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Inst.f.Chromatography

A selective summary, covering only µ-PLC for quantitative
or ‘within-each-other’compare analyses
including some additional little practical hints.

* Fundamentally new two-dimensional planar chromatography for the detection of counterfeits.
It is circular Micro-Planar Chromatography with three phases: a high quality thin layer stationary
phase, mobile phases of totally (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 or parallel
side by side µ-PLC is the first technique which analyses to be compared samples
INSIDE EACH OTHER.

*  Available for all soluble samples soluble in water, alcohol-ether-ester or mixtures of
nonpolar liquids.

*  The samples are transferred strictly near but not precisely into the plate center and after total removal of any solvent at room temperature by gas phase drying. After this step they are focussed into round bows or sharp circles with a volatile solvent like methanol, diethylether or other highly eluting liquids under chromatographic conditions resulting in
Rp = 1 (respective Rf = 1)

*  The total sample number for a single analysis is limited to 8 simultaneously and optimally to four samples.

* There are no repeat measurements needed for quantitation, as the comparative quantitation analysis reaches as minimum four and as maximum 16 local repetitions with a slight angle change (1 degree in minimum each) instead of by classical regulated standard analyses by two to three repetitions. Two to three photos are taken from the chromatogram plate under visible light, UV 254 and UV 366 nm, The pixel size, the figure area and the chromatogram position is optimized by PhotoImpact XL software and the multi integration is done by SORBFIL version II software. Thus all in-between data can be stored electronically in memory and the data completely documented far over ‘standard regulation’ procedures.
This results at least in a 0.5% - often in a 0.05 % standard deviation at N > = 4 .

* Thus one reaches a never-before practically applied statistical certainty in quantization. In sample COMPARE analysis done by the ‘within-each-other’ mode and detectable qualitative (Rp-values) and visible quantitative differences one ends up with 100% security - previously unknown in the material analysis.

*  Detection method is digital photography and software graphics analysis under light of certain wavelengths : - visible, UV 254 nm, 366 nm fluorescence. If there are no acceptable signals one has the possibility of specific gas phase reactions by the micro pump action with a flow of 2000 ml / min N2 bubbling through volatile reagents,  which may change the chemistry of the separated bows or circles making them detectable on the plate. If those reactions need elevated temperatures: no problem.
An electronically heated layer under the stationary phase plate will do it. The cover is a 1 mm thick gas phase layer and finally the thick glass plate with the PTFE-gas phase inlet in the center.

*  Separation by micro-chromatography is done with below 1 ml of mobile phase per run, more than >> 1000 specific mixtures are made well reproducible in the 1.0 ml micro bottle using 500 µl and 50 µl quality syringes.

*  The analyses require no classical calibration and validation, no regulated method of analysis is needed, as one can sample calibration test solutions overlapping the substance bow of interest on two sides by a calibration solution below the signal intensity at one side and a calibration solution above the signal intensity on the other side, thus allowing a nearly linear calibration line within a very narrow range of concentration.

*  Sample size limitations range from nanoliters up to 1000 micro liter per sample solution on-plate.

*  Sampling is done with very easily cleaned commercial micro brushes of size numbers zero to ten. Sample memory is no problem as brushes with ‘open’ capillaries can easily be cleaned by a quick manual rotation in the best sample solving liquid even if this is not very volatile, as a stepwise change from solvent to a next, more volatile one is possible and squeezing in porous paper does it.

*  Micro-brushes to have a quantitative accuracy of + - 2% in the 5 to 100 µl range at a cost of 50 cents to 2 € / pc

*  Space requirements of the complete set of tools: half a table. Power consumption low, and main electrical store (3rd world!)
 
*  Instrumental investment is at 1% to 10% over modern instrumentalized other planarchromatographic methods at 1500 € and reaches about 2600 € per complete set including computer, UV-lamp and digital camera. Analysis costs between 1 and 2 € per sample; analysis time at 15 - 30 minutes per 1 sample.

*  Overall, a very simple technique, but high mechanical precision is needed and best quality HPTLC plates. Three to six very pure solvents are required as minimum. But thin-layer chromatography expertise required.]

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