Online HPLC Training Course: About

Our HPLC courses are focused on HPLC method development and transfer. They provide the in-depth knowledge of HPLC and HPLC separation mechanisms that is required to develop the most high-performace & cost-effective HPLC methods like that >>


Our typical customers are R&D and QC departments of big pharma, food and agriculture enterprises as well as smaller pharma/biotech companies that develop their own routine HPLC methods or use HPLC in their research work.


We work with corporate groups only. The price is determined by the selected course program and the number of participants.

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Distinguishing Features

1. Special attention to development of highly specific routine HPLC methods is paid. It is shown that even the most complex multi-compound samples can be successfully analyzed using standard HPLC equipment with limited detection specificity due to application of specific HPLC modes like IC, HILIC, mixed modes, etc.  


2. It is shown how to succeed in analyzing extremely difficult matrices (like fatty food, ointments, oils, pastes, gels, syrups, etc.) using no sample preparation and at the same time not contaminating HPLC columns and HPLC equipment with matrix components. The main principles of developing matrix compatible HPLC methods are given and explained.


3. Highest attention is stressed to cost-effectiveness of an HPLC method. It is shown that throughput is not always the key attribute that defines cost-effectiveness. How to develop a cost-effective HPLC method is explained in detail.


4. Suitability is considered as a complex characteristic that also includes transferability and reliability. How to develop a reliable and transferable HPLC method is explained in detail.


5. How cutting-edge techniques like HILIC, mixed HILIC/IC, HILIC/IEX, RP/IC, RP/HILIC, RP/CT modes can be used in practice is explained. The approach to develop an MS-compatible HILIC, IC or mixed-mode HPLC method is given.   


6. Optimal mobile phase compositions for reversed-phase, HILIC, ion-exchange, normal-phase modes are given.


7. Optional modules include such topics as: chiral HPLC, qualification of HPLC packings, ion-pair RP HPLC and its alternatives, fine-tuning retention and selectivity in mixed HPLC modes.

Benefits of Online Training

1. Online distance learning can be done at any time or place; it is not affected by pandemic constraints.


2. The course can be followed from the workplace or even from home.


3. Training course program is very flexible; it can be adapted to fit requirement and work schedule of participants.


4. Participants can easily ask questions using the online chat. We will answer all questions of participants.  

Online HPLC Training Course: Program Modules

1. Three-step HPLC method development workflow

1.1. Basic chromatographic parameters. Factors that define the resolution of a peak pair.

1.2. What are the optimal retention, the optimal separation selectivity, and the optimal efficiency.

1.3. Three-step HPLC method development workflow.


2. Adsorption and exclusion HPLC modes. Mixed modes

2.1. Single adsorption (RP, NP, HILIC, IC, CT, LEC) and exclusion (SEC, IEX) HPLC modes.

2.2. Mixed modes that are widely used in practice: RP/HILIC, RP/CT, RP/IC, RP/SEC, HILIC/IC, NP/CT.

2.3. Predicting elution order in single and mixed HPLC modes. 


3. Contemporary HPLC stationary phases

3.1. Chemistries of contemporary HPLC stationary phases. Different types of mixed-mode stationary phase chemistries.   

3.2. An overview of commercially available stationary phases for reversed-phase (RP) chromatography. Classic RP stationary phases and mixed-mode (RP/HILIC, RP/CT) stationary phases. Main parameters that describe chromatographic properties of a reversed stationary phase.

3.3. An overview of commercially available stationary phases for ion-exchange HPLC (IC) and mixed-mode reversed-phase/ion-exchange (RP/IC) chromatography. SAX, SCX, WAX, and WCX stationary phases. RP/SAX, RP/SCX, RP/WAX, and RP/WCX stationary phases. Main parameters that describe chromatographic properties of an ion-exchange stationary phase.

3.4. An overview of commercially available stationary phases for normal-phase (NP), HILIC and mixed-mode HILIC/ion-exchange (HILIC/IC) chromatography. Main parameters that describe chromatographic properties of a HILIC stationary phase.

3.5. Quality of an HPLC packing. Chemical inertness and hydrolytic stability of a stationary phase. HPLC packing long-term stability.


4. Mobile phase composition: reversed-phase HPLC

4.1. Reversed-phase mechanism of retention.

4.2. Retention and selectivity fine-tuning in RP mode. Why mobile phase adjustment cannot be considered as the optimal way of RP selectivity fine-tuning.

4.3. Optimal reversed-phase buffers for UV and MS detectors. Typical reversed-phase mobile phases.


5. Mobile phase composition: normal-phase HPLC

5.1. Normal-phase mechanism of retention: two basic models.

5.2. What is a stationary phase modifier. Criteria of stationary phase modification.

5.3. Retention and selectivity fine-tuning in RP mode. Why normal-phase selectivity can be easily altered. Three simple empirical rules for selectivity fine-tuning in NP mode.

