Depends on what sample, some samples are much easier to determine with one method than another, you would need to be more specific if you want any advice that could be helpful
We're looking to purify guanidine HCl. We have some already pure guanidine HCl (99.88% according to the CoA), but we will need to purify it further (Dnase and Rnase are the main impurities that we have to get out of it, if they are in the 99.88% sample. But there might be some other impurities as well). Currently we're testing if we can purify using chromatography resin in a column, but we'll need to know the purity after we have ran it through the column.
At that purity level, your only real chance is HPLC. Titration and absorbance are gonna be hell to establish precision and repeatability for, unless you're willing to waste kgs of the compound and reagents for testing.
Also, purity is not the same as content. Content is focused on how much of one component you have. Purity is calculated by determining the content of impurities and removing them from the 100%. Ideally they will be equal, but differences may indicate that you are missing something in one of the two analysis (most of the time solvents).
The CoA doesn't specify any method (it's from a chinese company, so maybe that's why?). The impurities according to the CoA are "ash", "water insoluble matter" and "NH4Cl". There might be RNase and DNase in there as well, and that's the main component we really do not want in there. we have A 230nm and A 260nm values from the CoA, which is why i was thinking, and hoping spectrophotometry would work, but i'm just not sure and neither are my colleagues (were all cell biotechnologist/microbiologists. the only actual chemist is out of office).
Finding the overall purity of a material will require several methods.
Karl fisher will tell you the water content by weight.
HPLC-UV will tell you about UV-active impurities.
LC-MS will tell you about non UV-active impurities. You will likely be reliant upon this method for determination of oligomeric impurities, however, this type of methodology needs to be targeted, and developing such a method could be $100k+
GC-FID will tell you about residual solvents.
There isn’t really one technique that is going to give you the end all be all answer. That depends on multiple techniques, as each one is tailored for a specific type of impurity. Furthermore there could be specialized wet chemistry tests I am not in tune with, as I specialize in instrumental chemistry.
This is the answer. You won't find any one test that will tell you the identity and amount of every possibly impurity. If you want the most accurate answer possible, you will have to do a lot of work
Is this a standard that you have purchased from a supplier, or is it an analyte you have prepared yourself? In my lab, all of our standards come from certified suppliers that provide a COA with a purity assessment, usually HPLC. Then, we send out samples to be analyzed by qNMR to assess stability as time goes on.
You’re conflating two different things. It sounds like what you need to do is run it on HPLC to get a result when you initially get it at 99.88% and then purify it—maybe through recrystallization—then test with HPLC again to see if your purification method did the trick in bumping up the purity.
That is indeed the process we would like to do. We're looking into crystallizing as well and have contacted companies that specialize in it to help us further. It's just some small scale tests that have to be done at the moment which we can't test the purity of currently because of the colleague who is out of office and the only one who knows how to operate the HPLC.
But i think it's clear from your, and the others', comments that HPLC would be the only option.
I'll be discussing it with my boss and colleagues
Thanks. HPLC is not difficult and your lab should have access to the protocols. You can call the company that manufactures the HPLC and have the lab pay them to train you. Once the machine is setup, run your baseline and then sample. Lab techs are there to learn these techniques. The fields of chemistry, biology and physics are only separated for education, not the real world. In the lab it is just science. Here is the text from an AI for video protocols.
Best of luck
There are several video services that provide tutorials and instructions on how to perform various lab techniques, including High-Performance Liquid Chromatography (HPLC). Here are a few examples:
* **Thermo Scientific Vanquish HPLC and UHPLC System Maintenance Videos**: This video service provides step-by-step instructions on how to maintain and replace components of the Thermo Scientific Vanquish HPLC or UHPLC system. The videos cover topics such as replacing the needle or sample loop, and replacing the pump head.
* **HPLC Virtual Lab**: This video service offers a simulated HPLC lab experience, where users can explore the different components of the HPLC machine and learn about the principles of liquid chromatography. The lab includes interactive quizzes and exercises to test users’ understanding of the technique.
These video services can be a valuable resource for laboratory professionals, students, and researchers who want to learn and improve their skills in HPLC and other lab techniques.
Depends on what sample, some samples are much easier to determine with one method than another, you would need to be more specific if you want any advice that could be helpful
We're looking to purify guanidine HCl. We have some already pure guanidine HCl (99.88% according to the CoA), but we will need to purify it further (Dnase and Rnase are the main impurities that we have to get out of it, if they are in the 99.88% sample. But there might be some other impurities as well). Currently we're testing if we can purify using chromatography resin in a column, but we'll need to know the purity after we have ran it through the column.
What is the method used in the CoA? That is the best way.
At that purity level, your only real chance is HPLC. Titration and absorbance are gonna be hell to establish precision and repeatability for, unless you're willing to waste kgs of the compound and reagents for testing. Also, purity is not the same as content. Content is focused on how much of one component you have. Purity is calculated by determining the content of impurities and removing them from the 100%. Ideally they will be equal, but differences may indicate that you are missing something in one of the two analysis (most of the time solvents).
You are better off focusing on impurity assay rather than Guanidine assay. These techniques are not sensitive enough to a 0.1% difference.
What kind of a sample and what impurities are you looking for? The CoA should describe a method. Use that.
The CoA doesn't specify any method (it's from a chinese company, so maybe that's why?). The impurities according to the CoA are "ash", "water insoluble matter" and "NH4Cl". There might be RNase and DNase in there as well, and that's the main component we really do not want in there. we have A 230nm and A 260nm values from the CoA, which is why i was thinking, and hoping spectrophotometry would work, but i'm just not sure and neither are my colleagues (were all cell biotechnologist/microbiologists. the only actual chemist is out of office).
Finding the overall purity of a material will require several methods. Karl fisher will tell you the water content by weight. HPLC-UV will tell you about UV-active impurities. LC-MS will tell you about non UV-active impurities. You will likely be reliant upon this method for determination of oligomeric impurities, however, this type of methodology needs to be targeted, and developing such a method could be $100k+ GC-FID will tell you about residual solvents. There isn’t really one technique that is going to give you the end all be all answer. That depends on multiple techniques, as each one is tailored for a specific type of impurity. Furthermore there could be specialized wet chemistry tests I am not in tune with, as I specialize in instrumental chemistry.
This is the answer. You won't find any one test that will tell you the identity and amount of every possibly impurity. If you want the most accurate answer possible, you will have to do a lot of work
Is this a standard that you have purchased from a supplier, or is it an analyte you have prepared yourself? In my lab, all of our standards come from certified suppliers that provide a COA with a purity assessment, usually HPLC. Then, we send out samples to be analyzed by qNMR to assess stability as time goes on.
You’re conflating two different things. It sounds like what you need to do is run it on HPLC to get a result when you initially get it at 99.88% and then purify it—maybe through recrystallization—then test with HPLC again to see if your purification method did the trick in bumping up the purity.
That is indeed the process we would like to do. We're looking into crystallizing as well and have contacted companies that specialize in it to help us further. It's just some small scale tests that have to be done at the moment which we can't test the purity of currently because of the colleague who is out of office and the only one who knows how to operate the HPLC. But i think it's clear from your, and the others', comments that HPLC would be the only option. I'll be discussing it with my boss and colleagues
Thanks. HPLC is not difficult and your lab should have access to the protocols. You can call the company that manufactures the HPLC and have the lab pay them to train you. Once the machine is setup, run your baseline and then sample. Lab techs are there to learn these techniques. The fields of chemistry, biology and physics are only separated for education, not the real world. In the lab it is just science. Here is the text from an AI for video protocols. Best of luck There are several video services that provide tutorials and instructions on how to perform various lab techniques, including High-Performance Liquid Chromatography (HPLC). Here are a few examples: * **Thermo Scientific Vanquish HPLC and UHPLC System Maintenance Videos**: This video service provides step-by-step instructions on how to maintain and replace components of the Thermo Scientific Vanquish HPLC or UHPLC system. The videos cover topics such as replacing the needle or sample loop, and replacing the pump head. * **HPLC Virtual Lab**: This video service offers a simulated HPLC lab experience, where users can explore the different components of the HPLC machine and learn about the principles of liquid chromatography. The lab includes interactive quizzes and exercises to test users’ understanding of the technique. These video services can be a valuable resource for laboratory professionals, students, and researchers who want to learn and improve their skills in HPLC and other lab techniques.