What does it really take to be an analyst? Analysts require a diverse set of practical skills to be efficient in the lab, including basic lab skills such as weighing and pipetting, competency in correctly setting up instrumentation, understanding of the fundamental scientific principles of chromatography, as well as an organized and “right first-time mindset”. All of this combined ensures samples are run efficiently and with minimum errors and costly repeat analyses.

Whether you are graduating this year, are new to working in an analytical laboratory, or train analysts in the lab – this webcast is for you. We will discuss the skills that are needed and why it is so important to ensure that everyone has proper training in these skills. The options we have for training staff – mentor led, face to face, or online – and how to successfully combine these to minimize training time but get maximum results.

Topics include:

• Role of an analytical chemist
• Wet chemistry and instrumentation techniques used in an analytical laboratory
• What you really need to know to succeed in analytical chemistry
• How to successfully train to be an analytical chemist
• The advantages and disadvantages of mentor led, face to face, and online learning for training analysts

A fundamental understanding of chemistry will give chromatographers better insight into the chemical process which occur during sample preparation and chromatographic separation.

Topics include:

• The atom
• Ionic bonding
• Covalent bonding
• Organic chemistry
• Other important elements
• Intermolecular bonding
• Functional groups and molecular properties
• Hydrophobic or non-polar groups
• Hydrophobic interactions - solubility
• Polar groups
• Polar interactions - solubility
• Ionic groups
• Ionic groups - pH
• Ionic groups - Ka and pKa
• Ionic groups - ionic strength
• Ionic interactions - solubility
• Chelating groups
• Chelating interactions - solubility
• Common functional groups
• Acids and bases

Safety in the laboratory is of the utmost importance. Work carried out within a laboratory will often involve the use of chemicals and/or equipment which is potentially harmful to yourself or those working around you, therefore, proper training is required. Most laboratories will have an explicit standard operating procedure (SOP) associated with a particular test, piece of equipment etc. Most companies will have safety training unique to their site which will be mandatory for all employees. The following section will detail some common safety measures; however, all personnel should be familiar with the specific safety requirements of the laboratory in which they work.

Topics include:

• Laboratory safety
• Chemical storage
• Acid/base safety
• Risk assessments and control of substances hazardous to health (COSHH)
• Sample handling and storage
• Housekeeping

Working in analytical chemistry requires a high level of numerical competency. In this module we cover the common scientific units used, conversion between these units and how we use these units in the practical preparation of common reagents.

Topics include:

• Significant figures and rounding
• Units of weight
• Units of volume
• Density
• Units of concentration
• Solutions of known concentration

This module will consider the basic calculations required for reporting results.

Topics include:

• Single point (proportional) calibration
• Salt correction factor
• Standard verification
• Bracketing
• QCs/check standards
• Calibration lines and linear regression
• Internal standards
• Preparing dilutions

Most quantitative analytical determinations will involve a weighing stage. Any weight measurements are subject to error which must be minimized to ensure the lowest possible systematic quantitative deviation in the final result obtained. It is important that prior to carrying out any weighing task you are familiar with any governing bodies to whose standards you must comply or with any in-house SOPs for your company regarding the correct use of balances. This module focuses on the use of balances in the lab and discusses best practice on their use.

Topics include:

• Use of balances - introduction
• Selecting the correct balance
• Balance location
• Balance calibration
• Containers and types of substances to be weighed
• Weighing with balances
• Problems when weighing
• Balance specifications
• Microbalance weighing - hazard and risk
• Microbalance weighing - technique
• Microbalance practical exercises

Weighing is integral to the analytical workflow. But how do we improve our weighing technique? By asking questions and understanding what can go wrong. Here are 10 questions that we have been asked about balances that will provide information on how to look after your balance better and give tips on good weighing practice.

Analytical procedures often require liquids to be transferred quantitatively or solutions which need to be prepared for quantitative determination. When preparing solutions or transferring liquid quantitatively the type of equipment used will depend on the accuracy that is required, as well as the volume of liquid that is involved. This module will discuss the different types of volumetric lab equipment and how each piece should be used correctly.

Topics include:

• Volumetric glassware
• Volumetric glassware markings, grade, and cleaning
• Temperature, solution, and mixing effects
• Selection and use of volumetric glassware
• Parallax error
• Checking the accuracy of volumetric glassware and carrying out a calibration
• Solutions of known concentration

A traditional glass pipette is one of the most fundamental laboratory tools any analyst must become highly competent in using. They are used routinely to accurately dispense a known volume of solution. This module teaches you the main aspects of this essential skill.

Topics include:

• Automatic vs. conventional glass pipettes
• Glass pipettes
• Types of glass pipette
• General pipetting technique
• Using 'to dispense' type pipettes
• Using 'to contain' type pipettes
• Using graduated pipettes
• Cleaning and storage
• Practical exercises

Pipetting is one of the most common tasks carried out in the lab, yet it can be highly error prone which will adversely impact results. Good pipetting technique comes with training and practice - something that we can offer in this module.

