Practical Guide to HPLC

Introduction

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Our chromatography systems represent very valuable resources. If you’ve ever collected fraction with the old machine systems and tried adjusting gravity driven flow rates, you’ll appreciate the speed and ease with which purifications can be achieved on our systems.

This, however, comes at a cost, both in monetary terms and in levels of complexity. These details cannot be overlooked. It is essential that you become completely familiar with the separation techniques being used and the intricacies of the systems. The cost of most of the components run thousands of rands for the columns and tens-of-thousands of rands for the equipment. It is also equally important to bear in mind the fact that all system components originate overseas. We cannot afford to buy back-up columns and equipment and therefore any damage could result in lengthy delays to everyone’s research.

This document aims to cover the essential knowledge that users will require to operate the systems. It is by no means comprehensive and is no substitute for reading the actual manuals, which is highly recommended.

We can continuously update this document for our own reference and for future users.

Tubing

Tubing is specified according to its outer and inner diameter (OD and ID respectively). The OD determines what connectors can be used to join length of tubing. The ID determines the volume carried by the tubing and the backpressure.

The tubing used on our systems comes in two different sizes:

• 1/8″ OD tubing, 0.062″ ID
• 1/16″ OD tubing, 0.03″ ID

Connectors

Low pressure connections are formed between flat-bottomed receiving ports and threaded screws (nuts) that hold the tubing in place. The actual seal is created by a sealing cone or flange. The slope of the sealing cone faces the nut which has a cone shaped recess in its threaded end. The interface between these differently sloped surfaces forms the sealing surface. When the nut is tightened the flat-bottomed port exerted pressure on the ferrule and completes the seal.

Be aware that nuts and flanges are available for both 1/8″ and 1/16″ tubing. These are not interchangeable.

On some of our tubing from Gilson you will see flanges that have been created by heating and flanging the tubing. There are also metal washers between the flanges and the nuts in these cases. In this case the sealing surface is created between the flat flange and the flat-bottomed port. The washer ensures that the nut can be tightened without damaging the flange. The disadvantage of this system is that you need to reflange the tubing whenever the tubing configuration is altered.

We use a number of different connectors. The two most prevalent connectors we use follow the metric imperial divide:

¼-28″ – Imperial: The designation indicates that the threaded portion of the nut has a width equal to ¼ of an inch and has 28 threads per inch. These connectors are available for 1/16″ and 1/8″ tubing.

M6 – Metric: All the Amersham (GE healthcare) columns use these connectors. This designation tells you the nut has a thread diameter of 6mm and a pitch of 1 thread per mm. These connectors are generally black in colour.

Below the ¼” diameter the nut designation changes to grades. For instance the high pressure fitting 3/16″ is called a 10-32 nut. These longer nuts have a shorter thread pitch and are used for higher pressure applications. They are often needed to interface with Amersham adaptors.

You will soon learn to recognize the different connectors. If you’re not sure measure them with a ruler or compare them to the chart (print yourself a copy) in the fittings primer.

Materials

Don’t worry about the colour of the connectors and unions. They simply represent different materials with associated different tolerances to chemicals and pressures. All the materials we use are interchangeable and can withstand the aqueous solvents we employ (including 20% EtOH and 100% MeOH). If you intend using a different solvent please check the chemical resistance of the materials to this solvent in the Upchurch catalog or speak to me.

Making a connection

• First ensure that a previous user’s column has been stoppered correctly to prevent drying out before you disconnect it. Use the white Teflon plugs together with a standard ETFE union (¼-28). The black stopping caps are for metric threads (Amersham column connectors) and cannot be used.
• Identify the connectors. Is it ¼-28 or 10-32? If adding a new length of tubing follow the instructions supplied with the connector (these can be found in the fittings box in the EMU – next to the oven, on the shelf). In brief:
  • Slide the nut onto the tubing end with the thread facing the port to be connected.
  • Place a ferrule on the end of the tubing (with the cone facing the nut) ensuring that the tubing protrudes from the ferrule’s flat surface.
  • Screw the connector assembly into the port or union and tighten finger tight. Do not cross-thread the connectors. The nut should turn without effort – do not force it – if you have to use excessive force it’s cross-threading.
• If you’re connecting a column:
  • First remove the top stopper from the column-connector-union-stopper assembly.
  • Allow some buffer to fill the union (hold this upright obviously) i.e. connect drop-to-drop. Don’t allow air to enter.
  • Stop the pump.
  • Connect the system tubing by screwing in the second connector into the union.
  • Remove the stopper from the bottom assembly (don’t worry about air here – it will be flushed out).
  • Connect the bottom assembly to the spec.
• Disconnecting a column is the reverse procedure:
  • Stopper the bottom.
  • Disconnect the top.
  • Place some buffer in the union.
  • Stopper the top.

