796 SAMPLE PREPARATION organic solvents requires indirect heating via the placement of a microwave-absorbing device in the extraction vessel. These devices are usually rods or a powder of a microwave-absorbing material such as carbon black coated with an inert polymeric substance (e.g., PTFE) that will not contaminate the sample. Multiple samples can be simultaneously processed in a microwave oven, so the sample throughput can be very high compared to some of the other liquid–solid extraction techniques. Commercial systems have the capacity to treat up to 24 extraction vessels. Microwave extraction is a widely accepted alternative to Soxhlet, PFE/ASE, and other liquid–solid extraction techniques. For example, EPA Method 3546 [86] is a MAE procedure for extracting water-insoluble or slightly water-soluble organic compounds (pesticides, herbicides, PCBs, etc.) from soils, clays, sediments, sludges, and solid wastes. Extraction takes place at 100 to 115 ◦ C and 50 to 175 psi in a closed vessel containing the sample and organic solvent(s). Analyte recoveries are equivalent to those from Soxhlet extraction (Method 3540 [87]), but use less solvent and take significantly less time. 16.9 COLUMN-SWITCHING Column-switching was discussed in Sections 2.7.6, 3.6.4.1, 9.3.10, and 13.4.5.3. It is a powerful technique for sample preparation (discussed here), as well as for two-dimensional separation (Section 9.3.10). For sample preparation, a portion of the chromatogram from an initial column (column 1; e.g., the enrichment column of Fig. 3.23) is selectively transferred to a second column (column 2; e.g., the analytical column of Fig. 3.23) for further separation. Column-switching for sample preparation is used for: • removal of ‘‘column killers’’ prior to column 2 • removal of late-eluters prior to column 2 • removal of interferences that can overlap analyte bands in column 2 • trace enrichment The achievement of one or more of these goals often results in increased sample throughput due to the presentation of a cleaner sample to the column. Unwanted interferences can be directed to waste or backflushed to prevent their transfer to the HPLC column. The goal of column-switching is separation of the analyte from interfering compounds by the initial column; that is, the same goal as for SPE. While column-switching is similar to the HPLC analysis of fractions provided by SPE, several advantages exist: • SPE cartridges are used only once and discarded; the initial column in column-switching is used repeatedly • the initial column has a higher efficiency (e.g., 5-μm d p ) compared to an SPE cartridge (e.g., 40-μm d p ) • less sample loss occurs with column-switching • many valve configurations are possible for heart cutting, backflushing, diverting contaminants directly to waste, and so forth. 16.10 SAMPLE PREPARATION FOR BIOCHROMATOGRAPHY 797 See Section 3.6.4.1 and Figure 3.23 for further discussion of column-switching and an example for trace enrichment. 16.10 SAMPLE PREPARATION FOR BIOCHROMATOGRAPHY In the separation of biomolecules, sample preparation almost always involves the use of one or more pretreatment techniques. As is the case for HPLC of other sample types, no one sample-preparation technique can be applied to all biological samples. The sample preparation approaches used in modern biochromatography are often the same techniques that were used in classical biochemistry, such as dialysis, chemical precipitation, column chromatography, and centrifugation. Currently there is a growing interest in not only the application of these classical approaches but also newer sample preparation technologies to the fields of molecular biology, biotechnology, and the various ‘‘omics’’ (e.g., proteomics, genomics, metabolomics). In these areas the samples are often complex, are available only in small quantities, and require the utmost care in handling. The requirements for the recovery of biopolymers with structural and functional integrity often demand that the sample preparation be rapid and gentle. The complex nature of most biological samples necessitates some form of preliminary sample manipulation to achieve better separation results and to prolong the life of the HPLC column. The actual recommended sample preparation tech- nique(s) will depend on the nature of the sample (e.g., molecular weight, presence of additives, endogenous interferences, particulates, or other unwanted components). In addition