《美国FDA农残分析手册》（第一卷英文版）Chapter 6 HPLC

SECTION 601 Pesticide Analytical Manual Vol I System Leaks. Leaks are relatively easy to detect in LC instruments because liquid will be visible around a loose fitting. A loss of system pressure when using a constant volume pump is a common sign that a leak may be present. If this occurs, all fittings, especially sample valve and column fittings, should be checked and tightened if necessary with two open-ended wrenches. Care must be taken not to overtighten. If leaking does not stop, the faulty fitting must be replaced. 601D: SOL VENTS AND REAGENTS The mobile phase in HPLC is chosen for its ability, in combination with a particu- lar column, to provide the required separation of the analyte(s). The solvents used to prepare the mobile phase must be of high purity, most often HPLC grade, spectrophotometric grade, or distilled from all-glass apparatus. Other factors of importance include cost, viscosity, toxicity, boiling point, compressibility, UV trans- parency (if a UV detector is used), RI (if an ri detector is used), vapor pressure, flash point, odor, inertness with respect to sample compounds, and ability to cause corrosion. Choices of solvents and reagents cannot be made without careful con- sideration of the effect their presence can have on the entire HPLC system Solvents and reagents used in the hPlC determinative step and in sample prepa- ration procedures preceding HPLC should not: 1)cause degradation or unintended reaction of the analyte(s) 2)cause the solvent delivery system to malfunction; B)cause damage to the analytical column; 4)cause damage to the detector; or 5)contribute noise or increased or decreased detector response for the Potential Problems Many of the problems with mobile phases arise because of the presence of impu rities,additives, dust or other particulate matter, or dissolved air. Examples of some specific potential problems with solvents and reagents and suggested solu tions follow Degradation. Analytes can be degraded by solvents and reagents used in the ex- traction and cleanup steps of the analysis, or in the HPLC step itself. Analyte hemistry is usually known in advance, and reagents likely to cause degradation can be avoided. Unexpected reaction of the analyte(s) will usually be de strated by poor or no recovery of the compound (s)through the method, or by detection of additional reaction products in the determinative step The presence of impurities in solvents or reagents is often the cause of such unexpected reactions. For example, traces of oxidizing agents in solvents have been found to degrade N-methylcarbamates prior to their determination by HPlC Purity of all reagents used in trace-level determinations should always be as high as possible. 61-10

SECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–10 Form FDA 2905a (6/92) System Leaks. Leaks are relatively easy to detect in LC instruments because liquid will be visible around a loose fitting. A loss of system pressure when using a constant volume pump is a common sign that a leak may be present. If this occurs, all fittings, especially sample valve and column fittings, should be checked and tightened if necessary with two open-ended wrenches. Care must be taken not to overtighten. If leaking does not stop, the faulty fitting must be replaced. 601 D: SOLVENTS AND REAGENTS The mobile phase in HPLC is chosen for its ability, in combination with a particu￾lar column, to provide the required separation of the analyte(s). The solvents used to prepare the mobile phase must be of high purity, most often HPLC grade, spectrophotometric grade, or distilled from all-glass apparatus. Other factors of importance include cost, viscosity, toxicity, boiling point, compressibility, UV trans￾parency (if a UV detector is used), RI (if an RI detector is used), vapor pressure, flash point, odor, inertness with respect to sample compounds, and ability to cause corrosion. Choices of solvents and reagents cannot be made without careful con￾sideration of the effect their presence can have on the entire HPLC system. Solvents and reagents used in the HPLC determinative step and in sample prepa￾ration procedures preceding HPLC should not: 1) cause degradation or unintended reaction of the analyte(s); 2) cause the solvent delivery system to malfunction; 3) cause damage to the analytical column; 4) cause damage to the detector; or 5) contribute noise or increased or decreased detector response for the analyte. Potential Problems Many of the problems with mobile phases arise because of the presence of impu￾rities, additives, dust or other particulate matter, or dissolved air. Examples of some specific potential problems with solvents and reagents and suggested solu￾tions follow. Degradation. Analytes can be degraded by solvents and reagents used in the ex￾traction and cleanup steps of the analysis, or in the HPLC step itself. Analyte chemistry is usually known in advance, and reagents likely to cause degradation can be avoided. Unexpected reaction of the analyte(s) will usually be demon￾strated by poor or no recovery of the compound(s) through the method, or by detection of additional reaction products in the determinative step. The presence of impurities in solvents or reagents is often the cause of such unexpected reactions. For example, traces of oxidizing agents in solvents have been found to degrade N-methylcarbamates prior to their determination by HPLC. Purity of all reagents used in trace-level determinations should always be as high as possible

