be cutted therefore. Under the microscope one could see a promising separation effect with acceptable speed as long as it stopped below a 10 mm diameter circle run. There was however absolutely no hope to do anything quantitatively. The spots, bows and circles had been much too sharp. Mechanical linear scanners have still today much too broad light windows which react like old heat conductivity detectors with large dead volumes in gas chromatography. Their large volumes killed the separation efficiency of good columns and were useless for capillary gas chromagtography. D. Jaenchen from CAMAG - Muttenz helped in a few days to improve the circular mini chambers developed at IfC into something looking more industrial. The “high speed course” attendees were friendly enough stating after a week : well: by far not as fast as promised. From former TLC with one hour or 30 minutes run time down to 10 or 5 minutes. But fully new micro sampling systems and instrumentalized phase transfer by stepper motors and syringes had to be used and started to make TLC expensive. May be the early circular TLC / HPTLC tools from the seventies will in years become more than a toy. This may work from the day on quantitation works for the instrumentalized version of linear HPTLC.
A few years later the U-chamber from CAMAG and other circular multiphase and anti circular chambers disappeared from the market as still quantitation did not work correctly enough with the linear scanners available. Basically circular chromatography, although much more powerful than any linear mode of planar chromatography found no user, as basically linear scanning and circular separation did not cooperate prior
to 2007. It was only 2008, when the Russian company Sorbfil and Kaiser cooperated to make the photo quantitation compatible with circular planar chromatography.
To the history of circular PLC:
N.A.Izmailov and M.S.Shraiber (1938) started TLC from on the very beginning circular. Many later chromatographers had no problem with this semi quantitative technique. The plate separation speed desaster at IfC in 1976 pushed the research on precision of circular TLC already on 50 x 50 mm nanoplates from MERCK. By photo quantitation we found out:
* The phase flow into a circular plate is precisely constant.
* The repeatability of the mobile phase volume values over the area of wetted
stationary phase is so accurate and can so well be quantitized, that circular PLC
is (the only) digital chromatography. We could measure inter particle volumes,
inner particle volumes, the true position of the phase front and by this an accurate
and correct Rf-value. This could be translated into k-values of isocratic HPLC and
thus to the partition coefficient K. Comparing data it was shown that linear TLC
Rf-values have no accuracy and precision. But accurate PLC-K-values were equal
to HPLC-K-values in case of equal phases. Very helpful for MS-free identification.
* By our way to quantitize circular PLC using modified scanners with own software
we could show that the most critical systematic errors in narrow and wide range
calibration are well reproducible and false signals are caused by the particle structure
of the stationary phase.
* This depends on many technical factors based on the plate production concept and
not only on specific data of the spectroscopical systems. With top quality adjusted
scanners, in the dark and using dark clothes visible light scanning showed a factor of
ten sensitivity and accuracy improvement by removing the structure signal .
This is still today the analytically only correct way.
* Correct is to remove the structure signal by subtraction, not by electronic noise
smoothing. Structure signals have nothing to do with chromatography or noise.
* This way we could show that circular PLC is a quantitatively absolute technique. It
results in accurate weight data. In one or the other figure caption on page “TLC and
HPTLC“ details are given. Structure signal containing raw data make certain
trace analyses much less sensitive. For successful trace analysis structure reduction
is mandatory.
* The correct subtraction of the structure signal improved trace analytical data to a
level of +- 2...3 % repeatability standard deviation were without this correction
traces could even not be detected at the just mentioned level..
Here is the word “correct” in connection with subtraction often used.
* As PLC is planar, the subtraction of structure signal must be “area - position
- precise”, that is in both directions of X and Y by better than +- 0.1 mm .
This is possible only with the help of software which moves the data sets “structure
signal plus substance signals” versus “structure signals only” for an areal
subtraction. May be this hard way is the reason, why still today commercial top
scanner data are mathematically smoothed the wrong way giving away a factor of
ten for analytical accuracy and precision.
* There are also other technical and physical problems not yet solved by the certified
official linear TLC / HPTLC scanners.
