This document discusses analytical method development for pharmaceutical drug substances and products. It describes the importance of method development and outlines the key steps involved, including collecting sample information, selecting the chromatographic technique, stationary and mobile phases. It also discusses sources of impurities in drugs, control and qualification of impurities according to ICH guidelines, and pharmacopeial and ICH quality guidelines for impurity testing and thresholds.
Stability indicating method development and validation for the simultaneous e...pharmaindexing
Stability indicating method development and validation for the simultaneous estimation of rabeprazole sodium and ketorolac tromethamine in bulk and synthetic mixture by RP-HPLC
Formulation and evaluation of FDT and HPLC method development of Amlodipine b...pooja deshmukh
formulation and evaluation of fast disintegrating tablet by using superdisintegrants and RP-HPLC method development of prepered tablet of Amlodipine besilate and Candesartan cilexetil
Stability indicating analytical method development and validation for estimat...SriramNagarajan18
Stability indicating analytical method development and validation for estimation of Sacubitril and Valsartan in bulk and pharmaceutical dosage form using RP-HPLC
Purification method development for chiral separation in supercritical
fluid chromatography with the solubilities in supercritical fluid
chromatographic mobile phases
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
RP-HPLC method development and validation of ritonavir in bulk and pharmaceut...SriramNagarajan17
This document describes the development and validation of an RP-HPLC method for the quantification of the HIV protease inhibitor ritonavir (RIT) in bulk and pharmaceutical dosage forms. A simple isocratic RP-HPLC method was developed using a C18 column, mobile phase of 0.02M potassium dihydrogen phosphate buffer and acetonitrile (70:30 v/v), and detection at 237 nm. The method was validated per ICH guidelines and showed good linearity from 25-150 μg/mL, precision <0.5% RSD, accuracy of 99.3-100.6% recovery, and ability to quantify RIT in pharmaceutical tablets without interference from excipients.
This document discusses analytical method development for pharmaceutical drug substances and products. It describes the importance of method development and outlines the key steps involved, including collecting sample information, selecting the chromatographic technique, stationary and mobile phases. It also discusses sources of impurities in drugs, control and qualification of impurities according to ICH guidelines, and pharmacopeial and ICH quality guidelines for impurity testing and thresholds.
Stability indicating method development and validation for the simultaneous e...pharmaindexing
Stability indicating method development and validation for the simultaneous estimation of rabeprazole sodium and ketorolac tromethamine in bulk and synthetic mixture by RP-HPLC
Formulation and evaluation of FDT and HPLC method development of Amlodipine b...pooja deshmukh
formulation and evaluation of fast disintegrating tablet by using superdisintegrants and RP-HPLC method development of prepered tablet of Amlodipine besilate and Candesartan cilexetil
Stability indicating analytical method development and validation for estimat...SriramNagarajan18
Stability indicating analytical method development and validation for estimation of Sacubitril and Valsartan in bulk and pharmaceutical dosage form using RP-HPLC
Purification method development for chiral separation in supercritical
fluid chromatography with the solubilities in supercritical fluid
chromatographic mobile phases
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
RP-HPLC method development and validation of ritonavir in bulk and pharmaceut...SriramNagarajan17
This document describes the development and validation of an RP-HPLC method for the quantification of the HIV protease inhibitor ritonavir (RIT) in bulk and pharmaceutical dosage forms. A simple isocratic RP-HPLC method was developed using a C18 column, mobile phase of 0.02M potassium dihydrogen phosphate buffer and acetonitrile (70:30 v/v), and detection at 237 nm. The method was validated per ICH guidelines and showed good linearity from 25-150 μg/mL, precision <0.5% RSD, accuracy of 99.3-100.6% recovery, and ability to quantify RIT in pharmaceutical tablets without interference from excipients.
This document describes the development and validation of two stability-indicating methods for the quantitative analysis of rosuvastatin (ROSU) in the presence of its degradation products: a high-performance liquid chromatography (HPLC) method and a high-performance thin layer chromatography (HPTLC) method. Both methods were found to be precise, accurate, specific and sensitive for the analysis of ROSU in raw materials and pharmaceutical formulations in the presence of degradation products. The methods were fully validated per ICH guidelines and successfully applied to analyze ROSU levels in commercial tablet formulations.
Stability indicating rp hplc method development and validation for simultaneo...Rajasekhar
The document describes the development and validation of a stability-indicating RP-HPLC method for the simultaneous estimation of levodropropizine and chlorpheniramine maleate in bulk and pharmaceutical dosage forms. The method utilizes a C18 column with a mobile phase of 45% 0.1% orthophosphoric acid and 55% acetonitrile at a flow rate of 1 mL/min. The method was validated for parameters such as linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness according to ICH guidelines. Forced degradation studies were also performed to demonstrate the stability-indicating ability of the developed method.
This document describes the development and validation of a stability-indicating RP-LC method for the determination of famciclovir (FCV) in the presence of its impurities and degradation products. A gradient reverse phase liquid chromatographic method was developed using an Inertsil ODS 3V column with a mobile phase of a potassium dihydrogen orthophosphate buffer and methanol mixture. FCV was subjected to oxidative, acid, base, hydrolytic, thermal, and photolytic degradation and was found to degrade significantly under oxidative, acid and base conditions. The developed method was validated according to ICH guidelines and demonstrated specificity, linearity, accuracy, precision, and robustness. This stability-indicating method can
This document presents the development and validation of an RP-HPLC assay method for the simultaneous estimation of a muscle relaxant drug and analgesic drug in pure form and pharmaceutical dosage forms. It discusses conducting a literature review, determining drug profiles including solubility and wavelength absorption, optimizing the analytical method, and expected outcomes such as a simple, accurate, precise and cost-effective simultaneous estimation method. The method will be validated as per ICH guidelines and force degradation studies will be performed to evaluate the stability indicating properties of the method.
The document discusses quantification techniques for herbal drugs. It describes various chromatography techniques used for quantification including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). These techniques are used to separate, identify, and quantify marker compounds in herbal drugs. The document provides an example of using HPLC to quantify atropine and examples of GC and GC-MS analyses of herbal extracts to separate and identify various compounds.
This document describes the development and validation of an RP-HPLC method for the simultaneous analysis of diclofenac sodium and rabeprazole sodium without the need for an internal standard. The method utilizes a C8 column with a mobile phase of triethyl amine buffer (pH 5):acetonitrile (50:50 v/v) at a flow rate of 2 mL/min. Validation showed the method to be linear, accurate, precise, sensitive and stable. The method was applied to a pharmaceutical formulation containing both drugs with recoveries from 98-100%.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document presents a stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of Valsartan in tablet dosage forms. The method uses a Phenomenox C18 column with a mobile phase of methanol and phosphate buffer at a ratio of 65:35 at a flow rate of 1mL/min. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification and specificity. Stress studies showed Valsartan degraded under acidic, basic, oxidative and heat conditions. The developed method can be used for assay of Valsartan in tablets and its degradation products.
