Standard Peak elution

[brbabu_1979]

Dear all,
Greetings for the day...............
We were using YMC Hydrosphere C18, 150 X 4.6, 3 micron column for one of our product of MW is approx 1600. Our mobile phase is H3PO4 pH 2.0, Acetonitrile and Methanol. We are running the chromatograph with gradient elution. Regularly we used to get good peak of standard 6 ppm. All of sudden in the next sequence we are not seeing the peak in our standard run. We are getting clean blank base line. We do not know what happend. If any one faced like this problem please suggest us to get out from this issue. What we need to do? Please suggests us. Please provide the procedure.
The gradient program is as mentioned below.
Mobile phase A is H3PO4 pH 2.0; Mobile phase B is Acetonitrile and Buffer (pH 2.0) 70:30 and Mobile phase C is Methoanol: Buffer pH 2.0 50:50
Gradient Programme:

Time (Minutes) Mobile Phase A % Mobile Phase B % Mobile Phase C %
0 23 32 45
19 08 32 60
28 35 65 0
40 25 75 0
50 25 75 0
52 23 32 45
60 23 32 45

Peak RT is around 17.0 min.

with regards
Babu

[Consumer Products Guy]

Hmmm - pretty complicated gradient - I assume there are peaks from which you are trying to separate your sought-for component?

For fun I'd try going to a higher organic amount in the gradient, and/or also increase the post-run equilibration time.

If there's nothing around the sought-for peak, I think I'd use just one organic if that worked OK. Also, I think it's a little "much" to use phosphoric acid at pH 2; it would be way simpler and more robust to specify concentration of X Normality or Y ml/liter or Z g/liter for that. Then you wouldn't need to calibrate a pH meter, potentially contaminate your mobile phase, etc.

[tom jupille]

Just to clarify:

standard 6 ppm

Does this mean that you see the peak in standards at other concentrations?

And, is the 6 ppm standard always injected from the same vial, or do you have a separate vial for each injection.

[lmh]

If you sometimes have peaks, and sometimes just baseline, then my first suspicion would be an injection issue.

[brbabu_1979]

Just to clarify:

standard 6 ppm

Does this mean that you see the peak in standards at other concentrations?

And, is the 6 ppm standard always injected from the same vial, or do you have a separate vial for each injection.

Thanks for your replay. We are injecting standard from same vial. We have injected 1000 ppm standard solution. We were observed the peak with that. We also observing the peak with 50 ppm also. But not sharp one. The higher concentration standard also not in sharp. We are using the brand new column. Please suggest us what could be the reason behind of this.

Regards
Babu

[brbabu_1979]

Hmmm - pretty complicated gradient - I assume there are peaks from which you are trying to separate your sought-for component?
it would be way simpler and more robust to specify concentration of X Normality or Y ml/liter or Z g/liter for that. Then you wouldn't need to calibrate a pH meter, potentially contaminate your mobile phase, etc.

Thank you for your replay.

Our buffer is like this. 50 g of Phosphoric acid mixed with 50 g of water. Cooled to RT and from this 25 mL transferred in to 2000 mL of beaker and made up to 2000 mL with HPLC grade water. Finally pH adjusted to 2.0 with Ammonia solution. This is our final buffer. Any clue about this. Suggests us. What could be the reason behind this? Help us.

Regards
Babu

[DJ]

What is the nature of your solute?

[brbabu_1979]

What is the nature of your solute?

Nature of the solute is lipopeptide antifungal. MW is 1093 g/mol

[DJ]

What does the peak shape look like for a more well-behaved standard?

I think you first have to rule out instrumentation. Replace the column with a union and make some injections. Try preparing fresh standard and see if anything changes.

[tom jupille]

Some more suggestions (and questions).

1. Did the problem arise abruptly (in other words, one sequence looked OK and then the next sequence showed the problem)? If it did, were both sequences run the same day?
- What I'm looking for here is evidence for/against something like a partially plugged frit or a damaged column, which could occur suddenly. A gradual loss might point to a sample stability issue (DJ's suggestion of a fresh standard should answer that question).

2. Your posts indicate that there is also a problem with the higher level standards. If you compare the response factors (slopes of the calibration plots) before / after the problem arose, are they significantly different? Go back and measure peak widths and retention times on your higher level standards. Are they significantly different before / after the problem?
- What I'm looking for here is evidence for / against deterioration of the column.