Hey Everyone
I am having trouble collecting my sample from HPLC due to it's very short retention time of under two minutes. The way the machine is set up, it cannot be collected this early (despite many attempts at changing the gradient, set up, cut off etc.)
It is a water-soluble peptide (dissolved in water with 0.1% TFA) and the gradient is 0-100 water to ACN in 0.1% TFA over 30 minutes.
Has anyone here any advice on what I could try?
Any answers would be appreciated!
It sounds like the peptide is 'very' polar, so you might try another column, perhaps HILIC.
Hi Michelle
is your column a reversed phase?
if so, i would try isocratic elution first, folloewd by a dradient to clean your column , but not 100% organic, maybe 90
If that does not help, try normal phase columns before hilic,
Hi Michelle.
Bruce and Leonard are correct. You could try change your column for a HILIC if is avaible in your Lab or change your column for one more polar.
You could use a gradient with more time in polar solvent and use gradient curve in your HPLC pump. If you dont use mass spectrometer detector you could change TFA for another ion pair in mobile phase.
I hope this help you.
The retention time suggests the compound is eluting very close to the void volume, if not at the void.
What did the analytical HPLC look like?
How are you dissolving your peptide? If it is being dissolved in DMSO, try dissolving it in water since DMSO is a "stronger" solvent than water, and can cause the peptide to run down the column more quickly. This is very much a problem with compounds that elute early from C18. The attached images show an example. 462-51-E is a run with a sample dissolved in DMSO, 2.5 mL injected. 462-51-G is the same compound, dissolved in water at the same concentration. The sample dissolved in water gave good resolution with a much higher load. The "shark-fin" peaks indicate an over-loaded column which is fine for preparative HPLC.
What sort of column are you using? If you are using a regular C18 column, it may be subject to "phase collapse" at 0% acetonitrile (as mentioned above). The phase-collapse reduces the interaction of your compound with the column. Try using a C18Aq type column since this has end-capping that reduces phase collapse and allows running in 100% water. Alternatively, try using a C18 without end-capping; the unreacted silanols are also quite polar and help to resist phase collapse as well.
HILIC, as mentioned above, may work. It is a type of normal phase, and was called "aqueous normal phase". Good HILIC phases include bare silica, alumina, and diol.
An ion exchange resin may work for you too, as a water soluble peptide is either small or has a number of other groups to make it more polar.
Thank you so much for all of your answers!!
This has really helped me and I will get back to you all after I have spoken to my supervisor! I think the first (and easiest) thing to do would be to change my mobile phase A to 95% water 5% ACN with 0.1% TFA
As for the isocratic elution idea, did you mean in 95% Water 5% ACN?
The analyte is eluting under 2 minutes so yes during void.
Again, thank you all so much!
HILIC is a 'specialized' form of HPLC analysis which is for very water 'loving' compounds like your amino acid (which usually elute in the solvent front). Attached is some information;
http://www.chromatographyonline.com/separation-20-amino-acids-polymer-based-amino-hilic-column
http://www.waters.com/waters/library.htm?cid=511436&lid=10129985&locale=en_MY
Here is another HILIC column. In short, what works for your application.
http://www.restek.com/Landing-Pages/Raptor-SPP-Core-Shell-LC-Columns
i am having the same problem of retention time where my sample is eluting within 4 minutes and its natural product that am working on it i have have tried to use gradient method like A 95% and B 5% but am not getting the nice peak that i can use to identify the compound present in my extract, as you help Michelle please also help me so that i get one pure compound from my extract
Happy Nyirongo
What sort of column? We assume C18, but if you are using another column, our answers won't be useful.
What are your A and B solvents? Flow rate? Column size? Analytical or prep? What did you use to dissolve your sample? Are you using modifiers such as trifluoroacetic, formic, or acetic acid?
Some of my prep runs elute the sample in 4-6 minutes, so elution at 4 minutes may not be bad.
If you use a C18AQ type column, you might be able to run at a lower %B to start.
Am using C18 colum and and the solvent that am using is 0.1% ammonian in water and 100% methanol. my A is o.1% ammonia and i have also used 0.1% formic acid and my B is 100% methanol. my flow rate is 1ml/minute and the colum size is 250* 4.6mm and i used methanol to to dissolve my sample. and also how do i separate my sample that i have collected from the HPLC where i have used the above solvent system to separate on the HPLC?
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