- Email: [email protected]
Chromatography
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Chromatography: Thermo Electron Corporation on how chromatographers can optimise separations in the pharmaceutical industry. In the pharmaceutical industry, there is an increasing need to analyse quickly incoming raw materials where both identification and purity of results are critical. The accurate analysis of intermediates and final products is essential, and compliance with organisations such as the US Food and Drink Administration (FDA) – together with Good Manufacturing Practices (GMP) guidelines – must be clearly documented. Chromatography has emerged as a primary tool in pharmaceutical analyses.
What types of chromatography are available? Chromatography is a common method employed to separate a mixture into its constitutive fractions. The separation is normally achieved based on the difference in affinity of a sample constituent towards a mobile phase, in comparison to a stationary phase. Since 1906 when chromatography was first defined, several techniques have been successfully used in research and in industry, with different types being used for different purposes: • HPLC (high performance liquid chromatography) is one of the widely-used liquid chromatography techniques that employs high pressured liquid flowing through a column to achieve a high resolution of separation. Because of its dynamic nature, HPLC has found use in a wide variety of applications, but most especially in pharmaceutical analyses. The
most common type of HPLC, namely RPHPLC (Reverse Phase HPLC) is very useful in separating very similar constituents. This type of HPLC employs a non-polar column, commonly C-18, with a polar solvent as the mobile phase (one example of this technology is Hypersil GOLD from Thermo); • GC (gas chromatography) – specifically gasliquid chromatography – involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase that is adsorbed onto the surface of an inert solid. Since GC is a gas-based separation technique, it is limited to components that have sufficient volatility and thermal stability. A variety of sample types can be successfully analysed by GC. Unlike HPLC, which is used to separate larger molecules, GC is best suited for analysis of samples with smaller molecules. Thus, GC tends to be used for validation purposes of pharmaceutical samples (for example – TRACE GC Ultra from Thermo).
Pharmaceutical applications for chromatography HPLC has come a long way since the 1970s when it was initially used to separate a mixture of small molecules. Today it is employed not only to purify proteins from their native mixture, but also to
Figure 1: When the particle size is reduced, column length can also be reduced while keeping separation efficiency constant.
separate complex individual proteins from a sample mixture. The use of the HPLC system was further enhanced by coupling it to a mass spectrometer – like a Surveyor Plus from Thermo. A system that integrates both these technologies and automates the process of first separating the sample mixture into its constituents and then enabling analysis – in terms of identification and characterisation – has driven pharmaceutical analyses into new frontiers. Other applications that use chromatography in the pharmaceutical industry include proteomics, drug discovery, drug formulation, and quality control methods.
Using GC for QA/QC methods A major concern for the pharmaceutical industry is to confirm and ensure that final products do not contain impurities that come from the manufacturing process. One source of such impurities is from solvents used during the production of pharmaceuticals, which have not been removed prior to packaging. Gas chromatography is typically used to test for these compounds due to their volatility, and the powerful separating capability of the capillary column. A chief concern of the analyst using this instrumentation is how to convert the sample into a suitable form for introduction into the GC. A convenient analysis of residual solvents is by GC/MS with a headspace injection. This consists of dissolution of the drug in a suitable solvent, followed with
Figure 2 demonstrates the effect of particle size on the resolution of a mixture of 7 phenones, the half height resolution of the two last eluting components is annotated. The Y-axes of the five chromatograms have been normalised to illustrate the gain in sensitivity when more efficient peaks are obtained.
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introduction of the vapour above the resulting solution into the GC inlet. This is a fast and easy way of separating the volatile solvents from the nonvolatile matrix, which could interfere with the compounds of interest.
by a quadrupole mass spectrometer (from Thermo).
