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High-Performance Liquid Chromatography (HPLC)
HPLC Methods development

 For Normal Phase

The first step in NPC method development should consist of a review of the goals of separation, including reasons why NPC is being considered. Unless some problem can be anticipated for the use of RPC—or has been experienced in prior RPC separations of the sample—RPC is normally a best first choice at the beginning of method development. Some applications for which NPC might be considered initially include
-  the purification of crude samples
-  the separation of isomers
-  orthogonal separation
-  samples that contain hydrophobic interferences
-  samples that contain very polar analytes (e.g., unretained in near-100% water by RPC)

Reversed phase Chromatography for Neutral Samples
By a ‘‘neutral’’ sample, we mean one that contains no molecules that carry a positive or negative charge—usually as the result of the ionization of an acid or a base. Although a neutral sample implies an absence of acidic and basic solutes, this is not necessarily the case. Depending on mobile phase pH, any acids or bases in the sample may be present largely (e.g., 90%+) in the neutral (non-ionized) form—in which case their chromatographic behavior is similar to that of non-ionizable compounds. The separation of ‘‘ionic’’ samples (which contain one or more ionized compounds) by RPC is covered in Chapter 7.
RPC is usually a first choice for the separation of both neutral and ionic samples, using a column packing that contains a less polar bonded phase such
as C8 or C18. The mobile phase is in most cases a mixture of water and either acetonitrile (ACN) or methanol (MeOH); other organic solvents (e.g., isopropanol [IPA], tetrahydrofuran [THF]) are used less often. A preferred organic solvent for an RPC mobile phase will be water-miscible, relatively nonviscous, stable under the conditions of use, transparent at the lowest possible wavelength for UV detection, and readily available at moderate cost. Commonly used B-solvents can be ranked in terms of these properties as follows

Samples that contain acids or bases normally require a buffered mobile phase, in order to maintain a constant pH throughout the separation. Strongly retained, very hydrophobic samples may require a water-free mobile phase (nonaqueous reversed-phase chromatography [NARP]. Normal-phase chromatography can also provide acceptable separations of very hydrophobic samples, as sample hydrophobicity contributes little to retention for this HPLC mode. Preferred conditions for the isocratic separation of neutral samples by RPC are listed in Table below.

Compared to other forms of HPLC (normal-phase, ion-exchange chromatography, etc.;, separations by RPC are usually more convenient, robust,
and versatile. RPC columns also tend to be more efficient and reproducible, and are available in a wider range of choices that include column dimensions, particle size, and stationary-phase type (C1–C30, phenyl, cyano, etc.; Section 5.3.3). The solvents used for RPC tend to be less flammable or toxic, and are more compatible with UV detection at wavelengths below 230 nm for increased detection sensitivity. An additional advantage of RPC is generally fast equilibration of the column after a change in the mobile phase—or between runs when using gradient elution. Finally, because RPC has been the dominant form of HPLC since the late 1970s, a better practical understanding of this technique has evolved. This usually means an easier development of better separations. All of the foregoing reasons have contributed to the present popularity of RPC.

Many organic compounds have limited solubility in either water or the water-organic mobile phases used for RPC, but this is rarely a practical concern.
Thus very small weights (nanograms or low micrograms) of individual solutes are usually injected, so the required sample concentration is usually only
a few micrograms/mL or less. In those cases where sample solubility in water or water-organic mixtures is exceptionally poor (very hydrophobic samples), the use of normal-phase chromatography with nonaqueous mobile phases may be preferred.

Some samples are less well separated by RPC. For example, very polar molecules may be retained weakly in RPC (k   1), even with 100% water as
mobile phase; these samples may require a different approach. Similarly enantiomers require separation conditions that exhibit chiral selectivity
(Chapter 14). While many achiral isomers can be separated by RPC, these compounds are often better separated by normal-phase chromatography using
an unbonded silica column. Finally, normal-phase chromatography is often a better choice for preparative HPLC.

