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Selected papers from the latest issue:
Quantifying Injection Solvent Effects in Reversed-Phase Liquid Chromatography
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 7 March 2012
Bradley J. VanMiddlesworth, John G. Dorsey
Peak distortion due to the injection was measured as a function of injection solvent strength, volume, mass, retention factor, and column selectivity. The concept of a method's sensitivity (s) to injection solvent strength was mathematically defined as a vector of theoretical plate counts compared to an ideal vector that does not change with injection solvent strength. Near ideal sensitivity (s > 0.90) was measured on all columns with all analytes in low volume injections of 1.25 μL. Increasing the injection volume reduces the measured sensitivity from ideality to a greater extent than increasing the injection mass, with differing values for each column.Using column parameters measured from the hydrophobic-subtraction model and fitting parameters from the acetonitrile excess adsorption isotherm, differences among the columns studied are explained. Decreased ligand density and increased silanol activity provide a consistent peak shape with changes in injection volume or solvent strength. For method development, a quick test is suggested with the ratio of hydrophobic-subtraction column parameters, H/A, to predict the injection solvent sensitivity of a column. As H/A decreases, the sensitivity to injection solvent worsens. Sensitivity to organic modifiers other than acetonitrile are predicted with cited sorbed layer thickness, such that MeOH > EtOH > IPA ≈ THF ≈ MeCN, i.e., a strong MeOH diluent is more ideal (better) than a strong MeCN diluent.
Source: Journal of Chromatography A, Available online 7 March 2012
Bradley J. VanMiddlesworth, John G. Dorsey
Peak distortion due to the injection was measured as a function of injection solvent strength, volume, mass, retention factor, and column selectivity. The concept of a method's sensitivity (s) to injection solvent strength was mathematically defined as a vector of theoretical plate counts compared to an ideal vector that does not change with injection solvent strength. Near ideal sensitivity (s > 0.90) was measured on all columns with all analytes in low volume injections of 1.25 μL. Increasing the injection volume reduces the measured sensitivity from ideality to a greater extent than increasing the injection mass, with differing values for each column.Using column parameters measured from the hydrophobic-subtraction model and fitting parameters from the acetonitrile excess adsorption isotherm, differences among the columns studied are explained. Decreased ligand density and increased silanol activity provide a consistent peak shape with changes in injection volume or solvent strength. For method development, a quick test is suggested with the ratio of hydrophobic-subtraction column parameters, H/A, to predict the injection solvent sensitivity of a column. As H/A decreases, the sensitivity to injection solvent worsens. Sensitivity to organic modifiers other than acetonitrile are predicted with cited sorbed layer thickness, such that MeOH > EtOH > IPA ≈ THF ≈ MeCN, i.e., a strong MeOH diluent is more ideal (better) than a strong MeCN diluent.
Highlights
► Peak distortion from injection solvent effects was measured and quantified. ► Peak distortion occurs with increasing injection volume and solvent strength. ► A predictive test is proposed to estimate column sensitivity to these issues.Simultaneous Determination of Fluoxetine and Norfluoxetine Enantiomers using Isotope Discrimination Mass Spectroscopy Solution Method and its Application in the CYP2C9-Mediated Stereoselective Interactions
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 7 March 2012
Lushan Yu, Shenjia Wang, Huidi Jiang, Hui Zhou, Su Zeng
In this study, we developed an LC-MS/MS method based on an isotope discrimination mass spectroscopy solution (IDMSS) technology to simultaneously quantify enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) in a CYP2C9 incubation mixture.S-FLX andS-NFLX were labeled to formS-FLX-d5 andS-NFLX-d5. The method has several advantages over conventional chiral separation methods, in terms of the analysis period, resolution, and lower limit of quantification. The primary advantage of the method is that the two enantiomers can always be simultaneously determined by mass spectroscopy regardless if they are separated on column or not, owing to which it has high throughput and high sensitivity. The lower limit of quantification (amount on column) is 12.5 and 1.25 pg for FLX and NFLX, respectively. The retention time of FLX, NFLX, and the internal standard is only 1.9 min. The calibration curves were linear over the concentration range of 0.1-100 ng/ml for NFLX and 1-1000 ng/ml for FLX with an accepted reproducible (RSD < 10%) and accurate (CV < 10%). No significant kinetic isotope effect was found in the metabolism ofS-FLX-d5 catalyzed by CYP2C9*1 and CYP2C9*2. The half-maximal inhibitory concentration values betweenR-FLX andS-FLX catalyzed by CYP2C9*1 and CYP2C9*2 were determined in this study. The inhibitory effects ofR- toS-FLX were stronger than those ofS- toR-FLX in both CYP2C9*1 and CYP2C9*2. The IDMSS technology is useful for stereoselective study of chiral compound in vitro.
