Fundamentals of DryLab

Retention phenomena of reversed-phase chromatography (RPC) are described in many ways by different authors. One of the groups attempting to understand the fundamentals of RPC were the developers of the DryLab software, LC Resources Inc. in the United States, under the leadership of Lloyd Snyder, John Dolan, Tom Jupille, and Imre Molnár in Berlin, Germany.

Concept of the Solvophobic Theory

DryLab draws on the philosophy described in the "Solvophobic Theory" of Csaba Horváth, which was developed in the years 1975-1977 at Yale University, CT, USA, focusing on strong retention properties of water, which is ubiquitous in nature. Hydrophobic interactions are strongly participating and influencing chemical processes of life. The fundamental concept of this theory is that retention in RPC is enforced by water, as a component of the eluent. Nonpolar molecules in water require large amounts of energy for cavity formation to be dissolved in water. The retention factor k (also called the "capacity factor") is proportional to the energy needed for cavity formation in the dissolution process. In cases as shown with example-files of DryLab for %B-modeling

(s. also 8nitro.dlb in DryLab v.3.9 and 8nitro.dlproj in DryLab v.4 among others) and looking at the stronger retarded compounds on a C8 or C18-phase, their k is in water ca. 10-100.000 times larger, than in the organic eluent (f.e. acetonitrile or methanol). The factor is proportional to molecular weight, for proteins, it goes to millions. This means, that the retention force of water is extremely strong and therefore water is mainly responsible for the size of retention in the first place. However pH, temperature, additive and buffer concentration are also important. Nevertheless column chemistry is also influencing separation selectivity.

See presentation by A. Vailaya.

Principles of Gradient Elution in HPLC

Horváth and his team found that the only possible explanation for this extremely different scale of retention is the change in the surface tension between water and acetonitrile (AN) or methanol (MeOH). The explanation for this is, that water is strongly lipophobic, due to its high surface tension (72 erg/cm2). However the lipophobicity of water can be easily and continuously reduced by admixing MeOH or AN to it. That is what we do in gradient elution and this is the reason of the great success of gradient elution in HPLC. Gradient elution principle is applied in DryLab on a large scale. Gradient elution in RPC starts with water or with water-rich eluents. Upon injecting the sample into such a mobile phase (eluent), the water tries

to enforce a reduction of cavities around the more or less hydrophobic sample components and brings them onto the surface of the C8 or C18 column packings. At the moment of the attachment between the sample and the C-18-chain the contact surface area (ΔA) disappears and the corresponding energy (ΔAγ) is released. By increasing the amount of the organic component (MeOH or AN), the retention force from water will become weaker, as the surface tension of the eluent is reduced from approximately 72 erg/cm2 in water to ca. 20 erg/cm2 in MeOH or AN, and the retention time is reduced at the same time.

Applications of Gradient Elution

The process has tremendous capabilities for separating complex mixtures in a highly reproducible manner as the kinetics of the retention equilibrium is very fast (in opposite to ion-exchange). In gradient elution, DryLab calculates, based on only 2 gradient runs the retention precisely for every sample component. Isocratic conditions can be derived from gradient models. The amazing ease of

RP- gradient elution is exhibited in the continuous reduction of the retention force of water by increasing the amount of the organic eluent component (MeOH or AN). Fine differences in accessible solvophobic molecular surface areas, combined with steps in the gradient, are sufficient to achieve reasonably good separations with almost any mixture in life science applications.

Modeling HPLC Separations with DryLab

Modeling of RP separations by DryLab is based on the work of Lloyd R. Snyder and on the measurement of both: retention times and peak areas. The consequent calculation of sample positions in the corresponding chromatograms enables the chromatographer to look at experiments in a virtual mode and generate an overview of

all separation choices in a multifactorial space. If measurements are precise and reproducible, the retention behavior in complex mixtures can be modeled with 99%-retention-time-precision in seconds, instead of days or weeks, saving valuable time in the expensive laboratory environment.

Influence of Mobile and Stationary Phases

Changes in column selectivity can be explained by the influence of the free silanol groups or other entities bonded chemically to the surface of the silica. The Snyder-Dolan hydrophobicity substraction database is subtracting the hydrophobic contribution from all other terms in the total balance and is trying to focus on the silanol effects as the influence to change separation selectivity. In this way column similarities (and differences) can be predicted and a column substitution with equivalent stationary phases becomes possible. This database is included in DryLab. The

combined study of column- and eluent influences in DryLab allow the development of truly robust methods and enables the chromatographer to work with the best column at the most robust working point with 100% efficiency.

Based on the principles of gradient elution, DryLab can model not only RPC, but also ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), normal phase chromatography (NPC) and HILIC as well.