theor. practical details

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Inst.f.Chromatography




		This chapter contains: Special applications and theoretical details: A first example as special application: Checking chemical treatment of fruits . Is the grape fruit free from protection chemicals ? Are chemicals detectable on a chineese honey pomelo ? See the corresponding figure below. How was it done: With a micro brush no. 5...7 carefully cleaned in methylenechloride and saturated by CH2Cl2 an area of about one square cm is touched by a slow rotation of the wet brush -during two to three seconds. The tip of the brush contacts now quickly the 100x100 mm HPTLC plate about 2...3 mm apart from the plate center in a pre dtermined position - let say at “circular 45 minutes”. This transfer step of extracted fruit surface material ‘sampled’ onto the HPTLC plate is repeated at least six, better 12 times using always the feshly CH2Cl2 wetted brush. The ’brushed’ areas on the fruit surface change by position from extraction step to extraction step in order to transfer correctly and enough eluted material onto the “45 minute” position of the plate. The whole procedure is repeated with a second fruit or - the first used fruit but after washing in warm water. It is our believe and hope, that the chemical fruit treatment can be removed from the fruit by warm water - may be even after adding a droplet of washing up liquid. Now the surface of the ‘washed’ fruit is dried and extracted by CH2Cl2-brushing as mentioned above. These new extracts get the position “30 minutes” near the plate center. At Position “15 minutes” and “60 minutes” on the HPTLC plate we can sample extracts of other fruits or test substances of known concentration. After all sampling steps are done we dry the spot area by flowing air - as usual in the µ-PLC procedure - at least for 15 minutes, because the spots will be wet as well by condensed humidity from the surrounding air. Now we should make a detection check under UV and fluorescence. If there is a clearly visible signal at the extracts, we focus these quite wide spots. Most often they will have a size of 3...5 mm diameter. Focussing should be done with methanol or any other very volatile polar focussing mobile phase. Dryness is mandatory and we dry again after the focussing step. In many experiments CH2Cl2 plus hexane for a small elution power or CH2Cl2 plus 1...5...10 vol-% methanol for a large elution power will work as mobile phase allowing an acceptable chromatographic separation. Photo documentation in visible light, under UV and by fluorescence may produce the data for further decision making. In the figure below already a single run with 100% CH2Cl2 produced the shown document, from which we learned: the checked grape fruit was not a chemical free ‘bio’ product but on the chineese pomalo fruit we found no substance under UV,visible or fluorescence. Well: in Order to get the chemical formula for the grape fruitb treatement we would need compare chemicals or on-line-MS. The latter is now available without a next step of local extraction but by gas phase transfer from the plate into the MS. However to find out, if the often propagated wash up process with warm water works, we do not need any formula, only a quantitative compare analysis. So the figure below needs no further discussion. We see an interesting little application of the µ-PLC technique. Such tests may end up with results of the following type: 1. The used protection chemicals are clean or highly un clean. This may tell, that expensive fruit protection chemicals may come from less qualified producers or those who no longer check their own product quality py analysis. 2. The protection chemicals may not be removable from fruit surfaces, all though we may hope this as sometimes the fruit skin may be needed in the kitchen. 3. A special type of fruit may be qualified as ‘bio’ and sold as “free from chemicals” by guarantee but we may see the opposite by analysis. Those analytical results however MUST be correct in order to draw correct decisions. T = ‘Farbtest’ = colour test to only check the run - Left: UV 254 nm, right: Flu 366 nm.


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Multi focussing of critical samples

Some samples are highly complex with respect of composition, solubility, homogeniety etc. One special example was the compare analysis of original Viagra and a product, which was told to “be equal to Viagra” , named Kamagra Oral Jelly containing 5 mg Sildenafil Citrate equivalent to Sildenafil 100 mg”.

The Oral Jelly is deeply red colored and adsorbs on silicagel extremely strong. The compare analysis showed only after the ‘multi focussing effect’ discussed here Sildenafil and a by product in a nearly equal concentration as Sildenafil itself. Because of the strongly sorbing jelly red color we had to use several focussing steps after each other, starting with methanole, continued with ethanole, sec. propanole/water (70/30) and finally methylene chlorid always with drying steps in between. Now the original Viagra and the Kamagra sample could be compared chromatographically correct and showed no more problems in the overlapped zone. Well seen in the figure below it is of no question that Viagra is not Kamagra, but the latter contains Sildenafil plus an unknown substance of an equal amount as the Sildenafil itself - according to multi integration not shown here. This is the second circle seen right in the figure below at the Kamagra and the overlapped position.

A very strong improvement of the separation power of the used standard silicagel glass plate of Merck (TLC Silica gel 60 F254 [1.05729.0001]) could be reached resulting in a formely never possible separation number of 50 instead of 10...15 as usual. This shows basically how important is focussing in PLC - not only in the circular mode. However only in this mode multi focussing is practically applicable.

A next fact becomes understandable: Samples can have such a composition, that main products are locally nearly chemisorbed by special substances at the sampling position on / in the stationary phase, which would falsify a normal standard separation quantitatively. Many tailing effects could be overcome by specific multi focussing with solvents or gases, which allow preseparation from the position of primary application to a sharply focussed start circle. In case of the Kamagra sample it was partially ethanole but mainly the aquatic secundary propanole which finally allowed a correct compare chromatogram. Of course: WATER is not so easyly removed from silicagel. Thus a complete (room temperature) drying period by the on-plate-drying gas flow as used in µPLC for at least 15, better more than 15 minutes is a must .