Because very polar molecules interact more strongly with the polar mobile phase, these compounds are less retained and leave the column first. Similarly less polar compounds prefer the nonpolar stationary phase and leave the column last. Thus molecules of similar size are eluted in RPC approximately in order of decreasing polarity. An example is provided by Figure a below, where it is seen that the more-polar benzonitrile (1) appears in the chromatogram first, followed by the increasingly less polar anisole (2), and finally toluene (3). A more detailed example of RPC retention as a function of solute polarity is provided by Figure c.

Retention in RPC is largely the result of interactions between a solute molecule and either the mobile phase or the column. An increase in %B (volume-% of organic solvent in the mobile phase) makes the mobile phase less polar (‘‘stronger’’) and increases the strength of interactions between solute and solvent molecules. The result is decreased retention for all solute molecules when %B is increased. This is illustrated by the separation of Figure b (with a mobile phase of 60% B) compared to that of Figure a (40% B). An increase in temperature weakens the interaction of the solute with both the mobile phase and column, and decreases retention; compare Figure 6.1c (70 °C) and Figure a (30 °C). Finally, a decrease in column hydrophobicity weakens the interaction between the solute and column, and reduces retention; compare Figure d (more-polar cyano column) and Figure a (less-polar C18 column).

Solvent Strength

As noted in before, retention in RPC varies with mobile phase %B as

where kw refers to the (extrapolated) value of k for 0% B (water as mobile phase), S is a constant for a given solute when only %B is varied, and  is the volume-fraction of organic solvent B in the mobile phase (= 0.01%). for the final separation should provide values of k for the sample that are within a
desired range (e.g.,), while at the same time maximizing solvent-strength selectivity.

It should be noted that Equation is not an exact relationship but an approximation. For example, values of log k for a representative solute
(4-nitrotoluene; compound 2 in Fig below) are plotted against %B in Figure for both acetonitrile (ACN, ) and methanol (MeOH, •) as the B-solvent. Whereas
Equation predicts a linear plot, a slightly curved plot results for ACN as B-solvent. The data for MeOH fall closer to the linear curve in Figure that is fitted to these data. This behavior is typical of other samples and experimental conditions; more linear plots are usually obtained for MeOH, compared to the
use of ACN or other organics as B-solvent. However, over the usual range in k that is of interest (eg, .


The most effective way to improve the resolution (or speed) of a chromatographic separation is to initiate a change in relative retention (selectivity). For the separation of non-ionic samples by RPC, changes in selectivity can be achieved by a change in solvent strength (%B), temperature, solvent type (e.g., ACN vs. MeOH as the organic solvent), or column type (e.g., C18 vs. cyano). The relative effectiveness of a change in conditions for a change in selectivity varies roughly as

However, each of the four conditions above for changing selectivity can be useful for different samples or separation goals, as discussed next.

Variation of log k with%B for regular and irregular samples. (a) Regular sample (a
mixture of herbicides, separated on a C18 column with methanol-water as mobile phase
(c) irregular sample;


Column Selectivity

During the early days of HPLC, a change of column was often used as a means of varying selectivity and improving resolution. Indeed column selectivity represents a powerful means for altering relative retention and improving the separation of neutral samples. However, the use of column selectivity alone for the purpose of systematically improving separation has a serious limitation, compared to changes in %B, solvent type, or temperature.

We presently have a more complete understanding of the basis of column selectivity than for other kinds of selectivity (solvent strength, solvent type, temperature, etc.). THe column selectivity can be quantitatively defined by five different characteristics:

• column hydrophobicity H
• column steric resistance S*
• column hydrogen-bond acidity A
• column hydrogen-bond basicity B
• column cation-exchange capacity C

Method Development and Strategies for Optimizing Selectivity

The Perfect Method

1. The Perfect Method, Part I: What Is Your Goal?

2. The Perfect Method, Part II: Where to Start?

3. The Perfect Method, Part III: Adjusting Retention

4. The Perfect Method, Part IV: Contolling Peak Spacing

3. The Perfect Method, Part V: Changing Column Selectivity

4. The Perfect Method, Part VI: Make it faster

5. The Perfect Method, Part VII: The Gradient Shortcut 

All seven parts in a pdf file 2007 

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