Advanced Dryness Detection for Improved Throughput and Yield During Evaporation

In many laboratories across a wide range of disciplines, removal of solvent from dissolved sample is a routine, daily procedure. The majority of the methods employed to remove the solvent rely on the use of vacuum to achieve low-temperature boiling, normally to prevent damage to a temperature-sensitive sample. This article demonstrates the importance of paying sufficient attention to closely monitoring the drying process and presents solutions to save time and prevent sample loss.

Principles of vacuum evaporation

Vacuum-assisted evaporators (i.e., rotary evaporator, centrifugal evaporator, vortex evaporator, or traditional freeze dryer [or lyophilizer]) rely on boiling the solvents away at low temperature. Low-temperature boiling is achieved by pulling a vacuum on the sample and the boiling point of the solvent decreases proportionally to the pressure in the system (Figure 1). The pressure in the system controls the boiling point and therefore the temperature of the sample regardless of the temperature of the evaporator water bath or sample holders. In the case of the freeze dryer, a high level of vacuum is used so that the solvents begin to boil below their freezing point, causing the sample to freeze; thereafter the solvents sublime until dryness is achieved.

Figure 1 - Plot of boiling point versus pressure for some common solvents.

It can be seen from Figure 1 that to achieve low-temperature boiling of high-boiling-point solvents (e.g., dimethyl sulfoxide [DMSO] or dimethyl formamide [DMF]) better levels of vacuum need to be achieved. Methanol may be boiled at –200 °C using a pressure of 11 mbar. However, boiling DMF at –20 °C would require a pressure of 0.15 mbar, which might be achievable, although with a typical vacuum pump it is not possible to boil DMSO at anything below about +6 °C (even though it freezes at +18 °C).

Factors affecting speed of evaporation

Once a sufficient level of vacuum is achieved to cause the solvent to boil, there are two principal factors that affect the speed of evaporation—the specific latent heat of vaporization of the solvent and the effect of the dissolved sample.

The specific latent heat of vaporization of the solvent is the amount of heat energy that is required to boil one unit of a solvent. DMSO has a relatively low heat of vaporization (603 J/mL), whereas methanol requires 923 J/mL, and water, a massive 2441 J/mL. Therefore, water takes more heat energy than DMSO or methanol to boil and thus the samples will normally take longer to dry.

The effect the dissolved sample has on the boiling point of the solvent is highly variable. Some have little or no effect; others have a massive effect. The best known example is the use of common salt on the roads in winter. Sodium chloride dissolved in water boosts the boiling point but, more importantly, as in this example, drops the freezing point of water to approximately –15 °C. The problem for most laboratories in assessing the likely impact of these factors is the nature of their work is such that different and often unknown samples are processed each day.

Improving throughput

Efficiency savings are desirable in most laboratories—increases in throughput mean that more work can be achieved with the same resources, contributing to savings. While the use of fixed-length sample drying methods is commonplace, a notable exception is the rotary evaporator. Research shows that typical rotary evaporator users attend the sample during the drying process to ensure that the operation is trouble free. However, this is not good use of operator time when multiple samples need to be dried. In such circumstances, safe, unattended solvent evaporation of multiple samples can be achieved with a system such as the Genevac EZ-2 centrifugal evaporator (Genevac Ltd., Ipswich, U.K.) (Figure 2).

Figure 2 - Genevac EZ-2 centrifugal evaporator for parallel drying of samples.

The problem with fixed-length drying methods is that the drying time often varies between solvents and between samples. Therefore, to ensure that every sample is dry, users tend to overestimate the drying time. By automatically stopping the evaporation process when the samples are dry, the user can gain significant time savings.

Working with a laboratory that produces libraries of small molecules and undertakes many drying procedures daily (drying many different solvents), the effects on average drying time when switching from fixed-time methods to the Genevac automatic end-of-method detection software were studied. Despite the wide range of solvents used, the sample format is standard across all protocols—up to 6 mL of solvent in each of 24 vials per sample holder in an HT-12 series 2 evaporator (Genevac). This study was conducted over two key locations, where the solvent profiles differed significantly (Figure 3). The change in average drying times is shown in Table 1. The reduction in evaporation times achieved was significant and has contributed to increased throughput and productivity.

Table 1 - Change in average drying times

Figure 3 - Profile of solvents used in the two study locations.

Improving yield or reducing sample loss

Figure 4 - HT-4X evaporator in Genevac applications laboratory studying ibuprofen molecular structure.

Table 2 Molecular weights of some common drugs of abuse (source: http://pharmagkb.org)

Most samples can become volatile under the right conditions. Generally, the smaller the size of a molecule, the easier it is to volatilize—this is especially true for organic molecules. A number of Genevac users have reported that they have observed loss of sample when the sample is of low molecular weight (approximately 300 and below) and/or has high volatility (for example, a straight-chain organic molecule with few side groups).

Table 3 - Loss of ibuprofen during evaporation

The company has studied the effects of this problem on the common pharmaceutical ibuprofen in its applications laboratory using a HT-4X evaporator (see Figure 4). Ibuprofen has a molecular weight of 203, which is similar to many drug molecules or intermediates produced in drug discovery chemistry. However, ibuprofen may be far bigger than typical molecules studied within metabolism; adsorption, distribution, metabolism, and excretion (ADME); drug metabolism and pharmacokinetics (DMPK); or toxicology groups, which are usually looking for drug fragments. Another growing area where this effect may have impact is among the laboratories testing for drugs of abuse, either as part of routine prisoner, sports, or workplace screening or within forensic science laboratories. Table 2 illustrates the molecular weights of some common recreational and sporting drugs of abuse, demonstrating the potential problem for testers who might easily use a suboptimal evaporation protocol resulting in loss of some of the drug or drug metabolite under investigation.

In the Genevac applications laboratory, dried samples of ibuprofen were subjected to various levels of temperature and vacuum and the weight loss of sample was recorded (Table 3). Extrapolation of these data to show the potential sample loss is shown in Table 4 (note assumptions).

Table 4 - Sample losses due to overdrying at compound supply company*

Conclusion

Fixed-length vacuum drying methods are typically much slower than optimized procedures and, with lower-molecular-weight organic molecules, may lead to significant loss of the sample being dried. Compound loss is commonly due to sublimation of the dry compound following evaporation of the solvents in which they were dissolved. Preventing sample loss can be achieved by limiting the levels of vacuum and temperature used. However, this may have a knock-on effect in increasing drying times. Use of the automatic end-of-run software can help prevent overdrying and sample losses. In the authors’ case study, the average evaporation times were reduced by 25% and 28%. In cases in which a laboratory does not use such a system, it may be appropriate to revalidate the drying methodology and particularly the drying times used.

Mr. Griffin is Applications Engineer, and Mr. Darrington is Business Development Manager, Genevac Ltd., Farthing Rd., Ipswich IP1 5AP, U.K.; tel.: +44 1473 240000; fax: +44 1473 742987; e-mail: Rob.Darrington@Genevac.co.uk.

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