HPLC Basics, Equipment, and Troubleshooting
Contents
1. 60s switching to thin layer chromatography in the early 70s and then to HPLC and ion chromatography in late 70s His career has focused on instrument and column development and user support providing a broad foundation of practical experience to call on as an instructor He is probably best known as the moderator of the popular Chromatography Forum on line chromatography discussion group 1I IU O O DA Official Denmark Distributor ANALYTICAL TRAINING SOLUTIONS _ MD Scientific Premier Training for Analytical Chemists www md scientific dk COURSE DETAIL Below are listed each section and sub section of the class A brief summary of each sub section is given first After the subsection in parentheses is the approximate running time of that module Section 1 Ilsocratic HPLC Basics What is HPLC 15 mins A summary of the history of HPLC how it developed into the ubiquitous technique we use today and some of the standard vocabulary and terminology Separation Chemistry 15 mins An overview of the different chemical mechanisms used in HPLC e Reversed phase e Normal phase amp HILIC lon exchange e lon pair e Size Exclusion GFC amp GPC Key Measurements Retention and Selectivity 19 mins A number of parameters are used to describe an HPLC separation In this section we will define and explore the significance of k retention and alpha selectivity Key Measurements Efficiency and Resolution 16 mins A numbe2. compare selectivity systematically Section 5 pH amp lonization Effects Controlling lonization 15 mins By implication reversed phase chromatography works with neutral molecules Weak acids and bases which can be partially ionized complicate things In this section we will explore the impact of ionization and pH on reversed phase separation lon Pair Chromatography 15 mins As we saw in Section 1b ion pair chromatography behaves like a hybrid of reversed phase and ion exchange That makes it the ideal technique for mixtures of neutral and ionized compounds We ll look at how to control the separation chemistry and avoid or live with some of the pitfalls Section 6 Normal phase HILIC lon exchange amp Size exclusion Normal Phase Chromatography 15 mins Normal phase is the reverse of reverse phase In this section we ll look at the mechanism of normal phase its implications for selectivity and some of the reasons why it has proven to be less popular than reversed phase Hydrophilic Interaction Chromatography HILIC 7 mins HILIC is technically a subset of normal phase chromatography but it is used with solvents that we typically associate more with reversed phase That make it the ideal technique for the analysis of very polar molecules and often the best alternative to reversed phase lon Exchange Chromatography 10 mins In HPLC practice ion exchange is used primarily for the analysis of very small usually inor
3. operation We ll cover potential failure modes and how to avoid them The different designs of autosamplers will be reviewed along with a summary of the pros and cons of each Section 9 HPLC Equipment Columns Overview 4 mins A column is in fact a consumable item but many column die prematurely We ll look at the major causes of causes of column failure and early warning symptoms of impending column death Physical Problems 20 mins Physical causes of column death typically involve column inlet flow profile anomalies partially plugged frits trapped air amp head spaces We ll look at the symptoms of each possible fixes and prevention Non Column Problems Part 1 6 mins Sometimes we blame the column for things that are not the column s problem In this section we will look at the symptoms and prevention of flow variations and extra column volume Non Column Problems Part 2 11 mins Continuing the discussion of non column problems we ll look at temperature effects amp late eluting compounds Chemical Problems 19 mins Chemical causes of column death include contamination column degradation overload the strong diluent effect injection volume on column degradation mobile phase variations and sub optimal storage conditions We ll look at diagnostic symptoms for each as well as possible column regeneration techniques Documentation 12 mins Many problems are the result of pilot error and many of these pr