Pesticide Analytical Manual Vol. I SECTION 601 Dissolved Gases. The presence of dissolved gases in solvents composing the mo- bile phase is a major cause of practical problems in HPLC Gas bubbles can collect in pumps, the detector cell, or other locations in the HPLC system. This can affec the reproducibility of the volume delivered by the pump, or large bubbles may completely stop the pump from working. Detection can be affected in various ways. With the Uv detector, air in the detector cell can cause seriously increased detector noise or high absorbance Dissolved oxygen can interfere with detection at short wavelengths, as oxygen absorbs radiation at <200 nm. Solvents degassed, "a topic covered in Section 603 B, Mobile Phase Preparation nust be Damage to Columns. HPLC columns are easily damaged and expensive to replace Bases can remove the functional groups from bonded HPLC phases. Therefore, bases should not be used in analyses involving BPC unless their removal prior to chromatography can be assured Bonded phases are usually stable in the pH range of approximately 2-8 Microscopic particles and microorganisms can clog column frits or even the top of the column itself. If this happens, the pressure drop across the column for a iven flow will gradually increase, and the column may eventually become com- pIe letely blocked. Filtration of the sample solution and mobile phase to remove particles >5 um, and the use of an appropriate precolumn and guard column, are recommended to protect the analytical column. Particles <5 um may be of con cern with some columns and detectors Any mobile phase, especially one containing water or methanol, can dissolve silica gel in unmodified and bonded silica gel columns. A precolumn containing silica gel can be positioned between pump and injector to saturate the mobile phase with silica gel so that the analytical column is not dissolved. Both precolumns and guard columns are discussed in Section 602 E, Analytical Column Protection The potential for damage to the column by reagents used in post-column derivatization is unlikely but not impossible. If the flow of the mobile phase is stopped, post-column reagents can diffuse back through the column effluent onto the column. This can result in deterioration of the column packing Damage to Detectors. The potential for reagent damage varies with each detector As stated above, the compressibility of dissolved gases in solvents can cause bubbles to appear in the detector cell and interfere with the analysis. Traces of oxygen are incompatible with electrochemical detectors operating in the reductive mode, oxygen can also cause quenching in fluorescence detectors, leading to reduced detectors can be clogged by the presence of particles 20.2 um. Filtration of sol- vents through a 0. 22 um filter is essential when using this type of detector Solvent Impurities. Many reagent grade solvents contain levels of impurities that make them unsuitable for use in HPLC. Sometimes the impurities are added deliberately by manufacturers as antioxidants, stabilizers, or denaturing agents For example, chloroform usually contains up to 1.0% methanol or ethanol and tetrahydrofuran may contain butylated hydroxytoluene or hydroquinone. These impurities may cause increased or decreased detector response or change the mobile phase strength and /or selectivity 01-11

SECTION 601 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 601–11 Pesticide Analytical Manual Vol. I Dissolved Gases. The presence of dissolved gases in solvents composing the mo￾bile phase is a major cause of practical problems in HPLC. Gas bubbles can collect in pumps, the detector cell, or other locations in the HPLC system. This can affect the reproducibility of the volume delivered by the pump, or large bubbles may completely stop the pump from working. Detection can be affected in various ways. With the UV detector, air in the detector cell can cause seriously increased detector noise or high absorbance. Dissolved oxygen can interfere with detection at short wavelengths, as oxygen absorbs radiation at <200 nm. Solvents must be “degassed,” a topic covered in Section 603 B, Mobile Phase Preparation. Damage to Columns. HPLC columns are easily damaged and expensive to replace. Bases can remove the functional groups from bonded HPLC phases. Therefore, bases should not be used in analyses involving BPC unless their removal prior to chromatography can be assured. Bonded phases are usually stable in the pH range of approximately 2-8. Microscopic particles and microorganisms can clog column frits or even the top of the column itself. If this happens, the pressure drop across the column for a given flow will gradually increase, and the column may eventually become com￾pletely blocked. Filtration of the sample solution and mobile phase to remove particles ≥5 µm, and the use of an appropriate precolumn and guard column, are recommended to protect the analytical column. Particles <5 µm may be of con￾cern with some columns and detectors. Any mobile phase, especially one