This document presents the development and validation of an RP-HPLC method for the simultaneous estimation of a muscle relaxant drug and analgesic drug in pure form and pharmaceutical dosage forms. It discusses conducting a literature review on existing methods, determining the physicochemical properties of the drugs, optimizing the analytical method, and validating the developed method as per ICH guidelines. The expected outcomes are an accurate, precise, simple, cost-effective and fast method for simultaneously analyzing the two drugs.
Here are the key IR frequencies identified in the sample that match the reference standard of propafenone:
- C-C stretch at 1186 cm-1
- C=C stretch at 1651 cm-1
- C-H stretch (symmetric) at 2939 cm-1
- C-H bend at 1328 cm-1
- CH2 stretch (symmetric) at 1369 cm-1
- CH2 bend at 1485 cm-1
- CH3 bend at 1398 cm-1
- C-O stretch at 1100 cm-1
- C=O stretch at 1695 cm-1
- N-H stretch at 3417 cm-1
The IR spectrum
Development and validation of a stability-indicating HPLC method for the simultaneous determination of Losartan potassium, hydrochlorothiazide, and their degradationproducts
Development and Validation of Stability Indicating Assay Method of Montelukas...ijtsrd
Background A simple, precise cost effective stability indicating assay method of Montelukast by RP HPLC. Material Chromatographic separation was achieved on a Meteoric core C18 column 100mm x 4.6 mm, 2.7µm using a mobile phase 2 ml Trifluroacetic acid mixture of acetonitrile 250ml and methanol 400ml 350 650 at a flow rate of 1.5ml per minute. Wavelength was detection at 255nm. The retention time of Montelukast was 4 minutes. Results The method was found linear concentration the range of 120.39 802.57µg ml, tablet analysis of RSD 0.403, accuracy range 30 , 50 , 100 , and 200 , Precision RSD 0.3. The proposed method was validated as per the ICH guidelines. Conclusion The HPLC method was validated and demonstrated good linearity, precision, accuracy, specificity, robustness and stability indicating. The development HPLC method can be utilized for routine analysis stability studies for Montelukast. Miss. Harshada Ravindra Patil | Mr. Pavan Nathu Patil "Development and Validation of Stability Indicating Assay Method of Montelukast Tablet by RP-HPLC" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29809.pdf Paper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29809/development-and-validation-of-stability-indicating-assay-method-of-montelukast-tablet-by-rp-hplc/miss-harshada-ravindra-patil
Method validation in HPLC of omeprazole enantiomerscoolprashant33
Nilesh Kamble presented a seminar on the development and validation of a method for the enantiomeric estimation of omeprazole enantiomers in enteric-coated formulations using high-performance liquid chromatography. Omeprazole exists as two enantiomers but the S-enantiomer is metabolized more slowly and at lower doses. The method was developed using a chiral column with normal phase chromatography. Validation showed the method was specific, stability-indicating, linear, accurate and suitable for quantifying the undesired enantiomer in formulations. The developed method can help ensure only the desired enantiomer is present in marketed formulations.
The document describes the structure and logic of a prototype expert system called the Separation Synthesis Advisor (SSAD) for synthesizing separation sequences for gas/vapor mixtures. The core of the SSAD is the Separation Synthesis Hierarchy (SSH), which represents separation knowledge through a hierarchical, task-oriented framework. The SSH emulates the approach an expert process engineer would take. It divides the overall separation problem into four phases handled by distinct managers. This document focuses on the Gas Split Manager (GSM) which is responsible for separations involving predominantly gaseous mixtures. The GSM utilizes three specialized selectors to determine feasible separation methods and generate and sequence splits within a given gas mixture. Through a structured problem
This document describes a new approach for fingerprint analysis of Chrysanthemum morifolium Ramat combining ultra-performance liquid chromatography (UPLC) and chemometrics methods. UPLC allowed for chromatographic separation and detection of components in 10 minutes, much faster than conventional HPLC methods. Good precision, reproducibility, and accuracy were obtained for six typical components. Consistent results were seen based on cultivation source. The method provides a more rapid quality control technique for C. morifolium Ramat.
The document describes a method development and validation project for the analysis of drugs using UV spectrophotometry and HPLC. It provides an introduction, objectives, literature review, and plan of work. The literature review summarizes 15 research papers on method development and validation using these techniques. The instruments available for the project are also listed. The conclusion states that the project aims to develop analytical methods for quality assessment of drug products to ensure therapeutic effectiveness.
NOVEL SEPARATION AND QUANTITATIVE DETERMINATION OF LEVOFLOXACIN, PRULIFLOXACI...Dr. Ravi Sankar
The core AIM of the present study is to develop a novel, rapid, precise and accurate RP-HPLC method for simultaneous separation and quantification of six fluoroquinolones OF LEVOFLOXACIN (LEVO), PRULIFLOXACIN (PRFX), GATIFLOXACIN (GATI), SPARFLOXACIN (SPAR), MOXIFLOXAXIN (MOXI) AND BALOFLOXACIN (BALO) for the the day to day analysis.
The author felt that a novel single method for separation and quantification of all the above said drugs on single chromatographic system without any minor changes in detection wavelength and mobile phase composition.
To develop rapid, sensitive and economical analytical method based on HPLC for separation and estimation of six fluoroquinolones pharmaceutical dosage forms.
To develop method with shorter run time and better sensitivity.
Reducing the solvent consumption to make it more eco-friendly.
Avoid the column damage by minimizing the buffer strength and pH of mobile phase than reported methods.
To validate the method for different parameters like Accuracy, Precision, Linearity, specificity, Robustness International Conference on Harmonization ICH Q2(R1) guidelines..
To apply the developed RP-HPLC method in the analysis of pharmaceutical formulations.
This document describes the development and validation of two stability-indicating methods for the quantitative analysis of rosuvastatin (ROSU) in the presence of its degradation products: a high-performance liquid chromatography (HPLC) method and a high-performance thin layer chromatography (HPTLC) method. Both methods were found to be precise, accurate, specific and sensitive for the analysis of ROSU in raw materials and pharmaceutical formulations in the presence of degradation products. The methods were fully validated per ICH guidelines and successfully applied to analyze ROSU levels in commercial tablet formulations.