Source: Journal of Chromatography A, Available online 7 March 2012
Lushan Yu, Shenjia Wang, Huidi Jiang, Hui Zhou, Su Zeng
In this study, we developed an LC-MS/MS method based on an isotope discrimination mass spectroscopy solution (IDMSS) technology to simultaneously quantify enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) in a CYP2C9 incubation mixture.S-FLX andS-NFLX were labeled to formS-FLX-d5 andS-NFLX-d5. The method has several advantages over conventional chiral separation methods, in terms of the analysis period, resolution, and lower limit of quantification. The primary advantage of the method is that the two enantiomers can always be simultaneously determined by mass spectroscopy regardless if they are separated on column or not, owing to which it has high throughput and high sensitivity. The lower limit of quantification (amount on column) is 12.5 and 1.25 pg for FLX and NFLX, respectively. The retention time of FLX, NFLX, and the internal standard is only 1.9 min. The calibration curves were linear over the concentration range of 0.1-100 ng/ml for NFLX and 1-1000 ng/ml for FLX with an accepted reproducible (RSD < 10%) and accurate (CV < 10%). No significant kinetic isotope effect was found in the metabolism ofS-FLX-d5 catalyzed by CYP2C9*1 and CYP2C9*2. The half-maximal inhibitory concentration values betweenR-FLX andS-FLX catalyzed by CYP2C9*1 and CYP2C9*2 were determined in this study. The inhibitory effects ofR- toS-FLX were stronger than those ofS- toR-FLX in both CYP2C9*1 and CYP2C9*2. The IDMSS technology is useful for stereoselective study of chiral compound in vitro.
Highlights
► An isotope discrimination MS solution method was developed for chiral separation. ► Separated on column or not is not affect the enantiomers determination. ► The method has high throughput and high sensitivity. ► No significant kinetic isotope effect was found to theS-fluoxetine-d5 metabolism.Comparison of indirect and direct quantification of esters of monochloropropanediol in vegetable oil
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 7 March 2012
Mathieu Dubois, Adrienne Tarres, Till Goldmann, Anna Maria Empl, Alfred Donaubauer, ...
The presence of fatty acid esters of monochloropropanediol (MEs) in food is a recent concern raised due to the carcinogenicity of their hydrolysable moieties 2- and 3-monochloropropanediol (2- and 3-MCPD). Several indirect methods for the quantification of MEs have been developed and are commonly in use until today, however significant discrepancies among analytical results obtained are challenging their reliability. The aim of the present study was therefore to test the trueness of an indirect method by comparing it to a newly developed direct method using palm oil and palm olein as examples. The indirect method was based on ester cleavage under acidic conditions, derivatization of the liberated 2- and 3-MCPD with heptafluorobutyryl imidazole and GC-MS determination. The direct method was comprised of two extraction procedures targeting 2-and 3-MCPD mono esters (co-extracting as well glycidyl esters) by the use of double solid phase extraction (SPE), and 2- and 3-MCPD di-esters by the use of silica gel column, respectively. Detection was carried out by liquid chromatography coupled to Time of Flight mass spectrometry (LC-ToF-MS). Accurate quantification of the intact compounds was assured by means of matrix matched standard addition on extracts. Analysis of 22 palm oil and 7 palm olein samples (2- plus 3-MCPD contamination ranged from 0.3 to 8.8 μg/g) by both methods revealed no significant bias. Both methods were therefore considered as comparable in terms of results; however the indirect method was shown to require less analytical standards, being less tedious and furthermore applicable to all type of different vegetable oils and hence recommended for routine application.
