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Inst.f.Chromatography

A selective summary, covering only µ-PLC for quantitative
or ‘within-each-other’compare analyses.
 

*  Fundamentally µ-PLC is the new two-dimensional planar chromatography for the detection of counterfeits but not limited to this task - it also offers new ways to check for drinking water quality and to combine with column liquid chromatography to check for otherwise not detectable systematic separation and identification errors.

*  It is circular Micro-Planar Chromatography with three phases: a high quality thin layer stationary phase, a mobile phase of clean volatile solvents and a gas phase: usually air, nitrogen
or CO2.

*  Unlike all previous methods of analyses which treat sample by sample one after each other or several samples parallel  to each other µ-PLC is the first technique which analyses samples PARTIALLY INSIDE EACH OTHER.

*   µ-PLC is available for all samples soluble in water, in alcohol-ether-ester or in mixtures of
nonpolar liquids or for direct or indirect HPLC/HPTLC coupling..

*   The sample solutions are transferred  near but not accurately into the PLC plate center. The sample solvents are totally removed at room temperature by a 2 L/min gas phase flow. After this step the often quite broad sample areas are focussed into sharp round bows or sharp circles using a volatile solvent like methanol, diethylether or other highly eluting volatile liquids injected accurately into the plate center. Prior to the separation step(s) this ‘focussing’ solvent is removed completely at room temperature with a  2 L/min gas flow.

*   The total sample number for a compare analysis is limited to 8 and optimally to four samples of always partially paired: sample Q1 with sample A1, Q2 with A2, Q3 with A3, Q4 with A4  or Q1 with A1, A2, A3, A4 - see the figures in the µ-PLC picture section.

*  Identification or quantitation is not necessary if the compare analyses show clearly a qualitatively or quantitatively visible discrepancy in the overlapped region between the Q/A sample pair. This however must be clearly detected in visible light, UV 254 nm or UV 366 nm. In case no any discrepancy is  visible we have to change one, two or all three phases and start the compare analysis in the worst case from scratch. If however for instance only one phase - the gas phase - is changed after the run and now shows in an acid, basic or oxidized .state clearly compare discrepances, the analytical answer is by 100% sureness: “Q is NOT A”. This can also happen by using chemical reactions after the already done separation on the  plate by spraying with or dipping into specifically acting chemical reaction solutions. If however non of the mentioned modes show any discrepancy between Q and A in the overlap region, the analytical result is never “Q equals A” for sure. In this case we need the whole range of other analytical techniques.

* µ-PLC can be used for quantitative single sample analyses by using the internal or external standard technique at best again by some degree of overlapping. The samples however must offer a large enough bow length in order to apply the concept of MULTI integration - see this section for details. With always at least four or up to 16 partially overlapping scan tracks the main systematic error in PLC quantitation is reduced by a factor of 4 up to 16.
This results at least in a +- 0.5% - often in a +- 0.05 % standard deviation at N > = 4 . The analytical concept of partial overlapping - here by stepwise moved scan track angles - is a powerful  method improvement for quantitation and PLC trace analysis.

*  Quantitation is based on digital photography using the wide range of photo software for optimizing the software controlled photo integration. This allows for multiple inter run quantitation steps not necessarily only after drying away the former mobile phase.

*  Partial overlapping reaches in the qualitative analysis a never before possible level of 100 % sureness in case of detectable qualitative discrepancies. Quantitative single sample internal or external calibration analyses reach a high level  of statistical certainty, not yet possible by standard TLC or HPTLC which all suffer from the systematic and stable plate structure error.
 Whilst in the qualitative compare analysis the partial overlapping is done at sample positioning the partial overlapping in quantitative analysis is available by the software controlled integration track positioning.

*   Separation by micro-chromatography is done with about <1 ml of mobile phase per run. The more than >> 1000  possible specific mobile phase mixtures are made well reproducible in a one ml micro bottle using 500 µl and 50 µl quality syringes. The bottles are gas tightly closed. The phase enters with no composition changes under a constant flow into the stationary phase - a fundamental difference as compared with regulated standard TLC or HPTLC.

*   Sample size limitations range from nanoliters up to 1000 micro liter per sample solution on- plate. In case of water as sample the upper size of 1000 µl per run offers a new level of trace analysis in the ppt to ppq range for substances which offer UV- or fluorescence signals.

*   Sampling is done with very easily cleaned  micro brushes  of size numbers zero to six. Sample memory as known in precision syringes is no problem with  brushes, because  their ‘open’ capillaries can easily be cleaned by a quick rotation in the best sample solving liquid even if this is not  volatile, as a stepwise change from solvent to a next  now  volatile one is possible. Squeezing into porous paper is an easy final step.

*   Micro-brushes have a quantitative sampling accuracy of +- 2% in the 5 to 100 µl range. They  cost from 50 cents to 2 € / pc and show easily a life time of several years.

*   Space requirements of the complete set of tools: half of a standard  table. Power consumption is low for the UV lamp, the digital photo equipment and the aquarium air pump.
 
*   The total instrumental investment is limited to about 1% to 10% related to latest instrumentalized  planar-chromatographic equipments. The analysis costs reach 1 to 2 € per sample.  The analysis time per sample pair Q/A can be reduced to 15 minutes.

*   This simple µ-PLC technique however needs high mechanical precision, best quality HPTLC plates and very pure solvents for sample solutions and mobile phases. Thin-layer chromatography expertise is required.

*  The concept is optimal for roboterisation.  Manual micro brush sampling could easily be replaced by electronically driven precision syringes which allow for circular movements but this rises the hardware costs drastically.

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