In the minute we had circular PLC quantitation software, we could move to photo quantitation and multi integration. The latter uses the most simple way to reduce structure signal problems for precise circular chromatography: we “scan” the digital photo data at least 4 times at changed positions and divide the sum of the 4 times integrated data by 4. Or the 16 times integrated parts of a signal bow or circle we divide by 16. Details may be well understandable when looking into the chapter Multi Integration in this SITE.
This new way of digitizing planar chromatography made µ-PLC quantitative. Thus we could start to use µ-PLC as analytical tool with a most far reaching sample load: it ranges from a few nanoliters to more than one thousand micro liter sample volume.
As µ-PLC accepts not only original sample solutions but extracts, the extract enriching factor co-acts in quantitation. Therefore µ-PLC has the capacity for trace analyses down to the sub ppt-level. Today available digital cameras have every year higher resolution power, now a 3 mega pixel camera can be replaced by a 12 mega pixel camera surpassing fully the sensitivity / accuracy limits of the best optical linear scanner. µ-PLC is now in the hands of critical analysts and a powerful tool despite the fundamental limitations in TLC separation efficiency. The real power now lays in cooperation. Circular PLC can be life coupled with high resolution capillary GC or the new multi chromatography HPLC systems. It is directly compatible with micro HPLC columns.
This author developed and intensively used in the late forties / early fifties of the last century reversed phase circular PLC for the separation of synthetic fatty acids from C6 up to C26 made from alkanes by catalytic air oxidation. In linear TLC systems the mentioned acid separation did not work. (PhD-thesis, R. E. Kaiser 1953)
Linear versus Circular
Since the late fifties the author had a constantly critical look onto circular versus linear in planar chromatography. Linear is the standard mode of to days TLC / HPTLC and is supported by hardware and software. However there are some not too well known but serious drawbacks. The precision and accuracy for the qualitative analytical value - the Rf-value - is too often systematically falsified because the real position of the mobile phase front is in almost all cases systematically wrong - too “high” . The accuracy and precision of quantitative data is far below the level of other chromatography modes like capillary-gas-chromatography and high performance column liquid chromatography. The PLC data quality level is now far below the one qualified analysts need. Therefore the linear plate chromatography techniques of today are often disqualified as
semi-quantitative. Most of the modes of linear TLC / HPTLC are NOT really planar. Parallel linear substance tracks are isolated from each other and show chemically and physically differing unwanted gradients. Some more information about these and other problems with the linear standard techniques are mentioned below.
Here are results the author found when comparing linear with circular PLC techniques:
* Circular is the mother mode of Planar Chromatography.
* Circular PLC is the only fully digital chromatography mode, but not yet used as
such.
* Circular PLC has the minimum of mobile phase consumption.
* Circular sampling offers the widest volume range from nano- to milliliter.
* Already sampling - in any mode of chromatography - is circular.
* PLC sampling becomes perfect - qualitatively and quantitatively, by focussing.
* Circular focussing is the easiest, it repairs sampling problems optimal.
* The pressure less mobile phase flow is constant in circular PLC.
* Best phase purity is given and phase interference can be fully suppressed.
* Forced flow PLC is easiest when circular. No tightening efforts needed.
* Circular separation under forced flow conditions is the fastest.
* Anti circular separation is the fastest in all pressure less modes of PLC.
* Gas- or vapor phase application in PLC is easiest in circular modes.
* Circular gas treatment offers specific chromatography modulation best.
* Sample solvent and mobile phase removal is best under circular conditions.
* Quantitation is easiest and most accurate in circular modes of PLC.
* Circular modes allow to do qualitative compare analyses optimal.
* Partially overlapped circular separations offer a 100 % safe compare result.
* Life coupling of GC and HPLC with PLC runs easiest in the circular mode.
* The PLC plate material quality can easily be checked by circular tests.
* The real PLC-quality of mobile phases can most critically be checked circular.
* Trace analysis is most far reaching and promising by circular techniques.
These findings made the author to concentrate on circular modes of planar chromatography for many years, which resulted in his latest experimental work:
the development of µ-PLC.
To the history of µ-PLC:
As even tenth of a volume-% of specific mobile phase parts in a mix show often drastic selectivity effects for the PLC separation, the fast aging of freshly made mobile phase mixtures is an expensive drawback. The repeatedly use of a given mobile phase either by inserting plate by plate into the same TLC tank or by pumping the phase in-out-in-out in case of AMD systems cause growing systematic errors.
STOP: If you do not like this broken English text and the too long sentences, why not checking µ-PLC the “picture way” ? Pictures are international language. No misunderstanding (well: under each of the 40 colored and about 60 black-white pictures is still a sometimes longer figure caption part in broken English).
To go the “picture way” click PICTURES and use the right arrow ICON (right on top of the page). This way each single picture is available. If you only want to see pictograms, click PICTOGRAM and use the scroll bar. A click onto one of these “minis” brings you to the full size picture.
As in standardized PLC runs we have incomparably large quantity errors caused by many sources. We have time depending systematic qualitative errors because of the composition instability of mixed mobile phases. Although nearly no analyst likes discussing systematic errors and an author who nevertheless believes just in the importance of a hard discussion he takes the risk to loose friends by intensive reports about systematic errors. At the end of all stories however only the correct result pays back. Only this is for what we analysts work and can keep responsibility. So if you agree with me, have a look into summaries under qualitative systematic PLC errors, click here
www.interchromforum.com/html/ql_err_hptlc.html
and for quantitative systematic errors in PLC click here:
www.interchromforum.com/html/qnt_err_hptlc.html
Very many experiments led to a hardware solution and a processing concept for circular planar chromatography which fulfills the following conditions:
- drastic reduction of the mobile phase consumption to the technical minimum
- error free concept to make, store, use and re use the mobile phase;
- total removal of mobile phase from the stationary phase when cleaning the
plate and at the end of separation(s);
- wide ranging sample application from nano to milli liters;
(by now the maximum loadability is at 1.5 ml for extracts);
- memory free quantitative sampling of sample solutions even to soft plates;
- stress free sampling and multi sampling without any phase surface damage;
- total removal of the sample solvent from the plate at room temperature;
- optimal sample focussing and complete removal of the focussing solvent;
- single phase separation flexible in time, with or without a stationary phase;
- stationary phase impregnation by volatile acids or bases and/or other polar
vapor traces via the gas phase at room temperature;
- error free manual multi phase development;
- simultaneous multiphase separation;
- complete and stress free removal of the mobile phase after separation;
suppressing the non wanted secondary chromatography effects;
- pre quantitation during the separation run even in the wet state of the
plate;
- flexible and precise enough quantitation under UV, fluorescence, visible light
after drying;
- if necessary all steps from sampling through focussing, drying, separation
and quantitation under inert gas (for instance under carbon dioxide);
- simple enough but flexible statistics control of the digital data online the
data sets in the lab computer without any rewriting; complete reporting;
- qualified data transmission, data storage and electronic data retrieval.
Many such development steps resulted 2007 in
µ-PLC
The full potential of this probably new concept is not yet reached. Lots of work is still to be done mainly in the area of trace analysis, which because of the sample loadability at and above one milliliter is promising for environmental-, food- and water analysis. The author thinks of extracts made by auto circulation with volatile non polar solvents or by combining polar sorbents with displacement techniques.
The important differences between the new µ-PLC and TLC/HPTLC
sample preparation:
In µ-PLC we sample by micro brushes. This avoids spray techniques and the use of syringe sprayers. The raw sample can keep the original composition. That means: no sample falsification by filtering or centrifugation. Micro particulate matter in a sample may be of final importance when comparing products. As PLC detects sensitively substances, which are unknown to HPLC (because of chemisorption in the stationary phase) the inclusion of less soluble to insoluble substances into the final analysis may be important. Sample clean up by micro column separation prior to analyses results often in a specifically changed sample composition.
