Laboratory Chromatography Guide - Buchi

12Part 1 Flash Guide – Basics1IntroductionChromatography is a standard method used in preparative laboratories to isolate and purify substances. In the early days of chromatography simple glass columns were chiefly used, operated bymeans of the hydrostatic pressure of the solvent acting as an eluent. In a publication in 1978 Clark W. Still explored the possibilityof accelerating the separation process in simple glass columns,which was until then the commonly used method, and therebyconsiderably increasing the efficiency of the technique. The resultswere convincing and the foundations of modern flash chromatography were laid. It triumphantly established itself in laboratoriesas an indispensable purification method in preparative chemistry.Flash chromatography has since undergone constant development,and has been adapted to meet present day expectations in terms ofequipment and convenience.Figure 1:From the simple glasscolumn to modern flashchromatography.Modern flash chromatography systems are popular nowadaysbecause they are simple to handle, flexible and can be universallyemployed. The first part of this brochure aims to give simple, accessible advice, which should ideally instantly lead to effective laboratory elutions.

IntroductionThe following abbreviations are used in the first part:TLCRPNPUVSi%A%BRfCVΔCVRf1Thin-layer chromatographyReversed phase, modified silica gelsNormal phase polar silica gel phasesUltravioletSolvent strength (substitutes polarity)% solvent with low solvent strength% solvent with high solvent strengthRetention factor (from thin-layer chromatograms)Column volumesDifference in column volumesRetention factor of first substance (substance whichspreads onto the TLC plate the quickest. The indexincreases according to the time the substance takes tospread).13

14Part 1 Flash Guide – Basics2Principle of chromatographyChromatographic separation is based on a balanced state amongthe components to be separated, an adsorbent agent in the column( stationary phase) and a solvent flowing through it (mobile phase).When a component settles on the stationary phase this is definedas adsorption, while detachment by the mobile phase is defined asdesorption. A high adsorption capacity between the componentsof interest and the stationary phase means that there is a highretention of these components and that there is a considerabledelay in elution from the column. The separation of a mixture into itsindividual components is only possible if the individual componentsin a combination of stationary and mobile phases have differentadsorption/desorption properties.Figure 2:Adsorption undDesorption, schematicillustration of thechromatographicseparation process.

153Choice of the appropriate stationary phaseChromatographic separation can be carried out on both polar andapolar stationary phases, and suitable sorbents are available fromvarious manufacturers.“Standard” chromatography requires the use of polar stationary phases such as silica gel and nonpolar solvents. The individualcomponents are delayed as a result of a reaction between the polarfunction component groups and the polar groups of the sorbent.Low polarity substances are eluted first, followed by componentsof increasing size.In “reversed phase” chromatography, however, the stationary phase is nonpolar and elution is by means of polar solvents.These stationary phases are produced by modifying silica gel withnonpolar groups such as C-18 or similar substances. Substancesare eluted in order of decreasing polarity from reversed phase columns, i.e. the substance with the highest polarity appears first.Reversed phase materials are considerably more expensive thanstandard stationary phases, and this is one of the reasons whystandard stationary phases are primarily used in flash chromatography. If the substance classes to be separated allow, modifiedstationary phases can nonetheless be used without restrictions orproblems.Figure 3:Elution sequence fornormal silica gel.

16Part 1 Flash Guide – Basics4Evaluation of the chromatographic system bythin-layer chromatography (TLC)As mentioned earlier, most elutions in flash chromatography usenormal silica gel, or modified silica gel in special cases or for highlypolar substances. In all these cases it is advisable to carry out athorough TLC pre-elution so that, with a minimum investment oftime and material, promising elution conditions can be found, whichcan then be applied to the cartridge. The following applies:1. Define stationary phase2. Find mobile phase with best selectivity3. Set solvent strengthIdeally the sorbents on the TLC plate and in the cartridge shouldbe identical (type and pore size) so as to successfully apply TLCconditions to the cartridge!4.1Evaluation of the stationary phaseThe laboratory’s experience with TLC tests, with which most laboratories are familiar, can help to you make the right choice. IfTLC plates with normal silica gel are used for the tests, columnseparation can also be carried out using normal silica gel. If theresults of this prove unsatisfactory, it is then advisable to switch toRP plates.4.2Selectivity of the solventOnce the stationary phase has been established the mobile phasewith the most suitable selectivity needs to be found, i.e. the solvent or solvent mixture that isolates the substance of interest onFigure 4:Selectivity trianglewith various selectivitygroups.

