Chromatography Forum

Hello there...

friends, I am trying to analyse formic, acetic, propionic and butyric acids on a HPLC/PDA... mobile phase is acetonitrile/water 70%/30% 0.7ml/min.

The problem is:

While concentrated, they absorb (greatly) at 204nm, the peaks look great.
but when diluted they absorb (poorly but do) at 195 nm. Peaks are really messed etc.

Any advice on what to do?

Thanks

Which type of column are you using (RP or HILIC? column size?)?
What is the pH of the mobile phase?
What are the concentrations for both "diluted" and "concentrated" samples?
What are retention times under your current condition?

All four acids can be detected at 210 nm without problem at 50 ppm level (for 5-uL injection).

Which type of column are you using (RP or HILIC? column size?)?
RP column, it's a C18, 25cm x 4.6mm.

What is the pH of the mobile phase?
I don't know, all I can say is that I am using HPLC grade acetonitrile and ultra-pure water.

What are the concentrations for both "diluted" and "concentrated" samples?
max = 11000 mg/L and min = 10 mg/L

What are retention times under your current condition?
about 3.55 min

All four acids can be detected at 210 nm without problem at 50 ppm level (for 5-uL injection).
I am injecting 10 uL.

You are using a RP column, thus you need to control the mobile phase to be acidic, say pH 2.5 to 3.0. This way all acids of the interest are kept in their neutral form to enhance the RP retention.

The sample diluent should be highly aqueous, preferrablyin mobile phase.

You need an aqueous compatible column rather than a regular C18 column for this application to ensure reproducible result. Regualr C18 columns suffer phase-collapse or de-wetting under highly aqeuous conditions.

Please see Figure 6 in the attached link for the separation of the four acids using the Acclaim Organic Acid column (http://www.dionex.com/en-us/webdocs/259 ... OA_V22.pdf), FYI.

Use the parameters below.


7.1 HPLC Conditions:
Component Parameter
Equipment Shimadzu High Pressure Liquid Chromatograph or equivalent
Column C18 (Atlantis T3, 5µm, 4,6 mm x 25 cm column) or equivalent.
Temperature range 25 to 30 °C
Flow rate 1 ml per minute.
Injection volume 20 µl
Wavelength 210 nm
Relative Retention Times
Tartaric Acid : 1,0
Citrates : 3,0
Run time 15 Minutes






7.2 Mobile phase preparation (20mM NaH2PO4)
Dissolve 3,12 g of Sodium Dihydrogen Orthophosphate in 1000 ml of purified water and adjust the pH with phosphoric acid to a pH of 2,7. Pass through a 0,45 micron membrane filter and degas.

7.3 Standard preparation
Tartaric acid, Citric acid and Sodium Citrate standard solution
Accurately weigh 700 mg of citric acid working standard, 613 mg of sodium citrate working standard and 850 mg of tartaric acid working standard into a 1000 ml volumetric flask, dissolve in and dilute to volume with purified water. Dilute 10,0 ml to 100,0 ml with purified water.

7.4 Sample preparation
Transfer the contents of one sachet, accurately weighed, into a 1000 ml volumetric flask, add 50 ml of purified water to dissolve the powder and then dilute to volume with purified water. Dilute 10,0 ml to 100,0 ml with purified water.

thank you very much