Top 10 FAQ
- Which column is similar to column...?
- Direct comparison von 2-4 columns
- All details of column...
- "Best" column for separation of...?
- Find column for your criteria...
- Unknown sample - what now?
- Which column is different to column...?
- Which ones are the most polar columns?
- Which ones are the most hydrophobic columns?
- Columns for strong bases...?
Tips for use
- "Comparison" and "Selection" are the tools two most important functions. In daily routine the direct comparison of columns is probably most interesting ("I am searching for an alternative column to..."). In method development there is main emphasis on finding a suitable column ("I am searching for a column, which..."). For that you can start with the selectivity map. Here you can work with filters right away (limitation of manufaturer, functional group, porous/core shell, etc.). Or you decide to go for the start questions: "Similarity with...", "Difference to...", "Selectivity for...". Or you need to go for an unknown sample?
- It has been shown that the overview selectivity map portrays the character of phases (polar or apolar?) and therefore also the similarity quite well. Explanation: The bigger the alpha value EB/Fl (Selectivity facor Ethylbenzol/Fluorenon) in the y axis, the better is the hydrophobic selectivity of the corresponding column. This way for example Cortecs C18 or YMC Pro C18 RS have a good selectivity for the separation of apolar and polar molecules from neutral, non-ionic components and from weak acids and bases. The bigger the alpha value Tri/o-Ter (Triphenylen/o-Terphenyl) in the x axis, the bigger the stetic selectivity/molecular recognition of this column and in indirectly also its polar selectivity. This way for example Poroshell 120 PFP or Acclaim PA C16 separate planar/non planar molcules, isomeres or multi core, unsubtituted aromates and smaller polar molecules well. By removing them in the selectivity map the similarity/difference of columns becomes prominent. You can narrow down the selection of columns with the help of filters, e.g. only PFP columns, only core shell, columns of manufacturer X etc.
We deliberately chose substances that make it possible to evaluate the selectivity of stationary phases as differentiated as possible: from hydrophobic ethylbenzene to strongly basic benzylamine. This premise was also taken into account in the chromatographic conditions: isocratic runs, methanol/water mixtures, no buffer, no increased temperature etc.
Suitable substances and chromatographic conditions for the goal set with Colona
In RP-HPLC, in addition to the steric aspects (molecular recognition) hydrophobic and polar interactions play an important role with regard to selectivity The polar interactions can be hydrogen bonds, π-π- or ion exchange interactions. For a comprehensive comparison of columns, all these characteristics must be able to be revealed. The selected chromatographic conditions must also allow the differences present to be seen as far as possible. Thus, the use of buffers and gradients leads to "good-looking" and at times similar chromatograms, which, however, is against the goal we are aiming for here. This is also the case with acetonitrile as an organic solvent: tailing due to additional ionic interactions is more pronounced in a methanol/water eluent than in an acetonitrile/water eluent. In methanol/water shows pyridine a much stronger tailing than in acetonitrile/water.
Chromatographic conditions, tested substances
In the course of the tests, almost 50 substances with different properties were tested: Strong/weak acids/bases, polar/non-polar, small/large, aliphatic/aromatic, planar/non-planar molecules, complexing agents, etc. The tests were performed under different chromatographic conditions: methanol/acetonitrile, acidic/neutral/alkaline, different temperatures and different elution strengths. The comparison of the results finally shows the following: with the help of three isocratic runs (100% methanol, 80/20 methanol/water, 40/60 methanol/water) and certain analytes (see below), the different properties of the stationary phases with regard to selectivity can be determined quite accurately. The remaining tests with the remaining components and under other chromatographic conditions only confirm these results. The first five analyte pairs have the highest informative value; the remaining four are suitable for fine differentiation and are addressed to the very interested user:
- Ethylbenzene/fluorenone ("EB/Fl"); hydrophobic selectivity, separation of non-polar and polar components (analytes differ in hydrophobicity), the differences here: molecular size, aromaticity and presence of a =O group
- Triphenylene/o-Terphenyl ("Tri/o-Ter"); steric selectivity/molecular recognition, difference: planar/non-planar molecules, π-π interactions (with "similar" chemistry, analytes differ in spatial arrangement/molecular form)
- Perylene/chrysene ("Per/Chr"); aromatic selectivity, difference between two polyaromatic hydrocarbons (unsubstituted aromatics), π-π interactions
