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Frequently Asked TSK Questions Frequently Asked TSK Questions

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The following are some frequently asked questions for Tosoh Biosep TSK-Gel® columns and ToyoPearl® media, beginning with analytical issues and ending with more process-scale questions.
 

What is the maximum load for my SW or SWxl column (7.5mm or 7.8mm I.D. x 30cm L)?

Sample Weight - 1 to 3 mg
Sample Volume - 250ul, although 10-100ul is better.


How should I clean my column?

We recommend the following method to clean a column:
1. Clean in the reverse flow direction (it will not harm the column).
2. Clean at half the maximum flow rate.
3. Use 10-20% organic (MeOH, EtOH, IPA) for removing hydrophobic contamination.
4. Use 0.5M salt (NaCl, sodium sulfate) at pH 3 for ionically bound contamination.
5. Use 0.1M NaOH for ionically bound contamination (not for SW/SWxl columns).
6. Use 6M urea or Guanidine HCl if all else fails.


Which is better for my protein separation, PW or SW columns?

In general, SW columns provide better resolution than PW columns due to the pore structure. We recommend PW only for high pH applications (>7.5) or for high MW proteins such as viruses and DNA fragments. PW columns are also useful for synthetic polymers.


Which is better for my peptide separation, PW or SW columns?

Many peptide separations have been performed using the G2000SWxl column with a 45% acetonitrile, 0.1% TFA mobile phase. However, the G2500PWxl can also be used for certain peptide applications.


I ran my column dry overnight/over the weekend. What should I do?

Don't despair! Run the column in the reverse direction at half the maximum flow rate with 20% methanol in water for 1-2 days. When a stable baseline is achieved, the column should be ready.


Please recommend an SEC column for my protein separation.

Choose a column where proteins of interest fall towards the middle of your separation range.
For example, separating proteins of 25kd and 50kd could suggest either a G2000SW, G3000SW, or G4000SW. However, the G2000SW is the best choice because those two proteins are more towards the middle of the G2000SW separation range. In addition, the G2000SW separates within a narrower range. Both of these features mean that the G2000SW will provide better resolution of these two proteins.
Please understand the difference between the MW ranges given for PEOs, dextrans, and globular proteins. PEOs are proxies for linear compounds, dextrans are proxies for linear branched compounds, and proteins are proteins. Remember, it's the hydrodynamic radius that's important here, not the true MW.


Should I choose SW or SWxl?

For best resolution, choose SWxl. If you want resolution with higher capacity (remember capacity is proportional to column volume), try a 60cm length SW column or two 30cm length SWxl columns in series.


I'm not getting the resolution I need with my SEC column. What should I do?

Make sure you're not overloading the column.

Check to see if your compounds are within the column's separation range.

If you're using an SW, try an SWxl. Smaller particle size=better resolution.

If you're using an SWxl (or don't want to switch columns), try using a lower flow rate. A flow rate range of 0.3 ml/minute - 0.5 ml/minute is best; any lower and diffusion takes over and resolution suffers). You can always put two SWxl columns in series to improve resolution.


What's the best column for IgG and IgG fragment separations?

G3000SWxl will separate IgG from albumin and transferrin very well.


What about the new 2um Super-ODS column?

The column has a C18 functionality attached to a high-purity, metal-free, 2mm silica. It is useful for rapid, high resolution separations of nucleotides, peptides, low molecular weight pharmaceuticals, vitamins, and food and beverage components.


What is a good resin to desalt or perform a buffer exchange?

Toyopearl HW-40 is an excellent, inexpensive resin for desalting or buffer exchange. It can also be used as a process guard column.


I want to perform a DMT-on purification of a synthetic oligonucleotide. Which resin should I choose?

Amberchrom CG-300 has been used successfully for DMT-on purification (and detritylation on-column). This 300 angstrom, styrenic resin is stable from pH 1-14 and can withstand the wide pH ranges necessary for nucleotide purification.
How should Amberchrom/Toyopearl resins be stored?
With the exception of several Toyopearl affinity resins, all Toyopearl and Amberchrom resins can be stored in 20% ethanol at either room temperature or 4 degrees C.


How do I elute proteins in HIC?

Use a reverse salt (typically ammonium sulfate or sodium sulfate) gradient. If elution is not complete when the gradient is finished, 10-20% organic (MeOH, EtOH, IPA) can be used. If elution is still not complete, 1M urea or guanidine HCl (or another chaotrope) can be used. If all else fails, a detergent such as Triton, Emulgen, or CHAPs can be used, but detergents are very difficult to remove from hydrophobic interaction resins.


Which HIC resin should I choose?

Tosoh Biosep provides three different HIC resins, Ether, Phenyl, and Butyl Toyopearl. Ether is the least hydrophobic of the three resins, and useful for antibody and antibody fragment purification. Phenyl has intermediate hydrophobicity, and is useful for membrane protein and antibody fragment purifications. Butyl is the most hydrophobic of the three resins, and useful for growth factor and hydrophilic protein separations.


How do I pack Toyopearl/Amberchrom into a column?

Toyopearl is best packed under pressure (2-3 bar, 30-45 psi) in a 30-50% slurry. The highest ionic strength used in the separation cycle should be used as the packing buffer, and the packing flow rate should ideally be 2X the operating flow rate.
Amberchrom is also best packed under pressure (5-7 bar, 75-105 psi) in a 65-80% slurry. The lowest organic concentration used in the separation cycle should be used as the packing buffer, and the packing flow rate should ideally be 2X the operating flow rate.
Still have questions?
Send an e-mail directly to Tosoh Biosep technical service group.
 



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Last Updated: 12/02/16