containing water or methanol, can dissolve silica gel in unmodified and bonded silica gel columns. A precolumn containing silica gel can be positioned between pump and injector to saturate the mobile phase with silica gel so that the analytical column is not dissolved. Both precolumns and guard columns are discussed in Section 602 E, Analytical Column Protection. The potential for damage to the column by reagents used in post-column derivatization is unlikely but not impossible. If the flow of the mobile phase is stopped, post-column reagents can diffuse back through the column effluent onto the column. This can result in deterioration of the column packing. Damage to Detectors. The potential for reagent damage varies with each detector. As stated above, the compressibility of dissolved gases in solvents can cause bubbles to appear in the detector cell and interfere with the analysis. Traces of oxygen are incompatible with electrochemical detectors operating in the reductive mode; oxygen can also cause quenching in fluorescence detectors, leading to reduced sensitivity. Degassing of solvents is required. Porous flow-through coulometric detectors can be clogged by the presence of particles ≥0.2 µm. Filtration of sol￾vents through a 0.22 µm filter is essential when using this type of detector. Solvent Impurities. Many reagent grade solvents contain levels of impurities that make them unsuitable for use in HPLC. Sometimes the impurities are added deliberately by manufacturers as antioxidants, stabilizers, or denaturing agents. For example, chloroform usually contains up to 1.0% methanol or ethanol, and tetrahydrofuran may contain butylated hydroxytoluene or hydroquinone. These impurities may cause increased or decreased detector response or change the mobile phase strength and/or selectivity

SECTION 601 Pesticide Analytical Manual Vol I In some cases, incompatibility of a solvent or reagent with the HPLC system can be determined in advance and avoided. In the case of unknown impurities, prob- lems will be recognized only during use of the chemical; careful investigation will be needed to determine the cause of the problem. Even microorganisms in inad- equately purified water can cause a high background signal in some detectors(see Water, below). Whenever possible, HPLC grade solvents should be used to pre- pare mobile phases. Spectral or pesticide grade solvents may not be adequately pure for HPLC use Solvents should be adequately purified and tested before use Specific Solvents Water. Water is probably the most commonly used solvent in HPLC because of its role as the strength-adjusting solvent in RP mobile phases. It is also one of the most difficult solvents to purify and maintain in the pure state. Purity of water is especially critical in the determination of trace residues, when detectors are oper- ated at high sensitivity Purification of water by distillation, even triple distillation, is inadequate because volatile and codistilled organics will not be removed. Bonded RP columns will collect these impurities over long term use, which can alter the properties of th column or sometimes produce spurious peaks. Water can be purified by distilla- tion from potassium permanganate, by passage through a coarse grained C-18 bonded phase column that is periodically regenerated with acetonitrile, or by means of a commercial water purification system One widely used water purification system(Millipore Milli-Q pumps distilled water through a prefilter cartridge to eliminate particulates; then through sequential cartridges of charcoal, ion exchange resin, and Organex-Q; and finally through a 0.22 um filter. The activated charcoal cartridge removes organic impurities that can interfere with spectroscopic detectors. The mixed bed ion exchange resin cartridge(s)removes inorganics and ionized organics, as well as impurities leached from the charcoal; this removal is essential for proper operation of electrochemi- cal detectors. The Organex-Q cartridge eliminates any remaining organics, in addition to traces of material leached from the ion exchange cartridge. The final 0.22 um filter removes microscopic particles and microorganisms not eliminated by the previous cartridges. This filtration step protects column frits, columns, and porous flow-through detectors from particles that could clog them. It also mini- mizes the possibility that microorganisms will grow sufficiently to cause a back- ground detector signal. The quality of the feed water is improved and the life of the purification system is extended if a reverse osmosis system is included between the prefilter and carbon cartridges. This system lowers the base level of organics inorganics, and microorganisms Microorganisms such as bacteria and algae multiply rapidly in water. Therefore, en when using water purified in the manner just described, it is wise to discard all remaining water at the end of each week. The HPLC system should be flushed with methanol to destroy any microorganisms that have entered it during the week. At the beginning of a new work week, the water reservoir should be washed with methanol prior to filling with newly purified water. Growth of microorgan isms can also be prevented by adding 0.02% sodium azide or acetonitrile(which is present in many rP mobile phases) to the water Purified water is best stored in carefully cleaned glass containers. Plasticizers can leach into water stored in plastic containers, interfering with RP systems or 601-12 Form FDA 2905a(6/92]

SECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–12 Form FDA 2905a (6/92) In some cases, incompatibility of a solvent or reagent with the HPLC system can be determined in advance and avoided. In the case of unknown impurities, prob￾lems will be recognized only during use of the chemical; careful investigation will be needed to determine the cause of the problem. Even microorganisms in inad￾equately purified water can cause a high background signal in some detectors (see Water, below). Whenever possible, HPLC grade solvents should be used to pre￾pare mobile phases. Spectral or pesticide grade solvents may not be adequately pure for HPLC use. Solvents should be adequately purified and tested before use. Specific Solvents Water. Water is probably the most commonly used solvent in HPLC because of its role as the strength-adjusting solvent in RP mobile phases. It is also one of the most difficult solvents to purify and maintain in the pure state. Purity of water is especially critical in the determination of trace residues, when detectors are oper￾ated at high sensitivity. Purification of water by distillation, even triple distillation, is inadequate because volatile and codistilled organics will not be removed. Bonded RP columns will collect these impurities over long term use, which can alter the properties of the column or sometimes produce spurious peaks. Water can be purified by distilla￾tion from potassium permanganate, by passage through a coarse grained C-18 bonded phase column that is periodically regenerated with acetonitrile, or by means of a commercial water purification system. One widely used water purification system (Millipore Milli-Q) pumps distilled water through a prefilter cartridge to eliminate particulates; then through sequential cartridges of charcoal, ion exchange resin, and Organex-Q; and finally through a 0.22 µm filter. The activated charcoal cartridge removes organic impurities that can interfere with spectroscopic detectors. The mixed bed ion exchange resin cartridge(s) removes inorganics and ionized organics, as well as impurities leached from the charcoal; this removal is essential for proper operation of electrochemi￾cal detectors. The Organex-Q cartridge eliminates any remaining organics, in addition to traces of material leached from the ion exchange cartridge. The final 0.22 µm filter removes microscopic particles and microorganisms not eliminated by the previous cartridges. This filtration step protects column frits, columns, and porous flow-through detectors from particles that could clog them. It also mini￾mizes the possibility that microorganisms will grow sufficiently to cause a back￾ground detector signal. The quality of the feed water is improved and the life of the purification system is extended if a reverse osmosis system is included between the prefilter and carbon cartridges. This system lowers the base level of organics, inorganics, and microorganisms. Microorganisms such as bacteria and algae multiply rapidly in water. Therefore, even when using water purified in the manner just described, it is wise to discard all remaining water at the end of each week. The HPLC system should be flushed with methanol to destroy any microorganisms that have entered it during the week. At the beginning of a new work week, the water reservoir should be washed with methanol prior to filling with newly purified water. Growth of microorgan￾isms can also be prevented by adding 0.02% sodium azide or acetonitrile (which is present in many RP mobile phases) to the water. Purified water is best stored in carefully cleaned glass containers. Plasticizers can leach into water stored in plastic containers, interfering with RP systems or

Pesticide Analytical Manual Vol. I SECTION 601 contaminating the column. Leaching of metals from glass containers is also a possibility, but this is usually less of a problem than introduction of organic impu- HPLC grade water can be purchased from a number of commercial sources. This water can be used successfully as received for most applications The following purity check can be used to test water for applicability in HPLC Pump 100 mL water through C-18 column. With a UV detector in-line, run a linear gradient from 0 to 100% metha- nol at I mL/min for 10 min and hold for 15 min If the UV baseline shift at 0.08 AUFS is <10% and very few peaks of <3- 5% full scale deflection are observed, the water is pure enough for most applications Acetonitrile Acetonitrile is commonly used in RP HPLC mobile phases. Manufac turers specifications for HPLC solvent purity are usually based on acceptability for UV detectors. Specifications for fluorescence and electrochemical detectors are very difficult to define because of the complexity of instrumental parameters Methanol. Another of the more common solvents employed in RP HPLC is metha nol, which suffers from the same inadequacy of specifications as acetonitrile Methanol has the disadvantage of producing relatively viscous solutions when mixed with water, giving rise to much higher pressures than with other