Stability indicating rp hplc method development and validation for simultaneo...Rajasekhar
The document describes the development and validation of a stability-indicating RP-HPLC method for the simultaneous estimation of levodropropizine and chlorpheniramine maleate in bulk and pharmaceutical dosage forms. The method utilizes a C18 column with a mobile phase of 45% 0.1% orthophosphoric acid and 55% acetonitrile at a flow rate of 1 mL/min. The method was validated for parameters such as linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness according to ICH guidelines. Forced degradation studies were also performed to demonstrate the stability-indicating ability of the developed method.
This document describes the development and validation of a stability-indicating RP-LC method for the determination of famciclovir (FCV) in the presence of its impurities and degradation products. A gradient reverse phase liquid chromatographic method was developed using an Inertsil ODS 3V column with a mobile phase of a potassium dihydrogen orthophosphate buffer and methanol mixture. FCV was subjected to oxidative, acid, base, hydrolytic, thermal, and photolytic degradation and was found to degrade significantly under oxidative, acid and base conditions. The developed method was validated according to ICH guidelines and demonstrated specificity, linearity, accuracy, precision, and robustness. This stability-indicating method can
This document presents the development and validation of an RP-HPLC assay method for the simultaneous estimation of a muscle relaxant drug and analgesic drug in pure form and pharmaceutical dosage forms. It discusses conducting a literature review, determining drug profiles including solubility and wavelength absorption, optimizing the analytical method, and expected outcomes such as a simple, accurate, precise and cost-effective simultaneous estimation method. The method will be validated as per ICH guidelines and force degradation studies will be performed to evaluate the stability indicating properties of the method.
The document discusses quantification techniques for herbal drugs. It describes various chromatography techniques used for quantification including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). These techniques are used to separate, identify, and quantify marker compounds in herbal drugs. The document provides an example of using HPLC to quantify atropine and examples of GC and GC-MS analyses of herbal extracts to separate and identify various compounds.
This document describes the development and validation of an RP-HPLC method for the simultaneous analysis of diclofenac sodium and rabeprazole sodium without the need for an internal standard. The method utilizes a C8 column with a mobile phase of triethyl amine buffer (pH 5):acetonitrile (50:50 v/v) at a flow rate of 2 mL/min. Validation showed the method to be linear, accurate, precise, sensitive and stable. The method was applied to a pharmaceutical formulation containing both drugs with recoveries from 98-100%.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document presents a stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of Valsartan in tablet dosage forms. The method uses a Phenomenox C18 column with a mobile phase of methanol and phosphate buffer at a ratio of 65:35 at a flow rate of 1mL/min. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification and specificity. Stress studies showed Valsartan degraded under acidic, basic, oxidative and heat conditions. The developed method can be used for assay of Valsartan in tablets and its degradation products.
This document presents the development and validation of an RP-HPLC method for the simultaneous estimation of a muscle relaxant drug and analgesic drug in pure form and pharmaceutical dosage forms. It discusses conducting a literature review on existing methods, determining the physicochemical properties of the drugs, optimizing the analytical method, and validating the developed method as per ICH guidelines. The expected outcomes are an accurate, precise, simple, cost-effective and fast method for simultaneously analyzing the two drugs.
Here are the key IR frequencies identified in the sample that match the reference standard of propafenone:
- C-C stretch at 1186 cm-1
- C=C stretch at 1651 cm-1
- C-H stretch (symmetric) at 2939 cm-1
- C-H bend at 1328 cm-1
- CH2 stretch (symmetric) at 1369 cm-1
- CH2 bend at 1485 cm-1
- CH3 bend at 1398 cm-1
- C-O stretch at 1100 cm-1
- C=O stretch at 1695 cm-1
- N-H stretch at 3417 cm-1
The IR spectrum
Development and validation of a stability-indicating HPLC method for the simultaneous determination of Losartan potassium, hydrochlorothiazide, and their degradationproducts
Development and Validation of Stability Indicating Assay Method of Montelukas...ijtsrd
Background A simple, precise cost effective stability indicating assay method of Montelukast by RP HPLC. Material Chromatographic separation was achieved on a Meteoric core C18 column 100mm x 4.6 mm, 2.7µm using a mobile phase 2 ml Trifluroacetic acid mixture of acetonitrile 250ml and methanol 400ml 350 650 at a flow rate of 1.5ml per minute. Wavelength was detection at 255nm. The retention time of Montelukast was 4 minutes. Results The method was found linear concentration the range of 120.39 802.57µg ml, tablet analysis of RSD 0.403, accuracy range 30 , 50 , 100 , and 200 , Precision RSD 0.3. The proposed method was validated as per the ICH guidelines. Conclusion The HPLC method was validated and demonstrated good linearity, precision, accuracy, specificity, robustness and stability indicating. The development HPLC method can be utilized for routine analysis stability studies for Montelukast. Miss. Harshada Ravindra Patil | Mr. Pavan Nathu Patil "Development and Validation of Stability Indicating Assay Method of Montelukast Tablet by RP-HPLC" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29809.pdf Paper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29809/development-and-validation-of-stability-indicating-assay-method-of-montelukast-tablet-by-rp-hplc/miss-harshada-ravindra-patil
Method validation in HPLC of omeprazole enantiomerscoolprashant33
Nilesh Kamble presented a seminar on the development and validation of a method for the enantiomeric estimation of omeprazole enantiomers in enteric-coated formulations using high-performance liquid chromatography. Omeprazole exists as two enantiomers but the S-enantiomer is metabolized more slowly and at lower doses. The method was developed using a chiral column with normal phase chromatography. Validation showed the method was specific, stability-indicating, linear, accurate and suitable for quantifying the undesired enantiomer in formulations. The developed method can help ensure only the desired enantiomer is present in marketed formulations.
The document describes the structure and logic of a prototype expert system called the Separation Synthesis Advisor (SSAD) for synthesizing separation sequences for gas/vapor mixtures. The core of the SSAD is the Separation Synthesis Hierarchy (SSH), which represents separation knowledge through a hierarchical, task-oriented framework. The SSH emulates the approach an expert process engineer would take. It divides the overall separation problem into four phases handled by distinct managers. This document focuses on the Gas Split Manager (GSM) which is responsible for separations involving predominantly gaseous mixtures. The GSM utilizes three specialized selectors to determine feasible separation methods and generate and sequence splits within a given gas mixture. Through a structured problem
This document describes a new approach for fingerprint analysis of Chrysanthemum morifolium Ramat combining ultra-performance liquid chromatography (UPLC) and chemometrics methods. UPLC allowed for chromatographic separation and detection of components in 10 minutes, much faster than conventional HPLC methods. Good precision, reproducibility, and accuracy were obtained for six typical components. Consistent results were seen based on cultivation source. The method provides a more rapid quality control technique for C. morifolium Ramat.