The need for speed
Issues facing chromatographers in Pharma
One common need for most businesses is to perform their necessary functions faster. One way to speed up analysis time is to increase the flow rate through the GC column. Traditionally, the low pressures required by mass spectrometers discouraged analysts from using the high flow rates associated with other GC detectors. A study was conducted to test the use of a single quadrupole GC/MS (a DSQ mass spectrometer from Thermo) in handling the elevated flow rates in the analysis of solvents in pharmaceuticals: Samples and standards were analysed by headspace GC/MS using elevated gas flows in order to shorten run times. Vacuum fast chromatography, in which the vacuum of the mass spectrometer is used to pull carrier gas through the column, was initially tried. This method was compared to a more commonly used approach in which the GC flow module pushes carrier gas through the column. The eluting analytes were detected
The study demonstrated that the use of elevated carrier gas flows allowed for fast quantitation of the solvents.
The main challenge facing modern chromatographers is how to optimise chromatographic separations. This challenge can be crystallised down to a few core components, namely how to produce: • Faster analyses and improved resolution; • Enhanced sensitivity.
Obtaining faster analyses and improved resolution The aim of a chromatographic separation is to obtain good resolution in a short analysis time, i.e. resolution needs to be maximised while minimising analysis time. Sub 2 µm particles have been developed as a chromatographic media to achieve high resolution of the sample components, fast analyses and high sensitivity. A key tool in achieving faster analysis times has been developed in the form of highspeed chromatographic systems (for example the Accela from Thermo). Such instruments provide fast, efficient chromatographic
separations over an expansive range of flow rates and pressures. The systems are designed to optimise performance of sub-two micron particle columns, providing seamless operation spanning conventional LC pressures from short LC columns, up to 15,000 psi for long-column separations of complex bio mixtures. Another way of achieving faster analysis times can be accomplished with UltraFast chromatography. UltraFast analytical results are achieved in a fraction of the time required by conventional analytical techniques. UltraFast chromatography produces precise results, which meet or exceed current regulatory requirements. An UltraFast module from Thermo, for example, can be made with any of the common narrow bore capillary columns available from column manufacturers, making it usable with other headspace analytical techniques. The most important way to improve chromatographic resolution is to optimise the column that is used as part of the chromatographic system. Resolution and analysis time are determined by the ratio of column length to particle size. When the particle size is reduced, column length can also be reduced while keeping separation efficiency constant (see figure 1 on page 32).
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How can we alter the speed of analysis? Reducing column length
Column length 100 mm Column ID 4.6 mm Particle size 5 µm
Column length 50 mm Column ID 4.6 mm Particle size 5 µm
Reducing column ID, decreasing flow rate to maintain constant linear velocity Column length 50 mm Column ID 2.1 mm Particle size 5 µm
Flow rate 1 mL/min
Flow rate 1 mL/min
Flow rate 0.2 mL/min
Notes: Column: Hypersil GOLD, dimensions vary as shown; Mobile phase: H2O/ACN (50:50)+0.1% formic acid; Flow rate: varies as shown Temperature: 25° C Detection: UV@ 244 nm (2 µL flow cell) Tubing column - detector: 0.005" ID Injection volume: 60 nL
For example, if 13,500 plates (green line on figure 1) are needed to obtain the required resolution, a 150 mm column packed with 5 µm particles would be required. However, if the particle size is reduced to 1.9 µm, then only 50 mm of column is needed to obtain the same 13,500 plates. For a constant flow rate, analysis time would be reduced approximately 3-fold with this change in particle size and column length. Another effective way to increase the resolution of two chromatographic bands is to increase the selectivity factor by manipulating column chemistry or mobile phase composition; 1.9 µm columns are available in a range of chemistries, including C18 selectivity, polar endcapped C18 and perfluorinated phenyl, which allow more flexibility when developing high resolution, fast methods. A process for packing 1.9 µm particles into robust and reproducible columns has recently been developed and optimised. Packing bed homogeneity at higher pressures had to be ensured to maintain efficiency and column hardware, and end-fittings had to be re-designed to withstand high operating pressures.
