Nikolay Orlov asked: "Is it possible to inject a solution of pure acetone in water?" - ***Possible, yes. Should you do this on a RP column? Most likely NO, not at all.

Acetone (or most any other chemical) should never be injected onto a column undiluted. Samples must first be diluted to a concentration that is appropriate for: the column dimensions used, the detection method selected, and LC analysis method chosen. A good general rule that we teach to our students is to inject the smallest volume possible which allows for good detection signal, without overloading the column. Maximum injection volume should not exceed 3% of the column's void volume. Which volume should you use for your samples? No one can answer this for you. Injection volumes are selected during the method development process. Samples are diluted and dissolved in the mobile phase, then a "loading study" is run to determine the ideal amount. Be sure to select the best detection settings first, using a pure std (if available), before starting.

• Note: Acetone is not normally injected onto a RP column unless it is being used as a tracer for performance testing of gradient valves. It has a VERY strong absorbance in the UV/VIS range so can be injected at extremely low volumes for use in troubleshooting leaks. I do not know why you would want to inject it alone onto your column for sample analysis in water.

Use an unretained void marker first, measuring the column void volume. BTW: A compound such as acrolein is probably going to be very hard to detect, esp by UV (due to chem structure), so be careful you do not overload and damage (plug) the column. It should be easily retained in water though with a good K prime.

Please contact someone local who has professional experience in HPLC operation and analysis. HPLC analysis and especially, method development require many years of professional training in an industrial setting just to learn the basics.

William Letter Thanks for the answer! But I meant something a little different. We analyze air samples, in particular tests for acetone and acrolein content. Unfortunately, these two substances have very similar run times. I can’t understand how I can find out its run time exactly?

That makes no sense at all by HPLC. I suspect you may be looking at no retention at all so both compounds elute at the void volume. If you would share the complete HPLC method used to provide some context, then perhaps some useful suggestions could be offered.

William Letter Airborne aldehydes and ketones are collected by passing air through a cartridge containing 2,4-dinitrophenylhydrazine (DNPH). Carbonyl compounds react with the DNPH to form hydrazones, which are immobilized on the cartridge. These compounds can be eluted from the cartridge with acetonitrile and analyzed by HPLC with UV detection.

We determine the amount of formaldehyde, acetaldehyde, acetone and acrolein. The first peak is formaldehyde, the second is acetaldehyde, but the sequence of acetone and acrolein is not sure due to the close run times. Calibration using DNPH MIX standard.

Nikolay Orlov: Thank you for the additional information. What you are asking is completely different that what you posted. *This is why it is so important to describe your actual method in the initial post as you are not analyzing acrolein and acetone on a C18 column. That is not the case, and explains why your request made no sense at all. You are running a separate derivatization reaction first (DNPH), so the compounds that are later analyzed by HPLC are different than the native compounds. They are derivatized compounds, completely different from the originals.

To solve the problem: You will need to run the derivatized test standards of your compounds (formaldehyde, acetaldehyde, acetone and acrolein) to record the "normal" retention times for derivatized samples, using your system. If your samples do not match up with the actual retention times for the derivatized standards or can not be clearly resolved from each other, then you may have: (1) A fouled HPLC column (replace with a new, clean one); (2) Contaminated or "Changed" mobile phase (IOW: The % are not correct); (3) The sample contains some other compound that elutes near the two compounds resulting in invalid data (maybe from contamination or other species present); (4) Degraded derivatization chemical (DNPH derivatization agent). *Any of these issues can result in peaks which elute off the column at the wrong retention times or over-lap with other peaks, all resulting in an analysis with no usable scientific result. The cause must be found and corrected before any sample analysis can be continued. Most likely you have a fouled column, but it could be any of the other options too.

Contact an experienced chromatographer for help (local) to troubleshoot the steps and solve. Learning how to operate and use an HPLC system takes many years of full time professional training just to achieve a basic level of understanding (> 5 years for "basic" level).

William Letter Thanks for the detailed answer! We changed the gradient in the rest method and injection volume, which is probably why the retention times shifted. The standard mixture was introduced, but to be sure, I want to understand that the sequence of release of the substances will not be changed? How can I probably prepare this without the standard mixture?

For the method to be scientifically valid, it must be unchanged and repeatable. You can not just 'change' the gradient or injection volume at will. Those are variables. You must do the exact same thing, every time, through the collection and analysis (samples & standards). This includes the derivatization reaction, binding on the column, washing off of the bound material and HPLC analysis. Everything must be standardized, done the same exact way 100%, no changes allowed. Establish the method(s) and procedures first. Once the method has been standardized this way, then you can run samples, adding bracketed standards after some # of samples to check that everything is still working the same way. *These are all common principles so please contact an experienced chromatographer for assistance with your project. A web forum is no place for such basic questions as these types of issues can only be reviewed and solved on-site. Good luck.