to filtration for particulate removal, chromatographic principles (including affinity) can be used to clean up biological samples. Table 16.10 provides a listing of sample preparation techniques that may be used in the flow-through mode using cartridge, disk, or column format. Some of these techniques can be performed in a batch mode, where the media is poured into the sample in a liquid form, allowed to stay in contact usually with agitation, and then is removed by filtration or by pouring off the liquid phase leaving behind the compound(s) of interest sorbed onto the stationary phase or contained in the liquid phase. Although slower than the column approach, batch sorption is easier to perform. The flow-through format is more widely used. Although dilution is a possible consequence, the flow-through column approach is more useful for removing the last traces of the analyte of interest. Convenient, pre-packed cartridges and membrane disks, that offer less flow resistance due to their large cross-sectional areas, are readily available from manufacturers. Sometimes kits are purchased that contain all the media, chemicals and accessories necessary to perform a cleanup process. Liquids can be transported through the flow-through devices with applied pressure, vacuum, or centrifugation. Many of the sample preparation techniques of Table 16.10 use retardation of ionic species by means of ion exchange or ion retardation. Other procedures use hydrophobic interaction and adsorption to retain macromolecules, while letting ionic compounds and smaller molecules pass through. Besides the chromatographic principles covered in Table 16.10, cleanup of biological samples while maintaining biological activity can be accomplished using other approaches. For example, dialysis is a time-tested process using membranes to clean up and desalt biological samples. Miniaturized dialysis kits permit the 798 SAMPLE PREPARATION Table 16.10 Sample-Preparation Techniques in Biochromatography Requirement Most Frequently Species Retained Typical Applications Used Approaches Antibody purification Affinity chromatography; hydroxylapatite chromatography IgG and subclasses IgG concentration in serum, ascites, and tissue culture media; fluorescent labeled antibodies with unreacted fluorescent tag. Buffer and reagent ultrapurification Ion exchange Trace cations and anions Removal of ions that cause band broadening or high background. Adsorption Trace organics Neutral PS-DVB, alumina, and silica will remove polar organics from buffers; water can be removed from organic solvents. Deionization Mixed-bed ion exchange Ions Deionization of carbohydrates before HPLC; separation of ionic contaminants from proteins; reagent preparation; separation of anions from carbohydrates, dextrans, and polyhydric alcohols. Proteins Deionization of proteins containing hydrophobic molecules. Desalting and buffer exchange Ion exchange Cations, anions Desalting amino acids for better TLC and HPLC analysis. Gel filtration Large molecules are eluted before salts and small molecules Desalting proteins and nucleic acids with masses > 6000 Da. Ion retardation Cations, anions Removal of salts and ionic detergent from protein and amino-acid samples. Reversed phase Hydrophobic analytes Desalting of polypeptide solutions. Detergent removal Ion exchange Cationic detergents; anionic detergents From proteins, enzyme reactivation. Adsorption Nonionic detergents Triton X-100 from protein solutions. Ion retardation Anionic detergents Excess SDS from samples. Metal concentration or removal Ion exchange Cations Removal of metals and salts from aqueous medium. 16.10 SAMPLE PREPARATION FOR BIOCHROMATOGRAPHY 799 Table 16.10 (Continued) Requirement Most Frequently Species Retained Typical Applications Used Approaches Chelating resins Polyvalent cations Removal of copper, iron, heavy metals, calcium, and magnesium. Adsorption Metal-organic complexes Metals complexed with polar or hydrophobic complexing agents. Particulate removal Filtration Particulate matter Pretreatment to protect HPLC frits and valves; filtration of culture medium. Plasmid purification, probe cleanup Gel filtration Low-molecular- weight contaminants Removal of unincorporated radioactive nucleotides from labeling reaction mixture. Adsorption Large DNA Removal of RNA, protein, and other cellular compounds. Ion exchange Ethidium bromide or propidium iodide Removal from plasmid visualization