5 Steps to Successful Liquid Chromatography
Over the past few decades, chromatography has seen a significant amount of development and progress. Sophisticated equipment has been invented to improve the efficiency of chromatography-based testing, and progress has also extended to the software that acquires and processes the data. A quick look at the history of this technique1 shows how much this field has evolved; starting from large packed columns and mobile phases being moved by gravity, to 50 mm columns packed with sub-3 µm beads and mobile phases moved using powerful pumps.
This considerable jump has put the technique on the throne of analytical methods. You can easily find HPLC equipment of all shapes and brands in labs that analyze pharmaceutical, environmental and food samples, owing to HPLC’s remarkable ability to analyze complex mixtures without the need for extensive sample preparation steps.
It is worth noting that the term liquid chromatography includes several techniques like thin layer chromatography, preparative, and paper chromatography. This guide is focused on High Performance Liquid Chromatography (HPLC), as it is the most commonly used technique for routine testing. If you understand the principle and the nature of the instrument, yet find the testing process slightly confusing, then this guide can help you find your way in a fast-paced lab that uses HPLC routinely.
Analytical HPLC methods can be overwhelming for beginners. Such methods are usually packed with information and instructions related to each step in the experiment. Becoming familiar with the format of HPLC methods in the lab is important, so you can easily find your way through the process.
Most written procedures start with an introduction or a scope which describes the aim of the method. Most state whether the method is used to identify or quantify an active ingredient or impurities, whether an extraction or a derivatization step is employed to prepare samples, and whether normal phase or reversed phase chromatography is used.
This is followed by an item stating the exact features of the instrument needed for that method and the type of column (and pre-column, if needed). Since HPLC equipment and column types vary significantly, this item is very critical; it may dictate the use of a certain brand of HPLC, specific detector, or specific attributes concerning the plumbing and injection loop size. There is also a section that states the operation parameters as well as the chemicals required to prepare the mobile phase(s), reference (standard) solutions, and test sample solutions. In many methods, you may find another section stating some special instructions and rules regarding the preparation of various solutions and the sequence of injections.
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Once you have identified the necessary equipment, column, and chemicals, the next step is to start setting up your instrument for testing. It is very important to check the calibration status of the HPLC instrument. The chosen instrument must have passed all the calibration tests successfully; this is usually indicated by a sticker or a label on the instrument stating the result and date of the last and next calibration. Calibration tests are conducted to make sure that each part of the instrument is working within an appropriate range. For instance, the pump’s flow rate must be checked to match the digital input; similar tests are conducted on the detector, injector and column heater. The aim is to ensure that all the instruments in a lab are working properly and within a predefined margin of error.
After selecting your instrument, you must clean it and prepare the column. The cleaning step is important to remove any remaining residues from previous work; thus a steady baseline and accurate results are obtainable. Many of the methods state a special sequence for washing the system prior to testing. In general, the cleaning procedure involves running water first to remove any buffer residues, followed by pure methanol or isopropanol/methanol mixture. Finally, a 50% organic solution is flushed through the system. The composition of final flushing solution largely depends on the type of chromatography employed: reversed or normal phase. The column is also flushed based on its type, and the composition of the mobile phase to be employed. Consult the leaflet of the column to determine the best flushing and storage solutions for your column.
The first solution to prepare is usually the mobile phase(s). Sometimes you need to prepare more than one mobile phase for your analytical method when a gradient system is used. It is very critical to use HPLC grade water and solvents to prepare mobile phases.
Once you have prepared your mobile phase(s), it is time to start flushing your system with the respective solution, and start equilibrating your column. The majority of HPLC systems are quaternary in nature, i.e. can pump from four different inlet lines. If more than one line is to be used (i.e. the method involves a gradient), then the final solution to be flushed through each line should be a solvent that is compatible with the mobile phase.
For instance, if the mobile phases have high buffer content, then the inlet lines must have been flushed with at least 50% aqueous flushing solution to prevent salts in the mobile phases from precipitating out and clogging the inlet lines. All flushing solutions and mobile phases should be filtered through micro filters (0.45 µm or 0.2 µm) to remove bacteria and organic fragments.
