Hi friends,
I have a problem with HPLC analysis. I am using C18 column for isoquinoline extract analysis. Retention time of my standard sample is around 4.542, which is 4.595 at plant sample. But for the tissue culture samples (cell suspension) the peak with expected amount has a retention time at 5.4s. I am having a peak at between 4.542 and 4.595 but it is accepted as absent at reports. By the way, 5.4s peak does not exist at blank and plant samples. What should I do? Can I accept the band as my substrate?
Please provide us with the column name and dimensions (FULL name and measurements) and the actual detailed method (flow, %composition, pH, inj vol, detection signals, sample concentration etc). Your post is unclear in a few areas so more information would be very helpful (e.g. you report times with an 's' in them, which is probably a '5'?). You report two different times for your std.
QUESTIONS:
- What sample clean up steps have you taken with your cell suspension? Please provide details.
- What is the Tzero of your column and what is the K prime of your std using your HPLC method (basic parameters which should be known)?
Please clarify:
- Your std elutes at 4.542 minutes (is this correct?).
- Your sample elutes at 5.455 minutes (is this correct?).
Note: If your sample is still in matrix, then injecting it onto the column may result in a different retention time than a pure standard (because it may be bound to other compounds). Your method may not be complete or ready for use yet (but we still do not know what your method is). Contact someone local with experience in HPLC to help you. This is a training issue.
Dear William Letter,
My Column is Poroshell 120 , C18.
MPa= 1ml TFA in 1L H2O (Gradient 20/80)
MPb= 1ml TFA in 1L acetonitrile
Flow rate is : 1.5 ml/min
Inj. amount: 10ul
WL: 288nm
My control sample is cultivated plant tissue. Examination samples are plant cell suspension cultures. And my standart is 1000 ppm HPLC grade alkaloid sample purchased from Sigma.
Extraction protocol is very simple.
300mg freezedried samples are shaked with 50 ml of %10 acetic acid(in h2o) for 60 mins at RT. Filtered throught 0.45 microns syringe filter. No additional purifying steps.
Cell suspension culture is pre-desalted from ms media by PBS washing followed by rinsing with water.
My std elutes at 4.542 it is correct. Plant tissue extract elutes at 4.595. Cell suspension culture tissue elutes at 5.455.
Please provide your column dimensions. Length x ID x particle Size.
Also: Please provide the name of the standard (" my standart is 1000 ppm HPLC grade alkaloid sample").
Also, what is the pH of your mobile phase? pH is EXTREMELY important in plant alkaloid analysis. You wrote that you were using a solution of 0.1% TFA, which is an extremely strong acid and not a buffer. That alone may cause all kinds of problems with your method.
i guess you addition your sampel (cell suspension) with standard isoquinoline. if target peak in your sampel (cell suspension) become largest/highest, so that peak (5.4 min) is your target coumpond in your sampel (cell suspension). but if after addition standard isoquinoline in your sampel (cell suspension) having peak in 4.595 & 5.4, so 4.595 is isoquinoline & 5.4 is not isoquinoline.
sorry, i correct my first answer
I also agree with above suggestions. I think it is matrix effect, so put emphasis on clean up process and please monitor the pressure also!
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