Cost Minimization of Preparative Liquid Chromatography
Nowadays HPLC is one of the most effective methods for substances purification in the field of chromatography. Particularly the possibility for saving various costs, like for solvents or working time, demonstrates the strength of preparative HPLC. In the following article important factors are addressed, which may lead to cost reduction in substance isolation and purification.
The Column Packing Station
Preparative HPLC columns with an inner diameter of 25 mm or larger are expensive, often available only in a certain lengths and the separation performance can decrease rapidly due to instability of the column packing.
With a column packing station these problems can be avoided by packing HPLC columns with column material yourself. Silica gel or polymer phases are inexpensive and easy to obtain. A simple calculation shows the significant cost difference between a ready packed purchased HPLC column and a comparable HPLC column packed in the column packing station.
The column material C18 (RP18) with 10 µm particle size and 100 Å costs about 3,500 €/kg. For one HPLC column with 50 mm internal diameter and 250 mm bed length you need about 300 g, i.e. the costs for one 50 mm HPLC column in the column packing station add up to about 1050 € for each packing. The cost share for the column packing station are 2,400 € / year (€ 12,000 / 5 years). One ready packed HPLC column C18 (RP18) 50x250, 10 µm, 100 Å, from a reputable column manufacturer costs € 13,500 €.
The following comparison outlines the costs for an average consumption of three of these HPLC columns per year. The costs for HPLC columns packed in the column packing station include 1,050 € x 3 = 3,150 € for column material plus 2,400 € for the column packing station and add up to 5,550 € in total. In contrast, the costs for three ready packed columns amount to € 13,500 x 3 = € 40,500 per year.
In addition to these cost advantage, there are further benefits:
- Only pack the required bed length necessary for the separation and save column material and solvents.
- A dirty or clogged HPLC column can be unpacked and re-packed after cleaning the column material.
- Unpacking and packing can be done in less than an hour, i.e. you save time for no waiting of the ordered ready packed column.
Cost differences of the annual requirements of three self-packed HPLC columns (SP) compared to three ready packed purchased HPLC columns, 50x250 C18, 10 µm, 100 Å.
For those who want to purify only substance amounts, the costs of a system must be in proportion to the material throughput capacity of the system. However, an originally very inexpensive system can grow very expensive in the long run when it is difficult to use or the substance is not sufficiently purified. In this constellation solvent consumption and fast availability of the purified substance are the dominating cost factors. About 90% solvent costs have to be estimated for inadequately optimized or difficult separations.
Cycle Time Reduction for Preparative Separations
In preparative liquid chromatography optimized cycle time is the essential criteria. Analytical separations aim at separating as many components as possible, often resulting in long cycle times and use of large columns. For preparative separations, the cycle times should be kept short only due to isolation of certain sample components in the shortest possible time. Therefore, the HPLC column should be so heavily loaded that the target component starts overlapping with neighboring peaks.
For a high selectivity the column efficiency is crucial, which depends mainly on the particle size. It is, therefore, advisable to use short HPLC columns containing material with 5 or 10 µm particle size. This keeps cycle times short, resulting in high substance throughput and little solvent consumption. Although the prices for column material with fine granulation are higher compared to those for materials with larger paticle size from 20 to 100 µm. You can save up to 50% costs just by reduction of solvent consumption.
A further advantage of smaller column materials is their increased efficiency at high flow rates. For large particles smaller flow rates have to be used. This often means very long cycle times and large columns for compensating the reduced separation performance of the larger solid phase particles.
For larger scale purifications a different separation strategy is beneficial.
Instead of method optimization to baseline separation of the components of interest, the HPLC column is heavily overloaded. The pure material is collected and impure fractions are purified once again. With this method a 10 to 100-fold increased throughput is achieved because the effect of column overloading results in "Displacement Chromatography". The Peak bands no longer overlap as in classical chromatography where peaks have the form of a Gaussian curve. These bands show the same width over the entire peak height and are virtually separated from each other. This effect is particularly evident with small particle sizes; however, the optimum loading and the proportion of the pure fraction must be determined empirically.
In the past the lack of well-packed HPLC columns and HPLC pumps which could pump against high back pressures were compelling reasons not to use small particles in the preparative column chromatography. The application of dynamic axial compression in the column packing station and the use of HPLC pumps for high flow at high backpressure eliminate this problem.
