Author: Jiri Urban

Categories
Chromatography

Costless improvement of your HPLC instrument

The recent progress in the column development brought to the market new type of highly efficient columns. These columns can be use either with standard HPLC instruments or with the instruments allowing separations at ultra high pressure (900 MPa). The conventional size of the column (150 x 4.6 mm) has been decreased significantly. The typical size of column for ultra high pressure liquid chromatography (UPLC) is 50 x 2.1 mm with sub 2 μm ID particles.

Reduction of the extra-column volume of instruments

The decrease in the column size increases the significance of the extra column volumes – the volume of the liners between the injector and column, as well as column and detector, injector itself and detection cell. The contribution of the extracolumn volumes can be almost neglected using the conventional size of the column (150 x 4.6 mm) and flow rate as high as 1 mL/min (ok, almost neglected;). On the other hand, if we are using the identical instrument with small columns (50 x 2.1 mm) the influence of the extracolumn contribution is much higher and can devastate our efficiency and separation.

In recent paper, F. Gritti et al. compared several currently commercially available HPLC instruments in terms of the influence of the extracolumn volumes on their separation power. They found, that the only optimization of the extracolumn volume in standard Agilent 1100 HPLC system from 15.2 μl to 3.8 μl, together with the change in the volume of detection cell (13 μl to 1.7 μl) dramatically improve the columns efficiency. The average increases in the average column efficiencies were 28, 41, and 278% for the 100 x 4.6 mm, the 50 x 4.6 mm, and the 50 x 2.1 mm I.D. Kinetex columns, respectively.

Using this very simple and almost costless modification of the current instruments, the full performance of large diameter column can be achieved. However, other approach has to be applied in case of the resolution power of small diameter columns.

Sample focusing with weak solvent

In the same article, authors showed the possibility of column efficiency improvement with the injection of the weak solvent plug after the sample. After the sample injection, the weak solvent (water in reversed-phase chromatography) is injected and if there is no retention of this weak solvent on the stationary phase, the width of the sample band adsorbed at the column inlet can be reduced by diluting the sample with the weak solvent. Following picture (adapted from the discussed article) describes the whole idea.

Sample focusing with weak solvent
Sample focusing with weak solvent

With this approach, the sample dispersion on the column inlet is almost eliminated and it is possible to achieve apparent column efficiency close to the maximum possible for most columns currently available, including the short 2.1 mm I.D. columns packed with 2.6 μm superficially porous particles.

These examples show that it is possible to improve the HPLC instrument performance using very simple and costless approaches. However, the further improvement in the instrumentation (especially decrease in the instrumental extracolumn volumes) is necessary to be able to reach the full possible performance of the new, highly efficient, columns.

Categories
Monoliths

Four directions how to improve monolithic stationary phases

Georges Guiochon pointed out in his excelent reivew about monolithic stationary phases four directions from which we can expect a serious improvement in (monolithic) columns performance.

High temperature chromatography

High temperature chromatography, which causes a reduction in the viscosity of the mobile phase. So far, monolithic stationary phases have not yet been used at high temperatures but this is only a matter of time. High temperature liquid chromatography currently pioneered by Peter Carr and his group is going to be one of the major research areas in analytical chemistry for the next ten years. A significant reduction of analyses times by a factor between 3 and 4 is quite likely.

Increase in the pressure

An increase in the maximum pressure available to the analyst. Most commercial instruments can operate at inlet pressures of up to 40 – 50 MPa. A few of them can reach inlet pressures of 100 – 120 MPa and pumps able to reach 900 MPa are available. The use of high pressures requires far more caution than chromatographers are used to apply. This may create new, some times unexpected, safety hazards against which analysts should be forewarned. One advantage of monolithic columns is that extremely efficient columns, able to generate one or even several millions of theoretical plates could be operated with conventional HPLC instruments if long enough columns could be prepared.

Optimize the structure

A decrease in the minimum value of the height equivalent to theoretical plate (HETP) of the columns used. This will come from a reduction of the heterogeneity of the radial distribution of the flow-through pore sizes, also from a reduction of the average size of the domains of the monolithic column used and from a reduction in the variance of the domain sizes.

