In order to better understand your method, please provide us with the detailed column dimensions (Length x ID, particle size), the names of the solvent you are using in the mobile phase (co-solvent), the injection volume, all detection settings (e.g. wavelength/bandwidth) and state what your method's Tzero time is?

Now, just based on the initial info, here are a few general comments:

• 50% co-solvent concentration in SFC is VERY high for most SFC applications. *You may need to reduce this concentration, but we need to know what your actual method and sample are first. You also may be changing the concentration of co-solvent far too fast for the method (again, until we know what the column dimensions are and the actual conditions used, we can not say for sure).
• Peak "spiting" (I think you meant to write, Peak SPLITTING). This is often caused by sample overload or co-elution. Please review this linked article for more information, "HPLC Peak Splitting. Common Reasons For It".

http://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html

Dear Mr. Bill Letter,

First of all thank you for providing this information.

We are trying to screen some compounds in one single method so we tried a liner gradient. I used Viridis Silica column (150mm, 4.6 mm, 5um) which is plain silica column. I also tried to reduce the concentration but it is not effective. and we have PDA detector and we are scanning 200 nm to 400 nm. As co solvent i used 0.1% dea in Methanol. The retaliation time of my compound is 4.56 min.

I separated 10 molecules in this method and only one peak is splitting.

A few comments.

You did not include your injection volume or the injection solvent used and this info would be very useful to know (as it sounds like you may be overloading the column). Choice of inj solvent alone can result in splitting (as noted in the article I posted).

If you are experiencing detection issues and trying to solve them by overloading the column, then you may want to consider derivatizing the sample(s) or using a detector which is based on different chemical or physical properties (e.g. MS). Column overload will lead to poor peak shape which in turn leads to poor RSD and column fouling. If the detection issue is a minor issue, then sometimes just optimizing the flow path, detection settings and sampling rate can improve the results.

Based on the column dimensions and flow rate supplied, the compound eluting at 4.56 minutes is well retained (K prime ~ 9).

Amines are sometimes tricky on bare silica. Tailing is common. Ionization is often problematic too. They like to stick to the silica. Anionic ion-pairing methods on bonded phases or HILIC techniques on silica are often used (is your sample soluble in ACN?) with very good results. Why have you chosen SFC to separate them on instead of HPLC?

Can you include a chromatogram with answers to the above questions?

Dear sir,

I injected 20 ul sample and sample concentration is 1 mg/ml.

I used Methanol in sample preparation. sample pKa value is 7.9

my objective is to make a short screen method in SFC and want to show comparison between SFC and HPLC.

Thank you for the extra info. So, now we know that detection is not an issue at all and the sample concentration is high, but should be OK on-column. Here are some more comments:

With this method, you would not be able to directly compare the two methods (SFC vs HPLC) because the "HPLC" version would use a very different method. The comparison would instead be of two very different methods and techniques.

To determine if the peak splitting is due to overload, poor ionization or co-elution, try injecting 4 ul (or 2 ul, if your injector permits it) and compare the peak shape to the 20 ul injection to see if there is any change in peak shape. Since you are using a base additive in essentially pure methanol, pH will be a problem. The CO2 may also be acidifiying the solution too. All of these things may contribute to a poor quality method.

Ionization issues could cause the peak shape to look like that. This is one of the reasons we sometimes use a different column/method (RP or HILIC) and mobile phase to resolve them.

Since retention is good on silica, you may want to change your gradient method to incorporate a different co-solvent within the modifier (i.e. Ethanol;1-Butanol; 2-Propanol; 2-Butanol and even Acetonitrile). I have used many of these either with Methanol or alone, in place of it in thousands of SFC methods to improve resolution and peak shape. You will just have to try a few and see what happens.

Dear sir,

Is it preferable to use reverse phase C18 column in SFC?

can u provide a name of column which can i use?

Pranav: You stated that you wanted to compare "HPLC to SFC" for your sample. I mentioned RP columns in my response when I refereed to conventional HPLC amine methods, not SFC. If you just wanted to analyze amines, then you could easily do so using RP HPLC. Since your mobile phases will be different with HPLC/SFC, you will not use the same method (SFC uses CO2, HPLC does not). Please review the fundamentals of SFC to gain a better understanding of how this technique differs from HPLC.

Please give some of the provided suggestions regarding reducing the injection volume by a factor of 10 and also adjusting your modifier solvent a try.