Hi everyone..I tried to analyse toluene in HPLC for low level at less than 1% by HPLC at 190 nm.I prepared toluene standard by weighing 0.02 g in 25 mL diluted with acetonitrile followed by preparation of 5 standards for linearity at 4,8,18,35 and 50 ppm by vial dilution. I manage to get R square of 0.996.
Using this graph, I manage to determine toluen e content in sample to be at 0.3%.I understand a proper validation is important in order to evaluate the fitness of method for intended analysis and here I only manage to cover linearity.
Before proceeding to other validation parameters, I need to know if it is alright to proceed at this wavelength since I came across mixed response about analysing at wavelength lower than 200 nm.
Can kind souls outside there able to share your view?
I used 50 mm Eclipse plus C18 column with 2.1 mm internal diameter and 6 minute of analysis runtime.
Kanapathy Semaselaun writes; "I tried to analyse toluene in HPLC for low level at less than 1% by HPLC at 190 nm.":
This does not make sense. It appears you may be using HPLC without the years of formal training needed. Basic HPLC method details are needed to determine if the proposed method follows good chromatography principles. *Examples: The HPLC flow rate and particle size for the C18, 2.1 x 50mm column, critical values, and the composition of the proposed sample (besides toluene, what other chemicals may be present?) are needed at minimum.
- Can you share with us the specific goals of your project? Please be specific about the sample types, mixtures, desired information etc...
- Why are you using HPLC for this "measurement" (when GC is a more applicable technique)?
- If by HPLC, why are you using a C18 column when a silica or phenyl column (more specific for aromatic compounds) are more applicable to "Toluene" chromatography?
- Why are you using a NON-SPECIFIC wavelength such as 190nm for Toluene? At 190nm, nearly every compound in solution would be detected, including the mobile phase of ACN vs a wavelength that is selective for Toluene (~ 254nm, 10)? No selectivity plus high noise would result from such a choice. I would not recommend using 190, 200, 210nm or any similar wavelength(s) for such methods as doing so removes any value of using a UV detector (**you should be collecting full scans across an expected range, plus at least one single wavelength at the sample's lambda max).
- Linearity results (and the calibration table constructed) have little value when the proposed method does not follow good chromatography practices. Almost any sample can be injected onto an HPLC column, eluted quickly and measured by UV/VIS to show R2 of ~ 0.99999. By itself, this means nothing and does not demonstrate selectivity or that the method is fit for purpose.
- Does your company employ a professional chromatographer that you can work with to develop an analysis method that will meet the needed goals? If so, please discuss your project with them so you can find a scientifically appropriate method.
Dear William Letter , thanks for your time and valuable output.Regarding formal training, we undergone the training provided by the instrument vendor. But, I would say it was not in-dept. So, we need to learn on own,by finding literature online. But since there is not a single straightforward method, I am not sure which to be relied on.I have included my reply for each of your questions underneath.
- Can you share with us the specific goals of your project? Please be specific about the sample types, mixtures, desired information etc...
Reply: The sample I am testing is an agrochemical sample which uses toluene as a solvent during its synthesis. The purity of sample is 99% and toluene is expected to be present at trace level.
- Why are you using HPLC for this "measurement" (when GC is a more applicable technique)?
Reply:I am working on GC in parallel to HPLC. But I could not achieve the desired linearity and it looks more complicated. I am trying split condition at different ratio and different dilution factor. I can't share that much as it is a long way to go.
When you are saying GC is more applicable technique, are you saying that HPLC is still an applicable technique?
- If by HPLC, why are you using a C18 column when a silica or phenyl column are more applicable to "Toluene" chromatography
Reply:This column is what available with us currently and most of the articles I came across uses C18 column due to its high versatility.May I know why are you suggesting a silica or phenyl column?
- Why are you using a NON-SPECIFIC wavelength such as 190nm for Toluene? At 190nm, nearly every compound in solution would be detected, including the mobile phase of ACN vs a wavelength that is selective for Toluene (~ 254nm, 10)? No selectivity plus high noise would result from such a choice. I would not recommend using 190, 200, 210nm or any similar wavelength(s) for such methods as doing so removes any value of using a UV detector (**you should be collecting full scans across an expected range, plus at least one single wavelength at the sample's lambda max).
