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)
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).
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;
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?
The present section extends this treatment to the HPLC separation of ‘‘ionic’’ samples; these are mainly mixtures that contain acids and/or bases (with or without neutral compounds), but they can include compounds that are totally ionized between pH-2 and pH-12 (e.g., tetralkylammonium salts, sulfonic acids). In the early days of HPLC, ionic samples often presented special problems—partly the result of less suitable column packings that were available at that time but also because of a limited understanding of how such separations are best carried out. Although these past limitations have been largely overcome, the separation of ionic samples remains somewhat more demanding when compared with separations of neutral samples. Before 1980, ion-exchange chromatography (IEC, Section 7.5) was commonly selected for the separation of acids and bases, but today RPC and—to a lesser extent—ion-pair chromatography have become preferred procedures for the separation of ‘‘small,’’ ionizable molecules (<1000 Da).
Acid–Base Equilibria and Reversed Phase Retention
RPC retention of neutral samples decreases for less hydrophobic (more polar)
molecules. When an acid (HA) or base (B) undergoes ionization
(i.e., is converted from an uncharged to a charged species), the compound becomes much more polar or hydrophilic. As a result its retention factor k in RPC can be
reduced 10-fold or more:
Acids lose a proton and become ionized when the mobile-phase pH is increased; bases gain a proton and become ionized when mobile-phase pH decreases. The
Here [HA] and [A-] are the concentrations of the free and ionized acidic solute HA; [B] and [BH+] refer to the concentrations of the free and protonated basic solute B. The pKa value pH= -logKa) of an acid or base is given by the Henderson–Hasselbalch equation:
For example, the pKa value
in water of a (weakly basic) substituted aniline will fall within a range
of about ,
while the pKa of a (strongly basic)
aliphatic amine will usually lie between 9 and 11. Values of pKa in the literature for different acids or bases usually refer to solutions in buffered-water at near-ambient temperatures. If the mobile phase contains organic solute, or if the temperature is much different from ambient, values of both pH and pKa can change significantly.
Retention as a function of pH and sample ionization is illustrated in Figure 7.1 for the separation of a hypothetical sample composed of carboxylic acid HA (solid
Choice of Buffers
Whenever acids or bases are separated, it is necessary to buffer the mobile phase in order to maintain a constant pH and reproducible retention during the separation. The use of a pH meter to measure (and control) pH will be less precise when the mobile phase contains organic solvent because the electrode response tends to drift for organic-water solutions. Consequently we recommend that pH measurements be carried out for the A-solvent (aqueous buffer) prior to the addition of organic to form the final mobile phase. The pH of the final mobile phase (including organic solvent) can then be equated to (or labeled as) that of the A-solvent, although the actual mobile-phase pH will be somewhat different.
This uncertainty concerning
the final mobile-phase pH is unimportant for the routine application of
RPC. When the A-solvent is prepared in this way, different laboratories
should be able to obtain the same final mobile-phase pH within ±0.04
to 0.05 units. If a closer control of mobile-phase pH is required. When
to mobile-phase pH in this book, we will generally mean the pH of the A-solvent.
Directions for the preparation of buffer solutions of varied pH and buffer-type are given in other sites. In selecting a buffer for RPC separation, several buffer properties may prove relevant:
• UV absorbance (when UV detection is used)
• volatility (when mass-spectrometric or evaporative light-scattering detection is used)
• ion-pairing properties
• stability and compatibility with the equipment
The first four buffer properties are usually the most important.
Buffer pKa and Capacity
‘‘Buffer capacity,’’ or the
ability of the buffer to maintain a constant pH, depends on
• buffer concentration
• pH of the mobile phase
Just as for the ionization of a sample component in Figure 7.1a, the fractional ionization of the buffer as a function of pH can be expressed by Equations above; that is, buffer and solute ionization are identical functions of mobile-phase pH and pKa. Maximum buffering occurs when the concentrations of the two forms
of the buffer (e.g., HA and A-) are equal; that is, when the buffer pKa equals the mobile-phase pH. Buffer capacity decreases as values for the buffer pKa and
mobile-phase pH become more different. Consequently the first requirement of the buffer is that its pKa value should be within ±1.0 units of the selected mobile-phase pH (this requirement can be relaxed to ±1.5 unit for higher concentrations of the buffer). The buffer capacity of the mobile phase is proportional to buffer concentration, which typically falls within a range of 5 to 25 mM. To minimize the possibility of inadequate buffering of the sample during RPC separation, it is generally desirable for the sample to be dissolved in the mobile phase (or buffered to the same pH as the mobile phase); this practice becomes especially important for lower concentrations of the mobile-phase buffer, and/or for larger volumes of injected sample.
Table above provides a list
of buffers that can be used in RPC, along with pertinent properties such
as buffer pKa and the mobile-phase pH range in which the
buffer is effective. For separations with UV detection, and a mobile-phase pH < 8, popular buffers include phosphate, trifluoroacetate, acetate, and formate. In addition ammonium bicarbonate can also be considered. The pKa values of ammonia (9.2) and bicarbonate (10.3) overlap, hence somewhat extending the buffering range of ammonium bicarbonate . This buffer is volatile and therefore compatible with LC-MS; however, when the buffer pH < 8.5, loss of CO2 (e.g., from excessive degassing) may lead to an unintended increase in pH. Because of this instability it is recommended to prepare fresh ammonium bicarbonate buffers daily.