Flow rate 0.55 mL/min
Column length 50 mm Column ID 2.1 mm Particle size 1.9 µm
Analytes: Cortisone 11-α-hydroxyprogesterone 17-α-hydroxyprogesterone Progesterone
Surveyor MSQ Plus mass spectrometer) can provide a strong starting point for sample analysis by offering a quick and clear mass confirmation for chromatographic peaks. It eliminates the challenges facing the chromatographer using UV detection by: • Reducing time limitations; • Increasing sensitivity; • Identifying chromatogram peaks with confidence; • Providing high throughput mass confirmation. Unknown chromatographic peaks can appear during the development and manufacturing of drugs, chemicals and natural products. LC/MS enables the chromatographer to quickly and effectively suggest a number of possibilities for these unknown peaks. Sensitivity can also be enhanced by manipulation of the column technology; two simple ways to do this are to reduce the length and reduce the internal diameter of the column.
The key way of achieving enhanced sensitivity is to couple the GC or LC system with a mass spectrometer – LC/MS and GC/MS.
Decreasing column length results in a shorter run time. This in turn promotes a further small increase in the peak sensitivity. However, when decreasing the column internal diameter, it is necessary to adjust the mobile phase flow rate to maintain a constant mobile phase linear velocity through the column.
LC/MS solutions (for example Thermo’s Surveyor HPLC system coupled with the
By decreasing the particle size, efficiency is increased, which leads to more sensitivity.
Enhanced sensitivity
Reducing particle size and increasing flow rate
Using smaller particles also allows us to further increase the mobile phase flow rate, which also increases the speed of analysis. The box demonstrates these effects on the separation of four steroids (see box – ‘how can we alter the speed of analysis?’).
Conclusion Chromatography can provide a range of information that cannot be matched using other techniques; however it is still possible to optimise separations by employing a few simple measures: • Obtain faster analysis times by optimising modern column technologies; • In order to increase resolution, when the particle size is reduced, column length should also be reduced; • Increase the selectivity factor by manipulating column chemistry or mobile phase composition and this will result in higher resolution; • Coupling the chromatography system with a mass spectrometer will result in significant enhanced sensitivity; Manipulation of the column technology can also result in increased sensitivity.
•
Contact: Thermo Electron Corporation [email protected] www.thermo.com
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Feature
Filtration+Separation September 2006
Chromatography: Thermo Electron Corporation on how chromatographers can optimise separations in the pharmaceutical industry. In the pharmaceutical industry, there is an increasing need to analyse quickly incoming raw materials where both identification and purity of results are critical. The accurate analysis of intermediates and final products is essential, and compliance with organisations such as the US Food and Drink Administration (FDA) – together with Good Manufacturing Practices (GMP) guidelines – must be clearly documented. Chromatography has emerged as a primary tool in pharmaceutical analyses.
What types of chromatography are available? Chromatography is a common method employed to separate a mixture into its constitutive fractions. The separation is normally achieved based on the difference in affinity of a sample constituent towards a mobile phase, in comparison to a stationary phase. Since 1906 when chromatography was first defined, several techniques have been successfully used in research and in industry, with different types being used for different purposes: • HPLC (high performance liquid chromatography) is one of the widely-used liquid chromatography techniques that employs high pressured liquid flowing through a column to achieve a high resolution of separation. Because of its dynamic nature, HPLC has found use in a wide variety of applications, but most especially in pharmaceutical analyses. The
most common type of HPLC, namely RPHPLC (Reverse Phase HPLC) is very useful in separating very similar constituents. This type of HPLC employs a non-polar column, commonly C-18, with a polar solvent as the mobile phase (one example of this technology is Hypersil GOLD from Thermo); • GC (gas chromatography) – specifically gasliquid chromatography – involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase that is adsorbed onto the surface of an inert solid. Since GC is a gas-based separation technique, it is limited to components that have sufficient volatility and thermal stability. A variety of sample types can be successfully analysed by GC. Unlike HPLC, which is used to separate larger molecules, GC is best suited for analysis of samples with smaller molecules. Thus, GC tends to be used for validation purposes of pharmaceutical samples (for example – TRACE GC Ultra from Thermo).