experiments. Protein concentration Ion exchange Water Improve sensitivity of electrophoretic and HPLC analysis. Proteins Separation of proteins and low molecular weight substances. Adsorption Hydrophobic proteins C 18 solid-phase extraction to remove hydrophobic proteins from hydrophilic proteins. Removal or concentration of anions and cations Ion exchange Cations, anions Removal of ions from aqueous solutions; concentration of large proteins; removal of mineral acids. Removal or concentration of organics Adsorption Polar organic Removal of nonionic detergents and lipids; separation of ethidium bromide from nucleic acid preparations. Gel filtration (organic) High-molecular- weight compounds are eluted before small molecules Separation of soluble organic compounds with masses <150,000 Da from complex sample matrices. 800 SAMPLE PREPARATION efficient dialysis of small sample volumes. Electrodialysis is a more rapid approach for desalting or buffer exchange that is a gentle technique and provides excellent desalting without loss of biological activity of proteins. Ultrafiltration (UF) uses centrifugation as the driving force for membrane filtration. Membrane filters with molecular weigh cutoffs in the tens of KDa are held in a centrifuge cartridge that allows solvent, salts, and small molecules to pass through the membrane while macromolecules larger than the cutoff value are retained and concentrated above the membrane. Because the membranes are selected to show low nonspecific adsorption, UF results in good recovery and little loss of biological activity. Many of the sample-preparation techniques already covered, such as liquid– liquid extraction (Section 16.5), are directly applicable to the fractionation of biological samples. Often the combination of two sample preparation procedures results in an overall improvement in cleanup efficiency. For example, the use of chromatographic media combined with dialysis can provide excellent concentration of protein solutions. With the advent of proteomics, biological researchers are searching for new drug targets and biomarkers of disease at very low concentrations in human serum/plasma. Unfortunately, the predominant, less interesting proteins such as human serum albumin (HSA) and immunoglobulin (IgG) account for a high per- centage of protein in these body fluids and are orders of magnitude more concentrated than the low abundance proteins that need to be identified and quantified. Clas- sical techniques for removal of the high abundance proteins usually deplete only one or a few of them. New affinity-based products can deplete from 6 to 20 high-to-medium-abundance proteins leaving the thousands of lower-abundance pro- teins for further pretreatment. Affinity-based products contain antibodies that are specific for high-abundance proteins, and these products are available both as spin tubes and flow-through columns [65]. High-abundance proteins are retained on the column while low-abundance proteins pass through the column for collection and subsequent concentration by trace-enrichment techniques. The high-abundance proteins are then released by a change of buffer and can be either discarded or saved. Finally, the column is regenerated and can be re-used for several hundred injections. A combination of affinity depletion and multidimensional LC-MS/MS has been used to investigate trace levels of up- and down-regulated proteins in biological fluids [88]. 16.11 SAMPLE PREPARATION FOR LC-MS To obtain the maximum performance from LC-MS and LC-MS/MS, sample pre- treatment usually is required. When MS/MS was first introduced, it was widely believed that its specificity of would eliminate the need for sample preparation. Thus HPLC was regarded as little more than a sample-introduction tool. However, it was na ¨ ıve to assume that the MS alone would be able to solve all separation problems, and in subsequent years it was realized that both chromatographic separation prior to the detector and sample pretreatment prior to injection were required to avoid certain problems, such as previously unrecognized ‘‘matrix effects’’ in LC-MS and LC-MS/MS. 16.11 SAMPLE PREPARATION FOR LC-MS 801 Sample pretreatment can be used to minimize problems associated with the sample and/or sample matrix, such as: • spectral interference • system compromise • adduct formation • ion suppression Ions that appear at the same or nearly the same m/z value as the component of interest can