We can define the ‘‘effective
buffer capacity’’ of the mobile phase to mean that an increase in this
quantity will result in fewer problems due to insufficient buffering.
The effective buffer capacity of the mobile phase increases for:
1. a smaller difference between values of the buffer pKa and the pH of the mobile phase (change either the buffer or pH)
2. a greater difference between
the mobile-phase pH and the pKa of the solute (for large differences, the
solute is either non-ionized or completely ionized;
buffering is then much less important)
3. increased buffer concentration
4. smaller volumes of injected sample
5. a sample whose pH is adjusted to that of the mobile phase
Organic buffers are usually
adequately soluble in all organicwater mobile phases (0–100% B), but many
inorganic buffers have limited solubility
in mobile phases which are predominantly organic (high %B). Consequently there is a danger that combining the A- and B-solvents may result in buffer precipitation,
which could lead to blockage of the column or HPLC equipment. If there is any doubt as to whether a mobile phase might precipitate, the complete solubility of
the buffer in the final mobile phase should be confirmed first (over the intended pH range), especially when the A- and B-solvents are mixed by the HPLC pumping
system. Thus varying proportions of the A- and B-solvents can be combined manually in a container and observed over a period of 30 minutes or so. If any cloudiness develops, or a precipitate is observed for a given mobile-phase composition (%B), mobile phases of that %B or higher should be excluded or the buffer concentration should be reduced. Buffer solubility is of special concern for separations by gradient elution.
Buffer solubility is affected
by several separation conditions. The buffer counter-ion is one such factor;
for acidic buffers such as phosphate, buffer solubility
usually increases as
pKa as a Function of Compound Structure
When selecting a range of mobile-phase pH values within which to carry out method development (i.e., optimization of pH), it can be useful to know the approximate
Exact pKa values for sample
components are not required in RPC method development, and in many cases
the chemical structures of sample components may
not even be known. Values of pKa are determined by ionizable acid or base groups attached to the solute molecule, for example, –COOH, –NH2. This means that
estimates of pKa can be obtained from literature pKa values for compounds of similar functionality (e.g., benzoic acid, as a representative of aromatic carboxylic
acids). Table below summarizes pKa values in water for some common acid- or base-substituent groups present in typical sample molecules.
Mobile Phase Modifiers – Effects of Competing Acids on the Peak Shape of Acidic Compounds
Conditions: XTerra™ RP
18 , 3.9 X 150 mm, 5 µm, Mobile Phase: 65% 20 mM Buffers, 35% ACN,
Column Temp.: 30 °C, Flow Rate: 1.0 mL/ min, Detector: 210nm for pH
2. 5,pH 5.0, and pH 7. 0; 230nm for pH 10. 6 1: acetaminophen, 2: lidocaine,
3: doxepin, 4: imipramine, 5: nortriptyline, 6: ibuprofen
Tailing factors between 0.96 and 1.17
Mobile Phase pH
Usually it is recommended to control mobile phase pH whenever ionic or ionizable compounds are to be separated. To this end pure water as the polar component of the mobile phase is replaced by buffer. Rugged conditions can be expected if the pH is separated by at least one unit from the pKs of the compound of interest. In principle, although this is not obligatory, non-dissolved species are preferred. For complex mixtures it can be difficult to find the optimum pH.
The separation of nicotine and salicylic acid is not rugged around pH 6; it would be better to work at pH 5 or 7. At pH 5.65 nicotine is eluted in front of salicylic acid, at pH 6.05 the elution order has reversed, at pH 5.85 the two compounds merge to a single peak. The pKs1 of nicotine is 6.16 (15°C), the pKs of salicylic acid is 2.96 (25°C). If 67 mM citrate buffer is used (19.6 g/L of trisodiumcitrate dihydrate) the addition of 2.2 mL/L of 25% hydrochloric acid gives pH 6.05, 3 mL/L gives pH 5.85, and 4 mL/L gives pH 5.65. Needless to say, such differences in buffer preparation are quite large but errors of this kind can happen when work is performed carelessly. Note that the nicotine peak has strong tailing if a conventional, not base-deactivated, stationary phase is used.
Sample: nicotine and salicylic acid, Column: 4.0 mm x 25 cm, Stationary
phase: LiChrospher 60 RP-Select B, 5 µm
(reversed phase C8), Mobile phase: 67 mM citrate buffer pH 5.65, 5.85 or 6.05 / methanol , 6 : 4, 1.5 mL/min, Detector: 240 nm.
Effect of pH on Peak Shape at or Near the Sample pKa
Buffer Concentration Decreases Tf
Method Development Strategy
Estos son una serie de cinco (7) artículos que dan un marco de referencia a como mejorar los métodos que se desarrollan en HPLC.
Method Development Guides
The Secrets of Rapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times
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