Pharmaceutical applications for chromatography HPLC has come a long way since the 1970s when it was initially used to separate a mixture of small molecules. Today it is employed not only to purify proteins from their native mixture, but also to
Figure 1: When the particle size is reduced, column length can also be reduced while keeping separation efficiency constant.
separate complex individual proteins from a sample mixture. The use of the HPLC system was further enhanced by coupling it to a mass spectrometer – like a Surveyor Plus from Thermo. A system that integrates both these technologies and automates the process of first separating the sample mixture into its constituents and then enabling analysis – in terms of identification and characterisation – has driven pharmaceutical analyses into new frontiers. Other applications that use chromatography in the pharmaceutical industry include proteomics, drug discovery, drug formulation, and quality control methods.
Using GC for QA/QC methods A major concern for the pharmaceutical industry is to confirm and ensure that final products do not contain impurities that come from the manufacturing process. One source of such impurities is from solvents used during the production of pharmaceuticals, which have not been removed prior to packaging. Gas chromatography is typically used to test for these compounds due to their volatility, and the powerful separating capability of the capillary column. A chief concern of the analyst using this instrumentation is how to convert the sample into a suitable form for introduction into the GC. A convenient analysis of residual solvents is by GC/MS with a headspace injection. This consists of dissolution of the drug in a suitable solvent, followed with
Figure 2 demonstrates the effect of particle size on the resolution of a mixture of 7 phenones, the half height resolution of the two last eluting components is annotated. The Y-axes of the five chromatograms have been normalised to illustrate the gain in sensitivity when more efficient peaks are obtained.
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introduction of the vapour above the resulting solution into the GC inlet. This is a fast and easy way of separating the volatile solvents from the nonvolatile matrix, which could interfere with the compounds of interest.
by a quadrupole mass spectrometer (from Thermo).
The need for speed
Issues facing chromatographers in Pharma
One common need for most businesses is to perform their necessary functions faster. One way to speed up analysis time is to increase the flow rate through the GC column. Traditionally, the low pressures required by mass spectrometers discouraged analysts from using the high flow rates associated with other GC detectors. A study was conducted to test the use of a single quadrupole GC/MS (a DSQ mass spectrometer from Thermo) in handling the elevated flow rates in the analysis of solvents in pharmaceuticals: Samples and standards were analysed by headspace GC/MS using elevated gas flows in order to shorten run times. Vacuum fast chromatography, in which the vacuum of the mass spectrometer is used to pull carrier gas through the column, was initially tried. This method was compared to a more commonly used approach in which the GC flow module pushes carrier gas through the column. The eluting analytes were detected
The study demonstrated that the use of elevated carrier gas flows allowed for fast quantitation of the solvents.
The main challenge facing modern chromatographers is how to optimise chromatographic separations. This challenge can be crystallised down to a few core components, namely how to produce: • Faster analyses and improved resolution; • Enhanced sensitivity.
Obtaining faster analyses and improved resolution The aim of a chromatographic separation is to obtain good resolution in a short analysis time, i.e. resolution needs to be maximised while minimising analysis time. Sub 2 µm particles have been developed as a chromatographic media to achieve high resolution of the sample components, fast analyses and high sensitivity. A key tool in achieving faster analysis times has been developed in the form of highspeed chromatographic systems (for example the Accela from Thermo). Such instruments provide fast, efficient chromatographic
separations over an expansive range of flow rates and pressures. The systems are designed to optimise performance of sub-two micron particle columns, providing seamless operation spanning conventional LC pressures from short LC columns, up to 15,000 psi for long-column separations of complex bio mixtures. Another way of achieving faster analysis times can be accomplished with UltraFast chromatography. UltraFast analytical results are achieved in a fraction of the time required by conventional analytical techniques. UltraFast chromatography produces precise results, which meet or exceed current regulatory requirements. An UltraFast module from Thermo, for example, can be made with any of the common narrow bore capillary columns available from column manufacturers, making it usable with other headspace analytical techniques. The most important way to improve chromatographic resolution is to optimise the column that is used as part of the chromatographic system. Resolution and analysis time are determined by the ratio of column length to particle size. When the particle size is reduced, column length can also be reduced while keeping separation efficiency constant (see figure 1 on page 32).
