In-house HPLC Method Development Course
This unique 3-days HPLC course gives all necessary tools for quick and easy development of state-of-the-art, high performance, high-throughput & cost-efficient HPLC methods. It is the in-house course, and the price is fixed (10'000$), i.e. it does not depend on the quantity of participants in the group.
From this HPLC method development course you will learn:
1. How to predict behavior of a target molecule in any single or mixed HPLC mode using its structural formula.
2. How to select optimal stationary phase for a given HPLC separation in RP, HILIC, IC, NP or mixed HPLC mode.
3. What are the standard mobile phase compositions, and how to use mobile phase adjustments for fine-tuning retention and separation selectivity in PR, NP, HILIC and IC modes.
4. How to choose optimal flow rate, column length and particle size for the developed HPLC separation.
5. How to organize HPLC method development process in order to get an outstanding HPLC method just in few days.
6. How to take full control of your HPLC column’s chemistry, and understand it better than the column’s producer.
7. Why a HILIC separation can be hard to reproduce, and how to develop a really robust HILIC separation.
8. How to control selectivity and retention in ion-pairing RP mode.
9. How to avoid sample preparation and get rid of matrix components with use of HILIC, IC and NP modes.
10. How to convert an ion-pairing separation into a standard IC, HILIC, or mixed RP one,
11. How to convert a gradient RP separation into an isocratic one in mixed RP mode.
12. How to develop an MS-compatible HILIC or IC separation.
1.1. Specificity of an HPLC method.
- Chromatographic separation specificity and detection specificity.
1.2. Classification of chromatographic separation methods according to their purpose.
- Targeted and untargeted HPLC methods.
- Differences in chromatographic approaches to developing targeted and untargeted HPLC methods.
1.3. An overview of HPLC detection techniques.
- Specificity of optical, evaporative, electrochemical, and mass spectrometry detection methods.
- How to choose an optimal detection method.
2.1. Basic chromatographic parameters. Factors that define the resolution of a peak pair.
2.2. Three-step HPLC method development procedure.
2.3. The optimal retention.
2.4. The optimal separation selectivity.
2.5. The optimal efficiency.
3.1. Single adsorption (RP, NP, HILIC, IC, CT, LEC) and exclusion (SEC, IEX) single HPLC modes.
3.2. Widely used mixed HPLC modes: RP/HILIC, RP/CT, RP/IC, RP/SEC, HILIC/IC, NP/CT.
3.3. Specificity and flexibility of single and mixed HPLC modes.
4.1. The correlation between the chemical structure of an analyte and its ability to be retained (or excluded) in each adsorption (or exclusion) HPLC mode.
4.2. Predicting the elution order for the mixture of analytes possessing differing physical properties in single and mixed HPLC modes.
5.1. Modern trends in the development of HPLC stationary phases.
- Single-mode and mixed-mode chemistries of HPLC stationary phases.
- Fully porous and core-shell particles, their advantages and drawbacks.
5.2. An overview of commercially available HPLC stationary phases for RP chromatography.
- Classical RP stationary phases with monomeric and polymeric bonding.
- RP/HILIC mixed-mode stationary phases.
- RP/CT mixed-mode stationary phases.
5.3. An overview of commercially available HPLC stationary phases for IC chromatography.
- SAX, SCX, WAX and WCX stationary phases.
- RP/IC mixed-mode stationary phases.
5.4. An overview of commercially available HPLC stationary phases for NP and HILIC chromatography.
6.1. Testing reversed-phase and mixed-mode RP/HILIC & RP/CT HPLC packings.
- Why all single-factor separation selectivity models for RP packings are incorrect.
- Correct evaluation of HILIC-type and CT-type selectivity for a reversed-phase packing.
- evaluation of reversed-phase retention capability, HPLC packing chemical inertness and efficiency.
6.2. Testing silica-based HILIC, IC, and mixed-mode HILIC/IC packings.
6.3. Uncovering the chemistry of 'unique' stationary phases.
7.1. HPLC retention mechanism models.
- Solvent elution strength and eluotropic series.
- The mobile phase additive and the modifier.
- Acid/base equilibriums in HPLC.
