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Tosoh Bioscience LLC: The Chemistry of Innovation
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Hydrophobic Interaction
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Normal Phase/HILIC
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Size Exclusion


Questions:

How does SEC work?

 

What is GPC?

 

What factors should I consider when selecting the correct SEC column for my application?

 

What is the best column for IgG and IgG fragment separations?

 

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

 

Which is better for my peptide separation, TSKgel SWxl or PWxl columns?

 

What column do you recommend for my protein separation?

 

What TSKgel SW column should I use for my protein separation? 

 

What is the maximum load for my TSKgel SW size exclusion column?

 

How should I clean my TSKgel SWxl column?

 

What’s the difference between TSKgel SW/SWxl and TSKgel PW/PWxl SEC columns?

 

How do mixed-bed columns differ from your “Multipore” columns?

What causes peak tailing on a TSKgel SW/SWxl SEC column?

 

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

Can I use organic solvents with SEC columns?  What about surfactants like Triton or SDS?

What are the advantages and disadvantages when adding additives to my mobile phase for analysis of proteins by SEC?

When I use a new TSKgel SW/SWxl column I get low recoveries of my protein.  How can I avoid this?

Do the TSKgel PW/PWxl columns shed microparticles?

What endfitting do I use with my particular TSKgel SEC column?  

 

 

1. How does SEC work? (back to top)

 

Molecules in solution assume 1 of 3 possible configurations: linear, e.g. PEG; branched, e.g. dextran; and globular, e,g, protein. As such a given molecule will have a unique hydrodynamic radius in solution. It is the differences in molecular dimensions that enables separation of compounds in a mixture by means of size exclusion liquid chromatography. Whereas large molecules will have limited or no access to the pores in an SEC column, smaller species will fit in the pores and be retained. Thus, the molecules will elute from the column in reverse order of molecular weight. Unretained compounds will come out in the column’s void volume (the space between particles in the resin bed). A fully retained molecule will elute at the column’s included (or mobile phase) volume which is the sum of the void volume and pore volume.

 

 

2. What is GPC? (back to top)

 

Size exclusion chromatography (SEC) is a method in which polymers are separated based on their molecular sizes or strictly speaking hydrodynamic radii. Gel permeation chromatography (GPC) is a term that specifically applies to SEC when an organic solvent is used as the mobile phase. GPC is a procedure used in polymer chemistry for measuring the distribution of molecular mass.

Common to all GPC studies is that a polymer sample, which by definition consists of a distribution of (mainly) linear molecules that differ in the number of repeating chemical units, is delivered to a column that is packed with porous particles. Molecules go through the column at rates which are dependant upon their size. Larger molecules cannot enter the pores, therefore they pass quickly through the column and elute first. Smaller polymers enter the pores and take longer to exit the column. Various detectors are used to sense when the polymers elute from the column; the output of the detector is a molecular size distribution curve.

 

In 2008, Tosoh Bioscience introduced a dedicated GPC instrument. The EcoSEC GPC System is a semi-micro system that incorporates low dead volume and is engineered to produce superior results in semi-micro (4.6mm ID) columns that save time and money compared to wider-bore conventional columns that are filled with larger particle size packing materials. Semi-micro columns consume 1/6th the solvent of conventional columns (4.6mm ID vs. 7.8mm ID) and results are obtained in 1/2 the time when 6 micron particles are substituted by 3 micron particles.

 

 

3. What factors should I consider when selecting the correct SEC column for my application? (back to top)

 

  • MW and molecular configuration, e.g. globular: match this to the fractionation range of the column.
  • Solvent solubility: what mobile phase is required and is the column compatible with this solvent, e.g. TSKgel SW/SWxl columns operate in the pH range of 2.5-7.5.
  • Hydrophilic vs. hydrophobic: this will dictate the ionic strength of the eluent used in the mobile phase. Some proteins have hydrophobic sites that require a small amount of organic solvent in the mobile phase such as 5% methanol. This inhibits secondary hydrophobic interaction.
  • Other factors: temperature,  sample characteristics,  HPLC system requirements, fittings required.

 

4. What is the best column for IgG and IgG fragment separations? (back to top)

 

We recommend using the TSKgel G3000SWxl column for the separation of IgG from albumin and transferrin. For details, please download Separation Report 62.

 

 

5. Which is better for my protein separation, TSKgel PW or SW columns? (back to top)

 

In general, TSKgel SWxl columns provide better resolution for protein separations than TSKgel PWxl columns because of the higher pore volume of the silica-based SWxl packing material than the polymer-based PWxl packing material in high buffer and high salt concentration mobile phases. Although TSKgel PWxl and PW columns are ideally suited for analysis of water-soluble synthetic polymers, when it comes to biopolymers we only recommend PWxl columns for high pH applications (>7.5) or for viruses and DNA fragments.

