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Normal Phase Chromatography

Normal Phase Liquid Chromatography
for separation of isomers and compounds that are instable in aqueous environment

Page Index

  1. Principle
  2. When to use NPLC?
  3. Chemistry of stationary phase?
  4. Suppliers of Silica Phases
  5. Solvents for Normal-Phase Chromatography
  6. Separation of Molecules with Different Functional Groups
  7. Separation of Isomers
  8. Separation of Very Hydrophobic Molecules
  9. Separation of Very Hydrophilic Molecules
  10. Gradient Elution
  11. Stationary Phase Water Content
  12. Temperature Effects
  13. Summary of Advantages and Disadvantages of Normal-Phase Chromatography
  14. References

 

Normal Phase Liquid Chromatography

Principle

In normal-phase chromatography the stationary phase is polar and the mobile phase is nonpolar. Typical stationary phases for normal-phase chromatography are silica. There are also bonded normal phase material. They have organic moieties with cyano and amino functional groups.

In normal-phase chromatography, the least polar compounds elute first and the most polar compounds elute last. The mobile phase consists of a non-polar solvent such as hexane or heptane mixed with a slightly more polar solvent such as isopropanol, ethyl acetate or chloroform. Retention increases as the amount of non-polar solvent in the mobile phase increases.

Normal phase chromatography, an adsorptive mechanism, is used for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes, etc.

When to use NPLC?

For compounds that:

  1. Are too hydrophobic or too hydrophilic for separation using RPLC
  2. Are not soluble in water
  3. May decompose in water
  4. Isomers that require separation.
  5. For Prep and Process scale chromatography

 

Chemistry of stationary phase?

In normal-phase 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

NP_Acidity_Silicate
Fig 1.

 

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.

  1. Solvent Strength (Polarity) The strengths of various solvents are determined empirically and are listed in a eluotropic series  (See Solvents ). 
  2. 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
  3. 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. 
  4. 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

 

Gradient Elution

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.

Normal Phase Fig 2

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)

Temperature Effects

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.

NP Materials

Phase

Manufacturer

 Particle Size(μm)

Pore Size ()

Surface Area
(m2/g)

ACE  

ACT

3, 5, 10

100

 300

Chromolith

Merck

 

 

 

Cosmosil

 Nacalai Tesque

3, 5

120

300

Exsil

Grace Davison

3, 5, 10

100

 200

Genesis

Thermo Scientific

4, 7, 15

120

170

Hypersil GOLD Silica

Thermo Scientific

1.9, 3, 5

175

220

Inertsil

GL Sciences

3, 5

100

 450

Kromasil

Eka Chemicals

1 3.5, 5, 7, 10

60, 100

540, 320

LiChrosorb

Merck

5, 10

60, 100

490, 300

LiChrospher

Merck

5

 60, 100

700, 400

NUCLEODUR   -

Macherey-Nagel

3, 5, 10

110

340

NUCLEOSIL 

Macherey-Nagel

3, 5, 10

100, 120

 350, 200

Partisil172

Hichrom/ Whatman

5, 10

85

 350

Purospher STAR Si

Merck

5

120

330

Ultrasphere

Hichrom

5

80

 

Nova-Pak

Waters

60

120

Spherisorb

Waters 

3, 5, 10

 80

 200

YMC

YMC

 3, 5

120

300

Zorbax

Agilent

3, 5, 7

 70

330

Zorbax Rx 

Agilent

5

80

180

 

 

 

 

 

 

 

References

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.

 

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