7.2. Reversed-phase (RP) retention mechanism model.
- Adjusting the retention in RP mode.
- The correct way of separation selectivity fine-tuning in RP HPLC.
- Why peak tailing of basic compounds in RP mode is not a problem anymore.
- Typical buffers and mobile phases for RP HPLC separations.
7.3. Normal-phase (NP) retention mechanism model.
- Interaction between stationary and mobile phases in NP mode, and how it is taken into account by using the concept of the stationary phase modifier.
- The difference between the polar additive and the polar modifier.
- Selective solvation of analytes by the polar additive.
- Adjusting the retention and fine-tuning the selectivity in NP mode. Three simple empirical rules for fine-tuning NP separation selectivity.
- Using mixtures of several polar organic solvents as the mobile phase additives. Typical NP mobile phases.
7.4. Ion chromatography (IC) retention mechanism model.
- Adjusting the retention in IC mode.
- Typical IC buffers and mobile phases used in combination with strong and weak silica-based and polymeric IC stationary phases.
- The IC mode separation selectivity fine-tuning by adjusting the buffer pH.
- The IC mode separation selectivity fine-tuning by adjusting the composition of the organic component of the mobile phase.
- Myths about ion chromatography of pharmaceuticals: short column lifetime, poor peak shape, high salt concentration in the mobile phase.
- Improving peak shape and a packing lifetime in ion chromatography.
- How to develop an MS-compatible IC separation.
7.5. HILIC retention mechanism model.
- Similarities and differences between HILIC and NP retention mechanisms.
- Correct conditioning of a stationary phase in HILIC mode. Adjusting the retention in HILIC mode.
- Difference in conditioning procedures for neutral, acidic and basic HILIC stationary phases.
- HILIC/IC mixed-mode separations on polar silica-based columns. Switching between HILIC and IC modes.
- Improving loadability and improving peak shape for ionic compounds in HILIC/IC mode.
- HILIC/IC mixed-mode selectivity fine-tuning. Typical HILIC mobile phases.
8.1. Mixed-mode based approaches
- Converting an ion-pair RP separation into the non-ion-par mixed-mode separation.
- Converting a gradient RP separation into the isocratic mixed-mode separation.
- Using mixed exclusion-adsorption modes for fine-tuning separation selectivity.
8.2. Avoiding sample preparation by choosing an appropriate specific HPLC mode.
- Analyzing ointments and suppositories.
- Analyzing syrups and liquid formulation containing capsules.
9.1. Dynamic off-line and on-line modification of stationary phases.
- On-line stationary phase modification by the ionic mobile phase additive.
- Ion-pair modes. Reversed-phase ion-pair mode.
9.2. Two different chromatographic tasks solved by the reversed-phase ion-pair chromatography.
- Using ‘weak’ ion-pairing reagents. Compatibility of ion-pair mode with gradient elution.
- Using ‘strong’ ion-pairing reagents. Correct conditioning (modification) of RP stationary phase with the mobile phase that contains ion-pairing reagent.
- Why column washings are senseless in case of using ‘strong’ ion-pairing reagents.
10.1. Rate theory.
- Diffusion factors.
- Van-Deemter curves for different types of HPLC packings.
- Core-shell packings; their benefits and drawbacks.
10.2. Choosing an optimal flow rate for a given HPLC separation and packing type.
- Efficiency-optimal flow rate as the minimum working flow rate.
- Generally optimal and throughput-optimal flow rates.
10.3. The connection between the resolution and the analysis run time.
- Hydrodynamics of HPLC separation.
- Backpressure margin as the resource.
- Column impedance as a measure of an HPLC packing quality.
10.4. Choosing optimal column length and particle size.
11.1. Factors that contribute to additional peak broadening.
- Extra-column effects.
- Column overloading by mass and volume of injected sample. ‘Good’ and ‘bad’ sample diluents.
- How to minimize negative impact of additional peak broadening effects on efficiency and peak shape.
11.2. Correct method of estimating the throughput of an HPLC separation.
- Theoretical and effective peak capacity.
- Why a longer HPLC column can be faster than the shorter one. How to develop a real ‘fast’/‘ultra-fast’ HPLC/UHPLC separation without sacrificing too much HPLC method performance.