HPLC & UHPLC Method Development
Specificity, robustness, and cost-effectiveness – these three basic principles are at the core of our HPLC methods. A good HPLC method should provide the necessary accuracy, but at the same time should also be fast, inexpensive, and easy to validate and to transfer. And, certainly, it should work smoothly over the long term.
These objectives are achieved by using the state-of-the-art three-step method development procedure. We aim to solve every analytical challenge as interconnected problem in which each part is addressed separately, yet optimized with the entire solution in mind.
In the first step, a number of different separation solutions are applied to a given analytical task. Those selected meet two requirements: an HPLC separation solution should be done with minimal sample preparation, and at the same time it should provide the absence of HPLC column contamination.
In the second step, the stationary phase is carefully selected, and the mobile phase composition is properly adjusted in order to get the optimal selectivity.
The optimal selectivity corresponds to the situation in which the peak pair with the lowest selectivity is placed at the end of the chromatogram, and the peak pair with highest selectivity is placed at the beginning of the chromatogram. The first condition ensures high separation robustness and provides the opportunity for enhancing the method’s throughput. The second condition ensures high separation specificity.
In the third step, a number of technical parameters such as column length, adsorbent particle size, and flow rate are selected in order to achieve the best possible compromise between the method’s throughput and the separation robustness.
Such approach results in very robust and inexpensive HPLC methods that are the choice for routine HPLC measurements.
To provide the highest standard of HPLC method development, we apply a wide range of state-of-the-art HPLC columns and the most advanced HPLC techniques, including:
- different HPLC modes, single (RP, HILIC, IC, NP, CT, SEC) and mixed (RP/HILIC, RP/CT, RP/IC, HILIC/IC, NP/CT);
- different detection techniques (UV/Vis, FLD, RID, ELSD, CD, ECD, MS);
- HPLC column switching techniques (on-line SPE, heart cutting 2D-HPLC, gradient mode emulating isocratic systems, etc.).
We usually start by performing a diagnostic check-up of the analysis and develop the technical specification. In fact, it presents just a list of technical requirements for the problem at hand. However, in our practice, the correct technical specification is a key to success.
As a rule, technical specification sets threshold values for a number of HPLC separation characteristics such as:
- resolution of a critical pair(s);
- peak asymmetry factor;
- efficiency;
- maximum analysis time;
- maximum back-pressure;
- minimum retention k’;
- limits of detection (LOD) or quantification (LOQ).
As practice shows, it is hardly possible to resolve a complex mixture of structurally similar analytes using the conventional and widely used reversed-phase mode (RP) only. In many cases, to develop a robust, high-quality separation, our deep know-how of the entire range of HPLC modes is applied.
We are able to apply a number of different adsorption, exclusion and mixed HPLC modes, including:
- reversed-phase mode (RP);
- hydrophilic interaction mode (HILIC);
- ion-exchange mode (IC);
- normal-phase mode (NP);
- charge-transfer mode (CT);
- size exclusion mode (SEC/GPC);
- mixed modes: RP/IC, RP/HILIC, RP/CT, HILIC/CT, NP/CT.
To develop the most specific HPLC methods, we can apply various detection techniques: UV/Vis (DAD), FLD, RID, CD, ECD, ELSD, MS.
Every HPLC method development procedure is followed by the method’s specificity verification. Verification procedure is carried out for each of the developed methods; verification protocols constitute a part of the package we report to our customers.