Making a full recovery

The glass vial is often overlooked as part of the analytical pathway, says Brian King, technical manger at Chromacol, but its physical shape and surface properties may have important influences on the data quality or the efficiency of sample recovery

When dealing with the problems of carrying out analysis in HPLC and GC the last consideration may be the amount of sample available to carry out the assay. Often the sample presented in a QA or QC environment may be available in large quantities and the problem may be of obtaining a representative sample from the amount presented. This position has changed in two major areas.

In drug discovery the developments in synthesis have increased the numbers of samples presented for analysis by orders of magnitude. At the same time the amount synthesised may be in micrograms rather than milligrams. The sample is thus valuable in its own right and the quantities used for the assay should not deplete the small quantities of compound that may then be required for further structural or activity work.

best use

In order to carry out purity analysis or determine concentration the best use of the presented sample may be made by limiting the total amount used in the analytical phase without carrying out excessive dilutions that may reduce the overall sensitivity of the methods.

Routinely HPLC auto-samplers have been used to introduce volumes of between 20 and 100ml of sample. For routine analysis the performance of the auto-sampler may need validation in terms of repeatability of injection and this is normally carried out with the system injection volumes. When the move is towards lower volume injection the concern is that the injection is introducing the actual volume required and set, introducing this with a minimum of carryover and that there are no problems with sample loss with multiple injections from the same sample vial.

With some recently introduced systems for capillary HPLC the liquid injection volumes are now capable of nanolitre injection volumes with the requirement of extracting these from a few microlitres of sample.

The smallest individual vials for this purpose are Chromacol Sci-VI products that have total extraction volumes of less than 300ml. The smallest volume of 100ml is found with a controlled bore product with a syringe guide into the sample chamber.

When presented with the sample it is important to consider both the security and integrity of the sample during the injection phase. It is now possible to have a variety of closures available for the sealing of vials during this phase of analysis.

effect of evaporation

The first concern when performing an analysis may be that of sample evaporation. With volatile solvents this can have a very rapid effect on the concentration of a sample in an open vial or plate. For maximum sample integrity some evidence of tampering may also be required.

The conventional aluminium crimp cap with sealing septa is the method of choice for the closure of vials with maximum security. Removal of sample can occur only through piecing of the septa, while removal of the crimped unit is possible only using tools such as decrimping pliers or decappers. Correct crimping of the vials with such closures also gives a high level of sample containment with the possibility of storage with minimal sample loss. Correct crimping of such vials and the selection of the correct septa is also critical.

Over-crimping of the vial can lead to the septa being compressed with needle damage or coring occurring. This leads to septa tears that will release sample. Under-crimping does not give a firm enough seal with possible vapour loss around the loose septa within the cap.

The choice of septa will be dependent on both the sample composition and the instrumentation in use. For routine usage rubber septa coated with a Teflon film are commonly used. Where trace analysis is used and where multiple injections are required the use of silicone septa is recommended. The silicone elastomer is chemically cleaner and has better re-sealing properties. For instruments with delicate piercing needles the use of a pre-slit septa gives easier penetration while still retaining some sealing capability. Conventional vials often have crimp diameters of 11mm, while the smaller volume vials use 8mm crimp caps.

Where the permanence of crimping is not required, screw top vial gives the advantages of easily removable closures while using similar sealing septa. There are more variations in screw caps, with 8mm, 9mm and 10mm cap and vial combinations from most manufacturers. For low volume work there are fewer dedicated vials with screw closures, but glass inserts can be used with screw vials in a similar manner to crimp vials but with similar inconvenience. While less secure, the use of screw top vials allows easier work up of samples with no special tools.

It is also possible to automate some of the closure and opening of such vials. The type of cap will also be controlled by the method of transport used by the auto sampler. Screw top vials may not be able to apply the level of force found with a crimp cap and are thus not recommended for long term storage, but for routine analysis of small samples on a day to day basis they may have greater convenience.

96-well plates

Individual vials require individual closures. With smaller samples the use of 96-well microtitre plates allows a great density of samples to be processed from a single standard plate. For micro sampling applications the flexibility of the 96-well format may be combined with the inertness and solvent compatibility of glass micro vials. This is compatible with strong organic solvents such as acetone and acetonitrile while being inert towards acid and base residues that may have been formed.

