Laboratory Scale Lyophilization for Purification Laboratories—Developments in the Field

Freeze-drying, also known as lyophilization, has been used for many years to dry samples in the lab. This well-researched technique has become the preferred method for those researchers who need to dry only a few samples. Lyophilization is usually preferred as it enables a high level of dryness with low residual solvent levels. Moreover, the sample’s light, powdery/fluffy finish allows it to be easily removed and weighed out.

Yet, the traditional freeze-drying apparatus has several potential disadvantages. These include:

  • The process is slow
  • It is not possible to use volatile organic solvents and their mixtures
  • Samples have to be prepared in a restricted range of solvents, and usually, only water can be used.

As a result, researchers who have many mixtures of solvents or samples to process (for example, from preparative reverse phase HPLC separation containing acetonitrile and water), have started to use centrifugal evaporators. An example of this in other labs is how compound handling the aggressive nature of the organic solvents used makes a freeze drier unsuitable.

Even in such environments, advanced centrifugal evaporators (for example, the Genevac HT-4X, depicted in Figure 1), come with certain limitations. Issues reported include the following: samples are dried to a film and hence may be too complicated to re-suspend post drying; some samples do not dry with the majority; and some samples trap a small amount of residual solvent.

Genevac HT-4X Centrifugal Evaporator.

Figure 1. Genevac HT-4X Centrifugal Evaporator.

This analysis presents the results of a study which was carried out in the Genevac laboratory, that aimed to identify a “best of both worlds” solution, wherein quick parallel drying can be realized while offering: high levels of dryness, the popular “fluffy” finish preferred, and where each sample dries every time.

Problems with Purification

Within purification labs, samples are usually presented dissolved in acetonitrile and water, with a low level of a modifier present (typically 0.1% TFA). However, it is difficult to remove these solvents with a freeze drier. Firstly, a very deep vacuum is required to freeze the acetonitrile, or at least freeze drier that actively freezes the samples.

Acetonitrile freezes at –65 °C. If acetonitrile is not frozen, bumping can occur, which causes cross-contamination and sample loss. Secondly, acteonitrile present in the ice trap will spoil the vacuum and make it almost impossible to lyophilize the water.

Thirdly, it is a slow process, which is incompatible with the drive to bring down process times within a number of industries. With these issues in mind, the centrifugal evaporator has become the preferred technique because it enables rapid drying of many samples in parallel, and has been developed to regulate bumping while drying solvent mixtures.

However, there are two possible problems, and both are sample-related effects. It has been reported by users that they face difficulties when drying a few samples per batch. Firstly, the TFA might not have been removed completely and this residual TFA may affect the sample when in storage; secondly, an interaction may take place between the compound and water, which boosts the boiling point and makes drying difficult.

Residual solvent also shows up in nuclear magnetic resonance (NMR) analysis. While these issues can occur intermittently, the implications of picking some samples manually are prohibitive for several automated labs. Hence, the entire sample rack is processed again.

Lyophilization in a Centrifugal Evaporator

Lyophilization of samples prepared in water can be done in a centrifugal evaporator by pulling the best vacuum available in the Genevac HT-4X (see Figure 1) equipped with the solvent-resistant scroll pump. The ultimate vacuum is well below 0.5 mbar, which is more than sufficient for freezing water. This process is similar to regular freeze-drying, and for this reason it is comparatively slow.

Some years ago, Genevac created a process in which some of the solvent could be evaporated using the speed of a centrifugal evaporator - subsequently switching to a lyophilization mode when just a few milliliters of solvent were left, thereby providing the best of both worlds.

For one Genevac customer, the process time for 96 x 30 ml fractions reduced from 48 hours in a freeze drier to 16 hours (an overnight process) in a Genevac HT-12, which is equivalent to just 10 minutes per 30-ml sample, and where the samples dried in a sequential manner. Using this as a platform, the impacts of heating a sample at the time of lyophilization were analyzed to establish if this offered a productive advantage.

The analysis of water containing samples was done in two halves. At first, only water was used to develop ideal conditions, followed by acetonitrile and water to replicate the samples collected from HPLC.

Lyophilization of Water

Ibuprofen sodium salt was the standard sample used in all trials. First, a stock solution of 0.01M was prepared in water, and then 15 ml of the solution was loaded into each of 48 20-ml scintillation vials (Wheaton) and dried in a Genevac HT-4X evaporator under different conditions. A typical vial holder is shown in Figure 2.

Genevac 20-ml scintillation vial holder.

Figure 2. Genevac 20-ml scintillation vial holder.