4. 1b fore other et ten ANALYTICAL TRAINING SOLUTIONS Premier Training for Analytical Chemists A a m FER HPLC Basics Equipment and Troubleshooting HPLC Basics Equipment and Troubleshooting explains chromatography in practical terms from the ground up II IM D er tes Official Denmark Distributor ANALYTICAL TRAINING SOLUTIONS a MD Scientific Premier Training for Analytical Chemists www md scientific dk HPLC Basics Equipment and Troubleshooting HPLC Basics Equipment and Troubleshooting explains chromatography in practical terms from the ground up WHO SHOULD TAKE THIS COURSE If you use chromatography on the job and would like a better understanding of how it works HPLC Basics Equipment and Troubleshooting is the course for you It s designed for chemists and biochemists who use HPLC as a regular part of their jobs but technicians with some HPLC experience will also find the course valuable No previous HPLC training is assumed however much of the course will appeal to the experienced chemists who want a firm grounding in the basics of HPLC WHAT DOES IT COVER HPLC Basics Equipment and Troubleshooting explains chromatography in practical terms from the ground up Here s what you ll learn in this online course The basic principles of HPLC The five fundamental HPLC modes The easy and logical way to adjust experimental variables to get a good separation The best approach to use for various chr
5. ganic strong acid or base compounds or for the analysis of large biopolymers We ll look at how it works the mechanism and the major control parameters Size Exclusion Chromatography 13 mins SEC is arguably not chromatography at all but rather a first cousin my marriage We ll look at the reasons why the types of samples to which it is appropriate and how to minimize some of the accompanying problems Section 7 HPLC Equipment Reservoirs amp Pumps Reservoirs and Degassing 12 mins Why is degassing so important the pros and cons of different degassing techniques and quick checks for inlet blockages Pumps 19 mins The pros and cons of different pump designs potential failure modes and their symptoms and basic preventive maintenance Section 8 HPLC Equipment Tubing amp Injectors Tubing 15 mins The choice of tubing materials and dimensions good practice for cutting tubing Consequences symptoms of poor choice or technique 1I I O O D Official Denmark Distributor ANALYTICAL TRAINING SOLUTIONS P MD Scientific Premier Training for Analytical Chemists www md scientific dk Fittings 10 mins We ll look at the design of HPLC fittings with an emphasis on interchangeability from different vendors and good assembly technique We ll also cover the symptoms of poor assembly and ways to avoid the most common problems Injectors 16 mins We ll look at the way injection valves work and the consequences for reliable
6. inant sources of error including the pseudo internal standard trick 1b tele outer eb tes Official Denmark Distributor ANALYTICAL TRAINING SOLUTIONS a MD Scientific Premier Training for Analytical Chemists www md scientific dk Section 12 UHPLC What s Different 14 mins The latest buzz acronym is UHPLC Ultra High Performance Liquid Chromatography In many respects UHPLC is simply a continuation of the HPLC trend to smaller particles but there are some key differences that can complicate the transition Transferring Scaling Methods 10 mins We ll look at the steps required to adapt UHPLC methods to HPLC instruments and columns and vice versa Possible Surprises 9 mins An overview of the common pitfalls when moving from HPLC to UHPLC with an emphasis on Good Chromatographic Hygiene Section 13 Performance Qualification This section presents a detailed suite of tests used to verify performance of the HPLC system and its components Overview 5 mins Why PQ Relationship between PQ and System Suitability Pump amp Detector Checks 11 mins POST Siphon test Leak down test Flow accuracy Detector noise amp drift On line Mixing Checks 9 mins Step test GPV test Dwell volume measurement Linearity check Chromatographic Checks 9 mins Oven temperature accuracy amp range Detector wavelength accuracy Injector repeatability carryover amp linearity All up system test Section 14 Tro
7. king particle size solvent flow temperature and analyte molecular weight Future Directions 15 mins A brief look through the crystal ball at what HPLC might look like in the future with the potential impact of UHPLC solid core column packings and monolithic columns Section 4 Reversed phase HPLC Mechanism 11 mins Reversed phase chromatography is unquestionably the most widely used form of HPLC In this section we will take a look at the underlying mechanism and its implications for various types of samples bb I O er over Official Denmark Distributor ANALYTICAL TRAINING SOLUTIONS a MD Scientific Premier Training for Analytical Chemists www md scientific dk Solvent Selection 15 mins The most widely used solvents for reversed phase are water acetonitrile methanol and tetrahydrofuran In this section we will look at the reasons why and the general approach used for mobile phase selection and optimization Bonded Phase Columns 16 mins Most HPLC columns are based on silica gel to which has been grafted a relatively non polar moiety Here s why how it s done and the implications for separation mechanism and how this technology has contributed to the overwhelming popularity of reversed phase as a separation mechanism Column Selectivity 10 mins Even columns of the same nominal functionality e g C18 can vary tremendously in selectivity We ll look at the parameters that determine selectivity and how to