See the compare chromatogram below:

Theoretical details:

Rf values and the Pr data in µ-PLC.
Rf units versus k = (tm + ts) / tm units.
Application for a useful comparison HPLC versus circular PLC.
Qualitative µ-PLC by Chemical Reactions

By definition qualitative values in µ-PLC are given as POSITION data.
Unit POSITION is named = P
The plate center position has the Po value = zero.
The mobile phase front has the Pf value = 1.00

The Pr value of a substance s given from the plate center = Ps

Well known and often used in PLC are however LINEAR Rf-data. Their problem is an often falsified (too large) f-value, as the true front is nearly always below the visible front line.
Reason: compressed amounts of mobile phase sorb quickly in the stationary phase.

By definition the Rf value for a substance “s” equals the relation of the position measured as distance from start line to peak maximum “s” divided by the length of the mobile phase path from the start to the (true) front “f” thus

Rf = s / f See for this and the following formulas figures 31 to 33 HERE.

Position data into Rf (circular) values: Rf(circ) = (Ps - Po) / ( 1 - Po)

Rf (linear) values are always larger than Rf(circular) values, as

Rf (lin) = (Rf (circ))²

therefore [(Ps-Po) / (1-Po)]² = Rf (lin)

Application for a HPLC / PLC comparison in critical analytical conditions
To correlate PLC values with data based on strictly equal stationary / mobile phase systems in HPLC the partition factor k is used, which correlates with Rf according to
k = 1 / Rf - 1
but NOTE: ka(in HPLC) is NOT equal ka (in PLC). We can correctly correlate both ka values with an inter method correlation factor based on the formula

ka (in HPLC) / kb (in HPLC ) = ka (in PLC) / kb (in PLC).
From this correct relation we get

ka (in HPLC ) = ka (in PLC) * (kb (in HPLC) / kb (in PLC))

Thus we need two substances, which have differing k values and chemically equal chromatography systems.

HERE is the reason for such theoretical discussions:
Let us think of a substance “a” which in HPLC has a ka-value of 30. That means, the retention time of substance “a” is 30 * tm. The HPLC dead time tm may be 2 minutes, so the substance “a” is eluted after 60 minutes. The peak is broad, traces of “a” cannot be detected sensitively, the HPLC analysis time is at least a full hour. Let us assume, the inter method correlation factor kb(PLC) / kb(HPLC) equals 1.05,
so ka (PLC) = ka (HPLC) * 1.05 which results in ka(PLC) = 31.5.
From this HPLC value we get the Rf value in linear PLC, which is according to
k = 1/Rf - 1 ; Rf = 1 / (k+1) ; Thus Rf “a” = 1 / (32.5) = 0.031
That means, this substance “a” cannot be separated from the linear PLC start line, but at least we see “a” with high sensitivity hanging on the start line.
Now we repeat the analysis of the substance containing “a” by circular PLC.
As Rf (circular) equals the square root of Rf (linear), it follows
Rf ”a” (µ-PLC) = SQU (0.031) = 0.176
This means: the substance “a” is just visibly separated from the focussed start circle and detectable with high sensitivity. The analysis time is a few minutes as compared to a full hour in HPLC on equal phases at equal temperature.

CONCLUSION:
it is a good idea to check HPLC data with circular PLC, as all substances with a too high
k-value are not well detectable in HPLC after a long elution time. Such substances may even ruin the HPLC packing but are not visible to the analyst. By circular PLC with a well selected pair of phases we see the late comers or NO-comers of HPLC with high sensitivity. There may be even a chance for separation, if Rf (circ.PLC) is not too small. If there are substances hanging at the start circle we can continue by multi separation with mobile phases of growing elution power by liquids which are not available for HPLC. Example: phenol. This compound never useable in HPLC has a great elution power for substances which cannot be separated by any HPLC procedure. The separation would need higher temperature however.
µ-PLC can easily run on a heated ground board in case the cover glass plate is heated as well some tenth of degree centigrade above the ground board temperature. This way the author has also checked temperature programmed µ-PLC, but the cool down time is simply too long. Constant temperature µ-PLC is preferable.

Qualitative µ-PLC by Chemical Reactions
Whilst MS coupling with HPTLC plates in linear mode is meanwhile entering practice and spectra analysis based on data from linear light scanners is standard in many large laboratories both modes for qualitative PLC analysis are surely not the procedure together with the highly economical µ-PLC. There are however very valuable information about specific and sensitive identification procedures by chemical reactions prior or post chromatography in German language:

H.Jork, W.Funk, W.Fischer, H.Wimmer:
Duennschicht-Chromatographie, Reagenzien und Nachweismethoden, Band 1a (1989), 468 pages, and
H.Jork, W.Funk, W.Fischer, H.Wimmer:
Duennschicht-Chromatographie, Reagenzien und Nachweismethoden, Band 1b (1993), 499 pages
probably still available through MERCK order number from
E.Merck, Frankfurter Strasse 250, D-64293 Darmstadt (Germany),
printed at Wiley-VCH-Verlag, Boschstrasse 12, D-69489 Weinheim (Germany).

A huge amount of data about specific PLC reactions for identification and improved detection is found through CAMAGs literature service CBS, see CCBS in this Internet book.


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