We are trying to analyse organic acid production from bacteria supernatants by HPLC. We tried some methods that we found in the literature but we are having problems with some organic acids like with lactate and formate, they elute together.
Use Aminex organic acid (BIORAD) column with 0.05M H2SO4 mobile phase at 0.6 ml/min flow on RID detector. You can separate majority of organic acids (Formic,acetic, lactic, succinic, etc) as well as you can measure sugars and ethanol on the same method.
Please open this link, maybe it will help you to separate lactic acid and formic acid:
http://hitachi-hta.com/sites/default/files/appnotes/Organic%20Acids_LC100029_E.pdf
http://www.cfs.gov.hk/english/programme/programme_nifl/files/Analysis_of_Organic_Acids_2370.pdf
Use Aminex organic acid (BIORAD) column with 0.05M H2SO4 mobile phase at 0.6 ml/min flow on RID detector. You can separate majority of organic acids (Formic,acetic, lactic, succinic, etc) as well as you can measure sugars and ethanol on the same method.
Dear Dr. Torres-Fuentes:
Could you please provide us with your detailed method?
Which mobile phase do you use, which column, flow rate, gradient, temperature?
Slightly acid and very polar mobile phase is recommended for efficient separation of your target analytes. Anyway, if you provide us with more details about your methodology, maybe I can help you.
Best,
I know that organic acids come well by ionic chromatography.
After few experiments in HPLC with poor results, we use the GC-MS for fatty acids in water and liquid phase by direct injection and we have good results,
I'm sorry for can not help you.
hello -
As the previous colleague (Shrikant Dhoot) indicated organic acid HPLC columns in the form of H+ (reticulation 8%) can do the job. For better separation use 2 columns in line. Check with Phenomenex company. They make Rezex columns very good for these kind of separations. Supelco columns are equally good. It is just a question of price.
Hi all,
Thanks for all your answers and suggestions!
I will look at all of them in detail.
Regarding details of the method that we use:
- Column: 300 mmx 7.8 mm cation exchange column (Aminex HPX-87H)
- Mobile phase: 0.009 N H2SO4
- 0.7 mL/min at 65C
- UV detection at 220 nm
Dear Cristina:
From my point of view your column is a bit long. As your compounds are small (with low medium polarizability), a medium backpressure (or even low backpressure) will allow you higher retention times and most likely, a better separation.
For achieving a low backpressure, you'd better choose a shorter column (about 20 cm length) or the same lenght but with longer I.D. (about 8 mm maybe it is enough).
How about the particle size? Maybe you can diminish the backpressure by selecting a bit bigger size particle, without changing the dimensions of your column. By decreasing the temperature, you can also retard the elution of your analytes providing the backpressure is not too high.
On the other hand, maybe anion exchange will be more efficient than cationic exchange chromatography for separating organic acids due to anions are retained longer than cations onto analytical columns. In this case your analyte should be in anionic form, of course. This is easy to get by adjusting pH.
http://www.dionex.com/en-us/products/columns/ic-rfic/hydroxide-selective-packed/ionpac-as11hc-4um/lp-111520.html
Best,
The pH of your eluent (0.0045 M sulphuric acid) I calculate to be 2.24 (remember that the activity coefficient is about 0.64). Lactic and formic acids both have pKa values around 3.8, so in the conditions you're using, they are almost completely protonated. I think you need to use a column which will retain them somewhat - an anion-exchanger at higher pH (at least 5, 6 might be better) is probably easiest but there are other methods, as correspondents such as Gunawan Indrayanto and Shrikant Dhoot have suggested.
Dear Aurea and David,
Many thanks for your replies. Both really helpful. I will look at your suggestions and see if I sort out the problem,
Kind regards
Cristina
As you already know, the column and conditions you are using should be appropriate for separating formic and lactic acids. This is shown in the literature at http://www.bio-rad.com/LifeScience/pdf/Bulletin_1928.pdf, page 12. Since you are not obtaining the separation, you may have a defective column. I have used these columns and found them very reproducible and robust if operated using the manufacturer's suggested conditions and if your samples are free of interfering matrix. I would contact the column manufacturer for advice and possible replacement of the column. The first reference by Gunawan above also provides a good alternative.
You can also try Ion Chromatographic method. Using an anion exchange column and a weak eluent like borate it could be possible to separate close peaks.
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