The document describes a method development and validation project for the analysis of drugs using UV spectrophotometry and HPLC. It provides an introduction, objectives, literature review, and plan of work. The literature review summarizes 15 research papers on method development and validation using these techniques. The instruments available for the project are also listed. The conclusion states that the project aims to develop analytical methods for quality assessment of drug products to ensure therapeutic effectiveness.
NOVEL SEPARATION AND QUANTITATIVE DETERMINATION OF LEVOFLOXACIN, PRULIFLOXACI...Dr. Ravi Sankar
The core AIM of the present study is to develop a novel, rapid, precise and accurate RP-HPLC method for simultaneous separation and quantification of six fluoroquinolones OF LEVOFLOXACIN (LEVO), PRULIFLOXACIN (PRFX), GATIFLOXACIN (GATI), SPARFLOXACIN (SPAR), MOXIFLOXAXIN (MOXI) AND BALOFLOXACIN (BALO) for the the day to day analysis.
The author felt that a novel single method for separation and quantification of all the above said drugs on single chromatographic system without any minor changes in detection wavelength and mobile phase composition.
To develop rapid, sensitive and economical analytical method based on HPLC for separation and estimation of six fluoroquinolones pharmaceutical dosage forms.
To develop method with shorter run time and better sensitivity.
Reducing the solvent consumption to make it more eco-friendly.
Avoid the column damage by minimizing the buffer strength and pH of mobile phase than reported methods.
To validate the method for different parameters like Accuracy, Precision, Linearity, specificity, Robustness International Conference on Harmonization ICH Q2(R1) guidelines..
To apply the developed RP-HPLC method in the analysis of pharmaceutical formulations.
Development and validation of UPLC method for simultaneous quantification of ...Ratnakaram Venkata Nadh
A methodical design-of-experiments were performed by applying quality-by-design concepts to establish
a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the
presence of degradation products. Response-surface, central-composite design, and quadratic model
were employed for statistical assessment of experimental data using the Design-Expert software.
Response variables such as resolution and retention time were analyzed statistically for chromatographic
screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were
considered for method optimization. The method was developed using C8 [100 � 2.1 mm, 1.8 μ] UPLC
column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50
v/v, the flow rate of 0.4 mL minute−1 and UV detection at 285 nm for both Carvedilol and Ivabradine.
The method was developed with a short run time of two minutes. The method was found to be linear in
the range of 25.0–199.9 μg mL−1 and 8.9–21.3 μg mL−1 for Carvedilol and Ivabradine, respectively with a
correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8%
and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH
Q2 (R1) guidelines.
Nanoparticles have various applications in modern separation science techniques. They can be used in liquid chromatography, gas chromatography, capillary electrophoresis, microchip electrophoresis, and ion chromatography. Nanoparticles are relatively easy to synthesize and functionalize, and have large surface area to volume ratios ideal for separations. Common nanoparticles used include gold nanoparticles, silica nanoparticles, and magnetic nanoparticles. They have been shown to improve separation efficiency, selectivity, and resolution compared to conventional separation methods. However, while successful in research, nanoparticle-based separations have not been widely adopted in industrial settings.
1. A Box-Behnken design was developed to optimize the extraction of phenolic compounds from Chelidonium majus L. using four extraction parameters: percentage of methanol, extraction time, extraction temperature, and solid-solvent ratio.
2. The model showed that percentage of methanol and solid-solvent ratio significantly influenced extraction yield, while percentage of methanol and extraction time had the greatest influence on phenolic content.
3. HPLC-DAD-ESI/MSn analysis of the center point extract identified 15 alkaloids and 15 phenolic compounds, including 9 flavonoids and 3 hydroxycinnamic acids not previously reported in C. majus.
This document summarizes research on using commercial chiral anion-exchange LC columns packed with quinidine or quinine ligands to separate enantiomers of acidic drugs and related compounds using hydro-organic mobile phases. Key findings include:
1) Low pH mobile phases provided the best retention and enantioresolution. Selectivity was largely independent of mobile phase variables except at pH >5-6.
2) Enantioseparation was achieved for a range of drug acids including NSAIDs and mandelic acids. However, enantioselectivity was not sufficient to explore achiral-chiral separations in a single column.
3) Retention was very similar across the three columns tested,
Head space gas_chromatography_analysis_of_residual (1)DivvyaIndran
This document describes a study that developed and validated a gas chromatography method for simultaneously analyzing 16 residual solvents using an EC-5 column with headspace injection. The method was found to provide good separation and resolution between peaks. Key findings include:
- The EC-5 column was selected based on matching solute and stationary phase polarities to improve resolution.
- The method was validated according to ICH guidelines and showed the method to be specific, accurate, precise, and rugged.
- The retention times of the 16 solvents were reported and resolution between peaks was calculated, demonstrating good separation of the solvents.
Supercritical fluid (CO2) chromatography for quantitative determination of se...Ratnakaram Venkata Nadh
In the present study, two cancer therapeutic drugs (docetaxel and bortezomib) were separated from their
potential impurities on a chromatographic platform by utilizing CO2 gas (supercritical state) and quantified.
The chromatographic separations were achieved on two short columns BEH-2EP (100mm 3mm, 1.7 mm)
and CHIRALPAK AD-3 (100 mm 4.6 mm, 3 mm) for docetaxel and bortezomib, respectively. The present
work describes the role of organic modifiers in the separation of polar compounds by supercritical fluid
chromatography. The two new methods were fully validated in accordance with the current ICH
(International Council for Harmonization of technical requirements for pharmaceuticals for human use)
guidelines. The stability indicating power of the methods was demonstrated from the stress studies
conducted on the injection formulations of the two compounds. The methods are precise with % RSD of
0.4, linear with the correlation coefficient of r2 $ 0.999 and accurate in the range of 50–150% of the
target assay concentration. The two methods can be equally employed for the assay determination of
docetaxel and bortezomib APIs as well.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, history, types, instrumentation, procedure, advantages, disadvantages and applications of HPLC. Key points include that HPLC uses high pressure to separate compounds faster and more efficiently than other chromatography techniques. Silica is commonly used as the stationary phase because it can withstand pressures over 300-400 atmospheres. The major components of HPLC instrumentation are the solvent reservoir, pump, injector, column, detector and data collector. HPLC has various applications in fields like analytical chemistry, pharmaceuticals, forensics and environmental analysis.
