calibration of hplc

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mohd
mohd's picture
calibration of hplc

dear sir/ma
1. can u tell why will use caffeine fo wavelength  accuracy.(apart from its known maxima and minima and recemended by NIST .saffe for use) apart from these reasons there is any other reason.
2. why will use acetone fo gpv test in hplc.
3. other solvents for gpv test.

20101975
20101975's picture
I have worked over few hplc

I have worked over few hplc machines and what I know is that the calibration should be done by the service representatives and they dont tell as how the things work as those things are trade secret. What we do id validation.

Dr. Analytical
Dr. Analytical's picture
Mohd:

Mohd:
1. You are correct.  Caffeine is easy to use and that is the main reason.  Anything could be used for cailbration.
2. Also, acetone is used for the gradient programming valved (gpvv) because it is found in most laboratories and is not expensive.  It also absorbs in the UV region.  You could use any solvent that produces a response in your detector.  I use acetonitrile and set my absorbance detector to 210 nm.  Methanol would also work.  But it is best to prepare solutions of the solvents in water, rather than using the pure solvent, because there are some mixing effects that may make the data difficult to interpret.
 

fresher
fresher's picture
dear Dr. analytical

dear Dr. analytical
I was about to creat anew tobic on HPLC calibration but fortunately I found it here. in fact I'm new in dealing with HPLC I've recently bought a new column but the system is already there in the lab. my question is how can I calibrate the instrument to ensure that it's still working proerly? in addition, how can use acetonitrile as a solute to check the equipment? what's the eluent I must use in this case?
please reply to me as soon as possible  giving some tips I must follow. thanking you ,

mohd
mohd's picture
hi.

hi.
i thin this can help u lot
zahid

Greg Pronger
Greg Pronger's picture
Dear Fresher,

Dear Fresher,

What will help us a lot in assisting you, is if you give us some more information on your application. The more info we have the more tailored we can be.

Greg

abhigyan
abhigyan's picture
Hi,

Hi,

The other day I was running a Related Substances test by HPLC, for Tranexamic acid as per BP2009 monograph. The mobile phase I used contained 11 g NaH2PO4, 5 ml Triethylamine, 1.4 g SDS, 400 ml Methanol made upto 1000 ml with water , ph=2.5. The mobile phase was filtered and sonicated prior to use. All the column, instrument, VWD parameters were set as per the monograph. The column was washed prior to sample runs and baseline duly stabilised. However, the RT was found to be at 7.8 mins whereas the monograph specifies RT to be about 13 mins.
There are two options for me to set the RT at 13 mins; 1. change flow rate from 0.9ml/min ( specified in monograph) to about 0.57ml/min or, 2. tinker with the mobile phase composition. Both these options are deviations from the pharmacopoeial test procedure and cannot be done.

Kindly suggest what must have gone wrong. Are there any special considerations to be made when methanol is used in mobile phase?

kapsha
kapsha's picture
hi mohd

hi mohd
hw can i download ur attachment HPLC new.zip?

fatma_elzahraa
fatma_elzahraa's picture
dear

dear
it isn\t important to get the RT as u used a column with similar specifications not the same....
really from my experience... i found that it is greatly different to use a column with certain specifications but from different companies....
silica grade used and the perfection to make a column differ from one company to another...
so... it is good to have a good peak with the method u got.... as i sometimes made some changes in the mobile phase composition to give a good peak....
try to work on ur own conditions....
just take the method and if the retention time isn't the same... it doesn't matter....just build all ur quantitations on these conditions....

best regards

ajit_vaishu2004
ajit_vaishu2004's picture
No answer

No answer

fresher
fresher's picture
Dear abhigyansaha,

Dear abhigyansaha,

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From my experience I would suggest that you should check your pumps. does your pump work as per your set flow rate?

Santosh@India
Santosh@India's picture
abhigyansaha

abhigyansaha
Although you had set the analysis parameters as per BP, check the following:-
1)How old your column is, is it giving sufficient HETP's if not =>over used column efficiency decreases the sample retention capability for the analyte
2) Recheck the mobile phase pH with different calibrated pH meter.
3)Check the solvent purity that you had used for the mobile phase preparation.

revert back if you can able to find anything.

Satish Kumar
Satish Kumar's picture
dear sir/ma

dear sir/ma
1. can u tell why will use caffeine fo wavelength  accuracy.(apart from its known maxima and minima and recemended by NIST .saffe for use) apart from these reasons there is any other reason.
2. why will use acetone fo gpv test in hplc.
3. other solvents for gpv test.
4.why we take weight for the analysis of suspension or dry syrup
5.. why we pippte out in syrup
6.Why we use only potassium dichromate for the calibration of UV