 

 

6. Which is better for my peptide separation, TSKgel SWxl or PWxl  columns? (back to top)

 

Many peptide separations have been performed by using the TSKgel G2000SWxl column and a mobile phase containing 45% acetonitrile and 0.1% trifluoroacetic acid (TFA), while TSKgel G2500PWxl columns have been used successfully for peptides up to a molecular weight of 5,000 using the same mobile phase.

 

 

7. What column do you recommend for my protein separation? (back to top)

 

The best place to start is the overview page for SWxl-type products. Next, focus in on the column that would elute the protein of interest in or near the middle of the molecular weight separation range. For example, if you need to separate two proteins of 25,000 and 50,000 molecular weights, the calibration curves suggest either to use a TSKgel G2000SW, G3000SW, or a G4000SW column. However, the TSKgel G2000SWxl column is the better column to use as the molecular weights are closer to the middle of the separation range. In addition, the slope of the calibration curve for the TSKgel G2000SWxl column is less steep, which means that more pore volume is available to separate two proteins of similar molecular weight. Note that the calibration curves for polyethylene oxides (PEOs), dextrans, and globular proteins are quite different. PEOs are proxies for linear compounds, dextrans are proxies for linear branched compounds, and proteins are globular structures. Remember, it's the hydrodynamic radius that's important here, not the true MW.

 

 

8. What TSKgel SW column should I use for my protein separation? (back to top)

 

Tosoh Corporation has a long history of developing size exclusion chromatography columns for the separation of proteins. Historically speaking, the 10-13 micron TSKgel SW columns (G2000SW, G3000SW and G4000SW) were developed first, followed by 5-8 micron TSKgel SWxl columns (G2000SWxl, G3000SWxl and G4000SWxl), and most recently the 4 micron TSKgel SuperSW2000 and TSKgel SuperSW3000 columns.

 

  • For best resolution, select a TSKgel SuperSW2000 or SuperSW3000 column. However, your HPLC system needs to have minimal extra-column dispersion (read Separation Report 95 for guidance on how to make sure your system is optimized).
  • If you only have a limited mass of protein (see this FAQ for guidance), select a TSKgel SuperSW2000 or SuperSW3000 column.
  • If you want to isolate your protein of interest, use a TSKgel SWxl or TSKgel SW column, while keeping in mind that:
    • The mass (and sample volume) of protein that can be injected before losing resolution between neighboring proteins increases linearly with column length (constant diameter) and with the square of column diameter (constant length).
    • Standard TSKgel SW columns are available in 7.5mm ID x 30cm and 7.5mm ID x 60cm dimensions.
    • Columns with larger internal diameter (21mm ID and higher) are only available in the TSKgel SW product line. 

 

9.  What is the maximum load for my TSKgel SW size exclusion column? (back to top)

 

When it comes to analyzing how much of a component is present in a sample, we want to make sure to work within the linear range of the distribution isotherm, the shape of which describes the relationship between sample concentration in mobile and stationary phases. In preparative chromatography, where the goal is to isolate one of more sample components at a certain level of purity, we can always inject more as long as the component of interest can be collected at the required level of purity. Thus, the amount of sample that can be injected on a column is arbitrarily defined as the mass or volume of an injected sample that causes the efficiency of the peak to decrease by 10%. Again, from a practical point of view, as long as the compound of interest is well separated from its nearest contaminant, we can always inject more as long as we obtain at least the desired level of purity.

As indicated above and in the Tables, a column can be overloaded due to the mass of sample injected, but also by injecting a volume that is large in relation to the volume in which the component of interest elutes from the column. A good rule of thumb is not to inject a larger volume than one third the peak volume in which a mono-disperse component elutes from the column. Mass overload takes place when sample molecules no longer have free access to diffuse into and out of the pores, thus bypassing part of the column and thereby effectively reducing the length of the column that remains to fractionate the sample. 


The tables below provide rough guidelines of how much mass can be injected on a TSKgel SW size exclusion column for the most popular analytical and preparative TSKgel gel filtration columns. Included in the Analytical Table are the 4 micron TSKgel SuperSW3000 column, the widely used TSKgel G3000SWxl column in stainless steel hardware, the BioAssist G3SWxl column, which is packed in PEEK hardware, and the 15cm QC-PAK GFC 300 stainless steel column. The values for Mass and Volume will be similar for TSKgel G2000SWxl and TSKgel G4000SWxl columns.