- Phenol/ethylbenzene ("Phe/EB"); polar selectivity 1, OH- vs. ethyl group, hydrogen bonds possible (e.g. with acids)
- Benzylamine/phenol ("Benz/Phe"); polar selectivity 2, ratio of dissociated/undissociated silanol groups, ion exchange interactions (e.g. hydroxy acids, fatty acids/surfactants)
- Ethylbenzene/toluene ("EB/T"); hydrophobic selectivity, the difference is a methylene group, methylene group selectivity
- Fluorene/fluorenone ("Fluorene/fluorenone"); hydrophobic selectivity, the difference is an aromatic keto group, difference between a polyaromatic hydrocarbon and an extended aromatic system (conjugated double bonds, π system)
- Propyl-3-hydroxybenzoate/ethyl-3-hydroxybenzoate ("Propbenz/ethbenz"); hydrophobic selectivity, the difference is a methylene group in mononuclear polar aromatics (-O-, =O, OH)
- Phenol/toluene ("Phe/T"); polar selectivity, OH- vs. methyl group, hydrogen bonds possible (e.g. acids)
We use tools that have proven themselves from a practical point of view:
- Selectivity maps
These are x/y plots; alpha values of certain analyte pairs are plotted on the X and Y axes to determine the similarity/difference of the columns. Example "Overview selectivity map": The X-axis corresponds to the alpha values ethylbenzene/fluorenone and is a measure of the hydrophobic selectivity. The alpha values triphenylene/o-terphenyl are plotted on the y-axis (steric selectivity). It has been shown that with these two alpha values the columns can be assigned quite well according to their character (non-polar/polar). For other selectivity maps we have made finer differentiations for some of the columns.
Here the columns are sorted according to the alpha values (selectivities): e.g. ability of columns to separate polar and non-polar components - hydrophobic selectivity. Finally, we have created some bar charts for other model substances for interested users.
Here you can find the most similar/different columns to your column according to certain criteria (e.g. overall similarity / difference or steric similarity/difference). Use in this case the RP-Column-Finder
- Selectivity web charts
They are well suited to visually represent the similarity or difference of columns. The principle: the alpha values for the separation of certain analyte pairs (the more different the selected analytes are, the more reliable the statement becomes) represent the corners of selectivity web charts. It is normalized to the highest alpha value reached. You obtain the following two info’s directly: 1. the higher the alpha value, the more selective the column for these analyte types is; 2. the more similar/different the web charts of, for example, two columns, the more similar/different the selectivity characteristics of the columns concerned. This concerns first of all the character of the two columns (e.g. "hydrophobic" or "polar"). The comparison of the separation capability, on the other hand, is about how similarly "well" the two columns can separate certain analyte types - independent of the elution sequence. To do this, we consider only the magnitude of the alpha value, regardless of whether the value positive or negative is: the alpha values may be inverted.
- Polarity rankings
Here you can see the most hydrophobic and most polar columns at a glance.
By means of this application it is possible to directly compare any column with any other column by means of alpha values, or alternatively several columns.
Selectivity web charts are suitable for visualizing the similarity/difference of columns. The principle: the alpha values for the separation of certain analyte pairs (the greater the difference in the selected analytes, the more reliable the statement becomes) represent the corners of selectivity hexagons, pentagons or triangles. The charts are normalized to the highest alpha value reached. The higher the corresponding alpha value, the more selective the column is for these types of analytes. Note: the focus here is exclusively on selectivity, not peak symmetry. You can look at the web chart of a certain column or also the web charts of several columns at a glance.
Scale: The scale of the spectrum goes from 1 to alpha-max, the largest alpha value of all columns. The fact that the scale starts at 1 means that for all columns with elution reversal, the alpha value was inverted.
The dashes on the scale correspond to the individual alpha values of all measured columns. This gives an overview of the distribution and enables the classification of the column under consideration. Have a look at the table (Details)
Rank: In order to determine the rank, the alpha values were inverted again in the case of elution reversal. Columns with identical alpha values (rounded to two decimal places) share the rank. Therefore, there may be fewer ranks than columns. Or, for example, with "steric selectivity" fewer ranks than with "hydrophobic selectivity", although the same number of columns were measured.
