Principles and Advantages of Gel Filtration Chromatography
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Gel filtration chromatography (GF) (also known as molecular exclusion chromatography) relies on a stationary phase consisting of spherical gel particles whose size and porosity are carefully controlled during manufacture.
When the mobile phase is passed through a column of this material, molecules are fractionated on the basis of their sizes and shapes. Small molecules (shown in blue in the diagram on the left) are able to diffuse into the pores, while larger molecules (shown in red) are excluded. This occurs repeatedly as the mobile phase moves down the column, and as a result, during the elution process, the small molecules are retarded with respect to the larger molecules. This causes the smaller molecules to appear in the later fractions of the eluate.
Size is not the only factor that influences the rate at which solutes are retarded. For a given pore size, rod-like molecules will tend to be excluded with respect to spherical molecules of the same size. In addition, certain molecules may have some affinity for the stationary phase as a result of non-covalent interactions.
- Separation process is carried out under very mild conditions, from 37 ºC. to cold room conditions.
- High resolution can be achieved.
- A wide range of buffer systems may be used (buffer molecules and ions are generally excluded).
- Small molecules or ions that stabilize the biomolecules may be added to the buffer.
- Separation process is independent of ( ) ionic strength, so elution is carried out ( ) isocratically.
- Gel filtration can be used for desalting and/or buffer exchange.
- Gel filtration can be used to obtain an estimate of molecular size.
- A wide range of porous gels are available commercially.
A note on mass and volume:
The SI unit of mass is the KILOGRAM, kg, while the unit of length is the METER, m. It follows that the SI unit of volume is the cubic meter (m3). These units are far too large for practical work in molecular biology, so one use smaller compatible units: the GRAM, g, (1x10-3kg) and the cubic decimeter (dm3). For all intents and purposes, the LITRE, l is equivalent to 1 dm3, and that is the volume unit we will use.
You should be familiar with the use of SI prefixes:
In your laboratory work, you will become used to equipment capable of measuring masses with accuracies down to 1 μg and volumes as small as 1 μl.
The quantity of solute present in a solution is called the CONCENTRATION of the solute in that solution. A solution with an accurately known concentration of a particular solute is referred to as a STANDARD SOLUTION.
There are several ways in which concentrations may be expressed. Molecular biologists most frequently refer to the MOLARITY of a solution. This is the amount (number of moles) of solute present in 1dm3 (1000 cm3).
[NaCl] = 1.00 mol.dm-3 is a molar solution of sodium chloride. When "[ ]" are used to denote concentrations, the units of the concentrations are always mol.dm-3.
The symbol M is used to denote molarity. A 1.00 M NaCl is a solution of NaCl with concentration 1.00 mol.l -1. Since the relative formula mass (Mr) of sodium chloride is 58.5, one mole of NaCl expressed in grams in is 58.5 g, and a 1.00 M NaCl solution will contain 58.5 g of NaCl in 1.00 lof that solution.
Note that the following concentrations are all equal: 1 M = 1 mol.l-1 = 1 mmol.ml-1 = 1 μmol.μl-1.
Other ways of expressing concentrations are:
- Percentage by volume: 8% ethanol (8 volumes of ethanol in 100 volumes of solution). Usually written (v/v).
- Percentage by mass: 10% sucrose (10 g sucrose in 100 ml of solution). Usually written (w/v).
- Mass per volume: g.ml-1(mass of solute per unit volume of solvent).
- Mass per mass: g/100g (mass of solute per 100 g of solvent).
- Molality: Number of moles of solute per 1000 g of solvent.
Preparing Standard Solutions
Suppose that you need to prepare 500 ml of a 0.050 M solution of sodium chloride.
A 0.050 M solution will contain 0.050 moles of solute in 1.00 l of solution. Now, sodium chloride has a Mr = 58.44, so the solution will contain 58.44 x 0.050 = 2.922 g.l-1. Since we only want 500 ml (i.e. ½ l, we will only require 1.461 g of NaCl.
These calculations should be entered in your laboratory workbook.
Equipment and reagents required:
You will need:
- Analytical grade sodium chloride.
- Distilled water.
- A top-loading balance accurate to 0.01 g.
- Glassine paper.
- A spatula.
- A small beaker (50 ml).
- A 500 ml volumetric flask ( )
- A wash-bottle filled with distilled water.
Tip about 3 g of the sodium chloride into the small beaker. (As a rule, do not put spatulas into bottles of analytical grade reagents. This is important in order to maintain the purity of their contents. Close the bottle as soon as you have finished with it.
Place a square of glassine paper on the pan of the balance. Adjust the reading to 0.00 g. Using the spatula, gradually add small quantities of the sodium chloride from the beaker until a reading of 1.46 g is obtained. Switch off the balance. Make sure that no reagent has been spilled onto the pan. If you seen any reagent, clean the pan and start over.
Pour the contents of the glassine paper CAREFULLY into the volumetric flask. If any crystals stick to the paper, wash them in with the wash bottle.