Source: Journal of Chromatography A, Available online 7 March 2012
Mathieu Dubois, Adrienne Tarres, Till Goldmann, Anna Maria Empl, Alfred Donaubauer, ...
The presence of fatty acid esters of monochloropropanediol (MEs) in food is a recent concern raised due to the carcinogenicity of their hydrolysable moieties 2- and 3-monochloropropanediol (2- and 3-MCPD). Several indirect methods for the quantification of MEs have been developed and are commonly in use until today, however significant discrepancies among analytical results obtained are challenging their reliability. The aim of the present study was therefore to test the trueness of an indirect method by comparing it to a newly developed direct method using palm oil and palm olein as examples. The indirect method was based on ester cleavage under acidic conditions, derivatization of the liberated 2- and 3-MCPD with heptafluorobutyryl imidazole and GC-MS determination. The direct method was comprised of two extraction procedures targeting 2-and 3-MCPD mono esters (co-extracting as well glycidyl esters) by the use of double solid phase extraction (SPE), and 2- and 3-MCPD di-esters by the use of silica gel column, respectively. Detection was carried out by liquid chromatography coupled to Time of Flight mass spectrometry (LC-ToF-MS). Accurate quantification of the intact compounds was assured by means of matrix matched standard addition on extracts. Analysis of 22 palm oil and 7 palm olein samples (2- plus 3-MCPD contamination ranged from 0.3 to 8.8 μg/g) by both methods revealed no significant bias. Both methods were therefore considered as comparable in terms of results; however the indirect method was shown to require less analytical standards, being less tedious and furthermore applicable to all type of different vegetable oils and hence recommended for routine application.
Highlights
► We developed a direct method for MCPD esters quantitation by LC-ToF-MS in edible oil. ► Analytical standards were chosen rationally based on theoretical calculation ► 2- and 3-MCPD were also determined by indirect method using acid-hydrolysis and GC-MS ► Direct and indirect methods gave similar results analyzing 29 palm oil/olein samplesQuantification of Matrix Metalloprotease-9 in Bronchoalveolar Lavage Fluid by Selected Reaction Monitoring with Microfluidics nano-Liquid-Chromatography–Mass Spectrometry
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 6 March 2012
Laurette M. Prely, Krisztina Paal, Jos Hermans, Sicco van der Heide, Antoon J.M. van Oosterhout, ...
Quantitative protein analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease–9 (MMP-9; ∼ 90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.
Source: Journal of Chromatography A, Available online 6 March 2012
Laurette M. Prely, Krisztina Paal, Jos Hermans, Sicco van der Heide, Antoon J.M. van Oosterhout, ...
Quantitative protein analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease–9 (MMP-9; ∼ 90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.