mobile phases Chlorinated Solvents. Some chlorinated solvents are stabilized against oxidative breakdown by addition of small amounts of methanol or ethanol. Alcohol will increase polarity of mobile phases and shorten elution times in NP HPLC. Also, reproducibility will be affected because the concentration of stabilizer will vary slightly from batch to batch Chlorinated solvents can be purchased without stabilizer, or the stabilizer can be removed by adsorption onto alumina, or by extraction with water followed by drying. Unstabilized chlorinated solvents may slowly decompose, producing hydro- chloric acid, which degrades columns and corrodes stainless steel. The rate of decomposition may be accelerated by the presence of other solvents. Hydrochloric acid can be removed by passing the solvent through activated silica or calcium carbonate chips. Solvents can be stabilized with amylene to avoid these problems Gillespie et al. [4] noted problems such as increased detector response and discol- oration of equipment when ethylene dichloride or methylene chloride was used in HPLC mobile phases. The problems described were attributed to a reaction between solvent impurities and stainless steel upon prolonged contact. Ethers. Eth ntain additives to stabilize the de formation For example, tetrahydrofuran is often stabilized by addition of small amounts of hyd- roquinone. This compound absorbs UV radiation and so interferes with UV ab- sorption detection. It can be removed by distilling the solvent from potassium hydroxide pellets. Inhibitor-free tetrahydrofuran should be stored in a dark bottle and flushed with nitrogen after each use. Any peroxides that form should be periodically removed by adsorption onto alumina DA 2905a(6/92

SECTION 601 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 601–13 Pesticide Analytical Manual Vol. I contaminating the column. Leaching of metals from glass containers is also a possibility, but this is usually less of a problem than introduction of organic impu￾rities. HPLC grade water can be purchased from a number of commercial sources. This water can be used successfully as received for most applications. The following purity check can be used to test water for applicability in HPLC: • Pump 100 mL water through C-18 column. • With a UV detector in-line, run a linear gradient from 0 to 100% metha￾nol at 1 mL/min for 10 min and hold for 15 min. • If the UV baseline shift at 0.08 AUFS is <10% and very few peaks of <3- 5% full scale deflection are observed, the water is pure enough for most applications. Acetonitrile. Acetonitrile is commonly used in RP HPLC mobile phases. Manufac￾turers’ specifications for HPLC solvent purity are usually based on acceptability for UV detectors. Specifications for fluorescence and electrochemical detectors are very difficult to define because of the complexity of instrumental parameters. Methanol. Another of the more common solvents employed in RP HPLC is metha￾nol, which suffers from the same inadequacy of specifications as acetonitrile. Methanol has the disadvantage of producing relatively viscous solutions when mixed with water, giving rise to much higher pressures than with other mobile phases. Chlorinated Solvents. Some chlorinated solvents are stabilized against oxidative breakdown by addition of small amounts of methanol or ethanol. Alcohol will increase polarity of mobile phases and shorten elution times in NP HPLC. Also, reproducibility will be affected because the concentration of stabilizer will vary slightly from batch to batch. Chlorinated solvents can be purchased without stabilizer, or the stabilizer can be removed by adsorption onto alumina, or by extraction with water followed by drying. Unstabilized chlorinated solvents may slowly decompose, producing hydro￾chloric acid, which degrades columns and corrodes stainless steel. The rate of decomposition may be accelerated by the presence of other solvents. Hydrochloric acid can be removed by passing the solvent through activated silica or calcium carbonate chips. Solvents can be stabilized with amylene to avoid these problems. Gillespie et al. [4] noted problems such as increased detector response and discol￾oration of equipment when ethylene dichloride or methylene chloride was used in HPLC mobile phases. The problems described were attributed to a reaction between solvent impurities and stainless steel upon prolonged contact. Ethers. Ethers contain additives to stabilize them against peroxide formation. For example, tetrahydrofuran is often stabilized by addition of small amounts of hyd￾roquinone. This compound absorbs UV radiation and so interferes with UV ab￾sorption detection. It can be removed by distilling the solvent from potassium hydroxide pellets. Inhibitor-free tetrahydrofuran should be stored in a dark bottle and flushed with nitrogen after each use. Any peroxides that form should be periodically removed by adsorption onto alumina

