12/9/21

Dear Dokyeoung,

I have no experience w/ Toyopearl HW-50S. However, I have packed columns w/ molecular sieves. Below is some information about Toyopearl HW-50S from Sigma. It states that it can be cleaned in place w/ 0.5 M NaOH or 0.1 M HCl. To me this means that in can be cleaned in the column with either of these solutions. However, it should be OK to wash the resin in a beaker, then pour it into the column. I think that either way would be OK. My preference would be to do the washing in a beaker. After that, I would wash the resin with w/ DI-water several times to remove the wash solution (acid or base). Check the pH of the wash water to determine when the wash step is complete. This should be followed by washing and equilibrating the resin with the buffer you plan to use for your separation. It may be OK to do this in the column. The reason I would use a beaker is that when some resins (not all) are irrigated with different solutions, the resin shrink or expand. If the resin expands when it is in the column, the resin beads can be damaged.

According to the information below, the resin cannot be used at pressures above 3 bar (approx. 3 atmospheres, or ~45 psi). If you plan to use a peristaltic pump to pump your mobile phase through the column, be careful not to exceed this pressure. if you do, I suspect that you will cause the resin beads to collapse, in which case the resolution will suffer.

When pouring the resin in the column, it helps to fill the column approx. halfway w/ water or buffer, then add the resin slurry in stages. When you do it this way, the resin beads will get diluted and dispersed in the buffer, allowing any air bubbles in the resin to float to the surface and escape. If any air bubbles get trapped in the resin in the column, it can hurt resolution.

Something else you might want to do is go on Sigma's web site and present your questions to their Technical Service Department. Since they supply the resin, they should be able to answer your questions. I've done this before. Their Tech Service people are knowledgeable and helpful.

I hope this information helps you.

Bill Colonna, Ames, Iowa, USA [email protected]

PROPERTIES

Show More description HW-50Sproduct line TOYOPEARL®packaging bottle of 250 mLparameter 3 bar max. pressure application(s) LPLC: suitable matrix hydroxylated methacrylate matrix active group polymer phase particle size 20-40 μmpore size 500-20,000 Da MW range operating pH 2 - 13

Related Categories

LPLC Media & Accessories

DESCRIPTION

General description

Toyopearl HW-50 resin is a hydroxylated methacrylic polymer for size exclusion chromatography of proteins between 500-80,000 Da.

Specifications

Clean in place with 0.5 M NaOH or 0.1 M HCl.

Dear W.J. Colonna

I don't know how grateful I am for your kind explanation.

I finished packing the column today. If I had to ask you one more thing, how did you store the remaining resine? At first, 20% ethanol was with resine as a buffer buffer. Can I make 20% ethanol and store it with the resin in the same way?

Thank you so much for your help with the experiment.

have a good day!

When you test column performance, how did you test the packed column? Did you test it like a general SEC column performance test?

12/10/21

Dear Dokyeoung,

I believe that in your first e-mail, you mentioned that the resin came as a suspension in 20% ethanol. This is how it was supplied by the manufacturer, so it is an acceptable way to store the resin for future use. This solution keeps the resin in the "swollen" form so that it is ready for use. The ethanol serves to prevent the resin from being contaminated by microbes, e.g., molds. I have had contamination problems with resins before. If this happens, it is very difficult to remove the contamination, so it is better to discard it. If you prefer not to use ethanol, you can also use an aqueous solution of sodium azide (NaN3) at ~0.02-0.05%. This is another effective antimicrobial.

As I mentioned, I have never used Toyopearl HW-50S. However, I have used other SEC resins (molecular sieves). After preparing the column, I always tested it. I did this by using standards of known molecular weight. In my case, I was working with mono- and oligosaccharides, so I tested the column with a mixture of mono-, di-, tri- and tetrasaccharides. I observed good resolution of the sugars, so I knew that the column was working properly. I would suggest that you do this with your column using a mixture of standards with a suitable molecular weight range.

In the picture you sent, it looks like you are using a column manufactured by Pharmacia (I think they are now owned by General Electric.). They made good columns and different kinds of chromatographic media. I used their products and had good success with them.

From the picture, it looks like you did a good job pouring the resin!

I hope this information helps you. Good luck w/ your research.

Bill Colonna Ames, Iowa, USA [email protected]

12/10/21

Dear Dokyeoung,

I don't know if you have seen this (attachment). It may have been included with the Toyopearl HW-50S resin that you purchased. I got it from Sigma-Aldrich following an inquiry I made to their Technical Services department yesterday. I hope it helps you.

Bill Colonna Ames, Iowa USA [email protected]

Dear W.J. Colonna

Thanks to you, I was able to keep my resin poured and stored well.

Thank you very much.

And, I am trying to test it with the packed column.

I'm planning to do a SEC-UV-OCD setup, but the 3 papers I've seen don't have anything about oven temperature.

I'm not sure how to set the oven temperature for this packed column or not.

Could you possibly get some advice on this?

12/15/21

Dear Dokyeoung,

I'm not sure I understand your question. In doing SEC-OCD, the sample is first resolved into its components based on molecular weight by passage through an SEC column. After this, each component is mixed w/ H3PO4 which oxidizes it, releasing CO2 which is then detected. Are you asking if or how your column should be heated during the separation process? The papers you attached say nothing about heating the HPLC columns they used. The HPLC's that I have used had column heaters. These accepted relatively small columns (e.g., 250-300 mm x 0.8 mm) that could be heated (if necessary) at pre-determined temperatures to improve resolution of components in a mixture. Is this what you are trying to do, i.e., heat your column to improve separation of substances in your sample mixtures? If this is what you are trying to do, I don't see how it can be done. The column in the photo you sent is never going to fit in the column heater of an HPLC, as it is too large. If you are trying to heat your column, there is one way to do it. From the photo, it looks like your column has a water jacket. If so, you can circulate water through the jacket. You could set up a water bath at the desired temperature and pump it through the jacket w/ a peristaltic pump. Before you try this, you might want to test the column @ ambient temperature. You may get acceptable resolution w/o heating.

Have I misunderstood your question? Instead of heating the SEC column, are you asking about the oven temperature on the organic carbon detector? I have used a total organic carbon (TOC) analyzer in the past. This was not coupled to an HPLC. Instead, the sample was injected into the TOC which converted carbon in the sample into CO2. Heat was used in the reactor during the oxidation. I don't recall the temperature, but it was fixed (i.e., not adjustable). Does your device have an oven and can you adjust its temperature, or is it fixed? If it is adjustable, you may need to consult the instrument's manual or contact the manufacturer to find a temperature that will completely oxidize your material. Another option is to use a model compound at several temperatures to determine how much heat is needed to get complete oxidation. With the TOC analyzer I worked with, we used potassium acid phthallate.

I hope this information helps you.

Bill Colonna Ames, Iowa, USA [email protected]

I packed the column and tested whether it was packed with PSSs1.69, 5.58, 8.89, 16.5KDa, but the peak did not come out properly.

TOC STD 1mg/L, 5mg/L, and 10mg/L were prepared by diluting TOC STD (potassium hydrogen phthalate in H2O, 1000mg/L). And acetone was also measured as it was.

The analysis conditions were flow 0.7mg/L, 0.5Mpa, injection volume 300ul, and the temperature was measured without setting. Then, UV was measured using hplc and organic carbon was measured as the eluent. I want to know if the packing is correct, but I don't know.

question 1) Is the concentration of the sample to be analyzed now too low? How good would it be to do it?

question 2) Is the column packing good? If it doesn't work, how can I fix it?

question 3) Is there a better way to test the column?

question 4) What is the problem in the current situation?

question 5) Should TOC STD and acetone be filtered and analyzed?

12/27/21

Dear Dokyeoung,

I'm not sure if I understand what was done and your results.

From looking at the chromatographic profiles, your standards were detectable, so the concentrations seem to be OK. You can try increasing them to be sure.

You ran 3 standards at 1-, 5 & 10 mg/L (ppm). One thing that is disturbing is that the dose responses for the standards are poor. The peaks for the 1- and 10 ppm are nearly the same; the peak for the 5 ppm standard is hardly visible. The peaks for the 5- and 10 ppm standards should be 5- and 10 times that of the 1 ppm standard.

Also, the retention times (RT) of the 3 standards are different. The 1-, 5- and 10 ppm standards are at ~32-, 18- and 12.5 min, respectively. I replotted portions of the 3 chromatograms (See attached.) so that you can see this. In SEC, the RT's are based on molecular wt (MW). Are the molecular weights of the 3 standards the same? If so, they should have the same RT's even if the dose response is inaccurate. It looks like the 3 standards have different WT's, with the 10 ppm at highest MW and the 1 ppm at lowest MW. Acetone is not retained by this resin and would be expected to elute in the void volume (Ve) of the column you used. (Its RT was the same as that of the 10 ppm standard.) Also, any analyte with a MW greater than the exclusion limit of the SEC resin would also elute at the Ve, as is the case of the 10 ppm standard.

The investigators in the 3 references you sent me used TSK HW50S HPLC columns with dimensions of 20 x 250 mm. These were obtained from commercial sources of chromatographic equipment, and have ~3000 theoretical plates. You might have better success if used one of these commercially-available HPLC columns. They might give you better separations than the ToyoPearl resin you are using. Unfortunately, they are expensive.

Let me know what you think.

Bill Colonna Ames, Iowa USA [email protected]

Hi Dokyeoung,

I read your question about Toyopearl HW-50S. Is there any column available that is pre-packed with this resin? I am also looking for an HPLC column with this packing. I am not sure whether I can buy a pre-packed column or I have to pack a column by myself. I appreciate your advice.

1/12/22

Dear Saeideh,

You can try contacting the companies that make/sell HPLC columns, e.g., Bio-Rad, Supelco, Phenomenex, Agilent, etc. and describe to them what you are trying to do. They may sell this column, or an equivalent capable of the separations you want to accomplish. These companies have technical service departments that provide assistance to scientists with inquiries such as yours. I've done this in the past and have found them to be very helpful.

I hope this information helps you.

Bill Colonna Iowa State University, Ames, Iowa [email protected]

Dear W.J. Colonna

As you said, the response to standards is not good.

It seems that packing of the SEC column with Toyopearl hw-50s did not go well.

I don't know why the column packing is not properly done.

We would be grateful if you could share your experience with column packing.

1) Were you able to get good results at once when you did column packing?

2) What factors were considered when packing the column?

3) How was the packed column tested?

Thank you for your answer!

1/30/22

Dear Dokyeong,

The only types of columns that I have prepared myself contained either ion exchange resins or molecular sieves. They were prepared by pouring the resin slurry into glass columns that ranged in size from ~40-100 cm long and with inner diameters of ~2-3 cm.

I have never prepared an HPLC column, but rather used HPLC columns that were purchased from commercial suppliers. To determine if the column would successfully separate and quantitate the components of test samples, I first calibrated the column with standards of known molecular weights and/or concentrations. I separately ran 4-5 standards of known concentration and determined if the peak areas showed dose responses proportional to the amount of the analyte in the standards. That showed that the HPLC's detector could accurately measure increasingly higher amounts of the analyte in question. If I needed to separate analytes of different molecular weights (MW), I would run each analyte through the column individually (i.e., by itself) and determine its retention time (RT). After all of them had been run, it was possible to determine if the RT's followed their MW's, i.e., shorter RT for higher MW. If this occurred, I would then prepare a single solution containing standards with a range of MW's and see if separation of the analytes in the mixture gave the correct RT's. If so, then I knew that the column would separate these components in my unknown.

I hope this information helps you.

Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]