Reply:Based on the UV spectra of toluene, the lambda max is less than 200 nm as to why I chose 190 nm. Some agrochemicals like glufosinate ammonium also detected at 195 nm and supplier like Sigma quoted this in the certificate of the reference material.
When I analyse acetonitrile at 190 nm, I only see a small bump at the retention time of toluene as to why I dont see any specificity issue here.
- Linearity results (and the calibration table constructed) have little value when the proposed method does not follow good chromatography practices. Almost any sample can be injected onto an HPLC column, eluted quickly and measured by UV/VIS to show R2 of ~ 0.99999. By itself, this means nothing and does not demonstrate selectivity or that the method is fit for purpose.
Reply:But linearity is the first evaluation parameter when it comes to method validation, isn't?To my understanding, good chromatography practises is determined by the consistency of the retention time of the target analyte througout the runtime.
- Does your company employ a professional chromatographer that you can work with to develop an analysis method that will meet the needed goals? If so, please discuss your project with them so you can find a scientifically appropriate method.
Reply:Obviously my company didn't hire any professional chromatographer otherwise I would not be posting my question here.
Kanapathy Semaselaun wrote: "Regarding formal training, we undergone the training provided by the instrument vendor."
That is not HPLC "training". You can not learn chromatography from participation in a "vendor class" or "on your own". No one can. You can not learn chromatography from a one day, one week or one year long class or from reading a few papers or books. Note: It takes the average scientist at least FIVE years of industrial training, full-time, with an experienced mentor to achieve a BASIC level of proficiency (emphasis on the word BASIC). To learn basic HPLC method development and troubleshooting takes many more years of professional training and experience. To achieve an intermediate level of skill takes even longer. To learn how to use one specific HPLC instrument (all HPLC systems are different and have specific features and design aspects that require training to use properly. This is especially true between brands) and the specific CDS software also requires formal training and time too.
BTW: GC is much easier, but still takes a few years to learn the basics.
What you are doing makes no sense (your replies above make this clear) and does not follow good chromatography practices at all. Your "analysis methods", as described by you, are invalid and will not result in the collection of any valid data. Your explanations are also misguided. At this point in time, you are proceeding to take irrational steps (just look at your earlier RG posts and questions). You have not understood my comments because you do not yet understand the technique. If you wish to analyze samples using scientifically appropriate HPLC (or GC) methods, then please ask your company to hire scientists who have formal industrial training with experience to do the work you need. Simply purchasing a 'tool' such as an HPLC or GC system, does not in any way provide you with a push-button solution or answer to your analysis questions. It does not make you a chromatographer. BTW: Employers need to learn and understand this concept too. The instrument does not come with an operator or the years of training needed to use it. That is extra. Hiring someone out of school with NO experience or formal training to "figure it out" is a novice mistake by many employers. You can not "figure it out on your own". This implies no understanding of the technique or training needed.
BTW: Your statement makes no sense. You wrote: "Reply:Based on the UV spectra of toluene, the lambda max is less than 200 nm as to why I chose 190 nm." - ?????? **** You "chose" 190nm detector wavelength for Toluene? Try again. Look up Toluene or better yet, place a sample of it in a cuvette with ACN as the diluent and run it on a UV spectrophotometer to look at its spectra from 190 to 280nm. I think you will be surprised.
Hi everyone,..Just wanted to share that I manage to quantify the toluene under GC-MS method and surprisingly it is comparable to HPLC analysis I initially tried at 190 nm.
Kanapathy Semaselaun wrote: "Hi everyone,..Just wanted to share that I manage to quantify the toluene under GC-MS method and surprisingly it is comparable to HPLC analysis I initially tried at 190 nm."
- Glad to see that you finally used an appropriate analytical tool (GC-MSD, not HPLC), but that may be due to both "methods" being used, invalid.
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