surprising impact of temperature on N

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

6 posts Page 1 of 1
Hi folks

recently I tried running a reversed phase isocratic separation (8% organic) at higher temperatures to scope the effects on the separation. To my surprise I found that plate number decreased from 12000 to 7000 in response to raising the temperature from 30 - 45C. Mobile phase was water:ACN 92:8 with 0.1% H3PO4. The coulumn was an Agilent Zorbax Eclipse Plus with 5um particles.

This change seemed drastic to me. Why could this be?
What are the column dimensions and flow rate? What is the analyte? What are the retention times at 30 and 45 C? Is the data acquisition frequency sufficiently high?

If you work at the B-term dominated region of the van Deemter dependency, the plate height increases (N decreases) when the temperature inсreases.

Another explanation: the analyte retention factor (k) drops when the temperature is raised; consequently, the extra-column contribution to the band broadening becomes more important. There can also be an intrinsic (unrelated to the extra-column effects) retention factor dependency of the plate height (and N = L/H) resulting in smaller N at smaller k. Some analyte specific effects may also explain your observations.
After temperature control for HPLC column compartments was first developed, we started running column compartments a little higher than ambient (say 30C or 35C) to have controlled/same temperature for our automated assays 24/7.

Once such temperature controllers could also cool a column in addition to heating, if we had need for improved separation we would commonly try temperatures both lower and higher and document whether separation was better/worse/same. This was an easy way to adjust a single condition, and simpler than modifying mobile phase, column, etc., in the development stage.

Our manufacturing QC had same instruments as us, so their QC could be set up exactly the same.
Hi there,
in addition to the other comments. Is the change in temperature changing the pH of the mobile phase sufficiently to have any effect on your analyte properties.
Kevin
I remember 10-15 years ago looking through the Agilent catalog and they had one set of chromatograms for a Zorbax column where they showed the separation of about 12 analytes at maybe 5 different temperatures. It was surprising how several of the peaks actually changed order as the temperature changed. Temperature in LC affects each analyte differently, and is much less predictable than temperature change in GC from what I have seen.

In EPA Method 535 looking at ethanesulfonic acid (ESA) and oxanilic acid (OA) degradation products of acetanilide/acetamide herbicides, I found that running the analysis at 35-40C gave super wide/flat peaks but increasing the temperature to 65C gave nice sharp peaks. Other analytes will have just the opposite effect.
The past is there to guide us into the future, not to dwell in.
Hi folks

thank you for the replies. Unfortunately I cannot receive emails from chromforum, so I only looked at the forum again now.
The dimensions are 150x4.6mmx5um, vmu. The retention factors are about 3 at 30C and 2.5 at 45C – not a drastic change and k is not getting really low. Data acquisition frequency is definitely high enough. 10 Hz for a 6s FWHM peak. The peaks are symmetrical in both conditions.

The pH is 2.5 units below the pKa of the analyte, adipic acid, so I don’t think there can be a large enough change.

I don’t think I am in the B-term dominated region because I am running the 5um column rather fast at 1.5 mL/min (~3mm/s).

That all being said, we just went back to 35C and all is good now. We were just interested why the effect was observed.
6 posts Page 1 of 1

Who is online

In total there are 2 users online :: 1 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: Google [Bot] and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry