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《美国FDA农残分析手册》（第一卷英文版）Chapter 6 HPLC

In recent years, high performance liquid chromatography (HPLC) has grown in popularity as a determinative step for residue analysis, until today it is accepted as complementary to the more traditional gas liquid

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SECTION 601 Pesticide Analytical Manual Vol I system 601d Pump B Thermostatted Block Diagram of PLC Syster Column Reprinted with permission of West, C D(1987)Essentials of Thermostatted Quantitative Analysis Figure 14.1 Recorder or detector oven For analytical HPLC, typical flow rates of 0.5-5 mL/min are produced by pumps operating at 300-6000 psi. Although pumps are capable of high pressure opera- duce 1000-2000 psi at I mL/min High pressures should. packings apically pro- avoided because they contribute to limited column life expectancies Sample extract is applied to the column from an injector valve containing a loop that has been filled with sample solution from a syringe. After passing through the column, the separated analytes are sensed by visible/UV absorption, fluorescence, electrochemical, photoconductivity, or rI detectors. To minimize extra-column peak spreading, the instrument components must be connected using low dead volume(ldv) fittings and valves and tubing as short and narrow in bore as possible Analytical HPLC may use either isocratic or gradient elution methods. Isocratic elution uses a mobile phase of constant composition, whereas the strength of the mobile phase in gradient elution is made to increase continually in some prede- termined manner during the separation. Gradient elution, which requires an automatic electronic programmer that pumps solvent from two or more reservoirs, reduces analysis time and increases resolution for complex mixtures in a manner similar to temperature programming in GLC. Gradient elution capability is highly recommended for systems to be used for residue determination. However it is not always possible to employ gradient elution because some HPLC column/solvent systems and detectors are not amenable to the rapid solvent and pressure changes involved Stationary phases are uniform, spherical, or irregular porous particles having nominal diameters of 10, 5, or 3 um. Bonded phases produced by chemically bonding different functional groups to the surface of silica gel are most widely used,along with unmodified silica gel and size exclusion gels. Columns are usually stainless steel, 3-25 cm long and 4.6 mm id, prepacked by commercial manufac turers. There has been increasing use of microbore columns having diameters sz mm. Although many HPLC separations can be carried out at ambient tempera- ture, column operation in a thermostatted column oven is necessary for reproduc- ible, quantitative results, because distribution coefficients and solubilities are tem- perature dependent. Depending on the nature of the analyte(s), certain additional equipment may be quired. For example, apparatus and reagents for performing post-column 6o1- Form FDA 2905a(6/92]

SECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–6 Form FDA 2905a (6/92) For analytical HPLC, typical flow rates of 0.5-5 mL/min are produced by pumps operating at 300-6000 psi. Although pumps are capable of high pressure operation, state-of-the-art 25 cm × 4 mm id columns with 5 µm packings typically produce 1000-2000 psi at 1 mL/min. High pressures should be avoided because they contribute to limited column life expectancies. Sample extract is applied to the column from an injector valve containing a loop that has been filled with sample solution from a syringe. After passing through the column, the separated analytes are sensed by visible/UV absorption, fluorescence, electrochemical, photoconductivity, or RI detectors. To minimize extra-column peak spreading, the instrument components must be connected using low dead volume (ldv) fittings and valves and tubing as short and narrow in bore as possible. Analytical HPLC may use either isocratic or gradient elution methods. Isocratic elution uses a mobile phase of constant composition, whereas the strength of the mobile phase in gradient elution is made to increase continually in some predetermined manner during the separation. Gradient elution, which requires an automatic electronic programmer that pumps solvent from two or more reservoirs, reduces analysis time and increases resolution for complex mixtures in a manner similar to temperature programming in GLC. Gradient elution capability is highly recommended for systems to be used for residue determination. However, it is not always possible to employ gradient elution because some HPLC column/solvent systems and detectors are not amenable to the rapid solvent and pressure changes involved. Stationary phases are uniform, spherical, or irregular porous particles having nominal diameters of 10, 5, or 3 µm. Bonded phases produced by chemically bonding different functional groups to the surface of silica gel are most widely used, along with unmodified silica gel and size exclusion gels. Columns are usually stainless steel, 3-25 cm long and 4.6 mm id, prepacked by commercial manufacturers. There has been increasing use of microbore columns having diameters ≤2 mm. Although many HPLC separations can be carried out at ambient temperature, column operation in a thermostatted column oven is necessary for reproducible, quantitative results, because distribution coefficients and solubilities are temperature dependent. Depending on the nature of the analyte(s), certain additional equipment may be required. For example, apparatus and reagents for performing post-column Figure 601-d Block Diagram of HPLC System [Reprinted with permission of McGraw-Hill Book Company, from West, C.D. (1987) Essentials of Quantitative Analysis, Figure 14.1, page 346.] Pump A Solvent A Pump B Solvent B Sample injection system Gradient device Thermostatted column oven Column Thermostatted detector oven Detector Amplifier Recorder or readout device

Pesticide Analytical Manual Vol. I SECTION 601 derivatization, as used in Section 401 for N-methylcarbamates, may be needed to convert analytes to compounds that can be detected with the required sensitivity and/or selectivity HPLC System Plumbing Band broadening can occur not only in the analytical and guard columns, but also in dead volume in the injector, detector, or plumbing connecting the various components of the HPLC system. Thi effect, called extra-column dispersion Figure 601 must be minimized for high efficiency Column Outlet Fittings The proper choice and use of tubing and fittings are critical in this regard 14 Fittings. Figure 601-e illustrates three of column fittings. The conventi ional fitting (i)used in GLC and general laboratory plumbing has excessive dead volume. It has been modified to produce a zero dead vol 1/16→> ume (zdv) fitting (ii) in which the metal column and the tubing are butted up directly against the stainless (0) Conventional reducing union (dead volume is steel frit. There is evidence that the shaded): (] zdv union; (n]ldv union nature of the tubing connection in the ed with permission of John Wiley and Sons, Inc, from zdv fitting may lead to some loss in s[1987] High Performance Liquid Chromatography efficiency, especially if the connection 3a, page 28.] is not made carefully. The ldv fitting (iii)improves efficiency by use of a cone-shaped distributor connecting the gauze or frit at the end of the column with the tubing. a typical dead volume for the ldv fitting is 0.I HL Columns are usually d from Figure 601-f anufacturers with a 1/4-1/16"zdv or ldv Low Dead Volume Fitting outlet fitting and a 1/4"nut and cap or a reducing union at the inlet (i.e,,not 1/4·od 1/4" in size, but suitable for 1/4"tubing) Figure 601-f shows a complete ldv fitting connection between a column and a -Ferrule detector. The column fits snugly inside 2 um porous frit the stainless steel end fitting and is sealed y≤-1/4 end fitting by a high compression ferrule. A 2 um porous frit is firmly seated between the 1"d column and end fitting. The column and detector are connected by a short length of stainless steel (or polymer) tubing KTo detector The column is also connected to the jection valve using a zdv or ldv fitting from lc-102 audiovisual nd Sloane, Savant and a short length of stainless steel tub-

SECTION 601 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 601–7 Pesticide Analytical Manual Vol. I Stainless steel frit, 2 µm 1/4" stainless steel tubing 1/16" tubing (i) (ii) (iii) derivatization, as used in Section 401 for N-methylcarbamates, may be needed to convert analytes to compounds that can be detected with the required sensitivity and/or selectivity. HPLC System Plumbing Band broadening can occur not only in the analytical and guard columns, but also in dead volume in the injector, detector, or plumbing connecting the various components of the HPLC system. This effect, called extra-column dispersion, must be minimized for high efficiency. The proper choice and use of tubing and fittings are critical in this regard. Fittings. Figure 601-e illustrates three types of column outlet fittings. The conventional fitting (i) used in GLC and general laboratory plumbing has excessive dead volume. It has been modified to produce a zero dead volume (zdv) fitting (ii) in which the metal column and the tubing are butted up directly against the stainless steel frit. There is evidence that the nature of the tubing connection in the zdv fitting may lead to some loss in efficiency, especially if the connection is not made carefully. The ldv fitting (iii) improves efficiency by use of a cone-shaped distributor connecting the gauze or frit at the end of the column with the tubing. A typical dead volume for the ldv fitting is 0.1 µL. Columns are usually received from manufacturers with a 1/4–1/16" zdv or ldv outlet fitting and a 1/4" nut and cap or a reducing union at the inlet (i.e., not 1/4" in size, but suitable for 1/4" tubing). Figure 601-f shows a complete ldv fitting connection between a column and a detector. The column fits snugly inside the stainless steel end fitting and is sealed by a high compression ferrule. A 2 µm porous frit is firmly seated between the column and end fitting. The column and detector are connected by a short length of stainless steel (or polymer) tubing. The column is also connected to the injection valve using a zdv or ldv fitting and a short length of stainless steel tubing. (i) Conventional reducing union (dead volume is shaded); (ii) zdv union; (iii) ldv union. [Reprinted with permission of John Wiley and Sons, Inc., from Lindsay, S. (1987) High Performance Liquid Chromatography, Figure 2.3a, page 28.] Figure 601-e Column Outlet Fittings Column (1/4" od) Ferrule 2 µm porous frit 1/4" end fitting 0.01" id To detector [Reprinted with permission of Howard Sloane, Savant, from LC-102 audiovisual program.] Figure 601-f Low Dead Volume Fitting

SECTION 601 Pesticide Analytical Manual Vol I Figure 601-g External column end fittings( Figures 601-e and 601 Standard Internal Fitting f), which were formerly popular, are not durable during repeated attachments and removals. Thus, the internal fitting is practically standard today. This uses female threads in the fitting body and a male Colum nut( Figure 601-g) Unions. Unions are fittings that connect two pieces of tubing. The most commonly used type is the is not drilled through completely, but a short(0.02") web of metal is left between the twe Union ing with a small diameter (approximately 0.02 or 0.01) hole drilled through. Even though the tub ing ends do not butt against each other as in early zdv unions, there is essentially no dead volume added 1/16 tubing→> to the system through their use. For this reason they are commonly classified as zdv unions. This type of union has fewer assembly, re-assembly, and tubing interchange problems than the early butt- Inc.from Meyer. V.R.(1988 together zdv type High Performance Liquid raphy. Figure 6. 18, page B0. Assembly of Fittings. Fittings consist of four parts the body, tubing, ferrule, and nut. The nut and ferrule are slid onto the tube end, the tube is pushed all the way into the fittin body and held there securely, the nut is finger-tightened, and then another three- uarter turn is made with a wrench. This procedure should assure that the ferrule is pressed ("swaged")onto the tub- ing. To replace the ferrule, the tub- Figure 601-h ig m nd the fit Internal Thread Low dead volume Fitting made. When using fittings to con nect system components, the nut hould be finger-tightened and then ufficient to complete the seal Over- ightening of nuts can lead to fit ting distortion and leaks [Reprinted with permission of Aster Publishing Corporation, from Dolan, J.W., and Upchurch, P [1988]LCGC 6 Fitting components from different Figure 3. page 788. manufacturers have dissimilar de- signs,sizes, and thread types and are usually not interchangeable. Ferrules from different manufacturers have unique shapes, but they are usually interchangeable because the front edge is deformed when pressed onto the tubing. However, as a general rule, it is best to purchase all fittings and spare parts from one manufac- turer. Even fittings from a given manufacturer differ slightly because of manufac- tolerances. However. this is of concern onl ith microbore columns for which dead volume is a greater consideration. For these columns, it is best to not even interchange fittings from the same manufacti A variety of fittings are available that can be finger-tightened to the degree nec essary to seal stainless steel tubing at 2000-6000 psi. All of these are based on the use of polymeric ferrules, but some have a steel nut, whereas others are all plastic 6o1-8 Form FDA 2905a(6/92]

SECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–8 Form FDA 2905a (6/92) External column end fittings (Figures 601-e and 601- f), which were formerly popular, are not durable during repeated attachments and removals. Thus, the internal fitting is practically standard today. This uses female threads in the fitting body and a male nut (Figure 601-g). Unions. Unions are fittings that connect two pieces of tubing. The most commonly used type is the internal thread ldv type (Figure 601-h). The union is not drilled through completely, but a short (0.02") web of metal is left between the two pieces of tubing with a small diameter (approximately 0.02 or 0.01") hole drilled through. Even though the tubing ends do not butt against each other as in early zdv unions, there is essentially no dead volume added to the system through their use. For this reason, they are commonly classified as zdv unions. This type of union has fewer assembly, re-assembly, and tubing interchange problems than the early butttogether zdv type. Assembly of Fittings. Fittings consist of four parts: the body, tubing, ferrule, and nut. The nut and ferrule are slid onto the tube end, the tube is pushed all the way into the fitting body and held there securely, the nut is finger-tightened, and then another threequarter turn is made with a wrench. This procedure should assure that the ferrule is pressed (“swaged”) onto the tubing. To replace the ferrule, the tubing must be cut and the fitting remade. When using fittings to connect system components, the nut should be finger-tightened and then tightened a one-half turn more with a wrench. If leaking is observed, slightly more tightening should be sufficient to complete the seal. Overtightening of nuts can lead to fitting distortion and leaks. Fitting components from different manufacturers have dissimilar designs, sizes, and thread types and are usually not interchangeable. Ferrules from different manufacturers have unique shapes, but they are usually interchangeable because the front edge is deformed when pressed onto the tubing. However, as a general rule, it is best to purchase all fittings and spare parts from one manufacturer. Even fittings from a given manufacturer differ slightly because of manufacturing tolerances. However, this is of concern only with microbore columns, for which dead volume is a greater consideration. For these columns, it is best to not even interchange fittings from the same manufacturer. A variety of fittings are available that can be finger-tightened to the degree necessary to seal stainless steel tubing at 2000-6000 psi. All of these are based on the use of polymeric ferrules, but some have a steel nut, whereas others are all plastic. [Reprinted with permission of Aster Publishing Corporation, from Dolan, J.W., and Upchurch, P. (1988) LC-GC 6, Figure 3, page 788.] Column Frit Union 1/16" tubing Figure 601-g Standard Internal Fitting [Reprinted with permission of John Wiley and Sons, Inc., from Meyer, V.R. (1988) Practical High Performance Liquid Chromatography, Figure 6.18, page 80.] Figure 601-h Internal Thread Low Dead Volume Fitting

Pesticide Analytical Manual Vol. I SECTION 601 They are used mostly on frequently attached and detached high pressure connec tions, such as between the injector and column or column and detector, and for polymer tubing waste lines from the injector or detector Fittings must be kept free of silica particles, which may scratch surfaces between the ferrule and union and cause leaks Tubing. Stainless steel tubing is available commercially that is supposedly ready for immediate use in HPLC systems. It is machine cut, polished, and deburred to provide perfectly square ends. It is also cleaned by sonication, passivated, washed and rinsed with a solvent such as isopropanol to eliminate residual dirt or oils Despite this careful preparation, it is a wise precaution to rinse new tubing with mobile phase under operating pressure before using it as part of the HPLC system The most commonly used tubing for connecting components of the chromato- graph is 316 stainless steel, 1/16"od, with different inside diameters. Tubing with 0.01(0.25 mm)id is commonly used in areas where dead volume must be mini- mized to maximize efficiency, e. g, between the injector and column, precolumn damping spiral mns in series, and column and detector, and for preparing pulse and column coli Typical lengths of tubing connections are 3-6 c. Tubing with 0.005 or 0.007"id is used to connect microbore or short 8 um particle size columns to detectors and injectors Filtering of samples and solvents is especially critical to prevent clogging of this narrow bore tubing. Tubing with 0.02-005"id is available when ldv is not important and low resistance to flow and pressure drop is desirable. For example, I mm(0.04) tubing is often used between the pump and sample injector. Tubing can be cut to any required length in the laboratory, but it is important not to distort the interior or exterior during the process. The simplest method is to score the tubing completely around the outside with a file and then bend it back and forth while holding it on either side of the score with two smooth-jawed pliers The ends are filed smooth and deburred, and the tubing is thoroughly washed with solvent. If the bore should become closed by the bending and filing, the tube can be reamed out with an appropriate drill bit before final smoothing and wash ing. A number of types of manual and motorized tubing cutters are available from hromatography accessory suppliers. Proper cutting of tubing to make leak-free connections is an art that requires considerable practice lthough stainless steel tubing and fittings are standard for systems using organic and salt-free aque solvents, corrosion becomes a problem with buffers contain- ing salts, particularly halide salts at low pH. HPLC companies have available a ariety of accessories that can solve this problem. These include titanium high pressure system components, for use in the flow stream at all points of mobile phase contact, and titanium or polymeric fluorocarbon tubing with id values simi- is Tefzel(ethylene-tetrafluoroethyler copolymer), which can withstand pressures of 5000 psi or higher (Teflon is lim- ited to pressures <1000 psi. Titanium and polymeric plumbing components are especially valuable for biochemical HPLC and ion chromatography Reference 3 is a valuable source of information to help avoid many tubing instal- lation problems

