Gel Filtration Chromatography Media: What You Need to Know

Do you use pre-packed Superdex 75 or Superdex 200 columns for gel filtration chromatography? Ever wonder if other media could better address your purification needs? Look no further. Your quest ends here! But if you’re looking for information on gel filtration principles, check out this earlier article.

Choosing a Column for your Gel Filtration Experiment

All gel filtration chromatography columns separate components based on size. However, there are several factors you need to consider before choosing the column that’s right for you.

Size of the Analyte

First, keep its size in mind when choosing a column for purifying your analyte. Gel filtration matrices are not uniform; their pore sizes are variable, falling within a range. For example, Figure 1 shows a scanning electron micrograph of a 2% agarose gel.

Medin-agarose-gel-Usha-B-Nair-Know-Your-Gel-Filtration-Chromatography-Media-160704 — Reproduced with permission from Studies on Structure and Properties of Agarose, Anders Medin, PhD Thesis, Uppsala University, 1995.

Choose a medium with the correct range of pore sizes (or the correct fractionation range) to adequately separate the molecule of interest from the contaminants in your sample. For proteins, you may have prior molecular weight information from SDS-PAGE. However, for an oligomeric protein, the size information you have from SDS-PAGE may not represent its true size in solution. If you do not know the molecular weight of your analyte, start with a column with a broad fractionation range. Then, switch to a column with a narrower fractionation range to improve the quality of separation.

Sample Purity

Okay, so you chose your column based on the size of your analyte. What about the contaminants in your sample? Yes, you need to consider their sizes and relative amounts as well before picking your column. Ideally, choose a column that gives the best possible resolution. In case there is more than one option available, choose a column where the component of interest elutes earlier.

Sample Volume

Whether you choose a preparative grade or an analytical grade column depends on your sample volume and the purpose of your experiment. The larger volume preparative columns differ from analytical ones in terms of loading capacity and resolving power. Use preparative columns for fractionating large volumes of sample and analytical columns for fractionating small volumes or analyzing the composition of your samples. Keep your sample volume between 0.5–4% of the column volume because with increasing sample volume its spread over the column increases, which could adversely affect the resolution of peaks in your chromatogram.

Bead Size

We know that the distribution of pore sizes in the column matrix determines the fractionation range of the column. But, what about bead size? Yes, it turns out that bead size can affect the width of the eluting peaks and, therefore, resolution of the sample components. How so? You may ask. Beads that are smaller have larger surface area to volume ratios, which increases the contact area between the beads and the sample helping improve resolution. However, smaller beads increase column back pressure requiring you to reduce the flow rate, which extends your run time; this is why smaller beads are used more often in analytical columns.

Experimental Objectives

Finally, is desalting or buffer exchange the objective of your experiment? Or is it separating out the individual components of your sample? If it were the former, then a PD-10 column, or a column with resins, such as Sephadex G-10, G-25, Bio-Gel P-2, or Bio-Gel P6DG would suffice. In this case, all the macromolecules in your sample would elute as one group at the column’s void volume. The salt or small molecule contaminant that you were seeking to remove would elute very close to the column’s total volume. On the other hand, if your objective is to separate out the individual components in your sample, choose a column based on the principles we discussed above.

Column Efficiency

Another parameter you may want to note is column efficiency or the number of theoretical plates in the column, which reflects a column’s ability to produce narrow, well-resolved peaks; higher values mean better resolution. The concept of theoretical plates is widely used to determine the efficiency of separation processes, such as distillation. Imagine each theoretical plate as a segment of the column where equilibrium is established between the stationary and mobile phases. As you increase the number of equilibrium stages (theoretical plates) along the column, the separation of sample components and column efficiency improve. You can do this by increasing the contact area between the stationary and mobile phases. This is why decreasing bead size improves component separation and, in turn, column efficiency. Moreover, a column with a narrower distribution of bead sizes is more efficient than one with a broad range. But, if you think that a longer column should have a greater number of theoretical plates, think again. This need not be the case. Yes, longer columns may improve the separation of your sample components but their efficiency may be lower because of sample diffusion over their length, which could result in peak broadening.

Pre-packed Columns for Gel Filtration Chromatography

The table below lists a few popular gel filtration chromatography pre-packed columns and column characteristics that you must consider before picking one. Do bear in mind that the fractionation range of each column is based on data using globular proteins. If you use linear or branched chain polymers instead, this range is different.

Column	Bead Chemistry	Mean bead size (µm)	Column volume (mL)	Efficiency (m-1)	Fractionation range (kDa) (Mean pore size [nm])
Superdex 75 10/300 GL	Composite of cross-linked dextran and agarose	13	24	> 25000	3-70 (6)
Superdex 75 16/60 pg	Composite of cross-linked dextran and agarose	34	120	> 13000	3-70 (NA)
Superdex 200 10/300 GL	Composite of cross-linked dextran and agarose	13	24	> 25000	10-600 (13)
Superdex 200 16/600 pg	Composite of cross-linked dextran and agarose	34	120	> 13000	10-600 (NA)
Superose 12 10/300 GL	Cross-linked agarose	10	24	> 40000	1-300 (13)
Superose 6 10/300 GL	Cross-linked agarose	13	24	> 30000	5-5000 (25)
HiPrep 16/60 Sephacryl S-100 HR	Cross-linked copolymer of allyl dextran and N, N'-methylenebisacrylamide	50	120	> 5000	1-100 (NA)
HiPrep 16/60 Sephacryl S-400 HR	Cross-linked copolymer of allyl dextran and N, N'-methylenebisacrylamide	50	120	> 5000	20-8000 (NA)
PD-10 desalting column	Sephadex G-25 (dextran cross-linked with epichlorohydrin)	50-150 (dry)	8.3	-	1-5 (NA)
ENrich SEC 70	Hydrophilic polymethacrylate	10	24	>20000	0.5-70 (NA)
ENrich SEC 650	Hydrophilic polymethacrylate	10	24	>20000	5-650 (NA)
TSKgel-G2000SW (30cm)	Silica with hydrophilic diol functional groups bonded to silica surface	10, 13	13.25, 108.9	10000	5-150 (12.5)
TSKgel G3000SW (30cm)	Silica with hydrophilic diol functional groups bonded to silica surface	10	13.25	10000	10-500 (25)
TSKgel G4000SW (30cm)	Silica with hydrophilic diol functional groups bonded to silica surface	13, 17	13.25, 108.9	8000	20-7000 (45)
Zorbax GF-250	Zirconium stabilized silica functionalized with diol groups	4	4.15, 17.35, 88.25	>18000	4-400 (15)
Zorbax GF-450	Zirconium stabilized silica functionalized with diol groups	6	17.35, 88.25	> 10000	25-900 (30)

Hope you found the above information useful. You are welcome to post questions on this subject or share insights you gained during the course of your experiments. Also remember to check in on my next article about estimating molecular weight using chromatography.