N-alkylation methods, Characterization and Evaluation of antibacterial activi...IJERA Editor
A series of new 5-Chloroisatin derivates have been synthesized by the method of N-alkylation at room temperature, in the presence of a base and a catalyst with good yields. The chemical structures of these compounds were confirmed by NMR (1H &13C), these new compounds obtained were evaluated for their antibacterial activity. The final results revealed that the majority of the compounds exhibited good antimicrobial activity against various organisms
Caliberation and validation of High performance liquid chromatographySaBa SaBir
A brief introduction to HPLCX
its principle of working , types of column , detectors
and more importantly, the caliberation and validation of HPLC its column, detector , volume of injection , oven tempreture etc
This document reviews various analytical microextraction techniques, including liquid-phase microextraction (LPME), hollow fiber liquid phase microextraction (HF-LPME), single drop-phase microextraction (SDME), liquid-liquid-liquid microextraction (LLLME), dispersive liquid-liquid microextraction (DLLME), and ionic liquid dispersive liquid-liquid microextraction (IL-DLLM). It discusses the advantages and disadvantages of each technique and how newer techniques aimed to overcome limitations of older methods. The document also reviews ultrasound-assisted and microwave-assisted extraction techniques used to improve microextraction performance and solid-phase extraction techniques used for liquid sample preparation.
Chromatography is a technique used to separate components of a mixture through differential partitioning between a stationary and mobile phase. There are two main forms: planar and column chromatography. High performance liquid chromatography (HPLC) uses column chromatography with a mobile liquid phase. The basis is the distribution coefficient which describes how a solute distributes between two immiscible phases. HPLC is used for analytical separations and employs a pump to deliver the mobile phase through a column containing particles or bonded stationary phase, followed by detection of eluting analytes. Detectors commonly use UV-visible absorption to quantify separated components. Sample preparation and column parameters impact resolution of analyte peaks from complex mixtures.
This document provides an overview of chromatography. It begins with an introduction that defines chromatography and describes how it separates mixtures based on differences in solubility between components in mobile and stationary phases. The document then covers the history of chromatography, important technical terms, main types categorized by interaction with the stationary phase or physical state of the mobile phase. Applications are discussed in areas like drug development, food testing, and forensics. Advantages are noted as versatility in separation. Disadvantages include temperature sensitivity and ensuring solubility. References are listed at the end.
This paper will focus on Cooperative learning in science education.
Curcumin extract is subjected to 1H NMR, 13C NMR, and 2D -HSQC FT-NMR analysis for structure
the 2D NMR specra may be obtained that indicate coupling between hydrogens and carbons to which they are attached. In this case it is called heteronuclear correlation spectroscopy (HECTOR, HSQC, or C-H HECTOR).
Molecular Structural Elucidation of Coloumn Secondary Fraction in Malvaviscus...BRNSSPublicationHubI
Malvaviscus penduliflorus (Malvaceae) is a popular erect under shrubs. It is garden ornamental widely grown across tropical and subtropical regions. The plant widely grown as both garden ornamentals and medicinal plants with varied ethnomedical uses, mostly for wounds, fever, hypertension, sore throat, bronchitis, gastritis, and liver and gall bladder problems. The pharmacognostic studies such as moisture content, ash values, extractive values, histology, and powder analysis were carried out. Successive solvent extraction and phytochemical screening were carried out. The extracts showed the presence of alkaloids, glycosides, phenols, steroids, terpenoids, carbohydrates, and saponins. The fraction was extracted from leaves and purified by column chromatography. The phytochemical studies gave conformation of the above said results. Molecular Formula: C20H18O10, Formula Weight: 418.354, Composition: C(57.42%) H(4.34%) O(38.24%) 16-(25,27-dihydroxyphenyl)-8-hydroxy-11,12-dihydro-15H-7-benzopyran-10-one)-5-(hydroxymethyl)-3-hydrofuran-2,4,6-triol.
1. Chiral chromatography refers to the separation of enantiomers using a chiral stationary phase in HPLC. Approximately 60% of pharmaceutical drugs are chiral.
2. There are several types of chiral stationary phases used for separation, including polymer-based carbohydrates, Pirkle phases, cyclodextrins, chirobiotic phases, and protein-based phases. Each type interacts differently with enantiomers through mechanisms like hydrogen bonding and pi-pi interactions.
3. Being able to separate enantiomers is important for drug development since individual enantiomers may have different biological effects and safety profiles. Chromatographic techniques allow for the analysis and purification of single enantiomers.
Application of emulsion liquid membranes for removal of Cd ,Co,Ni and Pb from...IOSR Journals
The paper points to the presence of heavy elements such as cobalt, nickel, lead and cadmium ratios of small but very harmful to the environment as well as health harmful if used by people for agricultural purposes, etc. This is the heavy elements harmful if it exceeds the limit as it is then used as components of the value after the extract has found these elements mentioned sources such as Ismailia Canal - Manzala Lake and the Red Sea, has been used as comparison tap water ELM for the separation of these elements has been selected cobalt (III) dicarbolide Span surfactant 80/85 and the use of acid silicon tungestic stage stripping effect concentrations of the carrier and the amendment, has been selected Co(III) dicarbolide because metal organic compound with a larger surface area and the distinction between the structure of certain net structure.
This document describes Shimadzu's Nexera Method Scouting System for efficiently developing chiral separation methods using HPLC. The system allows up to 96 combinations of columns and mobile phases to be tested on three chiral compounds: Bromacil, α-Methyl-α-Acetyl-γ-Butyloractone, and Methylclothiazide. Data processing software quickly identifies the best separation conditions by comparing resolution and peak symmetry. Using the system and iCHIRAL columns, optimized methods for each compound were successfully determined. The method scouting system is useful for method development in pharmaceutical, chemical, food, and drug discovery industries.
1. Barnhart, Gahm, Tomas, Notari, Semin, Cheetham 619
Wesley W. Barnhart Supercritical fluid chromatography tandem-column
Kyung H. Gahm
Sam Thomas method development in pharmaceutical sciences
Steve Notari for a mixture of four stereoisomers
David Semin
Janet Cheetham
A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns was
Discovery Analytical Sciences, achieved for baseline separation of a mixture of chiral pharmaceutical compounds
Molecular Structure, Amgen Inc., (i. e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobile
Thousand Oaks, CA 91320, USA phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50 : 50
v/v). On the contrary, this mixture (mixture A) could not be baseline separated by
SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns.
The effects of various mobile phases on elution order, capacity factor, selectivity, and
resolution were determined with mixture A on the individual aforementioned columns
to develop the tandem-column method.