The focus of this module is to provide you with practical information to improve your pipetting technique - this encompasses the use of pipettes, how to select the correct pipette and tip, how to and why we calibrate pipettes, as well as how to maintain them in order to have confidence that they are functioning reliably. All of this is explained using video and animation to provide practical training. And once you have completed the module, test your pipetting technique using our practical exercises.

Topics include:

• Automatic pipette types
• Modes of pipetting
• Choosing the correct pipette
• Pipette tips
• Pipetting technique
• Pipette calibration
• Why calibrate?
• Pipette care and maintenance
• Practical exercises

Pipetting is an essential lab task, however, poor technique can contribute to large errors. The top pipetting mistakes are discussed in this Quick Guide with practical hints and tips on how to improve your pipetting technique, from selecting the correct pipette, using the correct pipetting mode, and ensuing that pipettes are properly maintained and calibrated.

pH is one of the most commonly determined parameters of aqueous (and non-aqueous) solutions because many chemical processes are dependent on pH. Altering pH can often significantly alter the rate of chemical reactions. The solubility and bioavailability of many pharmaceutically active compounds is also determined by solution pH. Furthermore, in chromatography, the retention and selectivity of ionizable analytes can be manipulated by changing the eluent pH. This module will discuss the equipment used to measure pH and how to use it.

Topics include:

• Introduction and theory
• pH paper and pH meters
• pH meter electrodes
• Buffer solution and pH electrode characteristics
• Correct use of electrodes, electrode care, and maintenance
• Calibrating a pH meter and making a measurement
• Buffer preparation using the Henderson-Hasselbalch equation
• Buffer preparation from stock solutions
• Calculating the masses of buffer components
• Calculating the pH of a strong acid solution
• Calculating the pH of a strong base solution
• Calculating the pH of a weak acid solution
• Calculating the pH of a weak base solution
• pH of common acids and bases

A titration is the determination of the specific quantity of an analyte by the addition of a reagent (titrant) of known concentration. The reaction involved in a titration must be fast, complete, unambiguous, and observable. The titrant is added until the reaction is complete, at which point the so-called end point, or equivalence point, is reached. The end point of the reaction is monitored using a color change indicator, pH meter, or electrochemical sensor. The volume of titrant added allows the quantity or concentration of the analyte to be calculated based upon the reaction stoichiometry. This module will discuss the different types of titrations, how to perform a titration, and how to overcome common problems.

Topics include:

• Titrations
• Acid-base titrations
• Titrant standardization
• Indicators for acid-base titrations
• Titration of a weak acid with a strong base
• Titration of a weak base with a strong acid
• How to carry out a titration
• Glossary of titration terms
• Errors in titration
• Moisture determination (Karl Fischer titration)
• Karl Fischer titration, side reactions, and rate control
• Bivoltametrc indication
• End point polarization
• Drift
• Calibration,. maintenance, and troubleshooting of Karl Fischer autotitrators
• Errors with Karl Fischer titrations

In 1935 Karl Fischer demonstrated that the reaction between iodine and sulfur dioxide could be used for water determination using methanol as a solvent. This module will detail the use of Karl Fischer titration apparatus for moisture determination.

Topics include:

• Water determination - Karl Fischer titration
• Karl Fischer titration, side reactions, and rate control
• Bivoltametric indication
• End point polarization
• Drift
• Calibration, maintenance, and troubleshooting of Karl Fischer autotitrators
• Errors with Karl Fischer titratrions

This module takes an initial look at the science behind the most commonly used sample preparation techniques.

By the end of this unit, you should be able to explain the concepts of protein precipitation, choose appropriate extraction approaches for various analyte and sample matrix types, decide when to use centrifugation (or ultracentrifugation) and understand what can be achieved, identify situations in which simple sample preparation techniques (dilution, filtration, centrifugation etc.) are sufficient to overcome practical problems.

Topics include: 

• Primary sample preparation techniques
• Sample dilution
• Sample filtration and ultrafiltration
• Centrifugation and ultracentrifugation
• Protein precipitation
• Extraction approaches to sample preparation
• Selectivity
• The partition coefficient
• Liquid/liquid extraction
• Support-assisted liquid/liquid extraction
• Solid phase extraction

Liquid/liquid extraction is one of the most widely employed and useful techniques in pharmaceutical sample preparation. This is due to a number of characteristics, including simplicity, rapid method development, and reasonable selectivity. In this module, the basic principles of liquid/liquid extraction (LLE) are described, as well as an overview of limitations and drawbacks of LLE protocols. Specific advice is given for various problems that are routinely encountered. Emulsions and how to deal with them is discussed, and support-assisted liquid/liquid extraction (SALLE) is presented as a useful variation on standard liquid/liquid extraction.