Evaluation of the chromatographic system by thin-layer chromatography (TLC)17the TLC plate with the greatest possible distance to the adjacentcomponents.In general every solvent has its own defined selective properties; some tend to be similar to each other, while others can differgreatly. L.R. Snyder and J.J. Kirkland investigated and comparedthe properties resulting from various solvents and grouped solventswith similar effects together into what are known as SelectivityGroups.The selectivity groups allow us to focus our search. There islittle point in comparing different solvents from the same selectivitygroup, as they all have the same properties. What we have to dois compare solvents from the various selectivity groups, as this isthe only way to see the difference immediately. The most importantsolvents for our separation are compiled in the following table. Thisonly shows solvents that are suitable for separation with UV detection, and do not make detection impossible as a result of highenergy absorption.SolventGroupStrength SiUV pyl etherI2.4220Diethyl drofuranIII4.0220Acetic acidIV6.0DichloromethaneV3.1250Ethyl Depending on the polarity of the components to be separated,the entire mixture can flow onto the TLC plate with the solventfront; the solvent is too strong and the TLC separation cannot beassessed in this form. In these cases the solvent strength is reduced by diluting the solvent with hexane, for instance, and the TLCseparation is then repeated.Table 1:The most commonsolvents with selectivitygroup allocation andsolvent strength Si .

18Part 1 Flash Guide – BasicsFigure 5:Reducing solventstrength so that theselectivity can beassessed in the firstplace. The TLC on theleft was developedin dichloromethane,and the TLC on theright in hexane/dichloromethane 3:1.Adding hexane reduces the solvent strength,but does not affect the selectivity!Figure 6:Evaluation of the optimal selectivity. In thisexample this is clearlyin the system of selectivity group VI, wherethe individual components have been separated most effectively.If there is only interestin component 1 (topmark), the choice is V.Ideal the other directly adjacent substances areseparated as well as possible from the substance ofinterest.4.3Solvent strengthEvery solvent has its own characteristic strength (which used tobe known as its polarity). The higher the figure, the stronger thesolvent and the quicker substances are transported through thechromatographic system. Rapid transport through the column doeshowever mean that there is less interaction between the stationary and the mobile phase, and that the separation is therefore notas effective. It is thus very important to have the correct solventstrength so as to achieve optimum separation results.

Evaluation of the chromatographic system by thin-layer chromatography –0.2Diisopropyl etherI2.4Diethyl II4.0Acetic acidIV6.0DichloromethaneV3.1Ethyl 2.4XyleneVII2.5ChloroformVIII4.119Table 2:Strengths of the mostcommon solvents.Using this table, solvents with different selectivity and usuallywith different strengths can be set at identical solvent strengths andthereby directly compared by mixing them with unselective solventssuch as hexane. The diagram in figure 8 shows the mixing ratios forthe most common solvents.To successfully transfer TLC results to the flash cartridge, theB Solvent with higher polarity4.51234.0Solvent strength 00Figure 7:Setting the solventstrength.1 Ethyl acetate2 Chloroform3 Tetrahydrofuran4 Dichloromethane5 Diisopropyl ether

20Part 1 Flash Guide – Basicssolvent strength should be set so that the resulting substances areapproximately 0.15 – 0.4.Figure 8:Optimum Rf rangeto transfer the resultsto the flash cartridgeare 0.15 – 0.4.Why such low Rf values? The reason for this is evident if we lookat the relationship between Rf values and column volume (CV).An Rf value of 1 in the TLC means that the corresponding substance with the solvent front is flowing. The substance would alsomove with the solvent front in a flash cartridge and after 1 columnvolume would leave the cartridge. At an Rf value of 0.1 the flowdistance is 1 10 of the front distance – the substance would need10 times longer to reach the front or in turn to reach the columnexit, i.e. 10 column volumes. The substance would be held back formuch longer and other components would therefore be separated.The following applies to the relationship between column volumeand the Rf value:– Column volume CV 1 Rf– Rf value ranging from 0.15 – 0.4, corresponding to 2.5 – 6.6column volume.Table 3:Correlation of Rfvalues and columnvolumes.Rf valueColumnvolume CVRf valueColumnvolume CVRf valueColumnvolume 30.42.500.110.0SummaryOptimize the TLC conditions by applying the following rules:1. Use identical silica gels if at all possible (same type and pore size)for TLC plates and flash cartridges. Different silica gels behavedifferently.2. Look for suitable selectivity. The ideal selectivity separates thecomponents of interest well before adjacent components or impurities. The greater the difference, the more efficient the flashseparation.3. Optimize the solvent strength. Ideal solvent strengths display

21Evaluation of the chromatographic system by thin-layer chromatography (TLC)Rf values ranging from 0.15 – 0.4 in TLC for the components ofinterest; the ΔCV 1.Apply these conditions to the flash cartridge.Figure 9Example of pre-elution using TLC and transferring the resultsto a Büchi cartridgeStep 1: SelectivitySubstanceTLC 110.54 1.8RfTLC 2CVΔCVRfTLC 3CV0.520.43 2.330.34 2.90.42 2.4CV0.31.41.00.35 2.98.70.08 12.5ΔCV0.45 1.90.27 3.83.30.16 6.2Rf0.61 1.60.60.64ΔCV0.56 1.82.30.19 5.2Figure 10:Evaluation of themobile phases in termsof selectivity andassessment.1 ethyl acetate2 diisopropyl ether3 chloroform4 dichloromethaneTLC 2 clearly displaysthe best selectivity.TLC 4 was not evaluated.

22Part 1 Flash Guide – BasicsStep 2: Solvent strengthFigure 11:Setting the solventstrength. The ratio ofhexane/diisopropylether 3:1.Components 2 and 3are of 430.128.416.840.0425.2Step 3: Applying result to the cartridgeFigure 12:Applying the conditionsto a Büchi flashcartridge 12 x150 mm.Eluent hexane/diisopropyl ether 3:1,flow rate 14 ml/min,detection UV 254 nm.012345678Time (min)910 11 12 13 14

235Injection/Column loadingInjecting the sample is usually a simple procedure in analytical chromatography. The quantities to be injected are low and solubility ishardly an issue.In preparative separations, on the other hand, the columns areoverloaded and the injection of the sample is of primary importance.When loading the column the mixture to be separated should beapplied to the column bed in as compact a form as possible, i.e.in a narrow horizontal band. Preparative separations are usually inlarger quantities, i.e. grams.For a long time the general rule for preparative separations wasthat a column can be loaded with an approximately 1% mixture,in terms of the silica gel level. The use of modern flash systemsand optimizing the mobile phase (Rf 0.04 – 0.4, CV 1) means thatnowadays the load can be increased to up to 10% – separation isfaster and more cost effective – more efficient all round!Approximate possible load in g*ΔVSCartridge12x75 mmCartridge12 x150 mmCartridge40x75 mmCartridge40x150 mm10.150.31.2220.30.62.5560.61.25Table 4:Approximate valuesfor loading atRf 0.15 – 0.4.10* Values are given as a guide and depend on the silica gel used and the percentilesample compositionExample of preparative separation at high loadOptimizing the conditions on the TLC plate silica gel 60:Hexane/diisopropyl ether 95:5 (CV 1, ΔCV 3Figure 13:Optimized conditionson TLC.