Giving samples and Focussing the spots, circles or lines:
If a micro brush is used to give samples then the following facts count:
No sample falsification as brushes are open capillaries. This brings micro brushes quickly into complete equilibrium with all substances of a sample. Just some seconds rotation of the brush inside the (possibly non homogenous) sample solution/dispersion will do it. No mechanical damage to the HPTLC layer happens even if sampling is repeated at the same position several times. Quite a quantitative volume transfer is given. The repeatability standard deviation of quantitative sampling by brushes is near to +- 2..3 %. After complete removal of the sample solvents an exactly positioned focussing starts in the plate center (see figure 12 ). This prepares an accurate and sharp start circle. As a result we get well reproducible substance bow / substance circle
positions. A level of often better than +- 0.2% is standard. Sharp focussing offers the best possible separation efficiency. A partially overlapped sampling offers 100% safe compare analysis in case there are detectable differences between the to be compared samples.
Separation, multi separation, multi phase separation:
The mobile phase “system” = 1.5 ml phase bottle is either closed by a plastic screw cap or - short before application - connected with a viscose felt wick inside a 5 mm PTFE-tube. The phase flow into the stationary phase is stabilized within a few seconds. The phase bottle sits in the cover glass plate and has been flow stabilized on a TLC waste plate . It is NOT touched by warm fingers but moved with the cover glass plate to the analytical plate by a handle into run position as shown in figure 21 )*. This needs only 1..2 seconds. It is important to move and position the bottle within the cover glass plate quick, especially important if the mobile phase is methylene chloride or an other highly volatile phase. Room humidity cannot condense this way on the open wick surface. If even smallest traces of humidity would make problems, the whole hardware and all waiting phase bottles could sit in a box flushed with dry air. The µ-PLC hardware is so flat, that this humidity protection is possible like an oxygen protection. A really simple task. Thus a quick change from phase one to two or three is possible either wet in wet or with drying steps in between. Just a next cover glass would have the gas tube inserted in its center hole and the gas pump running. It could sit also in a flush gas protected box. The pump soaks the air around itself for the gas flush. Simultaneously multi phase separation is possible with three or four mobile phases simply by using a cover glass plate with three or four 5 mm holes around the center, see figure 37 )*.
In between quantitation:
Under UV as well as in sun or flash light a quick photo shot takes data in between runs. In day light the photo can be taken without removing the cover glass - see figure 22 . If the mobile phase absorbs UV, the plate must be flushed dry and then the cover glass removed prior to the photo, see figure 6. In between quantitation offers many extra identification possibilities like by manual multi phase separations when chromatogram rings or bows move over each other or pass by.
Structure reduction by multi integration and Comparability versus Repeatability: click HERE
Overlap compare analysis and Combination GC / HPLC with µ-PLC - POP analysis: click HERE
)* = select the mentioned number within the pictogram page (use the scroll bar). If you want to see the figure in full size, then click onto this numbered pictogram. To return to this page, click the left arrow on top of your browser window twice.
A last word to Introduction:
Because of the top organizational help by my very good friend Sz. Nyiredy, Symposium Chairman of past Balaton Symposia in Siofok, Hungary and his late follower, I had the chance to report about steps by which µ-PLC was developed. First by thoughts and proposals, later more and more by facts. This way I could give three papers in a series from 2005 over 2007 up to 2009
Balaton 2005: Is the future of PLC (by part) Circular ?
Balaton 2007: Optimized Focussing in Planar Chromatography !
Balaton 2009: Drastic Reduction of the Structure Error in Quantitative PLC
In all three papers standard and classical PLC / TLC including overregulated modes of application (example: by linear calibration lines or not analyzed polynomial interpolation curves) is not forgotten but also quite critically treated, because we all pay for poor analytical results - excuse please.
ppt-papers are normally not available in books. But this Internet-only-book makes the papers available in a way an interested author may like as she/he even has more freedom to look through than in a life conference. Such material has the size of megabytes. The ppt structure is compressed and rewritten into pdf format. Thus a proper browser is necessary and latest pdf reading software like ADOBE reader 9, 10.... The quite long loading time depends on the hardware available. Use either Firefox 3.5.3 or later, MS-IE6...8 or later or SAFARI 4.04 or later and wait for complete loading. Three blue pages must show up. The author had no trouble under Windows XP SP3, MACINTOSH under OSX 10.5.8 or higher and even in virtual systems the papers could be read.
To jump to these papers click HERE.
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