SECTION 601 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 601–9 Pesticide Analytical Manual Vol. I They are used mostly on frequently attached and detached high pressure connections, such as between the injector and column or column and detector, and for polymer tubing waste lines from the injector or detector. Fittings must be kept free of silica particles, which may scratch surfaces between the ferrule and union and cause leaks. Tubing. Stainless steel tubing is available commercially that is supposedly ready for immediate use in HPLC systems. It is machine cut, polished, and deburred to provide perfectly square ends. It is also cleaned by sonication, passivated, washed, and rinsed with a solvent such as isopropanol to eliminate residual dirt or oils. Despite this careful preparation, it is a wise precaution to rinse new tubing with mobile phase under operating pressure before using it as part of the HPLC system. The most commonly used tubing for connecting components of the chromatograph is 316 stainless steel, 1/16" od, with different inside diameters. Tubing with 0.01" (0.25 mm) id is commonly used in areas where dead volume must be minimized to maximize efficiency, e.g., between the injector and column, precolumn and column, columns in series, and column and detector, and for preparing pulse damping spirals. Typical lengths of tubing connections are 3-6 cm. Tubing with 0.005 or 0.007" id is used to connect microbore or short 3 µm particle size columns to detectors and injectors. Filtering of samples and solvents is especially critical to prevent clogging of this narrow bore tubing. Tubing with 0.02-0.05" id is available when ldv is not important and low resistance to flow and pressure drop is desirable. For example, 1 mm (0.04") tubing is often used between the pump and sample injector. Tubing can be cut to any required length in the laboratory, but it is important not to distort the interior or exterior during the process. The simplest method is to score the tubing completely around the outside with a file and then bend it back and forth while holding it on either side of the score with two smooth-jawed pliers. The ends are filed smooth and deburred, and the tubing is thoroughly washed with solvent. If the bore should become closed by the bending and filing, the tube can be reamed out with an appropriate drill bit before final smoothing and washing. A number of types of manual and motorized tubing cutters are available from chromatography accessory suppliers. Proper cutting of tubing to make leak-free connections is an art that requires considerable practice. Although stainless steel tubing and fittings are standard for systems using organic and salt-free aqueous solvents, corrosion becomes a problem with buffers containing salts, particularly halide salts at low pH. HPLC companies have available a variety of accessories that can solve this problem. These include titanium high pressure system components, for use in the flow stream at all points of mobile phase contact, and titanium or polymeric fluorocarbon tubing with id values similar to stainless steel. One such polymer is Tefzel (ethylene-tetrafluoroethylene copolymer), which can withstand pressures of 5000 psi or higher. (Teflon is limited to pressures <1000 psi.) Titanium and polymeric plumbing components are especially valuable for biochemical HPLC and ion chromatography. Reference 3 is a valuable source of information to help avoid many tubing installation problems

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