Equipment required for low pressure liquid chromatography

1. The column

A sine qua non for column chromatography  is the  column.  Basically this consists of a glass tube with adapters -  preferably at either end  - to spread the liquid flow from the thin bore input tubing out to the relatively large bore of the column and back in to the thin bore of the output tubing (Fig. 1).  The column packing is supported on a sieve of some sort and a key element in the efficiency of the column is the “dead” volume between this sieve and the  output tubing, which should be as  small as possible.  The purpose of the column is to effect a separation of different types of solute molecules and the  whole purpose  is  defeated  if  the separated molecules arc allowed to remix, due to the dead volume  being too large.

Figure 1.  Schematic cross-section of a chromatography column.

Moveable flow-adapters enable different column bed volumes to be used.  It  is  useful  to  have  such flow adapters on both  ends  of the column.  After packing and equilibration of the  column  bed, the adapter on the  inlet side can be adjusted to  be in  contact  with the upper surface of the  packed resin bed.  This  is advantageous in that  it facilitates  sample application, as the sample can be simply introduced through  the inlet tubing.  It  is also necessary if any  form  of gradient  elution  is to be used.  In  the  absence  of  an upper flow adapter, the incoming buffer will mix  in  the  dead volume above the  packed resin bed and it will be impossible  to get smooth,  reproducible, gradient conditions.

The ratio between the internal diameter (i.d.) of the column and its length, the  so-called “aspect ratio”, differs depending upon  the  application of the  column.  Generally,  where adsorption occurs, such as in ion-exchange or affinity  chromatography, columns with an aspect ratio of ca1:10 or  less are used, whereas for molecular exclusion chromatography aspect ratios of about 1:50 are used.

Low pressure  liquid chromatography columns typically consist of a uniform  bore, thick walled, glass tube, with  plastic adapters etc.  Glass is favoured because it is chemically  stable, transparent, and has good thermal conductivity.  Obviously, all materials  used to  make  columns must be chemically stable  to  buffers  etc.  and  must  not  react  with  sample proteins. Proteins do adsorb to glass to some extent and this can be prevented by salinizing the  column before use, though this is only warranted for the most critical work.

2. Moving the mobile phase

The chromatographic process requires movement of the mobile phase and the simplest way of effecting this is by siphoning the buffer from a reservoir which is elevated above the end of the  outlet tube from  the column (Fig. 2).

Figure 2.  Simple chromatography, using a siphon to generate the mobile-phase flow.

The difference in potential energy (height) between the surface  of the liquid in the buffer reservoir and the end of the outlet tube  constitutes  the “pressure head”  which will cause the  mobile phase  to  flow.  A  problem with this simple set-up is that, as the level  of liquid in the reservoir drops, the pressure head will get smaller and the flow rate  will decline.  To  keep the pressure head constant a so-called Marriott  flask may be used  (Fig. 3).

Figure 3.  Use of a Mariette flask to maintain a constant pressure head.

Low-pressure column chromatography  occurs at a somewhat leisurely pace and it is too tedious to attend to the  column the whole time  it is running. On the other hand, using the simple set-ups shown in  Figs 2 and 3, there  is a danger that  an unattended  column might exhaust the buffer supply  and  run  dry.  If  this  happens  it  becomes  necessary  to remove the resin and repack the  column, which is tedious.  Gravity-flow columns can be protected  against running dry by arranging the  inlet tubing to loop down below the outlet from the outlet tubing (Fig. 4).

Figure 4.  A run-dry protection loop on a gravity-flow column.

Note that gravity-flow columns can be operated with the  flow going either downwards (Figs 56----58)  or upwards through  the  column  (Fig  5). An advantage of upward flow is that  it  is easier to  arrange  the  system  so that it will not run dry.  Upwards flow is recommended with very soft gels, such as Sephadex G-200, which otherwise tend to  be crushed by the combined effects  of gravity  and the  buffer flow.  With  ascending flow, the flow supports some of the weight of the gel.

Figure 5. Ascending flow, with run-dry protection, and a tap for sample application.