8. oblems in turn are the result of ambiguous documentation We ll look at some examples as well as ways to avoid the problem Section 10 HPLC Equipment Detectors UV Detectors 22 mins The most commonly used HPLC detectors are based on UV or visible light absorbance The Acronym soup includes UV VWD PDA DAD we ll cover what they mean how they work potential problems and preventive maintenance Gradient Baseline Issues 12 mins Using a UV detector with gradients poses a unique set of problems Here s how to diagnose and sometimes fix them Other Detectors 12 mins More acronym soup RI ECD ELSD CAD FD LC MS an overview of other detector types Section 11 Quantization Measuring Peak Area 15 mins Quantitative HPLC depends on relating the amount of analyte injected to the area of the resulting peak In this section we will cover how the area measurement integration is carried out and the consequences for accurate and precise measurement as well as how to allocate areas when peaks are not fully resolved Calibration Issues 8 mins Calibration is the determination of the relationship between peak area and amount injected We ll look at linearity response factors linear range Limit of Detection LOD and Limit of Quantitation LOQ Reproducibility Issues 15 mins In many analyses HPLC is only the final step in a more or less complex sample workup procedure We ll look at techniques for identifying the dom
9. omatographic applications How to maximize column life Special techniques such as gradient elution quantitation and sample pretreatment The operating principles of each module in an HPLC system Proven techniques for systematic problem solving and instrument maintenance The most effective timesaving money saving approaches to preventing common hardware problems and method failures WHAT WILL I GET FROM THIS COURSE You will acquire a good understanding of the fundamentals of chromatography and learn simple tips and guidelines to make your chromatography work easier and more efficient What you learn will demystify your instrument You ll find that all of the perplexing and frustrating problems your experience have simple and logical solutions And better yet you ll learn that most of these problems are preventable The techniques you learn from this course will make you more effective at work and save you big headaches If you ve never taken a formal course or if you need a refresher it s time to learn HPLC the right way Students tell us that this extremely practical course is a must for everyone who uses HPLC INSTRUCTORS The course was designed by John Dolan Tom Jupille and Lloyd Snyder This class is taught by Tom Jupille Tom has been a practising chromatographer for more than 30 years during which he has written more than 30 papers on chromatography and related subjects He worked primarily in gas chromatography in the late
10. r of parameters are used to describe an HPLC separation In this section we will define and explore the significance of N efficiency and Rs resolution Controlling Resolution 18 mins Resolution R is the key parameter that describes the quality of a separation In this section we will look at how R can be controlled by using k alpha and N Tailing and Fronting 6 mins The Gaussian peak shape is an idealization that is seldom seen in practice We will explore the standard measurements used to describe deviations from that ideal the USP Tailing Factor and the ASTM Asymmetry Factor Section 2 Gradient HPLC Basics Gradient is Just Like Isocratic 17 mins Gradient solvent programming HPLC is actually more similar to isocratic constant solvent than it is different Understanding those similarities can take much of the mystery out of gradients Gradient is Different From Isocratic 13 mins Despite the similarities there are some key differences between gradient and isocratic HPLC We ignore those differences at our peril Gradient Hardware Issues 12 mins In this section we will explore some of the hardware issues that complicate method transfer in gradient HPLC Section 3 HPLC Columns Dynamics of Column Performance 18 mins The performance of a liquid chromatography column is measured by the plate number N In this section we will explore the factors that influence N column dimensions pac
11. ubleshooting amp Diagnostics Preventive Maintenance Documentation and Troubleshooting 9 mins How to consistently do good chromatography A Troubleshooting Checklist 29 mins A comprehensive list of symptoms and what to look for as possible causes e No peaks e Missing peaks e Extra peaks e Wrong retention time e Poor efficiency or peak shape e Poor resolution e Poor precision e Baseline noise or drift e Pressure problems e Leaks e Column lifetime For more information and to discuss your training requirements contact MD Scientific MD Scientific ApS Klokkerfaldet 23 8210 Aarhus V Denmark 70 27 85 65 70 27 85 66 fax info md scientific dk www md scientific dk
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