After you have flushed your system and started equilibrating your column - which in some cases may take 3 hours or more depending on the column type and composition of mobile phase - you should start working on your sample and reference solutions. In many cases, you need to take special precautions when preparing these solutions, such as lower exposure to light or moisture, to prevent the analyte(s) from degradation and producing false results. These special precautions are usually mentioned in the analytical method. Reference and sample solutions should also be filtered through special filters called syringe filters (because they are mounted on a syringe). However, the type and material of filters used should be compatible with the analyte(s) and solvent to avoid adsorption or contamination.
It is now time to run your solutions through your pre-set instrument. Modern instruments are run via software that helps you create operating methods and sequences of injections. It allows you to determine the order of solutions to be injected, the size, and the number of injections. The order of injections is usually stated in the analytical method or the standard operating procedures concerning HPLC. The sequence usually starts with injecting a blank (the solvent) then one or more solutions to demonstrate the suitability of the HPLC system including the column and mobile phase(s). Several parameters from the “system suitability” solution(s), such as resolution or plate count, help you decide whether the column, mobile phase and the HPLC instrument are performing collectively as required by the method. It is also recommended to test the reproducibility of your system by performing replicate injections of one of the reference solutions (5 or 6). The reproducibility is indicated by calculating the relative standard deviation of the area for the main peak of interest.
At this stage you may face some issues that require correction in your system. Troubleshooting skills are very important for any analytical chemist in general; and it needs time and patience to gain such skills. Here is a summary of some of the most common troubleshooting incidents faced by HPLC analysts:
This considerable jump has put the technique on the throne of analytical methods. You can easily find HPLC equipment of all shapes and brands in labs that analyze pharmaceutical, environmental and food samples, owing to HPLC’s remarkable ability to analyze complex mixtures without the need for extensive sample preparation steps.
It is worth noting that the term liquid chromatography includes several techniques like thin layer chromatography, preparative, and paper chromatography. This guide is focused on High Performance Liquid Chromatography (HPLC), as it is the most commonly used technique for routine testing. If you understand the principle and the nature of the instrument, yet find the testing process slightly confusing, then this guide can help you find your way in a fast-paced lab that uses HPLC routinely.
Getting ready for liquid chromatography testing
Analytical HPLC methods can be overwhelming for beginners. Such methods are usually packed with information and instructions related to each step in the experiment. Becoming familiar with the format of HPLC methods in the lab is important, so you can easily find your way through the process.
Most written procedures start with an introduction or a scope which describes the aim of the method. Most state whether the method is used to identify or quantify an active ingredient or impurities, whether an extraction or a derivatization step is employed to prepare samples, and whether normal phase or reversed phase chromatography is used.
This is followed by an item stating the exact features of the instrument needed for that method and the type of column (and pre-column, if needed). Since HPLC equipment and column types vary significantly, this item is very critical; it may dictate the use of a certain brand of HPLC, specific detector, or specific attributes concerning the plumbing and injection loop size. There is also a section that states the operation parameters as well as the chemicals required to prepare the mobile phase(s), reference (standard) solutions, and test sample solutions. In many methods, you may find another section stating some special instructions and rules regarding the preparation of various solutions and the sequence of injections.
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Picking your HPLC instrument and setting up
Once you have identified the necessary equipment, column, and chemicals, the next step is to start setting up your instrument for testing. It is very important to check the calibration status of the HPLC instrument. The chosen instrument must have passed all the calibration tests successfully; this is usually indicated by a sticker or a label on the instrument stating the result and date of the last and next calibration. Calibration tests are conducted to make sure that each part of the instrument is working within an appropriate range. For instance, the pump’s flow rate must be checked to match the digital input; similar tests are conducted on the detector, injector and column heater. The aim is to ensure that all the instruments in a lab are working properly and within a predefined margin of error.
After selecting your instrument, you must clean it and prepare the column. The cleaning step is important to remove any remaining residues from previous work; thus a steady baseline and accurate results are obtainable. Many of the methods state a special sequence for washing the system prior to testing. In general, the cleaning procedure involves running water first to remove any buffer residues, followed by pure methanol or isopropanol/methanol mixture. Finally, a 50% organic solution is flushed through the system. The composition of final flushing solution largely depends on the type of chromatography employed: reversed or normal phase. The column is also flushed based on its type, and the composition of the mobile phase to be employed. Consult the leaflet of the column to determine the best flushing and storage solutions for your column.
Preparation of solutions used in HPLC testing
The first solution to prepare is usually the mobile phase(s). Sometimes you need to prepare more than one mobile phase for your analytical method when a gradient system is used. It is very critical to use HPLC grade water and solvents to prepare mobile phases.