Closures using an elastomer mat with Teflon facing give sealing properties similar to silicone septa. The narrow cross-section of the glass vials constricts the plugs to give an inert chamber with excellent solvent resistance and sample integrity. Where DMSO is used as a storage solvent, this technology has been further developed to give a film that resists the permeating vapours of this particular solvent.

Maximum recovery

With the degree of automation demanded by sample handling, the demands are in place to concentrate sample within the final injection or storage vessel. This requires that there is homogeneous concentration into the base of the vessel used for the concentration step to ensure correct sampling performance.

This concentration can be carried out in conventional flat bottom vials, either analytical or storage, but the resulting concentrate may not be extractable using conventional syringe needles or pipettes. For flat-based vials the sample may collect at the edge of the base and not be recoverable. This may lead to hundreds of microlitres of sample either being wasted or being unobtainable without re-dilution.

This problem may be overcome by the use of vials with a conical taper or a high recovery base. The vials with a conical taper may be used as conventional vials except that they require the use of a support. The high recovery vials are manufactured with a base that gives an internal reservoir while maintaining the external base profile as a self-supporting vial. Each vial's maximum recovery is dependent on the type of extraction or sampling needles used in the autosamplers routinely used in the laboratory. For different needle types and settings comparison residual volumes may be compared for different vial base profiles.

For larger volume storage the base of the vial may be critical when recovering sample for further investigation. With the development of automated sample processors the limiting factor is again the collection reservoir. This volume may be reduced by using rounded tube bases, but like test-tubes suitable racks must be used for filling and processing. The use of rounded profiles may improve recovery but reduce convenience. Again high recovery profile tubes have been produced for volumes of between 3cm³ and 6cm³.

reaction with glass

Many pharmaceutical research compounds contain chemical groups that are basic and will potentially bind to any acidic sites present on the surface of glass storage and analysis vials. For this reason the manufacture of most critical vials is from neutral borosilicate glass, which conforms to the USP Class 1 properties for surface activity and hydrolytic resistance.

However, in some cases even this is still not capable of preventing reaction with specific key basic groups. To address this problem Chromacol offers vials made from Chromacol Gold grade glass, a special range made from Type 33 glass with a reduced content of alkaline materials. These alkaline materials are known to produce active sites on the surface of the vials.

Chromacol Gold glass vials are produced under stringent conditions to maintain the low content of the alkaline components believed to be the source of the adsorption. Since it is known that if too high a temperature is used during the production stage then these components migrate from the centre of the glass to the outer surfaces.

All Chromacol's SCI-VI system vials are made from Chromacol Gold glass as these small vials are often used with samples that react adversely to active glass sites. Table 1 lists the alkaline material content of various glass types.

In some rare cases the use of deactivated glass is still not able to give the required assurance of analytical recovery. In these cases the application of a homogeneous hydrophobic layer onto the glass may be considered. This process may be carried out by a number of proprietary methods. The simplest involves the application of a silicone material that is physically absorbed onto the glass and forms a hydrophobic barrier, masking the still existing acidic surface on the glass. Strong solvents may affect this layer and it may also have an effect on any hydrophobic compounds in the sample solution.

reactive silanes

A more complete method of chemical deactivation is the reaction of reactive silanes with the glass. This silanisation gives a permanent bonding on the glass that chemically modifies the acidic groups capable of reacting with basic samples. The reaction converts any acidic silanols to inactive boned siloxanes. This introduces a small hydro-phobic group that is permanently attached, unaffected by strong solvents and gives permanent deactivation. The process may be carried out under liquid or gas-phase reactions, which give a similar degree of deactivation and permanence. Silanised vials may be used where sample recovery of basic compounds is absolutely critical.

All vials are produced in conditions that will minimise the possibility of contamination with organic materials. After production there is immediate transfer into packaging that as far as possible is low in extractable organics and plasticiser components. When used for ultra-trace determination of environmental pollutants it is, however, necessary to carry out further cleaning. This may be carried out following EPA protocols and can be supported by certification. Where this is necessary it is usual for vials to be supplied pre-assembled. In the field of water analysis vials of 20cm³ and 40cm³ capacity are routinely supplied in such cleaned forms.

The glass vial is often overlooked in laboratory analyses but its shape and surface properties are an important influence in achieving the quality of experimental information or the efficiency of sample recovery expected.