Table 5.10Approximate Plate Number for Well-Packed Columns under Optimized Test Conditions Particle Typea Particle Diameter μm Column Length mma Plate Number N Note: Small, neutral test
Trang 1Table 5.10
Approximate Plate Number for Well-Packed Columns under Optimized Test Conditions
Particle Typea Particle Diameter ( μm) Column Length (mm)a Plate Number N
Note: Small, neutral test-solute, low viscosity mobile phase, ambient temperature, measured at the plate-height minimum.
aEstimated values for 4.6 mm i.d columns.
bAn average for commercially available, sub-2- μm particles.
new column that met the manufacturer specifications If problems are encountered
with a routine method that might be caused by the column, values of N and either
‘‘bad’’ column can be confirmed as the cause of the problem
Every column used for a routine method has a finite lifetime (number of injected samples before column failure) that depends on separation conditions—especially mobile-phase pH and temperature For ‘‘clean’’ samples, 1000 to 2000 analy-ses should be possible for must silica-based columns, particularly reversed phase columns However, for other samples (with minimal sample preparation), such as extracts of blood, plant or animal tissue, or soil, 200–500 analyses is a more typical column lifetime
5.8 COLUMN HANDLING
The performance and life of the column depend on how it is used and handled During heavy use with ‘‘dirty’’ samples (especially samples from biological sources),
columns can develop severe peak tailing (Fig 5.26a) or double peaks for each component (Fig 5.26b)— usually the result of a partly blocked frit, a contaminated
Trang 2(a) (b)
peak tailing split peaks
Figure5.26 Examples of peak tailing (a) and split peaks (b).
column, or deterioration of the column packing The following restorative measures are sometimes effective, but the time and effort involved are often not cost-effective Usually it is more economical to simply replace the column
A blocked frit or contaminated column can sometimes be restored by periodi-cally purging the column with a strong solvent A 20-column-volume purge (about
0.1% ammonium hydroxide in 4% methanol is often effective for RPC columns Pure methanol or isopropanol can be used for normal-phase columns Flushing a RPC column (at least) daily with a strong solvent, such as methanol or acetoni-trile, can enhance column performance and lifetime for isocratic separations This approach removes strongly retained sample components that can build up at the
column inlet Back-flushing a column with a strong solvent at 0.2 to 0.5 mL/min
may be more effective, so as to avoid driving the column contaminants into the column However, some manufactures recommend against back-flushing because their columns are fitted with an inlet frit that has larger pores that might allow particles to be swept out (consult the column care-and-use instructions for a specific column) In gradient elution, clearing the column of strongly retained components can be accomplished by using a steep or step gradient (Section 9.2.2.5) The use of a
blockage of the column inlet frit, and is highly recommended
To reduce the possible impact of ‘‘dirty’’ samples on column lifetime, sample
pretreatment is commonly used (Chapter 16) A guard column can also be used to
protect the column, and it is recommended for routine analysis A guard column
is short (e.g., 10–20 mm) and preferably contains a packing that is the same as
or similar to that in the main column (Section 5.4.2) The guard column captures strongly retained sample components (and particulates), and prevents these from fouling the analytical column Guard columns must be replaced at regular intervals, before the column becomes saturated with strongly retained sample components that then pass into the analytical column However, because of their added expense and inconvenience, some users prefer to avoid guard columns and replace the main column more frequently The use of guard columns with low-volume, high-efficiency columns (e.g., sub-2-μm columns) requires special care, because of the greater importance of extra-column peak broadening when small columns are used For columns that are not well packed, a sudden pressure surge (as during sample injection) can cause a void at the column inlet, with a decrease in column performance Fortunately, this problem is no longer common for columns from
Trang 3silica-based columns, other types of particles (e.g., organic gels, graphitized carbon) are more fragile and less able to withstand sudden changes in flow, pressure, or temperature
Column performance can be reduced significantly by a loss of the bonded phase during use, leading to a short column lifetime The recommendations of the manufacturer, especially with regard to mobile-phase pH and temperature, should be
(Fig 5.16b) provide additional stability at low pH High-pH mobile phases (e.g., pH
> 8) can slowly dissolve silica-based packings, again resulting in a degradation of
column performance Columns of hybrid silica-silane particles (Section 5.3.2.2) and those based on zirconia (Section 5.2.5.1) are especially resistant to degradation by high-pH mobile phases; some other special stationary phases (e.g., bonded bidentate
silanes, Fig 5.14b or c) are also more stable for high-pH applications [44].
Stationary-phase loss from silica-based columns at low pH is accelerated at higher temperatures Therefore higher temperatures as a means of improving col-umn performance or separation selectivity should be used carefully Many workers
Degradation of silica-based columns at higher pH occurs via dissolution of the silica, and its degradation is also accelerated by higher temperatures Figure 5.27
0 5 10 15
500 400 300 200 100 0
Mobile-phase volume (L)
60 °C
40 °C
Figure5.27 Effect of temperature on silica-support dissolution Column: Zorbax Rx-C18,
15× 0.46 cm; continuous nonrecycled 20% acetonitrile/80% sodium phosphate buffer,
0.25 M, pH 7.0 Flow rate: 1.0 mL/min Adapted from [78]
Trang 4columns should be avoided because of rapid dissolution of the silica support [78].