Standard operating procedures

Log sheets are placed at each system to record usage statistics as well as provide accountability. The last block also serves to remind users of what needs to be done after each run. If the log sheet is full collect a new one from the EMU or print one from this document (see last page).

The booking sheets for each system have been moved from the central EMU station to the separate systems. The booking system is meant as a guide for planning experiments and not as an absolute cutoff. We should all understand that unexpected events can occur with these systems. Communication is the key. If you can tell your run is going to take longer than expected tell the next user inline so they can adjust accordingly. Ensure that they get the message – don’t email-and-forget. Equally, expect the next user to try usher you off the system if you did not plan your run times correctly (if you’re unsure discuss – we all have to share this system).

Know your column

Each matrix has different separation (obviously), wash, and storage conditions. It is absolutely essential that you read the technical notes for the matrix you are using. These are available from the Amersham web site or in the common directory Download\Chromatography. These provide important information on flow-rates, chemical stabilities, wash conditions, regeneration conditions, and storage instructions.

Every column is different — even Q sepharose and Q sepharose XL have different instructions.

Buffers and samples

Filter

You must use the in-bottle solvent filter especially if your buffer tends to form a precipitate. This filter requires the entire filter assembly to be submerged. Ensure that you have enough buffer at the end of the run to keep the filter submerged. If you are changing buffers during a run, air might get into the lines from the filter. Try to expose the filter to air for as short a time as possible and reverse the flow to remove any air if necessary. Also ensure that the tube is firmly in place – use tape if necessary. High flow rates will cause negative pressures in the filter due to the small pore size and this will result in outgasing of your solution. If this is occurring then you must degas your buffers prior to a run.

All buffers should be autoclaved to prevent contamination of the columns. All samples to be loaded should be filtered with a ≤ 0.45 μm syringe filter or centrifuged at high rpm (10 or 15 K for 10 min) to remove particulate material (transfer supernatant to another tube). For small volumes, 0.45 μm spin columns should be used.

Sample loops

Because the sample volume loaded onto a gel-filtration column is limited to 1-5% it is important to use the correct sample application loop (10 ml, 5 ml, and a 1.6ml loop are available). The most reproducible runs will be produced by completely filling the loops, however, this will result in sample loss. In our case, we are more likely to use partial filling. In the case of the latter it is important to know that the loops can only be filled to half their capacity before sample loss will occur. This is the result of the fact that the fluid in the centre of the tube travels twice as fast as the boundary layer near the tube walls. Hence, if you only want to load 200 μl use the 1.6 ml loop.

Injectors: These inject the sample into the column, but the sample is loaded into the injector with the syringe. The load position excludes the loop from the buffer flow. Be very sure of the injector position when loading.

Pay attention to the connections in the above diagram. Ensure that the connections to the pump and column are made to the correct ports. With suction loading and different loops the injectors are frequently disassembled and reattached.

Loading without a loop – When loading without a loop i.e. very large volume for concentrating matrices like affinity or IEX, wash the tube (add some buffer to the application tube and swirl the tube) before placing it back in you buffer. If you don’t you will contaminate you buffer and reduce its shelf-life. You will not likely be using the solvent filter in this instance so make every effort to ensure that your sample is free of particulate material.