cause spectral interference. Various MS detector designs exist (Section 4.14), with differing ability to distinguish between ions of similar m/z.TheMS detectors used for routine HPLC analysis are of sufficiently low mass resolution that sample pretreatment may be required to remove co-eluting compounds that have similar m/z values to the analyte(s) of interest. System compromise can occur when non-volatile sample or mobile-phase components precipitate in the LC-MS interface and degrade detector perfor- mance. The mobile-phase components that are most likely to precipitate in the atmospheric-pressure- or electrospray-ionization source are buffer salts and ion-pair reagents, so volatile buffers and reagents are used to solve this problem. Sample matrix elements, such as proteins, can also precipitate in the interface. Proteins are most commonly removed by precipitation, LLE (Section 16.5), or SPE (Section 16.6). Additional protein removal techniques are summarized in Table 16.11. Adduct formation between another ion and the component of interest shifts the m/z value at which the component of interest appears in the spectrum. Adducts can be beneficial or detrimental, but in either case the amount of adduct formation needs to be controlled. Adduct ions such as sodium, potassium, and ammonium can originate from the sample itself, from reagents, or even from the container holding the sample. Adduct formation also can be used as a way to improve signals for macromolecules. However, uncontrolled adduct formation generally is undesirable and may require specific sample preparation procedures to reduce or eliminate it. Techniques for the removal of ions are listed in Table 16.10. Ion suppression results when interferences are present that suppress (or com- pete with) the ionization of the analyte. Ion suppression is the most critical MS interference because it can be caused by components that do not appear in the mass spectrum. Phospholipids are one class of matrix components that are especially potent in causing ion suppression; titania sorbents can specifically remove these compounds [101]. In biological samples the natural variation in endogenous com- pound concentrations from one sample to another can cause varying levels of ion suppression. This variation in turn contributes to unacceptable variability in the sig- nal response for the compounds of interest. Another type of ion suppression occurs when very strong ion-pairs are formed that are not broken apart in the API interface. Ion-pairing agents, such as trifluoroacetic acid, have been shown to contribute to ion suppression [102], and therefore their use in LC-MS should be avoided where possible. LLE (Section 16.5) and SPE (Section 16.6) are two techniques commonly used to remove ion-suppressing materials from the sample. 802 SAMPLE PREPARATION Table 16.11 Techniques for Removal of Protein from Biological Fluids Protein Removal Principle Reference(s) Technique Precipitation Organic solvent (e.g., ACN), acid solution (e.g., perchloric), or salt solution (e.g., sodium sulfate) is added with agitation to a solution of plasma. The protein precipitates and forms a bead upon centrifugation. Supernatant can be analyzed by HPLC. 89–90 Restricted access media (RAM) Solution containing protein is injected into a RAM column to separate protein from small molecules. See Section 16.6.7.2. 50–54 Turbulent flow chromatography A large particle (∼50−um) small diameter bonded silica RP column (1-mm i.d.) is run at high linear velocities (up to 8-mL/min). Although not truly turbulent flow, these high linear velocities do not allow the slower diffusing proteins to penetrate the packing pores, and they are flushed to waste. Smaller molecules are retained within the pores by RPC and are eluted to an analytical column or the MS interface. 91–92 Ion-exchange chromatography Proteins are retained on an ion-exchange column at the proper pH; uncharged small molecules may pass through unretained onto an analytical column. 93–94 Size-exclusion chromatography By selection of the appropriate pore size, proteins can be excluded and elute from the column first while the small molecular weight compounds elute later. 95–96 Reversed phase chromatography The use of a C3 or C4 phase on wide-pore silica will retain proteins and have less retention of polar drugs, which can be eluted first. 