출처: 박사의 실험실.B 질 벤턴 – 버지니아 대학
이온 교환 크로마토그래피는 충전에 따라 문체를 분리하는 크로마토그래피의 한 유형입니다. 이온 교환 수지라고 하는 솔리드 지지대에서 충전된 고정 단계로 채워진 열이 사용됩니다. 강력한 양이온 교환 크로마토그래피는 음전하 수지를 사용하여 양이온을 우선적으로 분리하고 강력한 음이온 교환 크로마토그래피는 양전하 수지를 사용하여 음이온을 우선적으로 선택합니다. 크롬 토그래피의 이 모형은 단백질 또는 핵산 견본의 정화에서 예를 들면 견본 준비를 위해, 예를 들면 인기 있습니다.
이온 교환 크로마토그래피는 2단계 과정입니다. 첫 번째 단계에서 샘플은 로딩 버퍼의 열에 로드됩니다. 컬럼 수지에 충전된 샘플의 바인딩은 반대 전하의 샘플을 유치하기 위해 수지의 이온 상호 작용을 기반으로 합니다. 따라서, 수지에 반대 극성의 충전 된 샘플은 강하게 결합된다. 충전되지 않거나 반대 전하인 다른 분자는 결합되지 않으며 열을 통해 세척됩니다. 두 번째 단계는 수지에 바인딩된 별무를 엘로우트하는 것입니다. 이것은 완충제의 소금 양이 천천히 증가하는 소금 그라데이션으로 수행됩니다. 분수는 용출이 발생하고 이러한 분획 중 하나에서 정제 된 관심 샘플을 복구 할 수 있습니다으로 컬럼의 끝에 수집됩니다. 분광법과 같은 또 다른 기술은 샘플을 포함하는 분획을 식별하기 위해 필요할 수 있습니다. 이온 교환 크로마토그래피는 단백질 연구에서 특히 유용하며, 크기가 수지와의 상호 작용 수를 결정할 수 있으므로 특정 충전 또는 크기가 있는 관심 있는 단백질을 분리하는 데 특히 유용합니다.
이온 교환 크로마토그래피는 친화성 크로마토그래피보다 일반적인 분리 기술이며, 이는 항체가 하나의 특정 문체를 결합하기 위해 컬럼에 부착되는 단백질 샘플을 준비하는 데 자주 사용된다. 각 별무에 대해 새로운 친화성 컬럼을 구입해야 하며, 동일한 유형의 이온 교환 컬럼을 사용하여 동일한 충전의 많은 단백질을 정리하는 데 사용할 수 있습니다. 이온 교환 크로마토그래피는 다른 특성에 따라 분리되는 다른 유형의 크로마토그래피와 함께 사용할 수도 있습니다. 예를 들어 크기 배제 크로마토그래피는 크기에 따라 구분되며 이온 교환 크로마토그래피 전에 지정된 크기의 화합물만 선택하도록 사용할 수 있습니다.
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!