Increased Loading Capacity
The limiting factor for loading is often the sample solubility in the mobile phase. For many synthetic substances reversed phase chromatography is not suitable, since these often non-polar substances are better soluble in ethyl acetate than in water. The transition to normal phase separations usually leads to much higher throughput because the sample is much better soluble in normal phase solvents.
Proteins, peptides and biopolymers, however, are usually separated with reversed phase, ion-exchange or other methods. These kinds of samples must be diluted prior to injection in order to avoid precipitation of the substances on the column. The buffer type is often a decisive factor and shows highest solubility one pH unit above or below their isoelectric point. Mostly buffers such as trifluoroacetic acid (TFA), ammonium formiate, triethyl ammonium acetate (TEAA) and ammonium acetate are used because they can be removed by lyophilization and no demineralization is needed.
To avoid precipitation on the HPLC column it is also important to test the sample’s solubility in the solvents. A simple method may be helpful:
Linearly scale down the preparative separation method to an analytical column with 4.6 mm inner diameter and inject a small sample amount. Note the peak areas and increase the injection volume by a factor of 10 and so on. Should the peak area be substantially smaller than the x-fold value, this indicates a precipitation on the column. Also pay attention to solvent choice. Halogenated solvents are expensive and toxic. Methanol or ethanol should be given preference over acetonitrile in terms of costs and safety. Solvent recycling by distillation or by using isocratic methods should also be included in the considerations.
The nature of the sample prior to purification also affects overall costs. A sample, e.g. containing only 5% of the required product, requires 10 times the number of injections, as a sample containing 50%. Therefore, you should include precleaning steps, such as recrystallization, flash chromatography, solid phase or liquid-liquid chromatography. A preparative solid phase extraction cartridge costs less than 100 € and can save you thousands of euro by accumulating the target component. HPLC is an economical purification method, which is used to purify substances with 80 to 99% purity.
Separation MethodThe separation of substances can be carried out by liquid-liquid extraction, also referred to as distribution chromatography. In this case the sample separation takes place continuously in two immiscible liquid phases according to the polarity of the ingredients.
For this separation method the SCPC is used. It contains a rotor made of many round metal plates with more than a thousand small chambers interconnected via channels. Inside the rotor the liquid stationary phase is penetrated by the liquid phase. The centrifugal force causes the separation of the two liquid phases in the individual chambers of the rotor. When the liquid mobile phase is in equilibrium with the liquid stationary phase the sample can be injected into the rotor.
The separation process takes place in the two immiscible phases. The sample components are distributed according to their respective distribution coefficient KD in these two liquid phases and move at different rates through the chambers to the rotor outlet where they are fractionated. This innovative technology separats substance mixtures, from multigram to kilogram scale even for very problematic separation tasks.
The distribution chromatography of SCPC guarantees 100% sample recovery. This results from the fractionation of both the liquid mobile and the liquid stationary phase, in which all ingredients are contained. These features enable users to purify samples from different matrices quickly, easily and inexpensively.
In combination with HPLC components the SCPC accelerats the entire chromatographic process to the pure product. Upscaling from analytical to a productional scale is possible, as separation results are the same for all rotor sizes and sample amounts are scaled up by the volume factor.
The rotor design makes flow rate upscaling unnecessary. In total the costs for preparative separations are reduced by a factor of 10 compared to the exclusive use of HPLC with solid phase columns.
HPLC in combination with solid phase column and SCPC as a rotating column with liquid-liquid chromatography.
In this combination the SCPC is used for precleaning and the solid phase column for subsequent fine purification of substances. This method is particularly suited for separation processes which would be extremely difficult, or even impossible with the HPLC and solid phase column.
HPLC with solid phase column and SCPC with liquid-liquid separation column
In summary, many factors influence the costs for a preparative purification. Therefore, the various influencing factors must be considered, as for example, cost savings by increased substance solubility in a given solvent. The best method is to first model the preparative separation on an analytical column and to list all cost factors subsequently on this basis.
In this respect, the advantages of the column packing station and the SCPC become especially evident, as they can work with a large number of different separation phases and different separation conditions. This enables more cost-effective method development on analytical scale easier and also helps upscaling to the preparative scale.