Chromatography


Development of a Simple Technique to Automate Reverse Phase HPLC Fraction Pooling, Evaporation, and Reformatting


Dr Induka Abeysena & Rob Darrington, Genevac Ltd, UK.


Recently, the SampleGenie™ was developed by Genevac as a simple item of laboratory ‘automation’ to help scientists who work with, or store their dried samples in, small vials, but where their sample is initially dissolved in a large volume of solvent. SampleGenie (Figure 1) is essentially a glass fl ask to which a smaller vial can be attached, an inert seal ensures the integrity of the joint [1].


SampleGenie has been successfully implemented within both medicinal chemistry laboratories and dedicated purifi cation groups where samples are typically purifi ed by reverse phase high performance liquid chromatography (HPLC). Samples elute from the chromatography column in volumes larger than the average storage vial. Normal practice would be to dry the sample or fractions before pooling, and then re-dissolve in a minimal volume of solvent, such as dimethylsulfoxide (DMSO), transfer to the small vial, and dry to remove the DMSO. SampleGenie allows for the direct evaporation of large volumes (up to 250 ml) directly into a vial of choice.


SampleGenie has been developed for use with Genevac HT and EZ-2 evaporators as well as the Rocket™ Evaporation System (Figure 2) [2].


Early in the development issues were found with reverse phase HPLC fractions where a proportion of the samples stuck to the glass of the fl ask rather than fully entering the vial. This paper discusses the causes of this problem and the development of a solution that delivers more acceptable sample recovery.


Data from areas of applications outside of reverse phase HPLC has shown very high levels of sample recovery in the vial; two such being environmental analysis [1,4] and metabolic studies [3], where recoveries in excess of 90% or 95% were reported. For most scientists these are acceptable as any method of drying and transfer to a vial will have some losses. Losses can be minimised by washing out the fl ask, as could be done in this case, however this may reduce the gain in effi ciency obtained by using SampleGenie.


Figure 1. SampleGenie in section


Compounding this, in vacuum evaporation, the organic solvent evaporates more quickly than the water in a fraction. Once all the organic solvent has evaporated, non-polar molecules which are insoluble in water, will also crash out.


Solutions


A range of solutions were considered and evaluated, these can be subdivided into two categories, either, make the fl ask ‘non-stick’, or prevent the sample crashing out in the fi rst place.


One ‘non-stick’ solution considered was to end terminate the free silanol groups on the glass molecules, typically with a silane. Several commercially available treatments where tried, however, none of these coatings were that successful, the samples still crashed out, and still adhered to the side of the fl ask. The use of the silane treatment did help move all the sample closer to the neck of the fl ask but none was considered adequate. In addition, none were permanent, in that they were removed over time by washing of the fl ask. The fl asks were also coated with a Tefl on®


type material,


however, those providing this material had diffi culty in ensuring the adhesion between the coating and the glass, and delamination was a risk. This solution was dismissed.


Figure 4. Flask showing minimal sample sticking with added 1,4-dioxane


The solution showing the most promise was that of preventing the sample crashing out in the fi rst place by the addition of a solvent which would keep the sample in solution. Any such solvent would have to have a similar boiling point to water so that it did not all evaporate with the acetonitrile. Two were considered, toluene and 1,4-dioxane. Initial tests with toluene failed because it is immiscible with water, whereas 1,4-dioxane was much more successful.


Co-solvent Addition Experiments Figure 2. Rocket Evaporation System


Importantly, with reverse phase HPLC fractions the sample is usually dissolved in a mixture of water and organic solvent (acetonitrile or methanol), whereas, samples in the other applications are normally in a single organic solvent (or mixture of similar solvents). Also, in environmental or metabolism analysis the concentration of sample is typically low, a few milligrams (mg) of sample per 100 millilitres (ml), whereas a purifi ed sample may be as concentrated as 10mg/ml, or more.


Figure 3. Flask showing sample dried to the sides


With high concentratrations samples come out of solution before the volume is small enough to occupy only the vial. Evaporative drying is typically concentration until dryness, and at the point where the solution becomes saturated the dissolved sample will crash out, and stick to the fl ask (Figure 3).


A series of experiments were carried out using the Genevac Rocket evaporation system with SampleGenie. In this study three standard compounds were used, each was selected for a different solvent range based on solubility and where they are most likely to elute in gradient reverse phase HPLC:


Acetonitrile range 70-80% 50-60% 20-40%


Test substance Hydrocortisone Cimetidine Caffeine


These trials were done in two fraction volumes, 100 ml and 200 ml, with 100 mg and 200 mg of the standard compounds used, respectively. The HPLC fraction method at 40°C was selected for all experiments. The recovery levels were between 97-100% collected in the vial (Table 1).


The amount of 1,4-dioxane needed depends on the volume of the vial and the amount of water in the sample. To determine how much 1,4-dioxane is required to add, fi rst select the sample with the least amount of acetonitrile and, depending on the volume, select the corresponding amount of 1,4-dioxane from the look up table, Figure 5. Although 1,4-dioxane (BP 101°C) has a similar boiling point to water, because of the low latent heat it will evaporate quicker. So, hydrophobic compounds will need more care to avoid sticking to the fl asks.


LAB ASIA - NOVEMBER/DECEMBER 2017


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