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Feature
Filtration+Separation September 2006
How can we alter the speed of analysis? Reducing column length
Column length 100 mm Column ID 4.6 mm Particle size 5 µm
Column length 50 mm Column ID 4.6 mm Particle size 5 µm
Reducing column ID, decreasing flow rate to maintain constant linear velocity Column length 50 mm Column ID 2.1 mm Particle size 5 µm
Flow rate 1 mL/min
Flow rate 1 mL/min
Flow rate 0.2 mL/min
Notes: Column: Hypersil GOLD, dimensions vary as shown; Mobile phase: H2O/ACN (50:50)+0.1% formic acid; Flow rate: varies as shown Temperature: 25° C Detection: UV@ 244 nm (2 µL flow cell) Tubing column - detector: 0.005" ID Injection volume: 60 nL
For example, if 13,500 plates (green line on figure 1) are needed to obtain the required resolution, a 150 mm column packed with 5 µm particles would be required. However, if the particle size is reduced to 1.9 µm, then only 50 mm of column is needed to obtain the same 13,500 plates. For a constant flow rate, analysis time would be reduced approximately 3-fold with this change in particle size and column length. Another effective way to increase the resolution of two chromatographic bands is to increase the selectivity factor by manipulating column chemistry or mobile phase composition; 1.9 µm columns are available in a range of chemistries, including C18 selectivity, polar endcapped C18 and perfluorinated phenyl, which allow more flexibility when developing high resolution, fast methods. A process for packing 1.9 µm particles into robust and reproducible columns has recently been developed and optimised. Packing bed homogeneity at higher pressures had to be ensured to maintain efficiency and column hardware, and end-fittings had to be re-designed to withstand high operating pressures.
Flow rate 0.55 mL/min
Column length 50 mm Column ID 2.1 mm Particle size 1.9 µm
Analytes: Cortisone 11-α-hydroxyprogesterone 17-α-hydroxyprogesterone Progesterone
Surveyor MSQ Plus mass spectrometer) can provide a strong starting point for sample analysis by offering a quick and clear mass confirmation for chromatographic peaks. It eliminates the challenges facing the chromatographer using UV detection by: • Reducing time limitations; • Increasing sensitivity; • Identifying chromatogram peaks with confidence; • Providing high throughput mass confirmation. Unknown chromatographic peaks can appear during the development and manufacturing of drugs, chemicals and natural products. LC/MS enables the chromatographer to quickly and effectively suggest a number of possibilities for these unknown peaks. Sensitivity can also be enhanced by manipulation of the column technology; two simple ways to do this are to reduce the length and reduce the internal diameter of the column.
The key way of achieving enhanced sensitivity is to couple the GC or LC system with a mass spectrometer – LC/MS and GC/MS.
Decreasing column length results in a shorter run time. This in turn promotes a further small increase in the peak sensitivity. However, when decreasing the column internal diameter, it is necessary to adjust the mobile phase flow rate to maintain a constant mobile phase linear velocity through the column.
LC/MS solutions (for example Thermo’s Surveyor HPLC system coupled with the
By decreasing the particle size, efficiency is increased, which leads to more sensitivity.
Enhanced sensitivity
Reducing particle size and increasing flow rate
Using smaller particles also allows us to further increase the mobile phase flow rate, which also increases the speed of analysis. The box demonstrates these effects on the separation of four steroids (see box – ‘how can we alter the speed of analysis?’).
Conclusion Chromatography can provide a range of information that cannot be matched using other techniques; however it is still possible to optimise separations by employing a few simple measures: • Obtain faster analysis times by optimising modern column technologies; • In order to increase resolution, when the particle size is reduced, column length should also be reduced; • Increase the selectivity factor by manipulating column chemistry or mobile phase composition and this will result in higher resolution; • Coupling the chromatography system with a mass spectrometer will result in significant enhanced sensitivity; Manipulation of the column technology can also result in increased sensitivity.
•
Contact: Thermo Electron Corporation [email protected] www.thermo.com