At the end of this module you will understand how to maximize analyte recovery by optimizing liquid/liquid extraction conditions; maximize extract cleanliness by optimizing liquid/liquid extraction conditions; minimize the impact of some common problems in liquid/liquid extraction; and recognize practical aspects of support-assisted liquid/liquid extraction.

Topics include:

• Liquid/liquid extraction
• Drawbacks of liquid/liquid extraction
• Emulsions
• Support-assisted liquid/liquid extraction (SALLE)

Liquid/liquid extraction (LLE) is the most widely used extraction technique for liquid samples. The information provided in this article will mainly focus on liquid-liquid extraction (LLE) using a separatory funnel, however, many of the principles are applicable to the more modern miniaturized approaches to LLE i.e. single-drop microextraction (SDME), flow-injection extraction (FIE), membrane extraction techniques, and supported liquid extraction (SLE).

An interesting and useful variation of liquid/liquid extraction (LLE) is supported liquid extraction (SLE). Compounds are separated on the same basis of differential solubility, as with conventional liquid/liquid extraction. While the chemistry of this technique is very similar to that of LLE the physical nature of the technique offers distinct benefits which are detailed in this Quick Guide. 

Filter, filter, filter. It should be a common lab mantra. Whether preparing samples or mobile phase, filtration should be at the heart of these processes. From improving instrument up-time, to extending column lifetimes and improving chromatographic data, there are very few disadvantages to this quick and cost-effective sample preparation technique. Join us to learn how to correctly select a filter for your application allowing you to avoid some of the common pitfalls related to filtration.

Topics include:

• Mobile phase filters
• Syringe end filters
• In-line filters
• Filter compatibility studies
• Membrane materials
• Diameters
• Porosity
• Sterile/non-sterile filters
• Extractables/leachables
• Adsorption
• Troubleshooting

The first step to troubleshooting (or avoiding having to troubleshoot) any sample preparation or analytical method is to select the correct method and equipment in the first place. However, this does not guarantee that problems may not arise. The following article will look at selecting the correct syringe filter, where to find relevant information on selecting a syringe filter, and what common problems may arise during filtration using syringe filters.

Sample preparation is required for a number of reasons, including rendering the sample in the correct format for the analysis type (e.g. when the sample is too dilute, too dirty, or has an incompatible sample matrix), to reduce spurious peaks by removing interferences or matrix components, improvement of analyte peak shape, generation of robust analytical methods, increased column lifetime and instrument up-time.

This Quick Guide will provides hints and tips for optimizing filtration using syringe end filters.

Although Soxhlet extraction was developed in 1876, it is still widely used in many industry areas to extract analytes from solid matrices. It is, in fact, still seen as the gold standard technique for extraction from solid matrices against which others are compared. This quick guide details how Soxhlet extraction works, parameters that require optimization, and how improvements to the methodology and automation have allowed an increase in throughput and improved reproducibility. 

Modern microbalances allow us to accurately weigh small quantities of material, however, this requires a high level of operator skill. This module uses video content to teach best practices for microbalance weighing. This practical approach will ensure that common errors are avoided and accurate weighing can be achieved.

Topics include:

• Hazard and risk
• Microbalance weighing technique
• Practical exercises

Before starting any practical work, it's essential we take some time to review our method and plan our workload.

In this module an experienced analyst takes you step-by-step through the process of how we do this. We'll look in detail at every part of what appears to be a straightforward HPLC method, paying particular attention to the many areas which carry a high risk of error and how we can plan our work to minimize many of these common errors.

One of the most common reasons for re-analysis is when analysts make mistakes during the preparation of samples. In this module we will follow two very experienced analysts as they guide you through the process of managing your workspace an samples in a way that eliminates errors.

Topics include:

• Setting up
• Mixing solutions
• Preparing our internal standard
• Preparing our matrix / placebo / vehicle / blank samples
• Preparing our standards
• Receiving our samples
• Preparing our samples

A standard is a solution of exact and known concentration that we measure to calibrate the response of our instrument/method. Preparing standards for GC creates challenges which risk introducing accuracy errors into our standards due to issues with volatility, concentration, and the complexity of mixtures that are being analyzed. This quick guide details the optimum technique for successfully preparing GC standards.

Topics include:

• Practical considerations
• Preparing known and exact concentrations
• Measure by volume or measure by weight?
• Density and relative density
• Preparing low concentrations
• Choosing a suitable solvent
• Practical technique
• Preparing volume-in-volume standards
• Preparing weight-in-volume standards
• Preparing weight-in-weight standards
• Stock solution
• Worked example

The aim of this module is to give a brief history of liquid chromatography and compare and contrast high performance liquid chromatography (HPLC) with gas chromatography (GC). Liquid chromatography (LC) is explained, and the function of each component of the HPLC system discussed. The chromatogram is also introduced.

