Chromatography Forum

Hello dear all,

I would like to perform the metot given in the link.

http://www.hamiltoncompany.com/HPLC/app ... ?appID=306

Does anybody have an idea about this metot.
According to metot he pH of eluent should be adjusted to 8.9. Without any adjustment the pH at around 3.
I should increse the pH level.
Since I dont have much info about chemistry of chromatografy, I dont know which base I should use. Does it matter?
is it OK to use any base such as KOH, NaOH?
I neede your valuable suggestion
THanks

Yes, please follow this method, out of my experience it will work.

Yes, please follow this method, out of my experience it will work.

What is your suggestion for pH adjustment?

I use the PRP-X110S which is essentially the same chemistry for the column. We adjust the pH with NaOH, we use a mobile phase composed of 5 mM NaOH with 4 mM para-hydroxybenzoic acid, 0.1 mM NaSCN with 2.5% MeOH. Haven't had any problems with this column, much more inexpensive than others and so far it's very robust with the amount of abuse we've put it through.

Thank yolu very much

I recommend preparing this mobile phase as a concentrated stock (adjust pH top 8.5 with good grade NaOH or KOH), stopper and store at 4 C. Dilute to 1x daily since 4 mM is not much of a buffer. (The pH will drop over short time). The addition of 0.1 mM NaSCN is also a good idea as it really sharpens peak shape mostly for more hydrophobic anions (Br-, NO3,. You can use a UV detector and monitor negative peaks between 305-310 nm if you don't have a conductivity detector.

Please feel free to contact me at HPLC@hamiltoncompany.com for any assistance.

DJ

Can NaCN be used instead of NaSCN?
B.R.

Can NaCN be used instead of NaSCN?

No.

If you remember, 2 months ago you advised me to use NaSCN for anion measurments by indirect uv.
Firs of all I would like to thank for your help.

"I recommend preparing this mobile phase as a concentrated stock (adjust pH top 8.5 with good grade NaOH or KOH), stopper and store at 4 C. Dilute to 1x daily since 4 mM is not much of a buffer. (The pH will drop over short time). The addition of 0.1 mM NaSCN is also a good idea as it really sharpens peak shape mostly for more hydrophobic anions (Br-, NO3,. You can use a UV detector and monitor negative peaks between 305-310 nm if you don't have a conductivity detector."

As you said me, I ordered NaSCN and it has newly arrived. I did you what you say. Actually, it works. The peak shapes now ok. However there is a problem with sulfate. You can easily understand the problem from the attached file (please click the link). Shortly, when the concentration below 20 ppm, no problem. I saw a peak like shape just after the sulfate peak and it does not effect the accuracy. But, above 20 ppm, after the sulfate peak a big down peak I observed. And it effects the calibration curve.
Do you have any suggestion?
By the way since I changed the polarity NO2 and NO3 shown as negative peak.

http://s3.dosya.tc/server31/JUngt1/Hami ... _.doc.html

I do not have a lot experience with in direct UV detection. But it is for sure that all ions with no absorbance in the 300 nm range will show up as negative peaks. Any positive peak means that something with higer absorption than the eluent elutes.
With such systems with conductivity detetction we always find system peaks (usually after sulfate). My experience is that one gets one system peak per eluent component. I therefore would not be suppriced to see two system peaks with p-hydroxibenzoic acid and thiocyanide.
Usually system peaks get more pronounced with higher injected sample concentration.
I doubt that the peak you named sulfate really corresponds to sulfate.

If you remember, 2 months ago you advised me to use NaSCN for anion measurments by indirect uv.
Firs of all I would like to thank for your help.

"I recommend preparing this mobile phase as a concentrated stock (adjust pH top 8.5 with good grade NaOH or KOH), stopper and store at 4 C. Dilute to 1x daily since 4 mM is not much of a buffer. (The pH will drop over short time). The addition of 0.1 mM NaSCN is also a good idea as it really sharpens peak shape mostly for more hydrophobic anions (Br-, NO3,. You can use a UV detector and monitor negative peaks between 305-310 nm if you don't have a conductivity detector."

As you said me, I ordered NaSCN and it has newly arrived. I did you what you say. Actually, it works. The peak shapes now ok. However there is a problem with sulfate. You can easily understand the problem from the attached file (please click the link). Shortly, when the concentration below 20 ppm, no problem. I saw a peak like shape just after the sulfate peak and it does not effect the accuracy. But, above 20 ppm, after the sulfate peak a big down peak I observed. And it effects the calibration curve.
Do you have any suggestion?
By the way since I changed the polarity NO2 and NO3 shown as negative peak.

http://s3.dosya.tc/server31/JUngt1/Hami ... _.doc.html

What hardware size and particle diameter are you using? What is your flow rate? Back pressure?

Bear in mind that the PRP-x100 column is mixed-mode, and can also function as a RP column. With that in mind, consider sample, buffer, impurities that may have some capacity to bind a RP column. These build up over time, and will give spurious peaks, etc. Try regenerating the column by pumping 50-100 mL MeOH + dilute nitric acid, followed by copious water, then 50 mM tetrasodium EDTA. Flush again with water. Measure the pH of the effluent to ensure it is close to neutral. During water washes, you should replace the water (with fresh water, clean bottle) frequently. Consider passivating any metal components (such as inlet solvent filters "sinkers") in 6 M HNO3. If you pump is not "metal-free", passivate it, too with 6 M HNO3.

Ensure your mobile phase is prepared using HPLC-grade reagents (don't forget NaOH, KOH should be equally high grade). Install a guard column to capture hydrophobic impurities before they reach your analytical column. (wash the guard column with MeOH with dilute nitric as well). If I'm very superstitious, I will prepare a mobile phase stock, then pass through a (thoroughly cleaned) RP SPE cartridge. Idea is that hydrophobic impurities will be captured, but not buffers, salts.

If you still cannot move the sulfate peak away from what appears to be an artifact, I suggest trying another eluent- aminobenzoic acid + NaSCN. You will have to determine the wavelength empirically, but it is higher (> 360 nm) than p-HBA. Hopefully whatever is causing positive absorbance will be invisible at this higher wavelength.