Chemistry: analytical and immunological testing – Including sample preparation – Stabilizing or preserving
Reexamination Certificate
2000-04-17
2003-02-18
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Stabilizing or preserving
C436S183000, C422S105000, C422S091000
Reexamination Certificate
active
06521464
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns techniques for preventing sample loss from containers open to atmosphere, especially loss of liquid trifluoroacetic acid (TFA) due apparently to anomalous surface properties which, under certain circumstances, cause the liquid TFA and any solids in solution therein to creep up the walls of a container and even right out of, and down the external walls of a container.
BACKGROUND TO THE INVENTION
TFA has been found to creep in this way in partially filled tubes open to the atmosphere at temperatures of around 20° C.
This can be highly undesirable, particularly when it is used for cleaving synthesised molecules from resin beads in solid phase synthesis of organic molecules. In this application TFA, usually mixed with an organic solvent such as dichloromethane, is added to resin beads in a tube or other container, for example a microtitre plate, (container) on which organic molecules have been synthesised. These molecules are all attached, at some point on each molecule, to the beads and the TFA breaks the bond, which attaches them to yield the required molecule in solution in the TFA/solvent mix. The next step in the process is usually evaporation of the TFA/solvent mix (TFA mix) to yield the newly synthesised compound. If some of the TFA mix creeps out of the sample container it will take some of the compound with it and that amount of compound is normally lost. As synthesising such compounds can be very expensive this is highly undesirable. In multi-sample holders, such as microtitre plates, compound escaping from one well can migrate into an adjacent well causing cross-contamination.
SUMMARY OF THE INVENTION
According to one aspect of the present invention, creep of TFA may be reduced by the addition of dichloromethane to a sample so that the TFA content of the liquid is reduced to 20% or less.
According to another aspect of the invention, creep of TFA may be reduced by the addition of 10% water or another liquid to the sample. In general creep will be reduced but the compound may not cleave from its resin in dilute solutions of TFA.
It is of course a prerequisite that any added liquids will not react with the compound.
Creep of TFA liquid seems only to occur on surfaces which are in contact with concentrated TFA vapour. This form of creep may well occur in other liquids when in contact with concentrated vapour of the same liquid, and according to another aspect of the invention dichloromethane or water or other liquids may be added to reduce such creep of other liquids.
Five methods have been devised for preventing or minimising the effect of this phenomenon. In all these methods it is preferable though not essential to have a dry inert gas eg dry nitrogen, rather than air, filling that part of the container not filled with TFA mix. This is because the strong affinity of TFA for the water in atmospheric air causes other problems.
In a first method, the top of the sample container is sealed as soon as the TFA mix has been added. The TFA vapour, which is heavier than air, evaporates to form a layer over the liquid surface but cannot fill the rest of the empty space above the liquid because it cannot displace the air or other gas in the sample container above the vapour. If the TFA mix only partially fills the container the liquid film on the sides of the container will not rise to the top of the container. The sample containers need to be unsealed in a centrifugal evaporator only after they are subjected to enough centrifugal force (in the region of a few hundred G) to prevent creep when the sealing caps or closures have been removed.
In a second method a container is employed in which the top is formed with a central tubular entrance in the form of an unspillable inkwell. The container is filled with TFA mix to a level below that of the lowest level of the central downward pointing tubular entrance of the container. TFA vapours will occupy the central entrance tube up to a level slightly above that of the lower end of the entrance tube, but can go no further if gas or air, displaced in the container by the TFA vapour, is trapped in the annular space around the tubular entrance tube, below the upper wall of the container surrounding inwardly directed the tube. There is therefore no continuous path in the inside surface of the container wall, in contact with TFA vapour, up which the TFA liquid mix can creep. This solution to the problem is more convenient in that there is no need to cap the container after filling it with resin beads, TFA mix or other chemicals, so that there is no cap to be removed when the samples are to be evaporated as when rotated in a vacuum chamber.
The ring shaped top can be formed as an integral part of the tube or well, or a cap fitted to a straight tube, or a small cap fitted to each of the openings in a microtitre plate. In screw top vials (eg see
FIG. 9
a
) the ring can be formed by inserting a short tube and sealing it hermetically (
FIG. 9
b
).
In a third method the container is filled with an inert gas having a higher density than TFA vapour (such as sulphur hexafluoride) shortly before, or shortly after, adding the TFA to the container. The heavy gas is not so readily displaced as air or nitrogen and will prevent or delay the TFA vapour filling the container which, in turn, will prevent the liquid TFA mix from reaching the top of the container or delay the process sufficiently to prevent sample loss or cross-contamination arising from creep of TFA mix.
In a fourth method, the TFA vapour is continuously removed and replaced with air or an inert gas such as nitrogen. In this arrangement a tube may be inserted into the container to a position at which its lower open end is just above the TFA mix, and the top end of the tube is connected to a pump or reduced pressure collecting device, so as to suck out the TFA vapour as soon as it reaches the level of the lower end of the tube. A gas such as nitrogen or air, is admitted to the top of the container to compensate for the loss of gas volume caused by the removal of the TFA mix vapour. Under these conditions the TFA mix liquid rises as far as the lower end of the tube but no further, and again a barrier exists, above that level, up which the TFA will not in general creep.
This method has the added advantage that it promotes rapid evaporation of the TFA mix and can be used both as a method of preventing TFA creep and of evaporating the TFA mix. If this method is used to prevent creep, the suction flow rate is kept to the minimum required to reduce evaporation during the time the TFA mix is required to be in contact with the resin. For evaporation after the required period, the flow rate can be increased to give rapid evaporation.
In essence therefore, according to the invention, the phenomenon of liquid creep up the wall of a vessel containing the liquid, is prevented by creating a continuous region on the inside surface of the vessel which cannot come into contact with the liquid vapour which exists above the surface of the liquid in the vessel.
In a fifth method measures are taken to ensure that the whole of the surface of the container is already coated in liquid TFA mix but of a mix not containing the solid material in solution in the sample. Because the surface above the TFA mix sample level is already coated in TFA mix there is no tendency for further TFA mix containing the sample to creep up the walls.
This situation can be created, for instance, by spraying pure TFA mix onto the surface of the container before or after the containers are filled with sample. If the sample needs to remain in contact for a considerable time before evaporation can take place it may be necessary to repeat the application of pure TFA mix so that the walls above the sample do not dry out and draw up the TFA mix sample.
In most procedures for solid phase synthesis the TFA mix is evaporated in a centrifugal evaporator after it has achieved the required cleavage of the compound from the resin. Once sufficient centrifugal force has been applied to the sample, the tendency of
Gakh Yelena G.
Genevac Limited
Lee Mann Smith McWilliams Sweeney & Ohlson
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