Shown in Figure 3 is a plot of concentration and then lyophilization of water; this was developed to ascertain a baseline. Figure 4 shows a summary of the settings used. The total time taken for sample drying is about 8 hours, 4 hours of concentration in Stage 1 and 2, where the temperature of the sample is at roughly +8 °C and then Stage 2, the lyophilization stage, where the sample is frozen to –16 °C and warms up when dry.

Baseline method showing concentration and lyophilization of water-based sample—Trial 1.

Figure 3. Baseline method showing concentration and lyophilization of water-based sample—Trial 1.

Results of lyophilization trials with water.

Figure 4. Results of lyophilization trials with water.

While three stages are illustrated in Figure 3, the actual evaporation technique includes up to four stages:

  • Concentration of the solvent bulk using rapid evaporation
  • Cooling of the sample holders and samples in preparation
  • Freezing of the sample using deep vacuum
  • Lyophilizing the remaining solvent, with or without heat

The results of the method development for the water processing stage are depicted in Figure 4.

At the time of sequential runs, different heating levels were applied in the lyophilization stage, and higher heat levels were shown to decrease the total processing time from 8 hours in trial 1 to 5.5 hours in Trial 4. The results of Trial 4 are shown in Figure 5.

Results of concentration and lyophilization with heating, Trial 4.

Figure 5. Results of concentration and lyophilization with heating, Trial 4.

Since the exact processing time for each trial was not known, each sample was over-processed, and the endpoint was identified from the data. To ensure uniformity, the time at which the temperature of the sample became positive was considered as the endpoint. At this time, the samples warm up quickly, as there is no more cooling effect from lyophilization.

A few points to note from these data—it was believed that the best practice mandated a cooling stage, Stage 2. While this had invariably worked in the past, it has never been tested. In fact, the freezing stage achieves both cooling and freezing, and hence, this stage is not required (as shown by Trial 5). Yet, the freezing stage with no heat seems to be important, as demonstrated by Trial 6; in this example, the samples did not lyophilize and dried in the usual manner. From the data, it became apparent that the sample had not frozen at all. The Trial 3 results seem to be anomalous, in that higher heat usually reduces the processing time, but does not in this trial.

Lyophilization of Water and Acetonitrile

Lyophilization of water is insignificant for many users. While the time savings shown in the study are welcome, the problem remains of how to handle solvent mixtures. An alteration to the technique used for water is the inclusion of an earlier stage, Stage 0, to eliminate the acetonitrile prior to water concentration.

Stage 0 includes three parts:

  • Dri-PureTM—high rotor speed and vacuum ramping to prevent bumping
  • Draining the condenser—remaining acetonitrile will spoil the vacuum in later stages, and hence should be removed
  • Concentration—a 40 mbar stage to discard the acetonitrile without freezing the water, and at 40 mbar, acetonitrile boils at +2 °C

For such trials, a 0.01 M solution of Ibuprofen sodium salt was prepared in a 60:40 mixture of water and acetonitrile. The results are summarized in Figure 6. As is obvious from the data, the optimum conditions were realized only at Trial 10.

As with the water trials, the cooling stage was not needed. The process had to be adjusted to obtain the correct balance of lyophilization and concentration. 48 x 15 ml samples dried in 5 hours—equivalent to 6.25 minutes, provided the samples were dried in a sequential manner.

Results of lyophilization trials with water and acetonitrile.

Figure 6. Results of lyophilization trials with water and acetonitrile.

Shown in Figure 7 is the variation between a lyophilized result, realized in Trial 10, and conventionally centrifugally evaporated sample as per Trials 7, 8, 9, and 11. There is a stark difference, and the ease of re-suspension is higher with the lyophilized sample, while the dried sample does not fully dissolve.

Dried samples in scintillation vials.

Figure 7. Dried samples in scintillation vials.

Conclusions and Discussion

Lyophilization of the samples made it easy to re-dissolve the sample after drying. During the lyophilization stage, the addition of heat significantly reduced the lyophilization time. Although the traditional cooling stage is not needed when establishing the lyophilization method, the freezing stage has been shown to be important.

When evaporating mixtures of water and acetonitrile, the condenser should be drained after evaporation of the acetonitrile and before evaporation and lyophilization of the water. The presence of residual acetonitrile in the condenser spoils the vacuum and prevents achieving lyophilization conditions.

With the help of the GenevacHT-4X, the system had to be drained manually toward the end of Stage 0. Certain Genevac systems can automate this facility, thereby avoiding the need for user intervention mid-process. As a consequence of this work, an option to automate this on the Genevac HT-4X and HT-12 systems is being developed.

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Last updated: Aug 13, 2019 at 11:46 AM

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