Nucleic Acid Analysis with UV-vis and NMR - Spectroscopy

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32 Spectroscopy 24(11) November 2009 www.spectroscopyonline.com HETP (µm) 60 40 20 0 0 2 4 6 8 Flow Rate (mL/min) liquid chromatography (HPLC) flow rate for achieving the best separation can vary from 0.7 mL/min to 2 mL/ min, as shown in the Van Deemter plot in Figure 3. In this figure, the HETP label on the y-axis refers to the height equivalent to a theoretical plate — the lower the HETP, the better the separation. As shown in the Van Deemter plot, the best separations are achieved with 3-µm packings and a flow rate of 1.5–2 mL/min. ICP-MS can handle 1.5-mL/min sample introduction rates without a problem, so the best separations are achieved with a 3-µm packing and an LC flow rate of 1.5 mL/min. These conditions are well suited for standard-bore columns. If a 3-µm packing is used in a narrow-bore column, a flow rate of 1.5 mL/min will result in a very high back pressure, typically too high to be run safely. This is the result of the smaller internal diameter of the column. As a result, narrow-bore columns usually are run with LC flow rates of 0.2–0.5 mL/min. As seen in the Van Deemter plot, these low flow rates do not provide the best separation, which means that using a narrow-bore column may involve sacrificing some separation capability. This compromise may or may not be a problem, depending upon the spacing between peaks. However, this usually can be 10m 5m 3m Figure 3: Van Deemter plots for various LC stationary phase particle diameters. compensated for by changing the mobile phase conditions. Narrow-bore columns offer two advantages compared with standard-bore columns: sharper peaks and less reagent consumption. The sharper peaks are a direct result of the narrower column width — there is less radial dispersion, which leads to sharper, more intense peaks than those obtained using standard-bore columns. The increased peak sharpness may compensate for the loss of resolution resulting from the nonoptimal flow rates. The increased peak height may also mean that lower levels of the species can be seen. A main drawback of narrow-bore columns versus standard-bore columns is that their capacity is reduced. Because fewer stationary phase particles can physically fit into the narrower column, there is less surface area available for species to bind. As a result, these columns can become overloaded easily, which could be a problem when analyzing unknowns. Typically, this reduced capacity is compensated for by diluting the samples more or using smaller injection volumes. Because less sample is injected onto the column, this may offset the advantage of increased peak height. Therefore, it is not always possible to measure lower levels with a narrow-bore column. Another potential disadvantage of narrow-bore versus standard-bore columns is sample throughput. Because lower LC flow rates are used for narrow-bore columns, separations tend to take longer than with standard-bore columns, which will affect sample throughput. The advantage of a lower LC flow rate is that there is less consumption of reagents and less waste to dispose. When deciding whether to use a standard-bore or narrow-bore column, users must consider which combination of factors is best for a particular laboratory and application. There is no single correct answer — each situation is different. HPLC Versus UHPLC for Speciation Within the past several years, a new form of LC has emerged: ultrahighpressure liquid chromatography (UHPLC). The Van Deemter plot shows that the smaller the particle size in the column, the better the separation. UHPLC uses columns with particle sizes less than 3 µm, typically 1.5–2.5 µm. These smaller particles provide more surface area for mobile phase and species to interact, which leads to better separation. Or, another way to think of it is that shorter columns with smaller packings can provide the same separation as longer columns with larger packings. The advantage of the shorter columns is that the time for separations are reduced greatly. The other signifcance of the smaller packings is a much higher back pressure, typically more than 10,000 psi. This increased pressure requires a special HPLC pump capable of handling it. Normal HPLC pumps cannot be used with columns containing packing material with a diameter of
www.spectroscopyonline.com November 2009 Spectroscopy 24(11) 33 analysis times required for the separation of biological molecules (such as proteins, peptides, and so forth), which can take up to an hour or more. Most typical speciation analyses with either standard- or narrow-bore columns can be accomplished in less than 10 min. Therefore, there would not be a great time savings. Users of UHPLC also must be aware of other factors associated with these columns. First, because the particles are so small and packed so tightly, the columns can clog easily. To eliminate particulates, all mobile phases and samples must be filtered before analysis. Because ICP-MS is such a sensitive detection method, filtering can introduce conatmination, that could raise the baseline. Also, filtering samples may facilitate the interconversion of species in solution. Therefore, the effects of filtering on species conversion must be studied and understood. The additional active sites available on UHPLC columns provide the increased separation capability, but this also means that these columns take longer to equilibrate. While a typical 3-µm column may take 20 min to equilibrate, a UHPLC column could take upwards of 45 min for proper equilibration. This increased time would have consequences when starting the system, changing mobile phases, running gradient methods, and performing method development. Another aspect to consider are the types of UHPLC columns available. Because UHPLC is relatively new and was developed primarily for separation of biological substances, columns that will separate inorganic species of interest may not be available. For example, there are few ion-exchange UHPLC columns available. Where UHPLC could be advantageous is in the emerging field of metallomics. This field involves the separation of biological substances using ICP-MS to detect inorganic molecules associated with proteins, peptides, and other biomolecules. UHPLC columns may be available to separate the substances of interest. Summary When performing speciation analysis, there are many aspects that must be considered: separation scheme, column type, column diameter, mobile phase, species of interest, required measurement levels of each species, sample throughput, and reagent usage and disposal. There is no single right answer for every situation. Each user must consider these factors when undertaking speciation analysis. Kenneth Neubauer is a Senior Scientist at PerkinElmer LAS, where he works with both ICP-MS and HPLC–ICP-MS. He received a B.A. in Chemistry from Colgate University, Hamilton, New York, and a Ph.D. in Analytical Chemistry from the University of Delaware, Newark. Ken joined PerkinElmer in 1997. For more information on this topic, please visit: www.spectroscopyonline.com
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