By Anonymous on Thursday, November 7, 2002 - 11:32 am:
I'm trying to determine the amount of preservative (MIT and MIT-Cl) left in product at the end of shelf life. I am unable to get good peak seperation with the method I've found, mobile: 50% mehtanol 50% water at 1.3 mL/min, c18 column, 275 nm. My standard seperates nicely but I can't seem to get the peaks of my sample far enough away from everything else that elutes before the MIT. I'm very new to HPLC and wolud welcome any suggestions. I appreciate any help you can offer.
By Anonymous on Thursday, November 7, 2002 - 11:47 am:
Try decreasing the amount of organic in your mobile phase. Try using 60% water and 40% methanol. A rule of thumb in RP chromatorgraphy decreasing the amount of organic will increase the resolution between the two peaks of interest.
Is this a validated method and are you working in a GMP environment? If so, you can't change the method you are using without re-validating it.
Have you tried filtering your sample to get rid of the "junk"?
Hope this helps.
By Anonymous on Thursday, November 7, 2002 - 01:38 pm:
We use the following method for analysis and got just enough retention for MIT to be away from other non-retain compounds. The key is to use longer column. We used 150mm column and was not able to get enough retention for MIT. Try this method.
HPLC column: Waters uBondpack C18, 5 um 3.9 mm x 300 mm
Detector: Waters 996 PDA, 275 nm
Mobile phase: 50/50 methanol/water
By Anonymous on Friday, November 8, 2002 - 12:33 pm:
We do this assay all the time, in many different consumer product matrices including surfactants.
10 cm x 2.1 mm i.d 5u RP-18 column coupled with 3 cm x 2.1 mm i.d. 5u RP-18 guard cartridge
Solvent A = 0.4% acetic acid in H2O, Solvent B = HPLC CH3OH, Flow = 0.25 ml/min, Detector Wavelength = 273 nm, Column temperature = 27 *C, Run time = 17 minutes, Post-time = 5.0 minutes, Injection volume = 10.0 ul
HPLC Pump Gradient Conditions
Time (minutes) Event
Initial value 13% CH3OH
5.0 min. 40% CH3OH
7.0 min. 40% CH3OH
8.0 min. 100% CH3OH
13.8 min. 100% CH3OH
15.0 min. 13% CH3OH
17.0 min. stop run
Post-time 5 minutes for re-equilibration.
With these conditions the retention time for methylisothiazolinone was approximately 3.3 minutes and the retention time for methylchloroisothiazolinone was about 9 minutes.