Degassing

It is advisable to degas you buffers before (just before because they reabsorb gas) performing a chromatography run (all buffers run on the FPLC system MUST be degassed thoroughly). You can use the vacuum flasks and the pump in the EMU to achieve this. Also remember any runs performed in the cold room require buffers to be equilibrated to 4°C before the run begins. The temperature change causes dissolution of the dissolved gasses in the buffer and results in outgassing. For the same reason, a column cannot be removed from the cold room and run at room temperature without slowly bringing it up to temperature. Similarly, when washing with high organic solvent concentrations you should introduce the solvent with a gradient to avoid bubbles i.e. to get to 70% EtOH use a gradient from 0 to 70%. Also, you must always degas 20% EtOH.

Always check the flow rate

Flow rates can change with peristaltic tubing changes or even during it’s lifetime of operation. Do not exceed the maximum recommended flow rate (equivalenty the max flow rate used during packing) of your column, otherwise bed compaction and bead damage may occur. Flow rates are usually given in linear flow units i.e. cm/hr. The reason for this is that compaction and damage to the bed occurs in a vertical direction as a result of the flow through the column. Thus, it’s the vertical or linear flow that is important. A very wide column can be run at high volumetric flow rates but still not exceed its linear flow because it has such a large cross-sectional area. For example, the volume occupied by a 1 cm high region of a 5 cm diameter column = 19.6 ml. Compared to a 1cm high region of a 2.5 cm diameter column = 4.9 ml. Thus, if both columns are run at 1 cm/hr then their volumetric flow rates will differ by a factor of 4.

Always measure the ID diameter of columns. Do not include the water-jacket on the XK series of columns. The standard Amersham columns that we use have ID’s of 1.6, 2.5, and 5 cm.

Linear flow rate can be converted to volumetric flow rate as in the following example:

You will need to determine the actual flow rate using the measuring cylinder and a stopwatch. Remember that the longer the time period, the more accurate the flow rate reading will be. Do this every time you run your column!

Gel filtration: – lower-flow rates and smaller sample loadings give the highest resolution. Only load 1-5 % of the column volume (at around 40 mg/ml).

ALWAYS ensure that you have enough buffer for the required run. If you are uncertain rather make more buffer. Then you can rest peacefully knowing that even if something goes wrong and the computer reboots leaving the pump running, the column won’t dry out. It’s a lot easier to make more buffer than to repack a column (if at all possible).

UniPoint

Use the Unipoint system to wash or equilibrate your columns. By automatically stopping the pump there can be no mistakes with introducing air into the columns. Having said this, it is essential that the control method you intend to use be checked before each run. Things to check or change each run:

• The run times – does it correspond to your flow rate and the volume you intend to use.
• Pump speed and direction.
• Time per tube.
• Double check that there is a stop pump command, stop collection, home collection head, and turn off lamp (if over a weekend) command at the end of the run.
• When adding a new event do not forget that timed events sent to the same unit should be separated by 0.1 minutes.

To be sure, it is best to test the method. This can be done by reducing the run time to say 5 min i.e. change all the events that occur at the end of the run to occur at 5 min (again separated by 0.1 min delays). You will then be able to determine whether the pump reaches the correct speed, the fraction collector follows the correct pattern, and most importantly whether everything stops correctly.

Control methods

Open an existing control method or create a new one. Control files are *.gct

Check what instructions are included – add any omission and delete unnecessary instructions.

Check the times and parameters. Note – If inserting a new item you must click “insert” before “done”. Similarly if you are editing an entry you must click “change” then “done”. Save the control method – Do not overwrite someone else’s method. Long filenames are not permitted.

If you are starting from scratch you will need to define the data channels. This can be done from the device menu by selecting the data channels item. Select the first channel and give it a name. If you wish to measure both sensitivities at once (cover your bases) then define the second channel as well (give it a different colour using the options tab). The third channel can be left undefined because it measures the transmittance.

Operations:

Open an existing operation or create a new one. Operation files are *.gop.

Double-click the first block and change the control method input to the one you intend using.

Click “insert” and then “done”

Alternatively, if you created a new method, click “step” and fill in the required fields. For analysis method use “ucttrain”.

Results

To export you results open them using the “results” tab in the navigator.

Click export.

Choose channel data.

Choose a destination for the file and give it a name.

Type in 1200 for the export interval.

Click “export”.

