Problem with Retention time

[mk_3249]

Hi
I am Maharshi from Gujarat.
I am using JASCO PU1550 HPLC instrument with manual injector.
I've problem with the retention time in analysis of pharmacopoeial drugs.
One of my API is Adrenaline Bitartrate. I am using C18 RP-II 100X4.6mm, 3 micron column as per the pharmacopoeial requirement. Program is binary gradient and also as per the requirement and already setted in the instrument.
pH of mobile phase A is 2.2 and of mobile phase B is 2.4 as per the requirement.
One of our HPLC engineer advised us to reduce the length of tubings from injector to column and column to detector.
Before changing the length of tubing, I was getting the proper retention time. But after changing it to shorter length and small diameter, the retention is changed. Previously it was about 4 as per monograph but now I am getting about 1.4.
Not only this product but I am getting the same problem with the other products also.
Then I changed the tubings and again set the old one but still its not solved.
Column is also a new one. I've also verified it on old column but no difference I've got.
Now I am unable to understand what could be the problem.
Anyone please help me.

Thank you

[Hysol]

Shortening the length of tubing from your injector to column and then column to detector will decrease the total system volume. Therefore the sample will take less time to get from the injector, to the column and then to the detector. The final result is, as you have noted, a faster retention time. As long as the separation is okay there should be no problem with the new retention time.

[nonagon]

Something does not add up - a change from 4 to 1.4min is extream for just cutting dead volume. Usually cutting dead volume gets you better peak shape and resolution but does not significantly lowers your retention time.
Check your capacity factor [k'=(tR-t0)/t0]
A minimum acceptable k'=3 means that your unretained peak elutes at 0.35min with a linear velocity of 25.6cm/min does not sound like C18 on a jasco to me, how long were your tubings?
It seems as though there are other factors here. Were there any other modifications performed other than tube cutting?
I believe this is an ion-pair method, did you equilibrate your new column sufficiently with your ion pair modifier? (this could take very long), are you testing different products on an ion pair column?
Please provide all method parameters

[tom jupille]

Let me jump in here.

First of all, as nonagon suggested, simply reducing tubing volume should not have made that big a change in retention time. You didn't specify the flow rate, but 1 mL/min would be typical for a 4.6 mm i.d. column, and I can't imagine that you had 2.6 mL of tubing volume (that would be over 5 meters of 0.010" id tube). You confirmed that by replacing the original tubing without affecting the problem.

Second, nonagon's advice about k' is incorrect. That measurement applies to isocratic systems only, not gradients.

Since the problem is not simple physics (tubing volume) it must be chemistry: either the mobile phase is off or your column is dead. If it were my problem, I would do the following:

1. Prepare fresh batches of mobile phase and re-test.
2. If the problem persists, run performance test of the gradient proportioning system (and check the dwell volume at the same time). A brief description of the tests can be found here:
http://www.lcresources.com/resources/TSWiz/hs450.htm
http://www.lcresources.com/resources/TSWiz/hs410.htm
3. If 1 or 2 did not solve the problem, replace the column.

[mk_3249]

Thank you very much to all of u for suggesting me. Actually I m a beginer for HPLC. M using and analysing from last 1 year. There is no one with me who can teach or guide me. I've learned everything by trial and error.
By the way, the older tube length was approx. 30cm and new one is of about 15-20 cm. It was done by an Jasco HPLC engineer. Then the problem started. Then I set the old one back but the same results are coming.
The flow rate is 1.5 ml/min and as per gradient program it goes upto 4 ml/min. The run time is 30 min.
I am using freshly prepared mobile phases and even my column is a new one. I equilibrates it with mobilephase upto a straight n stable baseline. Pls. don't scare me by high-end technical language coz m a new one.

Thank you

[HPLCaddict]

You already rules out the changed tubing as source of the problem, since the changed retention times persisted when going back to the original tubing, correct? This leaves the mobile phase, the column or the HPLC-system itself as sources. You mentioned you have the same problem with other products, too. What does "other products" mean? Are using different columns and mobile phases? Then you may exclude the column and the mobile phase, too.
Some suggestions:
- double-check the method parameters (flow-rate, gradient composition)
- If you're NOT using different columns/mobile phases for the "other products", prepare a fresh batch of mobile phase and retest. Do you have a different column to test?
- Check the flow-rate of the system: Disconnect the column, pump 100%A with 1mL/min and collect the flow in a graduated cylinder. In 5 minutes you should get (surprise!) 5mL of eluent. Do the same with 100%B. If one of both severely misses the 5mL, there you are! Possible culprits are the gradient-proportioning-valves (in a low-pressure aka one-pump-system) or one of the pumps (in a high-pressure aka two-pump-system) is not working properly.
- If the problem is not fixed by now, run the gradient test as Tom already suggested.

BTW, how does the method look like? It contains a flow-rate gradient? this is, well, rather uncommon...