240Part 1 Flash Guide – Basics2468 10 12 14 16 18 2002468 10 12 14 16 18 200 2 4 6 8 10 12 14 16 18 20Time (min)Time (min)Time (min)Figure 14:The impact of increasing the load on theseparation. Cartridge12 x150 mm. Silica gel60, 40 – 63 µm, eluenthexane/diisopropylether 95:5, 14 ml/min,load (from left to right)300 mg, 600 mgand 1200 mg.Injection volume 1 ml.The volume must be low so that the sample can be compactlyapplied to the column bed. If the volume is too high, the band isconsiderably widened and the separation is less efficient.The sample that is to be separated can be brought into the separation system either as a solution or dry, adsorbed by silica gel.A classical solution sample injection requires that the sample canbe sufficiently dissolved in the starting eluent. The injection volumeshould be no more than 10% of the column volume. The followinginjection volumes apply to the Büchi cartridges:Table 5Recommended max. injection volumesCartridge12x75 mmCartridge12 x150 mmCartridge40x75 mmCartridge40x150 mm1.5 ml2 ml10 ml20 mlIf the sample cannot be sufficiently dissolved in the starting eluent, dry application can be carried out, where the sample is dissolved in any solvent and mixed with silica gel. The solvent is thendistilled off using rotation. This dry silica gel is packed into a precolumn and this is then fitted in front of the separation column into theeluent flow. The components to be separated are then constantlyeluted from the precolumn to the actual separation column. Thisprocedure is also advisable if there are sticky or solid impurities inthe sample which cannot easily be removed!Another slightly unconventional injection method for samples inthe form of solutions is not to dissolve the sample in the startingeluent but in a completely different solvent with excellent dissolvingproperties for the mixture. This “foreign” solvent is separated in theseparation flow as an additional component. Retention times areusually in the range of the solvent front. If the components of interest are optimized to an Rf range of 0.15 – 0.4, separating the frontis not a problem anyway.

25Injection /Column loadingToluene0246810121416Time (min)This simple and unconventional injection procedure is often usedto inject by-products that are not easily dissolved to the separation column. These substances are usually heavily adsorbed in thearea where they initially enter the column and remain there. This isnot, however, a problem if disposable cartridges are used, as thecartridge is changed anyway after the components of interest havebeen eluted.Special advice for injecting dissolved samplesIn preparative chromatography, it is often not possible to spendthe time pre-cleaning the samples and the mixtures are applieddirectly to the separation column with varying levels of accompanying substances. To ensure that the flash equipment operatessmoothly it is therefore very important to rinse the injection portclean after every injection, regardless of whether it is a quickstop valve or a device fitted with a tap system. This is the onlyway to avoid problems such as sample contamination or leakinginjection ports. The following should therefore be observed in theinjection process:1.2.3.4.Stop pumpInject sampleRinse injection portStart pumpFigure 15:Liquid sample injectionin toluol, eluent hexane/diisopropyl ether9:1.

26Part 1 Flash Guide – Basics6Gradient elutionThe examples given are all separated isocratically, i.e. the mobile phase is identical throughout the entire separation process. Inpractice this is, however, often not possible, as the substances tobe separated in adsorption often differ.Substances that cannot be successfully eluted isocratically canbe identified by pre-elution using TLC, and can be optimised accordingly. The following example explains the procedure:a) Establishing the suitable selectivity (see 4.2).Figure 16:Suitable selectivityusing ethyl acetate.b) Establishing the suitable solvent strengthThis is problematic – either the Rf values are so high that it is practically impossible to achieve separation with a preparative loading ofthe column, or the Rf values are so low that they can only be elutedfrom the column with a great deal of time and solvent.Figure 17:Different solventstrengths, achievedusing hexane withvarying levels of ethylacetate.

27Gradien

“Laboratory Chromatography Guide” – A close look at preparative liquid chromatography The present “Laboratory Chromatography Guide” is dedicated to preparative liquid chromatography, a common purification techni-que in most chemical or life science laboratories. T