You cannot export fraction information. Check your assignment of fractions based on the times given in the UniPoint trace window , i.e. choose a few fraction start sites and move the target cursor over the green lines – write down the times at which the different fractions started and adjust the trace from the channel export to reflect this. Remember the channel starts recording before the fraction collector starts.

Air in the columns

At present a small amount of air in the column lines and hence columns is unavoidable. For gel-filtration this, however, can lead to a degradation of separation quality. To remove small bubbles that entered the column during connection changes, reverse the flow of the column (slowly or you’ll remove matrix as well as air) by connecting the injector outflow to the bottom of the column and venting the top connection. Once the air bubbles have been expelled stopper the top and try again or quickly change injector back and connect the column inflow.

Whenever reconnecting the injector loop or if the system has been standing without buffer for a long time or if you are unsure what is in the loop, purge the injector and tubing. Make sure the injector is in the “inject” position. You can use the “rabbit” key on the pump for rapid priming but first choose a direction by pressing the normal run button and then the “rabbit” key.

Wash your column

If you’ve used an IEX column you need to wash the column to ensure no bound material remains in the matrix. This is called regeneration. For IEX wash with 5 column volumes of elution buffer (high salt or pH) or until the baseline returns to zero. Equilibration is then performed before the next separation — 5 column volumes of loading (IEX) or running buffer (GF).

For gel filtration columns it is recommended that the columns be washed using the Cleaning In Place protocol after every 5 runs or when peak resolution deteriorate. This will not be done for you. If you are unsure of the column’s state then it is advisable to wash it. Every time this is performed it needs to be noted in the booking sheet.

Note: extra care must be taken when reverse-flowing a gel-filtration column. If this is too vigorous the bed can become unpacked and lose separation capacity i.e. lower resolution runs.

Washing conditions are usually harsh and it is therefore important that the columns are not exposed to the cleaning agents for too long. The instruction manuals for each column will give a cleaning protocol with the allowable contact times, i.e. the permissible time that the matrix can be exposed to the cleaning solution. Following cleaning the column cannot be left in cleaning agent and needs to flushed with buffer in preparation for the next run or in storage buffer.

Store your column

Columns that are not used for more than a few days (especially at room temperature) need to be stored in such a way as to prevent biological growth. Remember, agarose and dextran (the two principal components of some of the matrices we use) can support microbial growth. It is therefore essential to store your column appropriately. Most can be stored in 20% EtOH, while others are better off in a bacteriostatic sodium azide solution

A distinction needs to be made between sanitizing, sterilizing and storage. If contamination due to microbial growth is suspected (discolouration, introduction of new proteins into a purification etc) then sanitization should be attempted. The first approach is usually 0.5M NaOH in a reverse flow direction. The only way to sterilize a column is to disassemble it and autoclave the matrix or leave the matrix in contact with 70 % EtOH in some instances. Which approach is adopted will depend on the matrix – again read the info for detailed instructions.

Cleanliness

As general good practice you should always wear gloves when handling proteins to avoid the introduction of contamination and proteases. Therefore, whenever connecting a column or placing the inlet tube into you buffer you should be wearing gloves. Wash these frequently with 70% EtOH.

Clean up any spills immediately and wipe the area with water or 70% EtOH. This not only makes the work environment more bearable but helps in troubleshooting. It is much easier to trace leaks back to their source if there is no evidence of past spills to confuse matters. If you find a mess tell whoever is responsible (hence the log) to clean it up.

After each run

Flush the loop and cuvette with 20 % EtOH or even better in case of blockage with 100% MeOH followed by distilled water. Try to avoid disassembling the cuvette. The parts are delicate and easily scratched. Try more forceful washing using the syringe with a yellow tip on the end. Rigorous cleaning protocols are given in the Gilson manual for the spec. The tube entering and leaving the cuvette has a very narrow ID. This is to minimize the volume between the detection cell and the collection arm, thus, preventing lag between signal and collection. If users find this tube is blocking frequently it can be changed for a larger ID one. Report any blockages so we can monitor this.

Pool your fractions and as soon as you can, wash the empty tubes

Write in the log sheet so that other users can track the state of readiness and condition of the columns.

Report any suspicious chromatographic behaviour. Most often tell-tale signs will appear that something is about to fail, for example, reduction in flow-rate or a puddle on the bench.