At the end of this unit you will be able to outline the main separation mechanisms used in HPLC, and explain the fundamental principles of separation. Also, you will be capable of highlighting the different modes of chromatography and explaining their uses and applications.

Topics include:

• Why is chromatography so important?
• Origins of liquid chromatography
• The basics of how liquid chromatography works
• Chromatography separation mechanisms
• The liquid chromatographic system
• The liquid chromatographic process
• The chromatogram
• Why choose liquid chromatography?
• Comparison with gas chromatography
• Suitable samples for HPLC
• Modes of chromatography

Join us for a little over an hour on this step-by-step guide through setting up an HPLC instrument. 

Avoid common errors and pitfalls and understand every step of the process to get your instrument set up correctly every time, decrease down-time, and reduce the risk of re-analysis. 

The HPLC Bootcamp uses video filmed in our lab to ensure you get the most realistic walk through of the instrument.

Topics include:

• Introduction to the analytical method we'll be using
• Mobile phase preparation
• System setup and equilibration
• Preparation of standards
• Verification of system performance
• Preparation of samples
• Sequence setup
• System checks and common mistakes

The aim of this module is to explain the purpose of the mobile phase in HPLC. The basic concepts of the liquid chromatographic process is outlined, as well as discussing the best way to prepare mobile phase components. Potential problems caused by improper preparation and use of mobile phases will be highlighted. We will also describe the way that mobile phase composition effects retention and selectivity in reverse phase HPLC. The various physicochemical properties of the typical solvents used for HPLC are described, and the various methods for degassing HPLC mobile phases introduced.

At the end of this module you will be able to demonstrate an understanding of the purpose of the mobile phase in HPLC and describe suitable methods to prepare and degas your mobile phase. The liquid chromatographic process will be easy for you to describe and you will be able to relate retention and selectivity of analytes to different mobile phase compositions. You should also be able to demonstrate an understanding of how certain physicochemical properties of mobile phase constituents may affect an analysis.

Topics include:

• The mobile phase - introduction
• The liquid chromatographic process
• Analyte retention processes
• The partition coefficient
• Retention and selectivity
• Solvent type and selectivity
• Optimizing selectivity using retention
• Gradient elution
• Mobile phase delivery
• Solvent miscibility
• UV cut off
• Other solvent considerations
• Mobile phase preparation
• Degassing
• Outlet degassing
• Degassing/column damage
• Methods of degassing
• Helium sparging
• Vacuum degassing

Mobile phase preparation - what could possibly go wrong? Weigh your buffer, quick pH check, measure out the organic, mix it all together and you have the perfect mobile phase ready to go. If only it were that simple. Let us walk you through a typical mobile phase preparation process highlighting how to avoid common pitfalls such as contamination from glassware, selecting the wrong buffer salt form, mixing organic and aqueous in the incorrect order, filtering with an inappropriate membrane and much, much more. After watching this webcast there will be much more right than wrong with your mobile phase preparation.

Topics include:

• Introduction – the role of mobile phase
• Why mobile phase preparation consistency is important
• Pre-preparation
• Glassware selection and cleaning
• Weight and volume calculation
• Reagent quality and salt form    
• Buffers
• Preparation
• Balance calibration
• Weighing and dissolving
• pH adjustment
• Mixing buffer and organic            
• Filtering/degassing

This Q&A session, based on the questions submitted in one of our most attended webcasts, will make you think about mobile phase preparation in a whole new way! We had nearly 100 questions submitted which allowed us to expand more fully on what was discussed on our webcast. Everything from how cleaning our glassware can impact analyses, to how to avoid or minimize contamination, what the best practices are for filtering mobile phases - and lots in between. This is a practical discussion that is a must see if you use HPLC.  

Topics include:

• Glassware
• Reagents
• Contamination
• pH
• Mixing
• Filtration
• Degassing
• Shelf life
• LC-MS

Learn about the most common bad habits that should be avoided with HPLC mobile phases!

The question of how long you can store HPLC mobile phases is asked on a regular basis to the CHROMacademy technical team. Here are our general guidelines.

The aim of this module is to introduce silica as a major stationary phase support for reversed phase HPLC separations and for normal phase applications. We explain how bonded phases are produced and highlight the deactivation process used. The use of bonded phases in various chromatographic applications, and the influence of surface silica silanol groups on chromatographic separations is illustrated. Modern bonded phases that give an advantage to today’s chromatographer are introduced – including water wettable phases, polar embedded phases, and phases capable of operating at extremes of pH. 

At the end of this section you will be able to highlight new and emerging stationary phase types, as well as explain how columns and stationary phases may be characterized.

Topics include: 

• Silica as a packing material
• Chemically bonded phases
• Surface treatment- endcapping
• Reversed phase stationary phases
• Silanol and separation
• Water wettable phases
• Aq type stationary phases
• Polar embedded phases
• Working at low pH
• Working at high pH
• Other stationary phase types
• Column test probes
• Column characterization

HPLC columns are manufactured in a variety of different internal diameter and length combinations, as well as having an assortment of particle sizes. HPLC column dimensions will affect the efficiency sensitivity, and speed of an analysis. The choice of column dimensions will depend on the chromatographic application; analytical, semi-preparative, preparative, number of analytes in the mixture etc. However, the dimensions can also be altered to improve chromatography by achieving more efficient, sensitive, and faster analyses. The primary column dimensions of particle size, length, and internal diameter will be discussed and their effect on chromatographic separations demonstrated, providing some practical insight into how to select the best column dimensions for a particular application.

System suitability is a series of checks built in to a sequence to ensure all sample data is correct and valid. This webcast will describe how we design and perform system suitability and which chromatographic parameters we should be assessing during the process.

Topics include:

• Why bother? Validation of a sequence
• Repeatability
• In-run repeatability
• Standard verification
• Quality control checks
• Bracketing
• Calibration lines and associated criteria
• Chromatographic assessment – resolution, asymmetry, efficiency
• Sensitivity

The aim of this module is to explain the purpose of injectors and autosamplers within the HPLC system. The principles behind which sample injection systems work will be described, as well as the individual components of an injection valve. The correct and incorrect ways to use loop injectors in manual injection mode will be highlighted, and we will discuss the major components and operating principles of a range of typical autosampler devices. Common autosampler problems are outlined, and some troubleshooting principles introduced. We also talk about how carryover may occur within the autosampler, and how to minimize it.

This module will teach the purpose of sample introduction systems within HPLC. The understanding of the components and operating principles of injection valves will be discussed. You will be able to select correct sample volumes to be aspirated into loop injectors to optimise quantitative accuracy, as well as describe the main operating principles of a range of typical autosampler devices. You will learn how to recognise advantages, disadvantages and potential problems of each type of autosampler, and outline strategies for reducing carryover in autosampler devices.

Topics include: 

• Injection valves
• Injection valve anatomy
• Manual injection systems
• Manual injection complete and partial filling
• Autosamplers
• Pull-to-fill auto samplers
• Push-to-fill auto samplers
• Integral loop auto samplers
• Autosampler contamination

The aim of this unit is to describe the role of the solvent delivery system (pump) within high pressure liquid chromatography (HPLC). The operational principles of the most popular solvent delivery types are explained, as well as the function of the components and potential troubleshooting and maintenance issues. We will describe good practice in pump use, care and storage, and discuss the operating principle of alternative pump designs such as preparative and micro-flow pumping systems.

At the end of this module you will be able to explain the working principles of the most popular solvent delivery systems in HPLC; demonstrate knowledge of the working principles of various solvent delivery system components and explain maintenance procedures; describe the advantages of dual piston pumps over single piston pumps; recognize system problems from characteristic baseline problems; and identify solvent delivery system problems associated with retention time variation.

Topics include: 

• Simple pumping systems
• Disadvantages of single piston pumps
• Dual piston reciprocating pumps
• Accumulator pumps
• Binary pumps
• Quaternary pumps
• Ternary pumps
• Check valves
• Pistons
• Seal wash
• Pulse dampers
• Purge valves
• Flushing gradients
• System dwell volume and time
• Troubleshooting - high back pressure
• Troubleshooting - check valves and pistons
• Troubleshooting - solvent mixing issues
• Preparative pumps
• Capillary (low volume) pumping systems
• Calibration and testing

This module will introduce the general terms and concepts that are applicable to all HPLC detectors; including selectivity, sensitivity, limit of detection and limit of quantification. The learning content will allow you to understand the function of an HPLC detector and what is required when selecting a detector for specific analytical outcomes.

Topics include:

• Introduction to HPLC detectors
• General terms and concepts
• Limit of detection/quantification (LOD/LOQ)

The most commonly used detector for HPLC analysis is the UV-visible detector. The detector fulfills many of the criteria for an ideal detector. This module will detail the workings of UV-visible detectors, including variable wavelength and diode array detectors. Key detectors settings that should be optimized are fully detailed.

Topics include:

• The flow cell
• UV-visible detection - quantitation
• External standard quantitation
• Calibration curve
• UV absorbance
• Variable wavelength UV-visible detectors
• Diode array detectors (DAD)
• Diode array detectors spectra
• Diode array detectors - bandwidth
• Diode array detectors - slit width
• Diode array detectors - response time
• Diode array detectors - reference wavelength
• Choosing sample and reference settings for DAD
• DAD - peak suppression

The CHROMacademy reversed phase chromatography module is a comprehensive guide to this ubiquitous technique. It will guide the novice, or seasoned chemist, from the basic concepts of the technique through to more complex topics on solvent and buffer selection, the effects of temperature on chromatography, and working with ionizable analytes. All of the topics will provide a fundamental understanding of the science behind the technique, often explained with the use of interactive learning tools and animations; as well as providing practical knowledge on how to develop reversed phase HPLC methods. 

The aim of this module is to give an overview of the mechanism of reversed phase high performance liquid chromatography (RP-HPLC), and explain the basis behind the retention mechanism. Retention order in RP-HPLC is described, and the influence of mobile phase composition on retention illustrated. The module will show how the mobile phase composition and constituents might be manipulated to optimize chromatographic separations. The principles used to select appropriate stationary phases and column geometry are explored. We will also look at how how mobile phase pH affects the retention of ionizable compounds. Typical RP-HPLC applications will also be highlighted. 

At the end of this section you will be able to interactively demonstrate how to use mobile phase pH to optimize separations, as well as choose buffers for RP-HPLC. You will learn how to effectively use the principles of ion-suppression to optimize separations.

Topics include: 

• Mechanism of reversed phase HPLC
• Applications of reversed phase HPLC
• Analyte retention in reversed phase HPLC
• Retention order in reversed phase HPLC
• Reversed phase mobile phase solvents
• Mobile phase strength and retention
• Changing the organic modifier
• Eluotropic series
• Selecting reversed phase columns
• Reversed phase HPLC of ionizable samples
• Controlling extent of ionization
• The 2 pH rule
• pH vs retention in reversed phase HPLC
• Basic analytes and ion suppression
• Buffers for reversed phase HPLC
• Henderson-Hasselbalch equation
• Buffer preparation using the Henderson-Hasselbalch equation
• Getting started with ionizable compounds
• Optimizing separation selectivity of ionizable analytes
• Effect of temperature on reversed phase HPLC

The aim of this module is to explain the problems that can be encountered when using isocratic HPLC, and demonstrate how these problems can be overcome using gradient HPLC. We will illustrate how gradient elution HPLC works, define its parameters and show how the retention and elution processes differ from isocratic HPLC. We will demonstrate why peak shapes in gradient HPLC are more efficient than those obtained from isocratic HPLC. The effects of gradient steepness will be examined, and it is shown how the various gradient parameters can be practically determined. We will interactively illustrate the use of scouting gradients in HPLC method development and optimization. Also, we will look at the pitfalls and advantages of gradient elution HPLC in a practical situation.

Topics include:

• Isocratic HPLC analysis
• Gradient HPLC analysis
• Gradient elution parameters
• Optimizing gradient parameters
• Gradient elution principles
• Peak shape in gradient HPLC
• Scouting gradients
• Optimizing initial gradient conditions
• Gradient steepness
• Optimizing gradient analyses
• Practical gradient HPLC
• Changing method parameters
• Gradient suitability
• Method transfer between HPLC systems
• Gradient method transfer calculator
• Non-linear gradients
• Segmented gradients
• Isocratic hold
• Peak capacity
• Estimating gradient parameters
• Gradient performance checks

This short module covers the history of gas chromatography (GC) and introduces the chromatographic process and the major components of a GC instrument. The technique is compared and contrasted with other analytical techniques such as high performance liquid chromatography (HPLC). The terms and appearance of a typical chromatogram are also explained. We will outline the fundamental basis for separation in GC, and indicate the major advantages of GC and the application areas in which it is used.

At the end of this unit you will be able to identify analytes suitable for GC analysis from physicochemical data, describe the function of the various components of a gas chromatograph, explain the fundamental basis of separation in GC in terms of solubility and vapor pressure of analytes, and recognize when the use of GC might be applicable to solving analytical problems.

Topics include:

• Why is chromatography so important?
• The basics of how gas chromatography works
• Origins of gas chromatography
• Why choose gas chromatography
• Sample types suitable for gas chromatography
• Gas chromatography separation mechanism
• The gas chromatograph
• The chromatogram
• GC advantages and disadvantages
• Typical GC applications

A step by step guide to setting up a GC instrument. From checking the cylinders, dismantling the inlet, to correctly installing a column and lighting an FID. All before shutting the instrument down correctly.

This practical module uses video filmed on a GC instrument in the lab to ensure that you get the most realistic walk through of the instrument; allowing you to correctly setup your instrument every time to get the most from it and decrease down-time by avoiding common mistakes. 

Topics include:

• Cylinders, regulators, and generators
• Gas filters
• Inlet
• Autosampler
• Column installation
• Electronic pneumatic control (EPC) setup
• Flame ionization detector (FID)
• Switching off

Prevention is better than cure. Avoid instrument down-time, reduce troubleshooting, and improve the data generated by your GC by following these eight tips for inlet maintenance. From the liner, to the gold seal, and everything in between we'll provide you with a maintenance schedule to follow and tips for carrying it out.