 

Analytical
TSKgel Column

Particle
Size (µm)

Dimensions
(mm ID x cm)

Column
Volume (mL)

Mass
BSA (mg)

Volume
BSA (µL)

SuperSW3000

4

4.6 x 30

4.98

~0.3 – 1

< 20

G3000SWxl

5

7.8 x 30

14.32

~1 – 3

< 100

BioAssist G3SWxl

5

7.8 x 15

14.32

~1 – 3

< 100

QC-PAK GFC 300

5

7.8 x 15

7.16

~0.5 – 1.5

< 50

 

Obviously, the more packing material in the column (i.e., the larger the column volume), the more sample mass can be injected before peaks start to overlap and purity decreases. The values listed in the Preparative Table below and the Analytical Table above are guidelines only – they are based on a 10% drop in efficiency of BSA (bovine serum albumin) eluting from the column. For the larger ID columns (21.5mm ID and up) the numbers are conservative, as we did not account for the fact that larger ID columns are filled with larger particle size supports, which results in lower column efficiency, which in turn results in higher mass and volume capacities.

Preparative
TSKgel Column

Particle
Size (µm)

Dimensions
(mm ID x cm)

Column
Volume (mL)

Mass
BSA (mg)

Volume
BSA (mL)

G3000SW

13

21.5 x 30

108.86

~7 – 22

< 1

G3000SW

13

21.5 x 60

217.72

~14 – 42

< 2

G3000SW

20

55 x 60

1,424.78

~45 – 143

< 10

G3000SW

20

108 x 30

2,746.87

~175 – 550

< 50

G3000SW

20

108 x 60

5,493.74

~350 – 1,100

< 100



10. How should I clean my TSKgel SWxl column? (back to top)

 

We recommend the following methods to clean a column. After each step wash the column with DI water to remove solvent, salt, or detergent. 

1. Clean in the reverse flow direction (it will not harm the column) at half the maximum flow rate.
2. Use 20% acetonitrile to remove contaminants that are held onto the column by hydrophobic interaction.
3. Use 0.5M salt (NaCl, sodium sulfate) at pH 3 to remove positively charged contaminants that are bound to the column by ionic interaction, such as with residual negatively charged silanol groups.
4. If the above steps do not work, use 6M urea or guanidine hydrochloride.

 

11. What’s the difference between TSKgel SW/SWxl and PW/PWxl SEC columns? (back to top)

 

The “S” in TSKgel SW and SWxl columns stand for silica so these columns are created using porous silica. The “P” in TSKgel PW and PWxl columns refer to a polymeric support of porous methacrylate. The  subscripted XL is used to denote smaller particle, higher efficiency columns. However, the pore size is the same for a given type of column, e.g. TSKgel G2000SW and G2000SWxl have the same fractionation range.

 

 

12. How do mixed-bed columns differ from your “Multipore” columns? (back to top)

 

Historically, when faced with the challenge of analyzing polymer samples containing a wide range of molecular weights one had two options, (1) connect two or more columns, each containing particles of different pore size, in series, or (2) use a so-called mixed-bed column prepared by blending particles of different pore size before the columns are packed. Although easiest, connecting different pore size columns in series seldom result in linear calibration curves, as the pore columns of the various columns do not always match exactly. Mixed-bed or ‘linear’ columns such as TSKgel GMHxl and GMHHR Series columns do not always provide linear calibration curves either. Imperfect blends will result in the appearance of inflection points in chromatograms due to a mismatch of pore sizes.

 

In the 1990’s Tosoh scientists pioneered a third option when they introduced Multipore GPC columns. The latest edition of Tosoh's innovative multi-pore particle synthesis technology is the TSKgel SuperMultiporeHZ Series columns. Multipore columns represent a new type of column packed with polystyrene or polymethacrylate gels having a broad pore size distribution within each particle. Multipore column share the advantage of mixed-bed columns in terms of having a wide range of pore sizes in the column. Multipore columns outperform mixed-bed columns by providing better linearity of the calibration curve. By manufacturing particles containing a very broad range of pore sizes, Tosoh scientists have been able to create a linear calibration curve within each particle. As a result, columns packed with multi-pore particles provide an extended linear calibration curve, without the need to mix particles of different pore sizes.

 


13. What causes peak tailing on a TSKgel SW/SWxl SEC column? (back to top)

 

The TSKgel SW/SWxl columns contain porous silica that has been chemically bonded with a proprietary hyprophilic material. This coating stabilizes the silica in aqueous solvents. When charged, strongly polar or hydrophobic molecules interact with the silica support, it is possible to have secondary electrostatic, hydrophilic or hydrophobic interaction between the solutes and the silica matrix. Thus, the separation involves two mechanisms, size exclusion and electrostatic, hydrophilic or hydrophobic interaction. When this occurs the chromatogram may exhibit peak tailing, as the number of adsorption sites that cause the secondary interaction is limited. The best way to avoid secondary interaction is to use a buffer with an ionic strength in excess of 200mM, e.g. 100mM phosphate buffer and 150mM NaCl, pH 6.8.

 

 

14. I'm not getting the resolution I need with my SEC column. What should I do? (back to top)

 

  • Make sure you're not overloading the column. Check to see if your compounds elute from the column in the separation range.
  • Following the saying “the smaller the particle size, the better the separation”, if you were using a TSKgel SWcolumn, try a TSKgel SWxl column, and if you if you were using a TSKgel SWxl column, try a TSKgel SuperSW column.
  • If you're using a TSKgel SWxl column (or don't want to switch columns), try using a lower flow rate. A flow rate range of 0.3mL/minute - 0.5mL/minute is best for 7.8mm ID columns and a flow rate of 0.10 - 0.15mL/min is better for the 4.6mm ID TSKgel SuperSW columns.
  • Finally, as in other modes of HPLC, in size exclusion chromatography you can always improve resolution by putting two or more columns in series. As is well known, column efficiency increases linearly with column length and resolution improves by the square root of the number of columns in series.


15. Can I use organic solvents with SEC columns?  What about surfactants like Triton or SDS? (back to top)

 

TSKgel SW/SWxl columns can tolerate up to 100% polar organics such as acetonitrile and methanol. TSKgel PW/PWxl columns can handle up to 20% polar organics in the mobile phase, and as high as 50% in special cases.  Please contact the technical service group for these special cases.  Using a surfactant in the mobile phase, e.g. 0.1% SDS will require that the column be dedicated to the application as the surfactant tends to irreversibly bind to the column and thereby alter the stationary phase.

 

16. What are the advantages and disadvantages when adding additives to my mobile phase for analysis of proteins by SEC? (back to top)


Additives are sometimes used in SEC mobile phases to promote the solubilization of proteins, to prevent secondary interaction of proteins with the stationary phase, or to denature proteins. Common additives used to analyze proteins by SEC include sodium dodecyl sulfate (SDS), urea and guanidine hydrochloride. All three of these additives enhance protein solubilization and each of them promotes denaturation by breaking non-covalent bonds that are part of the higher order structure of proteins. Adding a surfactant like SDS to the sample and mobile phase leads to a disruption of the native shape of a globular protein to form rod-like structures that are much smaller in size and thus chromatograph more efficiently. Guanidine hydrochloride and urea disrupt the protein structure to a randomly coiled (larger) structure.

The use of additives in SEC of proteins should be considered in cases where protein solubility is limited, where non-specific interaction is suspected between the sample and column matrix and where the addition of another buffer or water soluble organic solvent is not effective.

The performance of an SEC column is irreversibly modified when the column is subjected to most additives. This is a major disadvantage. For example, the retention time of a sample may shift and the calculated resolution between two peaks may change compared to a column run without additives.  Furthermore, it is almost impossible to completely remove the additive from the column. Therefore, the column must be designated for use only with additives in the mobile phase for its entire lifetime.


 

17. When I use a new TSKgel SW/SWxl column I get low recoveries of my protein.  How can I avoid this? (back to top)

 

There are active sites on the resin that require conditioning with either your sample protein or another material such as bovine serum albumin (BSA).  For the TSKgel SW/SWxl column we recommend 4 successive injections of 100µg BSA to condition the resin. The TSKgel PW/PWxl sizing columns are far less hydrophilic and normally do not require conditioning.

 

 

18. Do the TSKgel PW/PWxl columns shed microparticles? (back to top)

 

Microparticles do not interfere with most detection methods used in HPLC.  However, these particles can increase the noise level in laser light scattering detection systems. To minimize this effect using the TSKgel PW/PWxl columns run the column overnight in 10-20% methanol at one-half the normal  flow rate to clean any microparticles off the resin.

 


19. What endfitting do I use with my particular TSKgel SEC column? (back to top)  

Please visit the HPLC Columns - Replacement Parts page, which will provide a description of the available fittings, including the size of the frit in the fitting and the applicable endfitting to be used with your TSKgel column.  Question #10 within the General FAQ category includes additional information on fittings and connectors.

 


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