Fonte: Laboratorio del Dr.B. Jill Venton – Università della Virginia
La cromatografia a scambio ionico è un tipo di cromatografia che separa gli analiti in base alla carica. Viene utilizzata una colonna che viene riempita con una fase stazionaria carica su un supporto solido, chiamata resina a scambio ionico. La cromatografia a forte scambio cationico separa preferenzialmente i cationi utilizzando una resina caricata negativamente, mentre la cromatografia a scambio anionico forte seleziona preferenzialmente gli anioni utilizzando una resina caricata positivamente. Questo tipo di cromatografia è popolare per la preparazione del campione, ad esempio nella pulizia di proteine o campioni di acido nucleico.
La cromatografia a scambio ionico è un processo in due fasi. Nel primo passaggio, l’esempio viene caricato nella colonna in un buffer di caricamento. Il legame del campione caricato alla resina della colonna si basa sulle interazioni ioniche della resina per attirare il campione della carica opposta. Pertanto, i campioni carichi di polarità opposta alla resina sono fortemente legati. Altre molecole che non sono cariche o sono di carica opposta non sono legate e vengono lavate attraverso la colonna. Il secondo passo è quello di eluire l’analita che è legato alla resina. Questo si ottiene con un gradiente salino, in cui la quantità di sale nel buffer viene lentamente aumentata. Le frazioni vengono raccolte alla fine della colonna quando si verifica l’eluizione e il campione purificato di interesse può essere recuperato in una di queste frazioni. Un’altra tecnica, come la spettroscopia, potrebbe essere necessaria per identificare quale frazione contiene il campione. La cromatografia a scambio ionico è particolarmente utile negli studi sulle proteine, per isolare proteine di interesse che hanno una carica o una dimensione specifica, in quanto le dimensioni possono determinare il numero di interazioni con la resina.
La cromatografia a scambio ionico è una tecnica di separazione più generale rispetto alla cromatografia di affinità, che viene spesso utilizzata anche nella preparazione di campioni proteici, in cui un anticorpo è attaccato a una colonna per legare un analita specifico. Per ogni analita deve essere acquistata una nuova colonna di affinità, mentre lo stesso tipo di colonna a scambio ionico, spesso con condizioni di eluizione diverse, può essere utilizzato per ripulire molte proteine della stessa carica. La cromatografia a scambio ionico può anche essere utilizzata in combinazione con altri tipi di cromatografia che si separano in base ad altre proprietà. Ad esempio, la cromatografia ad esclusione dimensionale si separa in base alle dimensioni e potrebbe essere utilizzata prima della cromatografia a scambio ionico per scegliere composti di una determinata dimensione.
Ion-exchange chromatography is widely used in biochemistry to isolate and purify protein samples. Proteins have many amino acids with functional groups that are charged. Proteins are separated based on net charge, which is dependent on pH. Some proteins are more positively charged while others are more negatively charged. In addition, peptide tags can be genetically added to a protein to give it an isoelectric point that is not in the range of normal proteins, making it possible to separate completely. Ion-exchange chromatography is useful for separating multimeric protein complexes, as different configurations would have different amounts of charge and different interactions.
Another major application of ion-exchange chromatography is water analysis. Anion-exchange chromatography can be used to measure the concentration of anions, including sulfates, nitrates, nitrites, fluoride, and chloride. Cation-exchange chromatography is used to measure the concentration of cations such as sodium, potassium, calcium, and magnesium. A type of ion-exchange chromatography is also used in water purification, as most water softeners filter out magnesium and calcium ions in hard water by binding them to a resin, which releases bound sodium. Heavy metals, such as copper or lead, can also be removed from water using ion-exchange chromatography.
Ion-exchange chromatography is also useful in metal purification. It can be used to purify actanides, such as plutonium, and remove it from spent nuclear reactor fuel rods. It can also be used to scavenge uranium and remove it from water or other environmental samples.
Ion-exchange chromatography is widely used in the separation and isolation of charged compounds, particularly large biomolecules.
This type of liquid chromatography uses a column of packed stationary-phase beads, called resin. The technique separates analytes based on their affinity with the charged resin.
There are two main types of this technique. In cation-exchange chromatography, negatively-charged resin is used to bind positively-charged analytes. Similarly, in anion-exchange, negatively-charged analytes bind to positively-charged resin. The unbound compounds are washed through the column, and the analyte can then be collected in a separate container.
This video will introduce the basics of ion-exchange chromatography, and demonstrate the technique by separating a protein mixture in the laboratory.
The stationary phase is a key component to a successful separation. Strong cation-exchange resins typically feature strong acid functional groups, such as sulfonic acid. Weak cation-exchange resins feature weak groups, such as carboxylic acids.
