Isomers and compounds that are instable in aqueous environment are separated by Normal Phase Liquid Chromatography is used for separation of
The stationary phase is polar and the mobile phase is nonpolar to b e used in .In Normal Phase Liquid Chromatography. Typical stationary phases for Normal Phase Liquid Chromatography are silica. There are also bonded normal phase materials. Their organic moieties include cyano and amino functional groups.
The least polar compounds elute first and the most polar compounds elute last in Normal Phase Liquid Chromatography. As mobile phase we use non-polar solvent such as hexane or heptane mixed with a slightly more polar solvent. Polar solvents are isopropanol, ethyl acetate or chloroform. Retention increases as the amount of non-polar solvent in the mobile phase increases.
Normal Phase Liquid Chromatography has an adsorptive mechanism. It is used for analysis of solutes that are readily soluble in organic solvents and based on their polar differences such as amines, acids, metal complexes, etc.
When to use Normal Phase Liquid Chromatography
For compounds that:
- Are too hydrophobic or too hydrophilic for separation using RPLC
- Are not soluble in water
- May decompose in water
- Isomers that require separation.
- For Prep and Process scale chromatography
Chemistry of stationary phase?
In Normal Phase Liquid Chromatography, the stationary phase is more polar than the mobile phase. There are a number of stationary phases available for normal-phase chromatography. Silica is the most common of the non-bonded phases and can provide very high selectivity for many applications.
The adsorption is controlled by the Silanol groups on the Silica gel particle. The particles carry four different OH-Groups in its surface. Depending on manufacturing process some carry more acidic silanol groups than others as per animation below
The OH group have an strong affinity for water but water adsorption can make reproducibility difficult. Older less pure silicas (type A) have some trace metal content and more highly acidic sites on the surface. The strong acidic chelated silanol group that may cause esterification of Methanol.
Newer type B silica columns are made of high purity silica and have fewer acidic sites. They are recommended for separating highly polar or basic compounds.
Another non-bonded phase, 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.
Of the bonded phases, cyano columns are the best for general analysis because they are the most stable and are more convenient to use than silica columns. Diol and amino columns can offer different selectivities, but are less stable than cyano columns.
Solvents for Normal-Phase Chromatography
There are 4 main factors involved in the choice of solvents for normal-phase chromatography.
- Solvent Strength (Polarity) The strengths of various solvents are determined empirically and are listed in a eluotropic series (See Solvents ).
- Localization. This is a measure of the interaction of the solvent with the stationary phase. Solvent molecules with polar functional groups will prefer a specific position relative to nearby silanol groups (or other polar group on the stationary phase). Therefore the stationary phase is covered with a well defined layer of solvent molecules.
- The competition between analytes and these solvents for adsorptive sites is an important factor in normal–phase selectivity. Solvents that are not polar or weakly polar interact with the stationary phase very weakly and the coverage of the surface is random
- Basicity : Basicity is one of the axes of the solvent selectivity triangle. Selectivity can be changed by the used of basic solvents such as methyl tert-butyl ether or non-basic solvents such as acetonitrile.
UV cut off (See Solvents)
Separation of Molecules with Different Functional Groups
The hydrophobic portion of an analyte molecule has little effect on separation, for example, butanol, hexanol, and octanol cannot be well separated using normal phase chromatography (but they can be easily separated using reversed-phase).
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, and saturated hydrocarbons.
If a molecule has several functional groups, then retention is based on the most polar one. Normal-phase using silica is also an excellent method of separation compounds with different functional groups compared to reversed-phase chromatography using C18.
Separation of Isomers
Retention in normal-phase appears to occur by an adsorption process (analytes interact with the polar groups on the surface of the column packing). Because these surface sites are fixed, their location and spacing have an effect on separation. This feature allows for the separation of molecules that are chemically similar but physically different, so normal-phase chromatography is often used for the 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
One advantage of normal-phase chromatography is that organic solvents are used. 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. This may result in long chromatographic runs and selectivity can sometimes be low, resulting in poor separations. Very hydrophobic molecules can be analyzed using normal-phase chromatography.
Separation of Very Hydrophilic Molecules
Hydrophilic compounds are often not retained under reversed-phase conditions, but they are usually well retained under normal phase. One problem is that very hydrophilic compounds are not very soluble in the solvents used for normal-phase. This problem can be solved by the use of special normal-phase columns that can be used with aqueous mobile phases. Carbohydrates are often separated on an amino column with mobile phases consisting of 60-80% acetonitrile/water
Polar solvents can interact strongly with the surface of a silica or alumina column. 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). Because of this, gradient elution is generally not used with adsorption chromatography. An additional reason that gradient elution is difficult is a phenomenon called solvent demixing.
An illustration of solvent demixing can be seen with an example of a gradient of 100% hexane to 100% isoproanol. As the gradient changes from 100% hexane with the addition of isopropanol, all of 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.
Figure 2. 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).
Stationary Phase Water Content
Water is the most polar solvent commonly used in chromatography. It is also found in the air (especially in humid climates) and 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.
An example of this phenomenon can be seen with methylene chloride as the mobile phase and phenyl propanol as the analyte. Methylene chloride can be saturated with water at a concentration of 0.15% water. 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.
These large changes in k values with small changes in water content of the mobile phase makes obtaining reproducible retention times difficult with adsorption chromatography. There are two main ways to improve the reproducibility of retention times. One method is to add from 0.1% to 0.5% methanol or propanol to the mobile phase which can minimize the effects of changes in water content. Another method is to equilibrate the mobile phase with a certain intermediate concentration of water (such as 50% water saturation)
Changes in operating temperature do not generally have much of an effect on selectivity in normal-phase chromatography. However, some changes in selectivity with temperature can be observed with the used of localizing solvents such as acetonitrile. It is important to note that changes in selectivity due to temperature are not large, however, changes in overall retention time can vary with temprature so controlling column temperature may be needed to achieve reproducible retention times.
Summary of Advantages and Disadvantages of Normal-Phase Chromatography
Some of the advantages of normal-phase are, 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.
Some of the disadvantages of normal-phase are, 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 and gradient elution can be difficult because of water uptake by silica columns.
Meyer, V. R. Practical High-Performance Liquid Chromatography, 3 ed.; John Wiley & Sons Ltd.: West Sussex, England, 1998; 337 p.
Snyder, L. R.; Kirkland, J. J.; Glajch, J. J. Practical HPLC method development, Second ed.; John Wiley & Sons, Inc.: New York, NY, 1997; 765 p.