On the other hand, use of organic buffers (e.g., TRIS, HEPES, citrate) may increase column lifetime over that with phosphate and carbonate buffers when operating
at intermediate and higher pH [79] One speculation is that these basic, partially hydrophobic, organic buffer compounds tend to bind to unreacted silanol groups
on the packing so as to create an additional barrier to the dissolution of the silica support [79] However, the advantage of the organic buffers may be misleading, as the actual pH of the buffer/organic solvent mobile phase may be somewhat lower than that measured for the buffer solution itself (the pH of phosphate and carbonate buffers in organic-containing solvents is somewhat higher that the aqueous buffer [80]); see the further discussion of Section 7.2.3
The performance and lifetime of a column can also be affected by improper
microbial growth if they are stored for more than a day at room temperature
The resulting particulates can in turn block the column inlet, reducing N and
increasing the column pressure Therefore it is good practice to formulate buffers
absence of oxygen due to helium sparging, can inhibit bacterial growth and prolong buffer life
When removed from the system, the column is best stored in a nonprotic solvent such as acetonitrile (100% B) For short-term applications (overnight or
a few days) it is convenient (and acceptable) to leave the mobile phase in place However, prolonged storage with buffered solutions, particularly those with high concentrations of water or alcohols, should be avoided Prior to storage, the column should be flushed with 5 to 10 column volumes of the same aqueous-organic
mobile phase but without buffer before an additional 5-column-volume flush with
100% organic phase (this avoids precipitation of the buffer within the column) Flushing columns with pure water for long periods should be avoided because of
prevent columns from drying out, they should be tightly capped for storage
order to retain these very small particles Therefore both the sample and mobile phase should be passed through 0.2-μm filters to ensure that particulates do not block the frits and degrade column performance Small-particle columns are often
very narrow widths—measured either in time or volume This requires instrumental conditions that minimize potential extra-column effects that artificially broaden peaks:
• short, low-volume tubing that connects the sampling valve, column, and detector
• high data-capturing rate (at least 20 points/sec or 20 Hz)
For further details, see [81, 82]
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Trang 8CHAPTER SIX
REVERSED-PHASE
CHROMATOGRAPHY
FOR NEUTRAL SAMPLES
6.1 INTRODUCTION, 254
6.1.1 Abbreviated History of Reversed-Phase Chromatography, 255
6.2 RETENTION, 256
6.2.1 Solvent Strength, 257
6.2.2 Reversed-Phase Retention Process, 259
6.3 SELECTIVITY, 263
6.3.1 Solvent-Strength Selectivity, 263
6.3.2 Solvent-Type Selectivity, 265
6.3.3 Temperature Selectivity, 270
6.3.4 Column Selectivity, 273
6.3.5 Isomer Separations, 276
6.3.6 Other Selectivity Considerations, 278
6.4 METHOD DEVELOPMENT AND STRATEGIES
FOR OPTIMIZING SELECTIVITY, 284
6.4.1 Multiple-Variable Optimization, 286
6.4.2 Optimizing Column Conditions, 295
6.5 NONAQUEOUS REVERSED-PHASE CHROMATOGRAPHY
(NARP), 295
6.6 SPECIAL PROBLEMS, 297
6.6.1 Poor Retention of Very Polar Samples, 297
6.6.2 Peak Tailing, 298
Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R Snyder,
Joseph J Kirkland, and John W Dolan
Copyright © 2010 John Wiley & Sons, Inc.
253
Trang 96.1 INTRODUCTION
This chapter describes the separation of neutral samples by means of reversed-phase chromatography (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 behav-ior 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
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:
In a few countries, ACN is considered sufficiently toxic to limit its general use, but this is not true elsewhere See Appendix I for further information concerning solvent properties and the choice of B-solvent for a given application Samples that contain acids or bases normally require a buffered mobile phase, in order to maintain a constant pH throughout the separation (Chapter 7) Strongly retained, very hydrophobic samples may require a water-free mobile phase (nonaqueous reversed-phase chromatography [NARP], Section 6.5) Normal-phase chromatog-raphy (Chapter 8) 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 6.1
Compared to other forms of HPLC (normal-phase, ion-exchange chromatog-raphy, etc.; Table 2.1), 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,
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 (Table I.2
of Appendix I) 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 (Section 9.3.7) 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
Trang 10Table 6.1
Preferred Conditions for the Separation of Neutral Samples by Reversed-Phase
Chromatography
Dimensions: 100× 4.6-mm
particle size: 3 μm Pore diameter: 8–12 nm
Weight ≤ 50 μg
aAlternatively, use a 150× 4.6-mm column of 5-μm particles; flow rate, column dimensions, and particle
size can be varied, depending on the anticipated difficulty of the separation and the maximum allowable column pressure (Section 2.4.1)
bInitial values, which may be changed during method development (Section 2.5); a temperature 5–10◦C above ambient is suggested in most cases Also consult the column manufacturer’s recommendations for
a maximum column temperature.
cVaries with the sample; start with 80%B and adjust further as described in Section 2.5.1.
Many organic compounds have limited solubility in either water or the water-organic mobile phases used for RPC, but this is rarely a practical con-cern 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 (Section 8.4.1)
Some samples are less well separated by RPC For example, very polar
molecules may be retained weakly in RPC (k
mobile phase; these samples may require a different approach (Section 6.6.1) Similarly enantiomers require separation conditions that exhibit chiral selectivity (Chapter 14) While many achiral isomers can be separated by RPC (Section 6.3.5), these compounds are often better separated by normal-phase chromatography using
an unbonded silica column (Section 8.3.4.1) Finally, normal-phase chromatography
is often a better choice for preparative HPLC (Chapter 15)
6.1.1 Abbreviated History of Reversed-Phase Chromatography
Prior to the invention of RPC in 1950 by A J P Martin [1], the chromatographic separation of neutral samples was carried out with a polar column (or stationary phase) and a less polar mobile phase; such separations are now referred to as