Add about 200 ml distilled water to the flask, and swirl it gently until all crystals are dissolved.
Add distilled water carefully up to 2-3 cm below the mark on the flask. Make up to the final volume with the wash-bottle (Do not overfill!). Stopper the flask and turn it upside down a few times to thorougly mix the contents.
Pour the contents of the flask into a clean, dry blue-top reagent bottle.
Make sure that you label the bottle with its contents, your name, and date of preparation.
Rather than preparing small quantities of a standard solution every time you require it, it is better to prepare a relatively large volume of a concentrated stock solution, and dilute it as required. For example, suppose you have made up 2 l of a 3 M sodium carbonate solution, as described above. Then, if you need 100 ml of a 0.01 M Na2CO3 solution, you would need to dilute this solution 300 times:
Pipette 0.33 ml of your stock solution, using a Gilson pipette, into a 100 ml volumetric flask, and make up to the mark with distilled water. Use what you need, and discard the rest.
The use of Gilson pipettes is discussed in the next page.
Suppose you have a stock solution of a reagent, at 1.0 M. If you take 1.0 ml of that solution, and dilute it to 10.0 ml in a volumetric flask, you will have a new stock solution which will have 1/10th the concentration of the original solution. This process can be repeated, each time obtaining a solution which is 10 times more dilute than the previous one. Great care must be taken that the volumes are accurately dispensed, as errors increase exponentially as one makes several dilutions in this way.
Precision Volumetric Equipment
Variable volume precision pipettes:
The dispensing of small volumes of liquids with a high accuracy and repeatability is a routine operation in the bioscience laboratory. The equipment used for this purpose are costly precision instruments, which require careful handling. You will use the Gilson Pipetman® or the Labnet Biopette®, which allow one to deliver volumes down to 0.1µl. The information below is adapted from the Gilson Pipetman manual. The operation of the Labnet Biopette is very similar.
The Pipetman®, shown here on the right, is a volumetric instrument designed to measure and transfer liquids precisely and safely. Eight different models are available, which cover a volume range from 0.1 μl to 10 ml
A diagram of the outer features of the Pipetman is shown on the left. The labels A- F refer to:
A: The push button (which is labelled with the maximum volume for the instrument).
B: A knurled knob by means of which the volume
to be delivered may be adjusted.
The volume is read on a ( ) digital volumeter.
C: The shaft, which may be autoclaved at for 20 minutes at 120 °C and 1 bar pressure. Do not attempt to dismantle the Pipetman! Get a staff member to assist you!
Setting the volume:
The volumeter consists of three numbers, and is read from ( ) top to bottom. The three numbers indicate the number selected. Note that the sequence of number indicate different volumes for each model.
The volume of the pipette is set by turning the ( ) black knurled adjustment ring (marked B in the above diagram).
To obtain maximum accuracy when changing the volumeter setting, follow the recommendations below:
- When decreasing the volume setting, turn the adjustment ring slowly to reach the required setting, making sure not to overshoot the mark.
- When increasing the volume setting, turn the adjustment ring until you are about1/3 turn above the required setting. Turn the adjustment ring slowly to decrease the volume to reach the desired setting, making sure not to overshoot the mark.
Place a suitable tip on the shaft of the pipette. Press the tip on firmly using a slight twisting motion to ensure a positive, airtight seal. Never handle a liquid with a Pipetman that has not been fitted with a tip.
- Press the push button to the first positive stop (diagram A above).
- Holding the
pipette vertically, immerse the tip into the sample liquid. The
depth to which the tip should be immersed in the sample liquid
depends on the model
P2 and P10: £ mm
P20 and P100: 2-3 mm
P200 and P1000: 3-6 mm
P5000: 3-6 mm P10ml: 5-7 mm
- Release the pushbutton slowly and smoothly to aspirate the sample (diagram B above).
- Wait one second and then withdraw the tip from the liquid. Wipe any droplets away from the outside of the tip using a tissue. Avoid touching the orifice of the tip..
- Place the end of the tip against the inside wall of the vessel at an angle of 10 to 40 degrees. Press the pushbutton smoothly to the first stop (diagram C above). Wait one second. Press the pushbutton to the second stop to expel any remaining liquid (diagram D above).
- Keeping the pushbutton pressed to the end, remove the pipette by drawing the tip along the inside surface of the vessel.
- Release the pushbutton (diagram E above).
- Eject the tip by pressing the ( ) ejector button. It is only necessary to change the tip if a different liquid is being sampled or if the volumeter setting is changed.
When pipetting liquids that have a viscosity and density different to water, for example, organic solvents, a film of liquid is formed on the inside wall of the pipette tip. The film can create an error. Since the film remains relatively constant in successive pipetting operations with the same tip, this error can be avoided by forming the film before dispensing the first sample. This is done by aspirating a sample and dispensing it into the same vessel. Since the film is already formed, all the following samples will have better accuracy and repeatability.