Key Words: Supercritical fluid chromatography; Tandem-column; Diastereomer;
Received: January 4, 2005; revised: February 15, 2005; accepted: February 24, 2005
DOI 10.1002/jssc.200500005
1 Introduction can be overcome is to utilize tandem columns to obtain a
desired single-method separation. In this paper, an analy-
Supercritical fluid chromatography (SFC) is an important tical SFC method was developed by employing the cou-
analytical and preparative tool used in the pharmaceutical pling of Chiralpak AD-H and Chiralcel OD-H columns to
industry. Separation of chiral pharmaceutical compounds separate a mixture of pharmaceutical compounds (i.e.,
is an ever-increasing chromatographic field [1], and chiral- four stereoisomers); this separation was not achieved
ity is an important factor in drug development [2 – 5] due to using a single-column method. By understanding the elu-
the chiral specificity of biological processes [6]. Separa- tion order and effects of various mobile phases on the
tion of chiral compounds is vital because the activity of separation of the compounds by Chiralpak AD-H and
each enantiomer must be explored; it is possible that one Chiralcel OD-H columns separately, the development of
enantiomer may be inactive, an antagonist, or toxic [5 – 7]. the tandem-column method was made possible. A single-
A classic example is thalidomide. Originally marketed as a method separation is more desirable than multiple meth-
sedative, thalidomide was a racemic mixture; one enantio- ods, since it is less time consuming and cumbersome,
Original Paper
mer was a sedative, while the other was teratogenic [8, 9]. requiring no change of column or mobile phase conditions
SFC was the new and exciting chiral chromatographic between the analyses of different compounds.
topic in the early to mid 1980s [10, 11] when it was demon-
The use of coupled columns to separate compounds chro-
strated to be a technique capable of separating enantio-
matographically is not a novel concept [19, 22 – 27]; the
mers [12]. The use of SFC for the separation of enantio-
difficulty, however, is the determination of the correct
mers has been one of its most successful applica-
combination of columns given the large number of station-
tions [13]. Though the technique is over 20 years old, its
ary phases available on the market. According to San-
potential is continuing to be explored. SFC is a popular
dra [23], the selectivity and efficiency of a single column
chiral separation technique due to the inherent advan-
often does not provide adequate separation of a given
tages of using liquid CO2 in the mobile phase [1, 3, 11,
mixture of compounds. Recently, Phinney et al. [24]
14 – 21].
coupled achiral and chiral columns to separate a mixture
The development of a successful and facile single-column of achiral and chiral compounds.
analytical chiral separation method is not always achieved For our study, however, two chiral columns were coupled
due to limited in-house column selections or time restric- for the purpose of efficiently separating chiral compounds
tions imposed by the project cycle times of early discovery (a mixture of four stereoisomers). The initial choice to
chemistry projects. One way in which these limitations screen the Chiralpak AD-H and Chiralcel OD-H columns
was based on past successful experiences with these col-
Correspondence: Wesley W. Barnhart, Discovery Analytical
Sciences, Molecular Structure, Amgen Inc., Thousand Oaks, CA umns; Chiralpak AD and Chiralcel OD columns have been
91320, USA. Phone: +1 805 447 2055. Fax: +1 805 480 3015. shown to be very successful and widely used in chiral
E-mail: wesleyb@amgen.com. chromatography [1, 28, 29]. In addition to separating four
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2. 620 Barnhart, Gahm, Tomas, Notari, Semin, Cheetham
stereoisomers in a single method analytically, the mixture variable wavelength detector, and a waste containment
was also separated preparatively. As a proof of concept, vessel. The injector was a Modular Digital Pump (Model
approximately 1 mg of the four-stereoisomer mixture was XL3000) from Cavro Scientific Instruments Inc. (Sunny-
separated to baseline. The two coupled columns, though vale, CA, USA). The software used in the purification was
slightly cumbersome, did fit neatly into the column oven Berger SFC ProNTo v. 1.5.305.15.
heater and allowed the separation of all four stereo-
isomers in a single method. 2.4 Chiral packed columns
The analytical chiral packed columns were Chiralpak AD-
2 Experimental H and Chiralcel OD-H. Columns were purchased from
Chiral Technologies (Exton, PA, USA). The columns are
2.1 Materials
referred to as AD-H and OD-H throughout the paper.
The SFC-grade carbon dioxide was obtained from BOC Dimensions of the columns were 15064.6 mm ID with
Gases (Murray Hill, NJ, USA). Methanol (MeOH), ethanol 5 lm particle size.
(EtOH), and isopropanol (IsOH) were HPLC-grade from
Mallinckrodt Baker (Muskegon, MI, USA). The 1-propanol For preparative SFC, the chiral packed columns consisted
(nPrOH) and 1-butanol (nBuOH) were purchased from of Chiralpak AD-H (250621 mm, 5 lm) and Chiralcel
Sigma-Aldrich (St. Louis, MO, USA). All aforementioned OD-H (250621 mm, 5 lm) columns. Columns were pur-
solvents were of analytical grade. chased from Chiral Technologies (Exton, PA, USA). The
preparative columns are referred to as prep-ADH and
The compounds were synthesized in-house and dis- prep-ODH throughout the paper.
solved in methanol for analysis. The compound mixture
consisted of four stereoisomers and will be referred to
2.5 Analytical analysis conditions and
hereafter as mixture A. Individual stereoisomers of mix-
calculations
ture A were obtained by multi-step purification prior to the
development of the single-step, tandem-column prepara- For single-column analytical analyses, the mobile phase
tive method. The single-step purification method was consisted of 90% liquid CO2 and 10% organic modifier.
developed for the possibility of future purifications and to Organic modifiers were MeOH, EtOH, IsOH, nPrOH, and
provide a more efficient purification process than the nBuOH. For tandem-column analyses, the primary col-
multi-step method. umn order was AD-H and OD-H, respectively. These col-
umns were connected by a two-inch piece of stainless
2.2 Analytical SFC instrumentation steel tubing. Organic modifiers were EtOH, IsOH, and
EtOH:IsOH (50 : 50 v/v). Methods were isocratic with a
The analytical SFC instrument was a Berger SFC unit flow rate of 3.0 mL/min. Column oven and nozzle tem-
(Mettler-Toledo Autochem, Newark, DE, USA) with an perature were 408C, and the outlet pressure was 120 bar.