Highlights
► Highly sensitive and specific LC-MS/MS method for MMP-9 in BALF ► First application of LC-MS/MS in the SRM mode for protein quantification in BALF ► Accurate quantification at the amol level by microfluidics-nanoLC-MS/MS ► Application to real-life samples from lung transplantation patientsTrace Determination of 13 Haloacetamides in Drinking Water Using Liquid Chromatography Triple Quadrupole Mass Spectrometry with Atmospheric Pressure Chemical Ionization
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 6 March 2012
Wenhai Chu, Naiyun Gao, Daqiang Yin, Stuart W. Krasner, Michael R. Templeton
The haloacetamides (HAcAms) are disinfection by-products (DBPs) in drinking water which are currently receiving increased scientific attention due to their elevated toxicity relative to regulated disinfection by-products. A simultaneous determination method of 13 HAcAms, combining solid-phase extraction (SPE) enrichment, liquid chromatographic (LC) separation, and triple quadrupole mass spectrometry (tqMS) detection with atmospheric pressure chemical ionization (APCI) using selective reaction monitoring in positive mode, was developed to measure HAcAms, including chlorinated, brominated, and iodinated analogues. Ammonium chloride and Oasis HLB were selected as the dechlorinating reagent and polymeric SPE sorbent of HAcAm samples. The used tqMS apparatus showed higher sensitivity for the studied HAcAms in the APCI mode than electrospray Ionization. 13 HAcAms were separated by LC in 9.0 min, and the detection limits ranged from 7.6 to 19.7 ng/L. The SPE-LC/tqMS method was successfully applied to quantify 13 HAcAms in drinking water samples for the first time, and first indentified tribromoacetamide and chloroiodoacetamide as DBPs in drinking water
Source: Journal of Chromatography A, Available online 6 March 2012
Wenhai Chu, Naiyun Gao, Daqiang Yin, Stuart W. Krasner, Michael R. Templeton
The haloacetamides (HAcAms) are disinfection by-products (DBPs) in drinking water which are currently receiving increased scientific attention due to their elevated toxicity relative to regulated disinfection by-products. A simultaneous determination method of 13 HAcAms, combining solid-phase extraction (SPE) enrichment, liquid chromatographic (LC) separation, and triple quadrupole mass spectrometry (tqMS) detection with atmospheric pressure chemical ionization (APCI) using selective reaction monitoring in positive mode, was developed to measure HAcAms, including chlorinated, brominated, and iodinated analogues. Ammonium chloride and Oasis HLB were selected as the dechlorinating reagent and polymeric SPE sorbent of HAcAm samples. The used tqMS apparatus showed higher sensitivity for the studied HAcAms in the APCI mode than electrospray Ionization. 13 HAcAms were separated by LC in 9.0 min, and the detection limits ranged from 7.6 to 19.7 ng/L. The SPE-LC/tqMS method was successfully applied to quantify 13 HAcAms in drinking water samples for the first time, and first indentified tribromoacetamide and chloroiodoacetamide as DBPs in drinking water
Highlights
► Determination of 13 HAcAms by SPE + HPLC/tqMS with APCI mode was first developed. ► Ammonium chloride was selected as the dechlorinating reagent, compared to others. ► Oasis HLB sorbent has better enrichment effect than Oasis MCX, MAX, WCX and WAX. ► The tqMS showed higher sensitivity for 13 HAcAms in the APCI mode than ESI mode. ► Two HAcAms (CIAcAm and TBAcAm) were identified as DBPs in water for the first time.Micellar Electrokinetic Chromatography of Aromatic Anions and Non-ionic Aromatic Compounds with Stepwise Changes of the Concentration of Cetyltrimethylammonium Chloride
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 6 March 2012
Yukihiro Esaka, Miki Kobayashi, Hiroya Murakami, Bunji Uno
Micellar electrokinetic chromatography in which the concentration of cetyltrimetylammmonium chloride (CTAC) was sequentially changed in the separation system was investigated using 10 aromatic anions and 11 non-ionic aromatic compounds as model analytes. All separations were performed in the absence of electroosmotic flow (EOF), and thus, analytes were detected in the order of their strength of interaction with micelles in the system. In isocratic elutions without EOF, the model analytes could be separated better with lower concentrations of CTAC but migration times of the analytes possessing relatively higher polarities increased markedly, and thus, long analysis times were required. Therefore, we attempted to increase the concentration of CTAC during a single measurement to reduce the analysis time without hindering the resultant separation of analytes obtained with lower concentrations. Briefly, the present surfactant stepwiseelution can be performed by a sequential increase in CTAC concentrations of the running solution in the anodic reservoir from 30 to 50 mM for the anions and from 20 to 50 mM for the non-ionic compounds. Additionally, to perform expected gradient separations with good reproducibility, each running solution with a different CTAC concentration was treated with tetraethylammmonium chloride as an additive to adjust electric conductivities of each running solution to be equal. Under this condition, CTAC micelles of each zone of different CTAC concentrations would migrate with practically the same velocity. Consequently, by the present stepwise method, both the 10 anionic analytes and the 11 non-ionic analytes were well separated within reasonable periods which corresponded approximately to two-third and less than half of those by the isocratic elutions, respectively.
Source: Journal of Chromatography A, Available online 6 March 2012
Yukihiro Esaka, Miki Kobayashi, Hiroya Murakami, Bunji Uno
Micellar electrokinetic chromatography in which the concentration of cetyltrimetylammmonium chloride (CTAC) was sequentially changed in the separation system was investigated using 10 aromatic anions and 11 non-ionic aromatic compounds as model analytes. All separations were performed in the absence of electroosmotic flow (EOF), and thus, analytes were detected in the order of their strength of interaction with micelles in the system. In isocratic elutions without EOF, the model analytes could be separated better with lower concentrations of CTAC but migration times of the analytes possessing relatively higher polarities increased markedly, and thus, long analysis times were required. Therefore, we attempted to increase the concentration of CTAC during a single measurement to reduce the analysis time without hindering the resultant separation of analytes obtained with lower concentrations. Briefly, the present surfactant stepwiseelution can be performed by a sequential increase in CTAC concentrations of the running solution in the anodic reservoir from 30 to 50 mM for the anions and from 20 to 50 mM for the non-ionic compounds. Additionally, to perform expected gradient separations with good reproducibility, each running solution with a different CTAC concentration was treated with tetraethylammmonium chloride as an additive to adjust electric conductivities of each running solution to be equal. Under this condition, CTAC micelles of each zone of different CTAC concentrations would migrate with practically the same velocity. Consequently, by the present stepwise method, both the 10 anionic analytes and the 11 non-ionic analytes were well separated within reasonable periods which corresponded approximately to two-third and less than half of those by the isocratic elutions, respectively.
Highlights
► Stepwise-elution MEKC changing concentration of micelle during single separation. ► A gradient method changing phase ratio of distribution phases of chromatography. ► Shortening analysis time and improving separation for ionic and non ionic analytes. ► Stable operation by a supporting electrolyte maintaining a fixed electric field.Application of accelerated solvent extraction in the analysis of organic contaminants, bioactive and nutritional compounds in food and feed
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 6 March 2012
Hanwen Sun, Xusheng Ge, Yunkai Lv, Anbang Wang
Accelerated solvent extraction (ASE) has become a popular green extraction technology for different classes of organic contaminants present in numerous kinds of food and feed for food safety. The parameters affecting ASE efficiency and application advancement of ASE in analysis of organic contaminants, natural toxins compounds as well as bioactive and nutritional compounds in animal origin food, plant origin food and animal feed are reviewed in detail. ASE is a fully automated and reliable extraction technique with many advantages over traditional extraction techniques, so it could be especially useful for routine analyses of pollutants in food and feed.
Source: Journal of Chromatography A, Available online 6 March 2012
Hanwen Sun, Xusheng Ge, Yunkai Lv, Anbang Wang
Accelerated solvent extraction (ASE) has become a popular green extraction technology for different classes of organic contaminants present in numerous kinds of food and feed for food safety. The parameters affecting ASE efficiency and application advancement of ASE in analysis of organic contaminants, natural toxins compounds as well as bioactive and nutritional compounds in animal origin food, plant origin food and animal feed are reviewed in detail. ASE is a fully automated and reliable extraction technique with many advantages over traditional extraction techniques, so it could be especially useful for routine analyses of pollutants in food and feed.