Modern gas chromatography may require several different gases in order to operate. The nature and purity of the gas required is dictated by the type of inlet, column, and detector system used. The aims of this module include outlining the nature and quality of the gases required for GC analysis, as well as highlighting the various methods for controlling gas pressure and cleanliness including troubleshooting strategies. The use of gas generators as a viable alternative to cylinders for GC gas supply will be discussed. The principles of manual and electronic pressure control for GC carrier, inlet, and detector gas regulation are described as well as the differences between constant pressure and constant flow experiments.

At the end of this topic you will be able to recognize the gases required for carrier, inlet and detector operation, describe the required gas cleanliness and propose practical solutions for ensuring gas cleanliness requirements are met, describe which gases may be supplied by generator, illustrate various means be which gas flows are controlled and regulated, identify the two primary types of gas flow control and demonstrate that you understand the differences between them using schematic diagrams, and explain how GC analyses might differ when separations are carried out in constant flow or constant pressure mode. 

Topics include:

• Gases required for GC
• Gas supply management
• Quality of gas supply
• Gas generators - using hydrogen in the lab
• Manual pressure control
• Electronic pressure control
• Pressure/flow programming

The most common inlet for capillary GC is known as the split/splitless inlet, which as the name suggests, can be operated in two modes, spilt or splitless. This module fully details the working principles of this inlet and the parameters that must be set for successful sample introduction. Common problems are also highlighted, ensuring you can troubleshoot or avoid them all together.

Topics include:

• GC inlet systems
• Split/splitless inlet
• Split injection
• Setting the split ratio
• Sample discrimination
• Injection volume
• Optimizing injection volume
• Split injection experiments
• Splitless injection
• Optimizing splitless injection - purging the inlet
• Optimizing splitless injection - analyte focusing
• Optimizing splitless injection - solvent choice
• Splitless injection experiments
• Choosing an inlet temperature
• Liners for split/splitless injection
• Septa for split/splitless injection
• Septa problems

The correct setup of any instrument, method, or process in the lab is crucial to it performing to the best of its ability. Autosamplers are an invaluable part of chromatographic instrumentation as they are reliable and accurate and remove many of the errors associated with manual injection. However, we still need to take the time to set them up correctly. Over four short videos we will look at how to correctly setup a GC autosampler. We will consider parameters that can be changed by the user, including the syringe and needle, needle wash routines, vials, injection speed, viscosity delay, and dwell time.

Capillary columns were first used and patented by Golay in 1958/9, but were not widely used until the late 1970's, after which they have steadily grown in popularity. Today the vast majority of GC applications are developed or run using capillary columns. The selection of the correct stationary phase is one of the most critical parameters in the success of any GC method. The aims of this module include the comparison and contrast of packed and capillary columns. A revision session of of fundamental intermolecular interactions is included in order to relate the various types of interaction to retention in GC. We will explore various stationary phase types and explain the critical factors in choosing a phase. The important physical parameters of capillary GC columns and their relationship with retention, resolution and efficiency in GC separations is investigated. Column bleed and how to minimize it is introduced, and good practice for column installation and conditioning highlighted. 

At the end of this unit you will be able to recognize and explain the various interactions that occur between analytes and stationary phases in GC, select an appropriate stationary phase for various analyte and application types, select appropriate column dimensions for various application types, demonstrate a good understanding of how to manipulate column physical parameters in order to obtain good quality separations in a reasonable time frame, and explain the principles of good column handling, installation, and conditioning in a practical context.

Topics include:

• Open tubular capillary columns
• Comparison of packed and capillary columns
• Chemistry review - analyte and stationary phase polarity
• Electronegativity
• Dispersive interactions
• Dipole interactions
• Hydrogen bonding
• Stationary phases - polysiloxanes
• Polysiloxane classifications
• Stationary phases - polyethylene glycols
• Stationary phase selection
• Phase selection - dispersive phases
• Phase selection - dispersive interactions and polarity
• Phase selection - diople interactions and hydrogen bonding
• Stationary phase selection - PLOT columns
• Stationary phase selection summary
• Stationary phases for packed column GC
• Column dimensions- length
• Column dimensions - internal diameter
• Column dimensions - film thickness
• Column dimensions - phase ratio
• Carrier gas flow rate
• Column bleed
• Column installation and conditioning
• Exercises in column selection

This module will provide a brief overview of GC detectors, their function, and the main characteristics that are important when considering a GC detector; such as, sensitivity, linearity, noise, and minimum detectability.