Regarding polar selectivity: The polar character should be considered in a more differentiated way, since different polar interactions are possible. A first indication of polar selectivity is provided - in addition to the overview selectivity map - by the bar charts steric and aromatic selectivity (accessibility silanol groups, π-π interactions). The alpha value Phe/EB (Phenol/Ethylbenzene) provides an indication of stronger polar interactions or hydrogen bridge bonds - e.g. for acids. Finally, the alpha value Benz/Phe (benzylamine/phenol) would be a measure of the ion exchange/hydrogen bridge bond ratio: Thus, an irreversible adsorption of the strong base benzylamine (alpha-value Benz/Phe nominally greater than 20) indicates the tendency of the corresponding stationary phase to strong cation exchange interactions and thus exhibits a strongly polar character. Using the selectivity maps and the bar charts, alternative columns or, vice versa, quite different columns can be selected for orthogonal tests (e.g. to check peak homogeneity) or for equipping a column selection valve.
In the table you will find the physico-chemical data of the columns, the separation factors alpha, functional group etc. Of course you can sort the data as you like.
The chemistry behind the scenes
Selectivity is used as the classification criterion, indicated as the alpha value of meaningful analyte pairs, determined under isocratic conditions.
Justification for the use of alpha values as a criterion for the comparison of columns:
Firstly, alpha values represent a measure of the selectivity and that has by far the greatest influence on resolution (separation of peaks at the peak base). Under constant conditions (eluent, temperature), the influence of the stationary phase on the separation becomes clear. Furthermore, alpha values allow the comparison of stationary phases independent of actual column dimensions and flow. Secondly: we think that from the user's point of view of interest are questions such as: "which column separates like XYZ? Or: "which column is completely different from...?", "which columns show good selectivity for the separation of...?" It's about the separation, after all. From a practical point of view, it may not be so essential to know whether the interactions on these two columns are similar, i.e. whether the components elute with the same retention factor, k. The asymmetry factor also appears less useful to us as a criterion: if the packing quality of the column decreases or if there is a dead volume in the system, band widening/tailing can be incorrectly assigned to possibly silanol groups. Remark: Alpha = 1 means coelution. The greater the difference between the measured alpha value and "1", the more selective, i.e. the more "separable" the corresponding phase is for this type of analyte. The number can of course also be less than 1 (elution reversal) - the capability of the phase to separate these analytes is of course also given in such a case.
In the literature "hydrophobicity" (retention factor k of a non-polar component), sometimes also the asymmetry factor, mostly of a base, is often used as a criterion. So, if this tool gives you similarity/difference correlations of columns other than those given in the literature or on pages from column manufacturers, the cause could be the reason given above. Possibly also that the work carried out here is exclusively buffer-free and isocratic: buffers lead to a better peak shape - just like a gradient - but equalize differences between stationary phases, which would be counterproductive to the goal here. And finally: we carry out the tests with methanol/water mixtures, since acetonitrile leads to a "good-looking" peak shape due to its low viscosity. Methanol/water mixtures, on the other hand, show more "honestly" the differences between stationary phases.
In the following five cases, it is best to look at the bar charts:
- Large, unsubstituted aromatics: Aromatic selectivity
- Polar, uncharged molecules: Hydrophobic selectivity
- Non-polar/polar molecules: Hydrophobic selectivity
- Weak acids/bases: Hydrophobic selectivity
- Strong acids/bases, steric selectivity: Polar selectivity 1
- Very similar molecules: Steric selectivity
- Separation of molecules as varied as possible: General selectivity
Or else, you just don't know; then go to "Tips for an unknown sample"
Hydrophobic selectivity (α EB/Fl): Capability of the phases to separate analytes with different hydrophobic character; rough classification: α > ca. 1.4: Good hydrophobic selectivity, α > ca. 1.6: Very good hydrophobic selectivity, α < 1 (elution reversal): Right polar phases. Very large or very small (α < 1) alpha value α EB/Fl indicates the ability of the corresponding phase to separate different hydrophobic analytes regardless of the elution order, (overview selectivity map, bar chart α EB/Fl)
Steric selectivity (α Tri/o-Ter): Capability of phases to separate molecules with similar chemical character and similar molecular size but with differences in spatial arrangement (e.g. planar/non-planar). Good steric selectivity is shown by phases that are capable of polar interactions, i.e. phases with small α values EB/Fl. In addition, phases that have a cross-linked/polymer layer on the surface, e.g. Nucleodur ISIS, Nucleosil AB, SMT OD C18, Ultrasep ES C18 E (overview selectivity map, bar chart α Tri/o-Ter)
