Chromatography Forum

Hello,
I am trying to develop a method for CAPB using HPLC with ELSD Detection. I am using an Acclaim Surfactant Column, with Methanol and Ammonium Acetate buffer as mobile phase. As diluent i am using a 80:20 mix of Methanol and water. The ELSD parameters i am running are gain =6, temp 40 deg C, flow 1ml/min, with gas pressure not exceeding 3.5bar. However, no mater what ratios i run the MEOH and buffer at there is a huge peak that keeps splitting around 2-3 retention time. Is there any suggestions anyone could give in order to separate the CAPB from the sample. Thanks in advance for your time and consideration.

Hello,

The huge peak may indicate overloading or solvent effect from the sample diluent. When I ran a similar surfactant on the Acclaim Surfactant column, I prepared the standard with 0.1% concentration in D.I. water. The injection volume on a 4.6x150-mm column is 5 to 10 microliters. The method was straightforward for the standard.

If you are dealing with real-life samples, the sample matrix also matters. Because ELSD can "see" everything that is nonvolatile, matrix interference can be significant, such as the presence of salts. Probably what you saw was interference. When you were mentioning the high splitting peak, did you refer to the standard or a real sample?

It would be helpful if you provide the chromatographic conditions, such as mobile phase conditions, flow rate, injection volume. If you prefer, contact me at xiaodong.liu@dionex.com so that I can help you resolve this off line.

Xiaodong

Thank you so much for responding! The sample that I was analyzing was a raw material, which I will eventually use as the standard as well after the method is developed. The concentration of the active is 35%, and using conversions = 350000 ppm= 350000 ug/mL undiluted. I have tried several dilutions trying to determine the limits of linearity ranging from 100ppm to 2000ppm injecting 20uL each time, and neither of the concentrations gave me acceptable chromatography. The colum manual suggest concentrations of no less than 50ppm.

The sample matrix is tricky, it is classified as an amphoteric surfactant, but because of the quaternary ammonium compound on it, it carries a constant positive charge depending on the solution it's in. I'm currently using 50:50;MeOH:DI water as diluent because it's not soluble in Acetonitrile. Mobile phase conditions are still in progress, for this analysis i changed to the following parameters: 90:10; MeOH;NH4OAc buffer (ph=2.5) and pH'd with Acetic Acid to reach pH of 2.5. Flow is 1ml/min, gain=6, elsd temp = 40deg.

I can scan you a copy of my chromatography. THank you sooo much for helping, it's not a lot of literature on this process at all!

I looked through the data on coco amido betaine I ran a few years back. Here is the condition I used:
Column: Acclaim Surfactant 4.6x150-mm, 5-um
Mobile Phase: A - MeCN, B - 0.1 M NH4OAc, pH5
Gradient: 20%A to 80%A in 10 min and keep 80% A for additional 5 min
Flow Rate: 1 mL/min
Temperature: 30 C
Injection: 5-uL
Detection: ELSD (Sedex 85, evapration - 50 C; gain -
Sample: Coco amido bateine (0.2% in D.I. water)

The surfactant was a mixture of several components. It was soluble in water. The overall charge of an amphoteric surfactant is zero. Thus pH5 NH4OAc buffer works well. Here are my suggestions:
1. Dissolve the surfactant in water to make the concentration around 0.1%.
2. Run the gradient method described above. If methanol is preferred, modify the gradient from 15% to 90% methanol in 10 min, then keep at 90% methanol for additional 5 min.
3. Depending on the elution time of all peaks, work on an isocratic method if needed. I would expect the isocratic method should be around MeCN/buffer v/v 45/55.

If there are more components in addition to the surfactant specified, you can use a shallower gradient, like 20% to 85% MeCN in 20 min and keep 85% MeCN for additional 10 min, to screen almost all types of surfactants including cationics, nonionics, amphoterics and anionics. If a Sedex ELSD is used, you can set the Evap. T at 50 C and gain set at 8.

Hope it will help.

Thank you for your reply. Using those above conditions, you were able to get acceptable chromatography? Do you recall the retention time of the peak? I am getting a higher signal than normal around 2-3 minutes, not sure if that's the peak trying to elute or not.

Thanks for the c-gram. It looks that the background noise is very high. It is likely due to dirty mobile phase. The mobile phase needs to be very clean and free of "non-volatiles" such as inorganic ions. Please check the grade of the NH4OAc salt and acetic acid you used. The purity needs to be very good. I am using 99.99+% NH4OAc.

To test system and mobile phase suitability, you can replace the column with a coupler (or a piece of tubing). Let pump run and monitor the background level and noise. Compare the result with that in the manufacturer's manual. Once satisfactory background level and noise are obtained, you can try the chromatographic condition I suggested earlier.

When I used the gradient method before, I observed 3 to 4 sharp peaks ranging from 4 min to 9 min.