5.4. Typical normal-phase mobile phases.


6. Mobile phase composition: ion-exchange HPLC

6.1. Ion-exchange (IC, IEC) HPLC as the most underestimated HPLC technique for pharmaceutical analysis. Three popular myths about analytical ion-exchange HPLC: short column lifetime, poor peak shapes, high salt concentration.

6.2. Ion-exchange retention mechanism. Three general ways of fine-tuning the retention in IC mode.

6.3. Typical mobile phases for analytical ion-exchange HPLC on silica-based packings.

6.4. How to develop an MS-compatible ion-exchange HPLC method.


7. Mobile phase composition: HILIC & HILIC/ion-exchange HPLC

7.1. Correct conditioning of a stationary phase in HILIC mode.

7.2. Switching between HILIC/ion-exchange and ion-exchange modes.       

7.3. Retention and selectivity fine-tuning in HILIC/ion-exchange and HILIC/ion-exclusion modes.

7.4. Improving peak shapes in HILIC/ion-exchange mode.

7.5. Typical mobile phases for HILIC/ion-exchange HPLC. How to develop an MS-compatible HILIC/ion-exchange HPLC method.


8. Choosing optimal L, dp and F values

8.1. Rate theory. Diffusion factors. Van-Deemter curves for different types of HPLC packings.

8.2. Fully porous and core-shell packings, their benefits and drawbacks.

8.3. Choosing optimal linear velocity (flow rate). Efficiency-optimal and throughput-optimal linear velocities (flow rates).

8.4. Hydrodynamics of HPLC separation. The connection between resolution and run time. Backpressure as the resource that can be expended to improve resolution or to speed up a separation. Column impedance as a measure of an HPLC packing quality.

8.5. Choosing minimum necessary column length and particle size.

8.6. Factors that contribute to additional peak broadening. Extra-column voids. Column overloading by mass and volume of injected sample. ‘Good’ and ‘bad’ sample diluents. How to minimize the negative impact of extra-column effects on the resolution efficiency and peak shapes.
8.7. Choosing optimal column length and particle size.


9. Mixed HPLC modes: fine-tuning retention and selectivity

9.1. Flexibility of mixed modes. Shifting the balance between HPLC modes for fine-tuning separation selectivity in a mixed mode.

9.2. Switching on and switching off HPLC modes by the appropriate mobile phase adjustments.

9.3. Conversion of ion-pair HPLC methods into conventional HPLC methods with additive-free mobile phases. Ion-exchange-based mixed modes.

9.4. HILIC-based mixed modes. Using ion-exchange and ion-exclusion minor modes for fine-tuning retention and selectivity.

9.5. Conversion of gradient HPLC into isocratic HPLC methods. Reversed-phase-based mixed modes.


10. Ion-pair RP HPLC and its alternatives

10.1. Dynamic modification of a stationary phase. ‘Off-line’ and ‘on-line’ dynamic modification. Modification of a stationary phase by an ionic mobile phase additive. Ion-pair HPLC modes, reversed-phase ion-pair mode.

10.2. Chromatographic issues that can be resolved by means of reversed-phase ion-pair chromatography.

10.3. ‘Weak’ and ‘strong’ ion-pairing reagents, their benefits and drawbacks.

10.4. Critical disadvantages of ion-pair HPLC as an analytical method.

10.5. Why ion-pair HPLC methods can be sometimes so hard to reproduce and to use in practice. Correct conditioning of an intact stationary phase with the mobile phase containing ion-pairing additive. Correct handling of HPLC columns used in ion-pair mode.

10.6. Alternatives to ion-pair HPLC. 

10.7. Conversion of ion-pair HPLC methods into conventional HPLC methods that use additive-free mobile phases.  


11. Qualification of HPLC packings

11.1. Separation selectivity as a result of mixing several orthogonal HPLC modes.

11.2. Two parameter selectivity testing of reversed-phase and reversed-phase-based mixed-mode HPLC packings.

11.3. Two parameter selectivity testing of HILIC, ion-exchange and HILIC/ion-exchange mixed-mode HPLC packings.

11.4. Testing retention capability, chemical inertness and efficiency of reversed phases.

11.5. Testing retention capability, chemical inertness and efficiency of HILIC and ion-exchange packings.


12. Chiral HPLC

12.1. Main contemporary and outdated chiral selectors for CSPs.

12.2. Chiral HPLC in mixed normal-phase/charge-transfer mode.

12.3. Chiral HPLC in mixed HILIC/ion-exchange/reversed-phase mode.

12.4. Why racemate could not be resolved: four cases.

12.5. Possible ways to improve chiral separation in normal-phase mode.