Once you have prepared your mobile phase(s), it is time to start flushing your system with the respective solution, and start equilibrating your column. The majority of HPLC systems are quaternary in nature, i.e. can pump from four different inlet lines. If more than one line is to be used (i.e. the method involves a gradient), then the final solution to be flushed through each line should be a solvent that is compatible with the mobile phase.
For instance, if the mobile phases have high buffer content, then the inlet lines must have been flushed with at least 50% aqueous flushing solution to prevent salts in the mobile phases from precipitating out and clogging the inlet lines. All flushing solutions and mobile phases should be filtered through micro filters (0.45 µm or 0.2 µm) to remove bacteria and organic fragments.
After you have flushed your system and started equilibrating your column - which in some cases may take 3 hours or more depending on the column type and composition of mobile phase - you should start working on your sample and reference solutions. In many cases, you need to take special precautions when preparing these solutions, such as lower exposure to light or moisture, to prevent the analyte(s) from degradation and producing false results. These special precautions are usually mentioned in the analytical method. Reference and sample solutions should also be filtered through special filters called syringe filters (because they are mounted on a syringe). However, the type and material of filters used should be compatible with the analyte(s) and solvent to avoid adsorption or contamination.
Run the system suitability solutions and troubleshoot your system
It is now time to run your solutions through your pre-set instrument. Modern instruments are run via software that helps you create operating methods and sequences of injections. It allows you to determine the order of solutions to be injected, the size, and the number of injections. The order of injections is usually stated in the analytical method or the standard operating procedures concerning HPLC. The sequence usually starts with injecting a blank (the solvent) then one or more solutions to demonstrate the suitability of the HPLC system including the column and mobile phase(s). Several parameters from the “system suitability” solution(s), such as resolution or plate count, help you decide whether the column, mobile phase and the HPLC instrument are performing collectively as required by the method. It is also recommended to test the reproducibility of your system by performing replicate injections of one of the reference solutions (5 or 6). The reproducibility is indicated by calculating the relative standard deviation of the area for the main peak of interest.
At this stage you may face some issues that require correction in your system. Troubleshooting skills are very important for any analytical chemist in general; and it needs time and patience to gain such skills. Here is a summary of some of the most common troubleshooting incidents faced by HPLC analysts:
- Low pressure, high pressure, and erratic pressure: The back pressure is an important indication for how optimal your system is operating. Low pressure is a sign of a leak in one of your connections, while high pressure is a sign of clogging (which may happen due to incompatibilities of solvents as mentioned above) or a deteriorating column. Erratic pressure is mainly caused by the presence of air bubbles in the system. Special flushing procedures may be necessary to remove the bubbles and restore steady conditions. In some systems, erratic pressure could be a sign of worn out internal parts, such as the check valves or purge frits.
- Poor reproducibility of replicate injections: This is demonstrated by a high relative standard deviation. The first thing that should be checked is the injector; dirty injectors will not provide consistent results. In other cases, the presence of air bubbles in vials of solutions may be the cause; some methods recommend sonicating the vials before injecting to remove any air bubbles.
- Lost resolution between a critical pair of peaks that you are trying to detect: Maintaining the resolution between closely situated peaks is critical to the success of HPLC testing as it allows for accurate determination of different components. A decrease in resolution may be caused by a deteriorating column or in some cases - especially in gradient methods - a fault in the gradient proportioning valve (GPV) of the instrument.
Process your chromatograms, calculate and submit your results
Once you are sure your system is working properly, resume the sequence of injections to inject the sample solutions. After completing the sequence, you can use specific software to process acquired chromatograms so that meaningful results can be drawn. Some software programs allow you to “integrate” all peaks, regardless of how small they are. The area under each peak is computed, and the number generated is used to determine the results of the test.
There are two main types of calculations after an HPLC experiment: Firstly, to determine the content of one or more active ingredients through the use of an assay, e.g. calculating the content of paracetamol in tablets. Alternatively, you may wish to determine the chromatographic profile, i.e. determine the type of impurities and their relative proportions. Current software can conduct calculations and produce a final report, after you enter molecular weights and dilution factors.
Finally, submit all the paperwork and results to be checked and approved. Each lab possesses its own procedures for this process.
References
1. Evolution of HPLC Systems. (n.d.). Retrieved from http://www.labmanager.com/lab-product/2010/06/evolution-of-hplc-systems#.Wz22NvkzbIV