Graphical abstract
Highlights
► Parameters affecting ASE efficiency. ► Multi-residue analysis. ► Organic contaminants, bioactive and nutritional compounds. ► Application of ASE in analysis of animal origin foods, plant origin foods and feeds.Multiple, Simultaneous, Independent Gradients for Versatile Multidimensional Liquid Chromatography. Part I: Theory
08 March 2012, 00:09:05
Publication year: 2012
Source: Journal of Chromatography A, Available online 6 March 2012
Allen G. Hirsh, Latchezar I. Tsonev
The general method for constructing coupled dual gradients in liquid chromatography (LC) is to begin by filling a reservoir A with a solution of one mobile phase (MP) component at concentration [C1(A)] and a second MP component at concentration [C2(A)], followed by filling a reservoir B with a solution containing MP component one at concentration [C1(B)] and the second MP component at concentration [C2(B)]. In another scenario the reservoirs A and B are filled with solutions of only one MP component at different concentration [C1(A)] and [C1(B)] and the two solutions are titrated to a different pH value: pH (A) for the reservoir A and pH (B) for the reservoir B respectively. In either case, mixing of flows from the two reservoirs varies the concentrations of the two MP components (MP solutes) or the concentration of one MP component and pH along a particular compositional curve producing an eluent with two compositionally coupled gradients. This is a kind of a two dimensional LC utilizing dual simultaneous dependent gradients (DSDGs) wherein two parameters affecting the binding free energy of an analyte to a stationary phase (SP) are being altered simultaneously. Such a DSDG suffers from a significant limitation in that the gradient concentration of the two solutes or the concentration of one MP component and the pH cannot be varied independently. The only way to attain an optimal multigradient LC system, that promises a remarkable increase in chromatographic resolution of complex analyte mixtures, is to uncouple the multiple (dual) gradients, making each independent of the other(s). In this paper the theory of uncoupling ofnsuch gradients,n≥2 is developed. It is shown that fornsolutes 2reservoirs are required in concert with an LC eluent delivery system capable of freely apportioning the flows among the reservoirs according to equations we develop here. We go on to predict a substantial increase in chromatographic resolution when applying dual simultaneous independent gradients (DSIGs) of salt and pH to fractionate difficult to separate proteins. This prediction is naturally explained by the electrostatic interaction theory of protein binding to an ion exchanger. In subsequent experimental papers it will be shown that the algorithms presented here properly instruct a quad pump HPLC system to produce well controlled independent simultaneous gradients of pH and non-buffering solutes with attendant significant gain in chromatographic resolution of complex mixtures of protein isoforms.
Source: Journal of Chromatography A, Available online 6 March 2012
Allen G. Hirsh, Latchezar I. Tsonev
The general method for constructing coupled dual gradients in liquid chromatography (LC) is to begin by filling a reservoir A with a solution of one mobile phase (MP) component at concentration [C1(A)] and a second MP component at concentration [C2(A)], followed by filling a reservoir B with a solution containing MP component one at concentration [C1(B)] and the second MP component at concentration [C2(B)]. In another scenario the reservoirs A and B are filled with solutions of only one MP component at different concentration [C1(A)] and [C1(B)] and the two solutions are titrated to a different pH value: pH (A) for the reservoir A and pH (B) for the reservoir B respectively. In either case, mixing of flows from the two reservoirs varies the concentrations of the two MP components (MP solutes) or the concentration of one MP component and pH along a particular compositional curve producing an eluent with two compositionally coupled gradients. This is a kind of a two dimensional LC utilizing dual simultaneous dependent gradients (DSDGs) wherein two parameters affecting the binding free energy of an analyte to a stationary phase (SP) are being altered simultaneously. Such a DSDG suffers from a significant limitation in that the gradient concentration of the two solutes or the concentration of one MP component and the pH cannot be varied independently. The only way to attain an optimal multigradient LC system, that promises a remarkable increase in chromatographic resolution of complex analyte mixtures, is to uncouple the multiple (dual) gradients, making each independent of the other(s). In this paper the theory of uncoupling ofnsuch gradients,n≥2 is developed. It is shown that fornsolutes 2reservoirs are required in concert with an LC eluent delivery system capable of freely apportioning the flows among the reservoirs according to equations we develop here. We go on to predict a substantial increase in chromatographic resolution when applying dual simultaneous independent gradients (DSIGs) of salt and pH to fractionate difficult to separate proteins. This prediction is naturally explained by the electrostatic interaction theory of protein binding to an ion exchanger. In subsequent experimental papers it will be shown that the algorithms presented here properly instruct a quad pump HPLC system to produce well controlled independent simultaneous gradients of pH and non-buffering solutes with attendant significant gain in chromatographic resolution of complex mixtures of protein isoforms.
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