Aromatische Selektivität (α Per/Chr): Capability of phases to separate large unsubstituted aromatics. Separation via π-π interactions; polar phases are distinguished here with respect to good aromatic selectivity (bar chart α Per/Chr); there is a fairly good correlation with α Tri/o-Ter
Polar selectivity 1 (α Phe/EB): Capability of the phases to separate analytes with a difference of a CH3 - or -CH2 -CH3- vs. OH-group (see bar charts α Phe/EB and α Phe/T). Analogous to the hydrophobic selectivity, a high or low (α < 1) alpha value α Phe/EB indicates the ability of the corresponding phase to separate analytes with an acidic group - independent of the elution order
Polar selectivity 2 (α Benz/Phe): Large α values indicate strongly polar phases that can enter into ion exchange interactions
The alpha value Benz/Phe (benzylamine/phenol) can be seen as a measure of the ion exchange/hydrogen bond ratio. Furthermore, a strong tailing and a long retention time or even an irreversible adsorption of the strong base benzylamine (alpha value Benz/Phe nominally greater than 20) reveals the tendency of the corresponding stationary phase to strong cation exchange interactions, thus showing a strong polar character. The result is a peak form of benzylamine varying from a non-evaluable peak to a quite symmetrical peak, see example (left Fig. 15). On these 12 columns (exemplary!) the peak form of benzylamine is very good: there are no (accessible) acidic silanol groups on the surface and thus hardly any cation exchange interactions take place, the result: symmetrical peaks even with strong bases. Note: the benzylamine test is a very strict test.
All about Colona
No; the Benzylamine-Test is a very strict test. In the case of strongly polar phases such as PFP or older materials containing acidic silanol groups, irreversible adsorption is observed or benzylamine is eluted as a peak that cannot be evaluated. For this reason, for 35 columns a theoretical alpha value Benz/Phe for benzylamine/phenol of 20 is used in the preparation of the web charts. Unfortunately, we were unable to obtain the relevant information from either the literature or the manufacturer for the missing data on the physico-chemical properties of materials.
- The retention times and the alpha values calculated from them should be reliable: the data were recorded by us and have been confirmed by repeated measurements - even over a longer period of time.
- Strictly speaking, the statements refer to the substances investigated and the chromatographic conditions used. However, due to the large number of different analytes and the range of chromatographic parameters selected, the statements should have a certain general validity. However, under other conditions (pH value, gradient, etc.), other similarities may occur with the columns. Furthermore, although for several columns different batches were tested, the figures do not originate from statistical material. Basically: in order to judge the similarity of columns, you should consider several numerical values. The web charts and the xy plots, for example, are suitable for this purpose. For example, a numerical difference of individual alpha values - especially with polar analytes - can suggest a disproportionately large difference. A difference between alpha = 1.09 and alpha = 1.16 is a really small one, significant is basically a difference > approx. 15-20 %.
- The numerical values for the physico-chemical data of the columns (carbon content, pore diameter, etc.) are literature or manufacturer data. For the missing values, neither the literature nor the manufacturers were able to provide any information. If you have appropriate info, we are all of course thankful, please tell us the corresponding value. Thank you
The following people contributed to the realization of the project "Colona": Debora Grossmann, Antoni Kromidas, Hans-Joachim Kuss, Daniel Stauffer, Stefan Lamotte, Stavros Kromidas.
Software updates/grades are made irregularly as required. Regarding the addition of new columns, the procedure is as follows: The team observes the development in the market and announces interesting products; experience is gathered and the most interesting columns are tested. Every two years an expansion takes place, the number of new columns can naturally fluctuate.
No, not directly; most manufacturers have made their columns available to us for understandable reasons. We bought some columns. The database as such is strictly neutral.
- You can get detailed information about columns on the homepages of well-known column suppliers.
- The following book is older, but good and still recommendable: Uwe D. Neue, HPLC Columns: Theory, Technology and Practice, Wiley-VCH, ISBN-13: 978-0471190370
- Book chapter "Comparison and Selection of Modern HPLC columns" from Stavros Kromidas (Ed.) The HPLC Expert: Possibilities and Limitations of modern HPLC, Wiley-VCH, ISBN 978-3-527-33681-4