SECTION 601 Pesticide Analytical Manual Vol I Reagent blanks Blank samples should be analyzed to ascertain that no interferences from reagents (or glassware) occur during analysis Reagent blanks are especially important when using nonspecific optical detectors such as UV or ri detectors. Safety Precautions Beyond the concern over damage to HPLC systems that can be caused by reagents and solvents, it is important to protect the health of the analyst. An awareness of the toxicity of the chemicals in use is essential. Care must be taken to minimize exposure to toxic chemicals. See Reference 5 for more on laboratory safety for 601 E SAMPLE PREPARATION Sample Cleanup Extracts to be analyzed by HPLC must be cleaned up(i.e, interfering co-extrac- tives removed) sufficiently to permit identification and quantitation of residues, and to prevent contamination or harm to any part of the HPLC system. The column andyor detector may be impaired by injection of dirty extracts, especially when many samples are analyzed Cleanup procedures for trace residue determination by HPLC must be developed to accommodate the selectivity of the detector Dissolved interferences in the sample solution that appear in the chromatogram as extra peaks must be re- moved. Any materials that will be strongly adsorbed by the column must also be removed to prevent their affecting chromatographic characteristics of the column causing baseline drift, or appearing as spurious peaks in later chromatograms A recent innovation combines cleanup of the sample extract in-line with the HPlC determinative step [6]. A short column of SCX resin replaces the sample loop in a six-port HPLC injection valve, where it effectively removes the analyte, formetanate hydrochloride, from the extract Solvent flushing of the column while the short column is still off-line(disconnected from the analytical column) provides cleanup and substitutes for traditional separatory funnel partitionings. Subsequent switch ing of the valve places the cleanup column in-line with the analytical SCX column for elution and determination. This coupled column application and other mul- bidimensional variations [7] provide simple, rapid analysis with minimum solvent Sample filtration emoval of particulate matter in the sample solution is critical for HPLC stability Both column frits and the top of the colu B packing can become clogged by particles, leading to increased back pressure and adverse effects on chromato- graphic results because of decreased column efficiency, production of split peaks, At a minimum, samples should be passed through a commercial clarification apparatus, such as a syringe and a 5 um filter pad in a Swinny adapter, before injection In residue determination, passing samples through filters with <l um 601-14 Form FDA 2905a(6/92]

SECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–14 Form FDA 2905a (6/92) Reagent Blanks Blank samples should be analyzed to ascertain that no interferences from reagents (or glassware) occur during analysis. Reagent blanks are especially important when using nonspecific optical detectors such as UV or RI detectors. Safety Precautions Beyond the concern over damage to HPLC systems that can be caused by reagents and solvents, it is important to protect the health of the analyst. An awareness of the toxicity of the chemicals in use is essential. Care must be taken to minimize exposure to toxic chemicals. See Reference 5 for more on laboratory safety for HPLC analysis. 601 E: SAMPLE PREPARATION Sample Cleanup Extracts to be analyzed by HPLC must be cleaned up (i.e., interfering co-extrac￾tives removed) sufficiently to permit identification and quantitation of residues, and to prevent contamination or harm to any part of the HPLC system. The column and/or detector may be impaired by injection of dirty extracts, especially when many samples are analyzed. Cleanup procedures for trace residue determination by HPLC must be developed to accommodate the selectivity of the detector. Dissolved interferences in the sample solution that appear in the chromatogram as extra peaks must be re￾moved. Any materials that will be strongly adsorbed by the column must also be removed to prevent their affecting chromatographic characteristics of the column, causing baseline drift, or appearing as spurious peaks in later chromatograms. A recent innovation combines cleanup of the sample extract in-line with the HPLC determinative step [6]. A short column of SCX resin replaces the sample loop in a six-port HPLC injection valve, where it effectively removes the analyte, formetanate hydrochloride, from the extract. Solvent flushing of the column while the short column is still off-line (disconnected from the analytical column) provides cleanup and substitutes for traditional separatory funnel partitionings. Subsequent switch￾ing of the valve places the cleanup column in-line with the analytical SCX column for elution and determination. This coupled column application and other mul￾tidimensional variations [7] provide simple, rapid analysis with minimum solvent use. Sample Filtration Removal of particulate matter in the sample solution is critical for HPLC stability. Both column frits and the top of the column packing can become clogged by particles, leading to increased back pressure and adverse effects on chromato￾graphic results because of decreased column efficiency, production of split peaks, etc. At a minimum, samples should be passed through a commercial clarification apparatus, such as a syringe and a 5 µm filter pad in a Swinny adapter, before injection. In residue determination, passing samples through filters with <1 µm