97–98 High-abundance protein depletion High-abundance proteins (up to 20) from human and other plasma/serum samples are depleted by antibody affinity phases. See Section 16.6.7.4. 99–100 16.12 DERIVATIZATION IN HPLC Derivatization involves a chemical reaction between an analyte and a reagent to change the chemical and physical properties of the analyte. The four main uses of derivatization in HPLC are to: • improve detectability • change the molecular structure or polarity of analyte for better chromatog- raphy • change the matrix for better separation • stabilize an analyte 16.12 DERIVATIZATION IN HPLC 803 Ideally a derivatization reaction should be rapid, quantitative, and produce minimal by-products. Excess reagent should not interfere with the analysis or be easily removed from the reaction matrix. With the increased popularity of LC-MS and LC-MS/MS, especially in the field of bioanalysis, many laboratories prefer this approach to high sensitivity and selective detection, rather than contend with the relatively time-consuming, labor-intensive approach of compound derivatization. Derivatization often is a last Table 16.12 Functional Group and Derivatization Reagents Functional Group UV Derivatives a,b Fluorescent Derivatives a,c Carboxylic acids PNBDI BrMaC Fatty acids Phosphonic acids DNBDI BrMmC PBPB Alcohols DNBC Dabsyl-Cl NIC-1 Aldehydes PNBA Dansyl hydrazine Ketones DNBA Amines, 1 ◦ Fluorescamine OPA Amines, 1 ◦ and 2 ◦ DNBC NBD-Cl SNPA NBD-F SDNPA Dansyl-Cl Dabsyl-Cl NIC-1 Amino acids (peptides) SBOA Fluorescamine SDOBA OPA Dabsyl-Cl NBD-Cl NBD-F Dansyl-Cl Isocyanates PNBPA DNBPA Phenols DNBC NBD-Cl Dabsyl-Cl NBD-F NIC-1 Dansyl-Cl Thiols Dabsyl-Cl NBD-Cl NBD-F OPA a See Table 16.13 for list of derivatives. b Typically aromatic derivatives enhancing UV detection at 254 nm. c Typically aromatic derivatives for enhanced fluorescence detection. 804 SAMPLE PREPARATION resort when developing a method. The introduction of pre- or post-column reaction that provides sample derivatization adds complexity, other sources of error to the analysis, and increases the total analysis time. While these procedures can be automated, the analyst must ensure that the derivatization step is quantitative (if necessary) and that there are no additional impurities introduced in the analysis. Although derivatization has its drawbacks, it may still be required to solve a specific separation or detection problem—as when mass spectral detection is not available. Reagents are available that react selectively with specific functional groups to form derivatives with enhanced UV- or fluorescence-detection characteristics. Some of the more common functional groups that can be reacted are listed in Table 16.12; the reagents are listed in Table 16.13. Figures 4.13, 4.36, and 4.37 show examples of derivatization to enhance fluorescence detection. In addition to derivatization to enhance detection, derivatization is used to enable separation of enantiomers, such as by the use of the reagents listed in Table 14.1. For more information on the use of derivatization in HPLC, see Sections 4.16 and 14.3, or consult one of the books dedicated to derivatization [103–108]. Table 16.13 Derivatization Reagents UV Derivatives Fluorescent Derivatives Dabsyl-Cl 4-Dimethylaminiazobenzene- 4-sulphinyl NBD-Cl 7-Chloro-4-nitrobenzo-2-oxa- 1,3-diazole DNBA 3,5-Dinitrobenzyloxyamine hydrochloride NBD-F 7-Fluoro-4-nitrobenzo-2-oxa- 1,3-diazole NIC-1 1-Naphthylisocyanate Fluorescamine 4-Phenylsprio(furan-2(3H),1 - phthalan-3,3-dionePBPB p-bromophenacyl bromide PNBA p-Nitrobenzyloxyamine hydrochloride OPA o-Phthaldehyde Dansyl-Cl 5-Dimethylaminonaphthalene-1- sulfonyl chloridePNBDI p-Nitrobenzyl-N,N - diisopropylisourea BrMmC 4-Bromomethyl-7- methoxycoumarinDNBDI 3,5-Dinitrobenzyl-N,N - diisopropylisourea BrMaC 4-Bromomethyl-7- acetoxycoumarinPNBPA P-Nitrobenzyl-N-n-propylamine hydrochloride DNBPA 3,5-Dinitrobenzyl-N-n- propylamine hydrochloride SNPA N-Succinimidyl-p- nitrophenylacetate SDNPA N-Succinimidyl-3,5- dinitrophenylacetate DNBC 3,5-Dinitrobenzyl chloride Note: Functional group reactivity is listed in Table 16.12. REFERENCES 805 REFERENCES 1. P. 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Unwanted interferences can be directed to waste or backflushed to prevent their transfer to the HPLC column. The. endogenous interferences, particulates, or other unwanted components). In addition to filtration for particulate removal, chromatographic principles (including affinity) can be used to clean up biological