Topics include:

• GC detectors overview
• GC detectors characteristics

The flame ionization detector (FID) is the most widely used GC detector and was invented specifically for GC applications. Its high sensitivity and linear range for carbon containing compounds make it very popular in organic analyses. This module comprehensively details the working principles of this detector, giving you the knowledge and understanding to operate and optimize the detector for your analyses.

Topics include:

• The flame ionization detector (FID)
• Operating and optimizing
• Uses and performance

Temperature is one of the two most important variables in GC and affects the retention, selectivity, and to a lesser extent the efficiency of all separations. As temperature is convenient to manipulate within a GC experiment, and as it has a large effect on the selectivity (and hence resolution) of a separation, it is a primary variable in method development and optimization. The aims of this module include illustrating the relationship between temperature and retention in GC, and highlighting the advantages and disadvantages of isothermal and temperature programmed GC separations. The theory behind temperature programmed GC and several useful rules of thumb is discussed, as well as the usefulness and optimization of scouting temperature programs for GC method development.

At the end of this section you will be able to recognize the advantages and disadvantages of using temperature programmed GC, explain how to implement a scouting temperature program for an unknown sample, make decisions on whether samples can be run isothermally or need a temperature program, suggest reasonable isothermal temperatures, develop a temperature program for an unknown sample, and optimize the separation obtained from the temperature program suggested.

Topics include:

• The role of temperature in GC separations
• Isothermal and gradient temperature GC
• Theory of temperature programmed GC
• Scouting temperature programs
• Optimizing isothermal conditions
• Initial temperature and hold times
• Adjusting the ramp rate
• Mid-ramp holds
• Peak capacity
• Final temperature and time

Learn how to carry out batch integration. We will provide you with a generic approach that can be applied to your data.

Integration of chromatographic peaks (determination of height, area, and retention time) is the first and most important step in the data analysis of HPLC and GC methods. This information is used for all subsequent calculations, including construction of calibration curves and calculation of unknown concentrations. This first installment will define what chromatographic peak integration is and the primary chromatography data system (CDS) settings which are required.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).

Not every method will result in peaks being baseline resolved, or have peaks all the same size. In the second installment of this series we will define some of the situations which most commonly occur and result in difficult integration.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).

When confronted with a chromatogram which contains peaks which are difficult to integrate what can be done? In this final installment we will look at the use of advanced integration options for integrating problematic peaks.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).

This module will provide an understanding of the mathematical methods which are used to quantitate and calibrate chromatographic data. Using a practical approach applied to relevant applications we will provide you with the tools to generate better data for your applications.

Topics include:

• Introduction
• Glossary
• Overview of chromatographic peak integration
• Single point (proportional) calibration
• Salt correction factor
• Standard verification
• Multi-point calibration
• Bracketing
• QCs/check standards
• Calibration lines and linear regression
• Internal standards
• Introduction to non-linear calibrations and weightings
• Standard additions
• Relative response factors
• Surrogates (internal QC)
• System suitability

The aim of this module is to define quantitative HPLC and explain the information that can be derived from this type of HPLC analysis. We highlight the principles of peak identification and analyte characterization from a practical perspective. We also explain the use of peak height or area in quantitative calculations and investigate integration of chromatographic peaks. The use of calibration and calibration curves in quantitative HPLC is discussed, as well as the principles of single and multi-level calibration in quantitative analysis. The practical uses of external standard, internal standard and normalization methods of quantitative analysis will be investigated. Example calculations for various quantitative methods are outlined. 

Upon completion on this section, you will be able to define qualitative HPLC and explain the information that can be derived from this type of HPLC analysis. you will also be able to define and explain the principles of peak identification and analyte characterization from a practical perspective.

Topics include:

• Qualitative analysis overview
• Peak identification and assignment
• Sample spiking
• Spectral peak identification
• Quantitative analysis overview
• Chromatographic requirements
• Peak integration
• Peak height or peak area
• Principles of quantitative analysis
• Area% and height % - normalization
• External standard quantitation
• Calibration curve
• External standard multi-level calibration
• Internal standard analysis
• Internal standard multi-level calibration

Learn how to carry out batch integration. We will provide you with a generic approach that can be applied to your data.

Integration of chromatographic peaks (determination of height, area, and retention time) is the first and most important step in the data analysis of HPLC and GC methods. This information is used for all subsequent calculations, including construction of calibration curves and calculation of unknown concentrations. This first installment will define what chromatographic peak integration is and the primary chromatography data system (CDS) settings which are required.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).

Not every method will result in peaks being baseline resolved, or have peaks all the same size. In the second installment of this series we will define some of the situations which most commonly occur and result in difficult integration.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).

When confronted with a chromatogram which contains peaks which are difficult to integrate what can be done? In this final installment we will look at the use of advanced integration options for integrating problematic peaks.

This article was written by Dr. Dawn Watson (Technical Expert, Crawford Scientific).