FCM1200 flow control module, a dual pump control mod-
ule, a TCM2100 thermal column module (temperature is Retention times used for calculating capacity factor and
controlled from 7 – 1508C), a column selection valve cap- resolution were obtained from the chromatographic data
able of switching between six columns, and a solvent con- generated via MassLynx v. 4.0 SP1. Void volume was
trol valve permitting selection of up to six modifiers. The estimated by using the retention time of the peak that
SFC was equipped with an Agilent 1100 photodiode array resulted from the change in refractive index from the injec-
detector with a high-pressure flow cell (Agilent Technolo- tion solvent. Peak widths were measured manually.
gies, Palo Alto, CA, USA). The autosampler/injector was
a CTC LC Mini PAL from Leap Technologies (Carrboro, 2.6 Preparative analysis conditions
NC, USA). A Waters (Milford, MA, USA) ZQ benchtop sin- The mobile phase consisted of 90% liquid CO and 10%
2
gle quadrupole mass spectrometer with an atmospheric EtOH:IsOH (50 : 50 v/v). The method was isocratic with a
pressure chemical ionization (APCI) source was coupled flow rate of 55 mL/min. Column oven and nozzle tempera-
to the SFC. The software packages used in the analyses ture were 408C, and the outlet pressure was 120 bar.
were Berger MassWare v. 4.01 and MassLynx v. 4.0 SP1.
3 Results and discussion
2.3 Preparative instrumentation
The preparative SFC was a Berger Multigram II from Met- 3.1 Single column analyses of mixture A
tler-Toledo Autochem (Newark, DE, USA). The compo- To gain a better understanding of the effects of organic
nents were the Separator Control Module (SCM)-2500, modifiers (i. e., MeOH, EtOH, IsOH, nPrOH, and nBuOH)
Electronics Control Module (ECM)-2500, CO2 solvent on the separation of the four stereoisomers, mixture A
delivery module, modifier solvent delivery module, direct was screened overnight with the OD-H and AD-H columns
expansion probe chiller, ventilated collection cabinet, UV separately (Figure 1 and Figure 2, respectively). The
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3. SFC tandem-column method development 621
Figure 1. Analytical chromatograms of mixture A with the OD-H column. The mobile phase consisted of 90% liquid CO2 and 10%
of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were
408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomeric
pair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)
goal was to develop a single method to separate all four with EtOH and nBuOH used as the organic modifer. Over-
stereoisomers. No single-column method, however, all, however, the elution order was not predictable with
yielded baseline separation of all four stereoisomers. respect to the various organic modifiers that were studied.
Therefore, the individual stereoisomers (designated as A closer look at individual peaks and their elution order
Peaks 1 through 4) were screened by OD-H (Figure 1) with respect to modifier (Figure 2) indicated three different
and AD-H (Figure 2) columns to determine the identity of cases. One such case was the elution order of Peak 2. Its
the peaks noted in the screening of mixture A. The stereo- elution order remained the most varied throughout the use
chemistry of the stereoisomers represented by Peaks 1, of the various modifiers. When MeOH was used as the
2, 3, and 4 are S,R; S,S; R,R; and R,S, respectively. The modifier, Peak 2 eluted first. With IsOH as the modifier,
peak number is based on the peak order found with the though, Peak 2 eluted last. Only with EtOH and nBuOH (2
OD-H column, since the peak order remained constant out of 5 modifiers) did Peak 2 have a consistent elution
regardless of the modifier. order. The second case is the elution of Peak 1. Peak 1
Considering the OD-H column results (Figure 1), no remained most consistent with its elution order. Except
change in peak order was observed throughout the use of when MeOH was used as the modifier, Peak 1 eluted first
the various mobile phases. Overall, adequate separation (4 out of 5 modifiers). Lastly, the elution order of Peaks 3
of all four components was not achieved with the OD-H and 4 remained the same for three of the five modifiers
column. Further exploration with IsOH via the OD-H col- (MeOH, EtOH, and nBuOH). Overall, as observed with
umn was performed, but baseline separation of Peaks 2 the OD-H column, no single AD-H method provided ade-
and 3 was not achieved. Decreasing the amount of IsOH quate separation of the four stereoisomers.
in the mobile phase did not improve separation.
Figure 2 clearly shows the elution order varied for Peaks 1 3.2 Capacity factor (k 9) of individual peaks of
through 4 via the AD-H column with the use of different mixture A for OD-H and AD-H columns
organic modifiers in the mobile phase. The only two ana- The capacity factor (k 9) for the four individual stereo-
lyses that yielded the same order of elution (1-2-4-3) were isomers was calculated and plotted (Figure 3). When
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4. 622 Barnhart, Gahm, Tomas, Notari, Semin, Cheetham
Figure 2. Analytical chromatograms of mixture A with the AD-H column. The mobile phase consisted of 90% liquid CO2 and 10%
of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were
408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomeric
pair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)
comparing k 9 values for Peaks 1 through 4 for both the
OD-H and AD-H columns, it appears that there was a
clear difference between columns in the trend found for k 9.
The k 9 values of the AD-H column with nBuOH (7.2, 9.4,
11.5, and 10.0 for Peaks 1, 2, 3, and 4, respectively) were
greater than the OD-H column with nBuOH (4.5, 5.8, 6.1,
and 8.3 for Peaks 1, 2, 3, and 4, respectively). However,
IsOH provided the largest k 9 values (i. e., 12.7 and 22.0 for
Peak 4 via AD-H and OD-H, respectively) with both col-
umns.
All four stereoisomers showed the same trend with the
OD-H column. The k 9 increased from MeOH to IsOH, with
the maximum value (8.0, 11.2, 13.3, and 22.0 for Peaks 1,
2, 3, and 4, respectively) found using IsOH as the modi-
fier. Unlike the AD-H column results, Peaks 2 and 3 main-
tained the most similar k 9 values between the use of the
various organic modifiers with the OD-H column.
For the AD-H column, all of the peaks did not follow the
same pattern with respect to the k 9 values; however, simi-
larities did emerge. Peaks 1 and 3 had very similar pat-
terns of k 9 vs. modifier (Figure 3). Also, Peaks 2 and 4
Figure 3. Capacity factor (k9) of all four peaks with various
showed similar trends and k 9 values. Peaks 2, 3, and 4 mobile phase components utilizing OD-H and AD-H col-
show very similar results from IsOH through nBuOH as umns. Liquid CO2 is 90% of the mobile phase, with the
the modifier. Both the pattern and k 9 values are very simi- remaining 10% as MeOH, EtOH, IsOH, nPrOH, or nBuOH.
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5. SFC tandem-column method development 623
3.4 Analytical scale AD-H-OD-H tandem column
analyses of mixture A
Individual AD-H and OD-H columns did not provide an
adequate method for the baseline separation of all four
stereoisomers. The individual column results previously
discussed, however, indicated the possibility of separat-
ing all four stereoiosomers if the AD-H and OD-H columns
were coupled. Organic modifiers were chosen based on
the results of the individual AD-H and OD-H column
screening. When considering tandem-column LC, the
retention time noted for a particular analyte is the sum of
the retention times of the analyte for the individual col-
umns; this is independent of the column order [25]. It was
by adding the peak retention times for the individual AD-H
and OD-H columns (with EtOH and IsOH as the organic
modifiers) that there appeared the possibility of resolving
all four stereoisomers through the coupling of the two col-
umns.
