Just one beautiful example of circular chromatography

The Core Methodology,

Which I hope can be used by many of us, to test a variety of substances, using the same standard protocol.

I hope that in that way, we can "scientifically" compare our results, for "the same substance", be that a soil sample, or orange juice, or whatever.

This protocol has been taken from the work conducted in Padua University, in Italy. I have also included the full paper of results in the materials below.

The Core Protocol and Practice.

Figure 1. Graphical representation of the performance of the Pfeiffer’s circular chromatography tests: a circular filter paper sheet with a wick insert into the central perforation is located on a plastic dish in order that the wick’s end is immersed in the solution (liquid phase) to be imbibed.

Pfeiffer’s circular chromatography test

Ten gram of soil were placed in a flask containing 100 ml of 1% aqueous solution of NaOH for extraction. The flask was agitated by hand at the beginning of the extraction, and after 15 min, 1, and 2 h. After the last agitation the flask was left for another 2 h for extraction and sedimentation. After a total of 4 h the supernatant was collected. In the meantime, as depicted in Figure 1, a circular Whatman 1 filter paper (150 mm diameter) was pierced in the middle and a cylindrical wick, folded up from a 15 mm × 15 mm filter paper, was inserted into the perforation. The circular filter paper was put on a plastic dish (60 mm diameter) in order to rest on its edges; whereas the wick was manipulated in order to touch the dish’s bottom. The so prepared filter paper was imbibed twice: (i) in the dark, with 0.6 ml of a 0.5% aqueous solution of AgNO 3 , and, after the filter paper dried, (ii) in artificial light, with 1.2 ml of the supernatant collected after the soil extraction, by placing the imbibition liquid at the bottom of the dish. After the second imbibition the filter paper was left in the light for ca. 12 h to dry and to let the forms and colors fully develop. After this time the wick was detached. All the experimental steps were made at room temperature.

Figure 2. Example of a PCC pattern with described zones and pattern characteristics (on the top) and pattern sections (on the bottom) illustrating, from (a) to (d), an increasing development of radial features, (e.g. channels and spikes) and color intensity, and a decrease of concentric features (e.g. concentric rings).

From each soil sample 3 PCC patterns (filter papers) were prepared, the whole experiment was repeated three times on different days (in total nine PCC patterns per sample). PCC patterns were scanned (resolution 3440 × 3440 pixel) in color; the PCC paper patterns were stored in the dark.

Visual check of the PCC patterns.

All PCC paper patterns obtained in the present experimentation were subjected to a visual check, when pattern zones (central-, median-, and outer-zone; denoted as CZ, MZ, and OZ, respectively) and differentiating pattern characteristics were identified and described (Figure 2).

Measurement of PCC pattern zones

Zone measurement (Figure 2) was performed on PCC paper patterns by means of a ruler. On each pattern the following measurements were performed: total radius, CZ radius, MZ breadth, and OZ breadth; as the border between MZ and OZ the bases of the spikes were considered. All measurements were taken in vertical and in horizontal, and the mean values were calculated.

Visual PCC pattern evaluation

The visual PCC pattern evaluation was performed on PCC paper patterns by two schooled evaluators and consisted in scoring of the development degree of the differential pattern characteristics (channels, spikes, color intensity, and concentric rings; Figure 2) selected upon the results of the visual check. A maximum of 5 points was accredited to characteristics channels, spikes, and color intensity. For channels and spikes 1 point was accredited when these characteristics were absent and 5 points when their development was full; for color intensity 1 point was accredited when the color was visually sensed as blurred and 5 points when it was sensed as intense. Intermediate points (2, 3, or 4) were accredited in cases of intermediate characteristic development. For the characteristic concentric rings (score from 1 up to 4 points) the number of rings was counted and 1 point was accredited to each ring visible on the pattern.

Computerised PCC image analysis

The computerised analysis consists in the measurement of the pattern’s texture and was performed on scanned PCC images (3500 × 3500 pixels) by means of the software ImageJ (Collins 2007) with the installed plug-in Texture Analyser (Cabrera 2003–2005). Out of each image a rectangular region of interest (1000 × 200 pixels) of the median zone was randomly selected (in such a way as not to contain parts of the central or outer zone [OZ]), cropped, and converted to 8-bit type (pixels only in grayscale). On such prepared image selections the texture analysis was performed. In particular the parameter entropy was considered for further analysis, since it is sensitive to differences in brightness intensity between the pixels and thus also to the presence or absence of channels.

Chemical compound analysis

The following soil compounds and characteristics were selected and analysed in accordance to the Italian Official Regulations on soil analysis (MIPAF 1992, 1999): organic matter (Wolkley and Black method), total nitrogen (Kjeldahl method), cation exchange, assimilable phosphorus, assimilable bromine, exchangeable calcium, exchangeable magnesium, pH, sand, clay, and silt. All the parameters were analysed for the 16 soil samples.

Statistical analysis

The data-set was analysed by means of analysis of variance (ANOVA) followed by the post hoc multiple mean comparison with LSD test using the CoStat statistical software (v. 6.4, CoHort Software). Also the visual evaluation data were subjected to the ANOVA analysis, even though they were not continuous (the corresponding statistical output should be therefore interpreted only in a generic way). In addition, the Bravais–Pearson linear coefficient of correlation r was computed to determine the degree of correlation between the PCC data (visual evaluation, pattern zones measurements, and computerised analysis) and the chemical soil parameters.

Results

PCC pattern description All PCC patterns obtained in the present experimentation consisted of three ring-shaped zones located around the central perforation (Figure 2): (i) the central zone (CZ), located nearest to the hole and characterised by rather light color, then (ii) the darker middle zone (MZ), and, on the pattern periphery (iii) the very light and in many cases only weakly visible outer zone (OZ).

The visual check of the pattern set showed that the most prominent differences concerned the presence of concentric and radial pattern features, which appearance seemed to be inversely related (e.g. the stronger the concentric features, the lesser and weaker the radial ones, and vice versa; Figure 2). As shown in Figure 3(a), patterns with strongly marked concentric features contained up to four concentric rings located in CZ and MZ. In these patterns the coloration seemed blurred