We have to be able to control (and suppress) monolith heterogeneity. My small prediction: one who is able to prepare the (monolithic) stationary phase with no or limited heterogeneity will be able to achieve unimaginable efficiency and column performance. Like for example homogeneous pillars.

Higher column permeability

Internal heterogeneity of organic polymer monolith
Internal heterogeneity of organic polymer monolith

An increase in the column permeability. This requires an increase in the average flow-through pore size. Since this size is included in the domain size, this requirement is in conflict with the previous one. Both can be achieved only by decreasing the average size of the porons, which would increase the external and total column porosity at the expense of the internal column porosity and the total surface area of adsorbent in the column. There is no clear limit here but it does not seem that much can be gained. Most probably, a reduction in the variance of the domain size accompanied by an increase in the degree of radial homogeneity of the monoliths constitute the most promising avenues for the monolith designers and makers.

Solutions?

One of the possible ways how to connect these last two conflicting requirements can be preparation and optimization of hypercrosslinked monolithic stationary phases. The porous structure (flow through pores) can be optimized independently on the structure of the thin hypercrosslinked layer prepared on the surface of the monolith (micro- and mesopores). Firstly, the generic monolith is prepared (flow through pores) and then the surface of the stationary phase is modified with the hypercrosslinking reaction and thin layer of small pores is formed.  Then, only the general models connecting the preparation and modification of the hypercrossllinked monoliths with their chromatographic properties have to be developed and understand.

What do you think about these suggestions?

PS: if you haven’t done yet – look at the review written by Georges Guiochon. There is all you need to know about monoliths but were afraid to ask.

Categories
Monoliths

History of monolithic stationary phases

Analyst 1957, 77, 964 - 969.
Analyst 1957, 77, 964 - 969.

Although the monolithic stationary phases suitable for separations were introduced in the 1990s [1,2,3], the idea of using a “continuous block of the porous gel structure” as stationary phase was discussed in Analyst by R. L. M. Synge, A. J. P. Martin, and A. Tiselius as longs ago as in 1952.

Both equilibrium and kinetic aspects of the molecular-sieve properties of zeolites have been studied in detail by Barrer, and it is clear that these equilibria could be used for the separation of small molecules on chromatographic columns of zeolites. Zeolites could not be used with larger molecules, as the spaces in them are too small. However, from dialysis and ultrafiltration studies enough is known of the properties of membranes and gel structures to suggest that these, though their pores could not be expected to possess the regularity of those of zeolites, could nevertheless be used for more refined separations than have hitherto proved possible. If used as powders in ordinary chromatograms, however, these substances would exhibit the disadvantages already discussed, namely that adsorption, increasing with molecular weight, would work in the opposite sense to molecular-sieve effects. An alternative possibility, suggested in discussions between Dr. A. J. P. Martin, Prof. A. Tiselius and one of us (R.L.M.S.), is to use electro-endosmosis to move a solution through a continuous block of porous gel structure. In this way the equivalent of movement of liquid through a very thick ultrafiltration membrane is attained without the necessity of great hydrostatic pressures, which would destroy the membrane structure. Here adsorption and molecular-sieve or frictional effects would all act in the same sense, tending to retard more the larger molecules.

And conclusions?

  1. Smart people have smart ideas.
  2. Each idea we are reading today in scientific journals may have a huge impact in comming years. At least in same way, as monoliths have changed the chromatography.
Categories
General

Writing for publication in research journals

Writing for publication
Writing scientific paper

Berkeley Lab organized yesterday short workshop focusing on writing for scientific journals. The workshop took 2 hours and it was mainly focused on the “schedule” we should follow during the publication preparation.

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Categories
Theory

Column permeability

One of the most important characteristics describing the column properties is column permeability. Term permeability refers to the column packed with a stationary phase (particles or monolith) and describes how easy flows the liquid (mobile phase) through the column.

Continue reading “Column permeability”