Chromatography Forum

I've developed a method for determining Thiamine HCl and Pyridoxin HCl.

Analysis condition :
column : L1 C18 endcapped 250 x 4.6 mm, 5 um
Mobile phase : (1.4 g Hexansulfonate + 5 mL Triethylamine + 14 mL acetic acid in 1000 mL water, adjusted with phosphoric acid ad ph 2.65) : methanol = 80:20

Flow : 1.2 mL/min
temperature : 30 C
Diluent : water
Injection volume : 10 uL
Detection : 280 nm

Retention time: Pyridoxin HCl : 6 min
Thiamine HCl : 10 min,

At the beginning all of peaks were looked good. The problem occured after several injections of standard (approximately 20 injections). Thiamine HCl showed Shouldering peak whereas Pyridoxin HCl didn't show any peak problems. This problem was still occured even though I've changed column batch (from the same manufacturer).

Does anyone have any suggestions?

Thanks in advance

Does a freshly prepared standard show the same thing?

Yes, it shows the shouldering peak even with a freshly prepared standard.

You aware that hexane sulfonate is an ion-pairing agent and can change the regular RP column chemistry with its column load.

Triethylamine might be creating problems. Since, you have an endcapped column - there is no need for the Triethylamine if not anticipating tailing interactions.

Plus, this is not a regular RP but an ion-pair method. I assume that the Triethylamine (being a very strong base) can compete with your analyte at low pH conditions, in ion-pairing - which makes the analysis very difficult to predict - since you have mixed mode interactions in place.

With the same condition, I considered using Triethylamine since thiamine HCl showed tailing peak (Tailing factor = 2.3). Retention Time Thiamine HCl 20 min, Retention Time Pyridoxine HCl 7 min (Tailing factor = 1.5).

Triethylamine can reduce tailing for regular Reversed-phase methods.
However, for ion pairs - the protonated form can ion pair.

Again, I find it very odd using Triethylamine when using Ion pair methods.

Chicay, which of your results are correct? Are you constantly changing, maybe even inadvertendly, the conditions? I just wonder whether either the ion pairing agent or triethylamine is necessary. The first thing I would do is optimize the pH using a good buffer. You need a good buffer as you inject the hydrochlorides in water, among other reasons. You should be aware that this is certainly not a chromatography of the hydrochlorides.
Incidentally, the second results that you quote make more sense to me, as they seem to indicate a pH mismatch. The first results could be a combination of this + severe equivocacy (non-robustness) which seemed to be pointed out by mohan.