The tandem-column methods explored utilized mobile
phases that consisted of 90% liquid CO2 and 10% EtOH,
IsOH, or EtOH : IsOH (50 : 50 v/v) (Figure 5). Looking at
Figure 4. Resolution of Peaks 1 & 4 (D) and 2 & 3 (F) utilizing
different mobile phase components (MeOH, EtOH, IsOH, the tandem-column separation with EtOH and IsOH indivi-
nPrOH, and nBuOH) with the OD-H and AD-H columns. dually, it was predicted that a combination of the two
Liquid CO2 is 90% of the mobile phase, with the modifier would provide the desired separation. A combination of
(MeOH, EtOH, IsOH, nPrOH, or nBuOH) as the remaining EtOH and IsOH (EtOH : IsOH (50 : 50 v/v)) was then used
10%.
to achieve the separation of the four stereoisomers with
an analysis time of less than 15 min. Since baseline sep-
lar, indicating the AD-H is a column that had very similar
aration was already achieved with the 50 : 50 mixture,
affinities for all three peaks with IsOH, nPrOH, and
further exploration of EtOH : IsOH combinations for
nBuOH as the modifiers.
method optimization was not conducted. Also, reversing
the order of the tandem-columns did not result in a notable
difference in the separation of the stereoisomers.
3.3 Resolution (R ) of enantiomeric pairs of By utilizing the separation characteristics of individual
mixture A for OD-H and AD-H columns Chiralpak AD-H and Chiralcel OD-H columns, it was pos-
sible to obtain baseline separation of all four of the stereo-
The resolution (R) for the enantiomeric pairs was calcu- isomers through the coupling of the two columns; this was
lated and plotted (Figure 4). The resolution plots in Fig- not achieved with a single column. In addition, the base-
ure 4 show similar patterns found for the selectivities (not line separation was only accomplished by combining two
shown), as expected. For the OD-H column, the resolution mobile phase components (EtOH and IsOH) for the
patterns of both peak pairs (Peaks 1 & 4 and 2 & 3) were coupled column method.
very similar; however, Peaks 1 & 4 demonstrated much
greater R values than those found with Peaks 2 & 3, thus
indicating that the OD-H column was much better suited 3.5 Application of the analytical tandem-column
for separating the enantiomers 1 & 4 than 2 & 3. Peaks 1 & method
4 were best separated on the OD-H column using MeOH The analytical tandem-column SFC method was success-
as the organic modifier. fully applied to quickly determine the results of a reaction
A closer look at the AD-H results showed that each of the designed to transform a precursor into two of the four
enantiomeric pairs had much different behavior. The stereoisomers found in mixture A. Figure 6 shows the
modifier that resulted in the greatest R value for enantio- chromatograms indicating the precursor, the two stereo-
meric pairs 2 & 3 was MeOH. The lowest R value for enan- isomers formed, and a chromatogram of the original mix-
tiomeric pairs 2 & 3 was obtained through the use of IsOH ture A. The precursor could be easily separated from the
as the organic modifier. For 1 & 4, the greatest R value stereoisomers in mixture A, which would provide a quick
was observed with nPrOH as the modifier, and the lowest and facile procedure to monitor both the presence of the
R value resulted from the use of MeOH as the modifier. precursor and the product of the reaction utilizing a single
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
6. 624 Barnhart, Gahm, Tomas, Notari, Semin, Cheetham
Figure 5. Chromatograms of the AD-H and OD-H column coupling for the separation of the four stereoisomers. The mobile
phase consisted of 90% liquid CO2 and 10% EtOH, IsOH, or EtOH : IsOH (50 : 50 v/v). The temperature and outlet pressure were
408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indicates and correlates the order of elution with
the OD-H column.)
method. This was critical for a timely analysis of the single-column method. However, with the columns in tan-
results of the reaction. A single column could have been dem, a single method was successfully established. The
employed to resolve Peaks 1 and 2; however, both benefit of a single method was quickly realized when a
Peaks 3 and 4 would not have been identified (i. e., reaction (that converted a precursor into two of the stereo-
resolved) if they had also been formed during the reac- isomers of mixture A) was efficiently and effectively moni-
tion. tored; this provided critical data in a timely manner to
easily fit within the timelines provided for project support.
3.6 Preparative scale AD-H-OD-H tandem-column Overall, the order of elution of the four stereoisomers with
separation of mixture A the AD-H column was difficult to predict, while the order of
After establishing a tandem-column analytical method, elution remained constant with the OD-H column. This
the next step was to attempt a preparative separation. To indicates the separation mechanism on the AD-H column
demonstrate proof-of-concept, the same mobile phase for these compounds is more complicated compared to
composition (90% liquid CO2 and 10% EtOH : IsOH the OD-H column. Since both columns contain the same
(50 : 50 v/v)) was used for the preparative separation (Fig- derivative (tris-3,5-dimethylphenylcarbamate [30, 31]),
ure 7) as that used on the analytical scale. The overall the behavior of the stereoisomers with the AD-H column is
separation time was 24 min. This example illustrates how most likely due to the difference in the structure of amy-
a single method with coupled columns can be used to lose (AD-H) compared to cellulose (OD-H). The helical
baseline separate four stereoisomers and facilitate the nature of amylose allows for better inclusion when com-
purification process. pared to the more planar cellulose [3]. With the AD-H col-
umn, different alcohol modifiers in the mobile phase can
alter the chiral cavities and could modify the elution order
4 Conclusions of the peaks [32]. The AD-H column may therefore allow
Method development to separate a mixture of four stereo- more diverse interactions than the OD-H column resulting
isomers with AD-H or OD-H columns did not produce a in varied elution order with change of organic modifier.
J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
7. SFC tandem-column method development 625
Figure 6. Application of the tandem-column method for the analysis of the end products of a reaction. Chromatograms of a pre-
cursor, peaks 1 & 2, and mixture A are shown. The mobile phase consisted of 90% liquid CO2 and 10% EtOH : IsOH (50 : 50 v/v).
The temperature and outlet pressure were 408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indi-
cates and correlates the order of elution with the OD-H column.)
Figure 7. Preparative chroma-
togram of 1 mg of mixture A uti-
lizing the tandem-column
method. The mobile phase con-
sisted of 90% liquid CO2 and
10% EtOH : IsOH (50 : 50 v/v).
The column oven and nozzle
temperature were 408C, and
the outlet pressure was
120 bar. Total flow rate was
55 mL/min. (Peak number indi-
cates and correlates the order
of elution with the OD-H col-
umn.)
An advantage of analyzing the effects of organic modifiers References
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