With a gravity  flow system  the  sample  is most  easily applied using a three-way tap on the  inlet side of the column, as  shown in  Fig.  5. Better than  a Marriotte  flask, if the  budget allows, is a peristaltic pump. “Peristalsis” refers  to  the  rhythmic,  wave-like, contractions  that pump the gut contents along the digestive tract.  By analogy, a peristaltic pump is one in which a flexible  silicone  tube  is pinched  by a roller which runs along the tube, pumping the tube contents in the same direction  as it does so (Fig. 6).  A  peristaltic  pump  gives  a  smooth,  almost  pulse-free, flow.

The advantage in using a pump is that  it gives more  precise flow control and greater freedom in the chromatography lay-out, i.e. the buffer reservoir  does not  have  to  be higher than  the  column  outlet.  To prevent the column running dry during unattended operation when using a peristaltic pump, a timer switch is required.  The timer can be  arranged to switch off the pump - and any other associated apparatus - after a pre-set time. Note that because the peristaltic pump rollers pinch the silicone tube, there can be no flow of liquid through the  pump  when  it  is switched off.

Figure 6.  Schematic drawing of a peristaltic pump.

When using a peristaltic pump, the sample can be applied simply by stopping the pump, transferring the inlet tubing from the  buffer reservoir to the sample container,  restarting the  pump until all of the  sample is sucked up, wiping the tubing and returning it to the buffer reservoir.

3 .Monitoring the effluent and  collecting fractions.

The purpose of column chromatography is to  separate solute molecules and it follows that  some means is required of monitoring  the separation achieved, and of separately collecting the  resolved solute molecules. The separated fractions are most easily collected using an automatic fraction collector.  Fraction collectors are available in different versions that collect the effluent stream in fractions  on  the basis of time,  volume,  or  number of drops.  Time-based fraction collectors are the simplest and most economical and, if the mobile phase flow rate is accurately controlled with a pump, the fractions collected will be of equal volume.

The simplest means of monitoring the separation of proteins is to collect the  column  effluent for the  whole run  in  a convenient  number of fractions - say, 100  - and to  measure the A₂₈₀ of each  fraction  in  a spectrophotometer. The results can be used to construct a so-called elution profile,  in which A₂₈₀ is plotted against the elution volume. Such manual  reading  of the  elution  profile  is  inexpensive  in  capital terms, but it consumes operator time and, perhaps more importantly, some detailed information  is lost.  A  better  way, again  if the  budget can afford it, is to use a flow-through UV-monitor, that continuously reads the absorbance of the effluent stream at 280 nm.  Such a monitor is plumbed into  the  effluent  line,  between  the  column  and the  fraction collector. Besides a power source, it requires two other electrical connections;  an output to  a recorder and an event-marker connection between the  fraction  collector  and the  recorder.  Each  time  the  faction collector changes tubes, it sends a pulse to the  recorder  so  that  the  event - the tube change  - is recorded.  In  this  way it  becomes possible to subsequently correlate  the  recorder trace  of A₂₈₀ with the  collected fractions, so that  fractions corresponding to the  required peaks can be harvested. A flow-through UV-monitor constitutes  a time-saving automatic system which also captures the fine detail of the elution profile.

Figure 7. A typical elution profile of A₂₈₀ vs elution volume with event marker pulses.

4. Refrigeration

Proteins are structurally labile and are susceptible to microbial degradation. For these reasons, wherever possible, protein solutions are maintained at low temperature and preservatives are added to  the  buffers. Denaturation and degradation are both minimized by keeping the  proteins cold and protein  separations  are therefore  usually carried out at  about 4C.

Chromatography is usually done in coldrooms, but working in coldrooms is miserable and unhealthy. A better and more  versatile system is to have  a refrigerated cabinet with some components  of  the chromatography set-up being kept at 4C  and others  at room temperature (Fig. 8).  In  Fig.  8,  items  labelled  on the  left are within the cabinet at 4C  and those  labelled on the  right are at  room  temperature.

Electrical apparatus kept in a coldroom  or fridge can be damaged by condensation of moisture onto  its circuits.  For  this  reason  it  is  best  to keep  as much as possible at  room  temperature.  The  fraction  collector must, however, be kept in the fridge.

Figure 8. A protein chromatography system in a glass-fronted, refrigerated cabinet.

Remember if any electrical  item  is ever  removed  from  the  fridge for servicing, it must be allowed to  warm up to  room  temperature  and all moisture must be allowed to dry off before it is switched on.  If this is not done, short-circuits caused by condensed moisture may burn out the electronics.

References

-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .