I'm trying to isolate keto acids from a matrix, and was recommended to use a special SPE filter for the job by both a mentor and a Waters representative. Problem is, I am unsure how to justify its necessity vs. purification via hplc or other chromatographic methods.
Why is SPE filtering preferred?
Edit: Thanks for the answers. I want to point out that I have access to and in fact use hplc quite a bit. I suppose my question is more along the lines of necessity of the SPE filter when I have access to hplc and fplc; if I have to use organic solvents for washing with the SPE filter (which seem to result in salts), what is the benefit of buying SPE when I can just purify with hplc and desalt with fplc before entering the sample(s) into hplc/LC-MS/MS? It just seems like a steep learning curve for something I can already do, but I want to make sure I'm not missing something. Thanks.
Dear Rex,
In addition to the comment provided by Wahab, Some SPE cartridges have a filtration disk bottom of it. For such occasion, it is not requited additional filtration before HPLC injection. However, some SPE eluent, additional cleanup step is require to remove interfering compounds.
Piyal Aravinna
Dear Rex,
I have some experience regarding SPE. Actually SPE is act as selective tool and it helps to avoid the metrix effect in some extent. Best wishes for your work.
A SPE cartridge is basically a disposable HPLC column. It is designed to be used and abused then thrown out. It has a filter but also contains resin containing a functional group or providing a charge that retains the sample. You pick a preparative column stationary phase much the same way you pick the SPE cartridge stationary phase. This allows for a cleanup and or concentrating of the sample at minimal cost. The downside to SPE is it retains everything in the sample that has similar retention characteristics to your analyte. Even if you are looking to purify the sample to point of collecting one analyte in a sample you probably should use SPE to remove the matrix.
Michael Toner , M. Farooq Wahab
thanks. I did add that I have anytime-access to hplc/fplc, so the larger cost for me would actually be in procuring this SPE filter. I also have a SAX column, which I have used for purification before. I just don't understand the benefit of the SPE filter if I can purify with SAX on hplc/desalt with fplc before mass spec (besides maybe having 1 step instead of 2, but I'm not even sure with all the organic washing if the SPE won't result in salts, anyhow) and the learning curve for all of this SPE equipment seems steep.
Dear Rex,
As I have mentioned at the beginning, in addition to the comment provided by others, Some SPE cartridges have a filtration disk bottom of it. For such occasion, it is not requited additional filtration before HPLC injection specially for the suspended particles . However, For some SPE eluent, it is required additional cleanup step to remove interfering compounds.
Otherwise you have to use strong mobile phases to remove those from the column.
Piyal Aravinna
It is best to perform your work based upon the advice of your mentor and worry about modifying those type of procedures later when you are in the position to make such decisions. Trainees listen and do. Scientists decide what to do and do it.
SPE is a sample preparation tech. If your sample have matrix, you may use SPE for cleanup steps.
Hey Kiko,
the SPE technique is only a fast clean-up technique. This reduces only the number of compounds which will be injected later on the analytical column.The cartidged are much lower expensive than HPLC-columns. If you have no problems with your matrix, which could overload or irreversible change the HPLC-column it is of cause the best way to have a seperate HPLC for clean-up. The SAX-column is a ion-exchanger which seperate the anions from the other compounds. This could also be done by SPE without HPLC-system, only to understand the differences.
SPE is something more than a fast cleanup step. It allows you to concentrate the sample to levels that are compatible with the instrument technique you are using as well as removing problem components in the sample matrix. It can also take quite a bit of time since there are multiple steps involved (at least four) and high flow rates can result in reduced wetting or rinsing of the resin as well as retention and elution of the analyte. It does require some method development as the default methods recommended by the vendors are not necessarily adequate for your sample.
Michael Toner w.r.t. high flow rate during SPE, would you recommend gravity filtration over using a vacuum manifold for the reasons you mentioned?
We always used a vacuum manifold simply because we do multiple samples simultaneously. Whether or not we use a vacuum pump is another matter. if all you are doing is eliminating matrix interferences a vacuum makes quick work of the samples. It also can ensure that each sample has the same amount of elution time on the column.
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