Similarly, strong anion-exchange resins utilize strong bases, like quaternary amines, while weak anion-exchange resins use secondary or tertiary amines. The selection of resin will depend on the properties of the sample mixture, and the analyte of interest.
The buffers used, collectively called the mobile phase, are also important to separation, particularly in terms of pH. For proteins, pH is selected based on its isoelectric point, or pI. At a pH equal to the protein’s pI, the protein is neutral. Above the pI, it will have a net negative charge, while below the pI, it will have a net positive charge. The buffer pH must be selected so the protein is properly charged and able to bind to the stationary phase.
Ion-exchange chromatography is generally a four-step process. First, a packed column containing either anion- or cation-exchange resin is equilibrated using buffer. For anion-exchange columns, this involves protonating the resin, ensuring it is positively charged.
Next, the sample is loaded on the column. The buffer must have low conductivity, as charged species can compete with the sample for interactions with the resin. Compounds of opposite charge bind to the resin. Molecules that are not charged, or carry the same charge, remain unbound.
In the third step, the column is washed with additional buffer to remove the unbound components from the column, leaving the bound behind.
Finally, the fourth step is the elution of the bound analyte. This is accomplished either by using a salt gradient, where the salt concentration is gradually increased, or using a high salt elution buffer.
Molecules that are weakly bound will be eluted first, as the low salt will most easily disturb their ionic bonding to the resin. Compounds that are more strongly bound will elute with higher salt concentrations.
Now that the basics of ion exchange chromatography have been outlined, lets take a look at its use in the separation of two proteins.
First, to prepare the protein mixture for separation, add 0.2 mL of binding buffer, and vortex to mix thoroughly. Then, centrifuge the mixture to remove any froth. To prepare the cation-exchange column, clamp it vertically onto a ring stand, and allow the resin to settle.
Open the top cap of the column, and then the bottom. Allow the buffer to drip out under gravity into a tube below.
To prepare the column, equilibrate it by loading a column-volume of buffer, in this case 0.3 mL. Let the buffer drip out of the column into a waste vial. After a column-volume of buffer has exited, repeat the equilibration step.
To run the experiment, place a 2-mL centrifuge tube labeled “Unbound 1” below the column. Carefully load 0.1 mL of the protein sample onto the top of the column.
Once the sample has been loaded, wash with a column-volume of buffer and allow it to flow all the way through. Repeat for a total of 5 washes. Collect each wash in its own tube, labeled “Unbound 1” through “5”. For the last 2 washes, centrifuge the column for 10 s to make sure that all unbound species wash off the column. Put the column in a new 2-mL centrifuge collection tube, and label it “Bound 1”. Load 1 column-volume of elution buffer on top of the column. Centrifuge for 10 s at 1,000 x g.
Repeat the elution step 2 more times to ensure collection of the bound analyte. Label the tubes “Bound 2” and “3”. Record any color changes or observations about the fractions.
In this example, hemoglobin and cytochrome C were separated. Hemoglobin has a pI of 6.8, while cytochrome C has a pI of 10.5. In the pH 8.1 buffer, hemoglobin is negatively charged and does not bind to the column. Conversely, cytochrome C is positively charged at pH 8.1 and binds to the column.
Hemoglobin, a brownish colored protein, was found in the unbound fractions, while cytochrome C, a reddish colored protein, was observed in the bound fraction.
There are many forms of liquid chromatography, each with different abilities to separate components of a mixture.
In this example, column chromatography was used to separate a mixture of single and double stranded DNA. Hydroxyapatite, or HA, is a crystalline form of calcium phosphate commonly use as a stationary phase due to its positively-charged calcium ions. In this case, the HA column was ideal for the separation of DNA as it can bind to DNA’s negatively-charged backbone.
Another form of column chromatography frequently used to separate proteins is immobilized metal affinity chromatography, or IMAC. In IMAC, the stationary phase possesses a ligand with a metal ion, which binds to a histidine tag on the protein of interest.
All other components of the mixture exit the column. The protein is then eluted with a solution of imidazole, which has a similar structure to histidine, and binds more strongly with the metal ion.
A common application of column chromatography is high performance liquid chromatography, or HPLC. HPLC is widely used in analytical chemistry for both the identification and separation of biological and non-biological compounds in a mixture.
HPLC is similar to the column chromatography demonstrated in this video, except that it is automated, and operated at very high pressures. This enables the use of smaller stationary-phase beads, with a higher surface area to volume ratio. Thus, improved interactions between the stationary phase and components in the mobile phase are possible.
You’ve just watched JoVE’s introduction to ion-exchange chromatography. You should now understand the concepts behind it, the 4 steps involved, and some related techniques.
Thanks for watching!
