Transcript Lecture 17
Normal-phase chromatography Normal-phase chromatography is really not that normal. That is to say that it is used much less frequently than reversed-phase chromatography. The main reason for this is simply that many separations can be accomplished using either reversed-phase or normal phase chromatography, but reversed-phase is easier, and hence more common. Separation in Normal-Phase Chromatography -- samples with many different functional groups -- samples with molecules containing double bonds -- samples containing isomers -- samples that are very hydrophobic -- samples that are very hydrophillic Normal-phase chromatography is also called adsorption chromatography or liquid-solid chromatography. Retention of analytes occurs by an adsorption process rather than by a partition process (as in reversed-phase chromatography). Silica is the most common of the non-bonded phases and can provide very high selectivity for many applications, but water adsorption by the silica can make reproducible retention times difficult. Comparison of different types of silica columns. (a) acidic type A silica; (b) less acidic type B silica. 1, toluene; 2, benzanilide; 3, phenol; 4, benzyl alcohol; I, impurity Other Stationary Phases Alumina, has unique selectivity, but it is little used because it has problems such as low theoretical plate number (N), variable retention times, and low sample recovery. Cyano columns are the best for general analysis because they are the most stable and are more convienent to use than silica columns. Diol and amino columns can offer different selectivities, but are less stable than cyano columns. Parameters for Commonly Used Normal-Phase Solvents. Solvent Hexane Chloroform Methylene chloride Ethyl ether Methyl t-butyl ether Ethyl acetate Acetonitrile Tetrahydrofuran 1- or 2-Propanol Methanol Strength(o) 0.00 0.26 0.30 0.38 0.48 0.48 0.52 0.53 0.60 0.70 Localization no no no yes yes yes yes yes yes yes Basic? UV-cutoff Not relevant 201 Not relevant 247 Not relevant 234 yes 219 yes 225 no 256 no 192 yes 230 Proton donor 214 Proton donor 210 Separation in Normal and Reversed-Phase Chromatography In normal-phase chromatography, the stationary phase is polar and it interacts with the polar parts of the molecule, therefore it would not be an effective method for separating molecules such as butanal, hexanal and octanal. In reversed-phase chromatography, the stationary phase is non-polar and therefore it interacts with the non-polar portion of molecules. Reversed-phase chromatography would be a good choice for separating butanal, hexanal and octanal. O BUTANAL O HEXANAL O OCTANAL The adsorption of analyte molecules decreases in the following order: carboxylic acids amides amines alcohols ketones aldehydes esters nitro compounds ethers sulphides organic halogen compounds aromatics olefins saturated hydrocarbons Separation of Isomers The adsorption of an analyte is based on the type of functional group present and also steric factors which makes is similar to chiral or affinity chromatography, the difference being that the adsorption sites on silica are not very specific. Separation of Very Hydrophobic Molecules Hydrophobic analytes are more soluble in these solvents than they would be in the aqueous mobile phases used in reversed phase chromatography. Very hydrophobic molecules are strongly retained in reversed phase chromatography often resulting in poor separations. These molecules can be analyzed using normal phase chromatography. Separation of Very Hydrophilic Molecules Just as there is non-aqueous reversed-phase chromatography there are also aqueous mobile phases used in normal phase chromatography. In this case, very hydrophyllic samples that are not retained in reversed phase conditions can be chromatographed. Carbohydrates are often separated on an amino column with mobile phases consisting of 60-80% acetonitrile/water. Normal phase separation of carbohydrates using an amino column and 75% acetonitrile-water as the mobile phase. 1 = fructose 2 = glucose 3 = sucrose 4 = maltose Gradient Elution Polar solvents can interact strongly with the surface of a silica. This strong interaction makes changing solvents difficult because it takes a long time for the column and solvent to come to equilibrium (typically from 45 min to 1 hour). Solvent Demixing - Example: 100% Hexane --> 100% Isopropanol As the gradient changes from 100% hexane with the addition of isopropanol, all the added isopropanol is adsorbed to the column surface and 100% hexane continues to elute from the column. After a while, the column becomes saturated with isopropanol, and a sudden jump in isopropanol concentration is seen in the mobile phase. This rapid change in mobile phase solvent strength will elute sample components with low k values and poor separation. Stationary Phase Water Content What else is silica used for? Even fairly non-polar solvents will adsorb some water from the air. The dissolved water will be adsorbed on the surface of the column during the chromatographic run, changing the mobile phase polarity which can have a drastic effect on analyte retention times The k value (relative retention time) for phenyl propanol when there is no water in the mobile phase is about 18 and at 100% water saturation of the mobile phase (0.15% water), the k value is about 4. Improving reproducibility of retention times: add from 0.1% to 0.5% methanol or propanol to the mobile phase equilibrate the mobile phase with an intermediate concentration of water Temperature Effects Changes in operating temperature have little effect on selectivity. Some changes in selectivity with temperature can be observed with the used of localizing solvents such as acetonitrile. Changes in overall retention time can vary with temperature so controlling column temperature may be needed to achieve reproducible retention times. Summary of Advantages and Disadvantages of Normal-Phase Chromatography ADVANTAGES: -the sample can be dissolved in a non-polar solvent -it can be used for analytes that may decompose in water -it is good for separating isomers and very hydrophopic or hydrophillic analytes -it can use higher flow rates due the use of low viscosity solvents. DISADVANTAGES: -higher costs for purchase and disposal of solvents, -difficulty in controlling solvent strength, -lower boiling point solvents are subject to evaporation and bubble formation, -retention may be variable - gradient elution can be difficult.