HPLC trouble shooting - help regarding HPLC (Apr/18/2007 )
dear all
i am using HPLC with diod detector for analysing some secondary fungal metabolites like pinicilic acid, ochratoxin, etc for the standerd of all these metabolites the peaks i am getting are normal but for the extracts the peaks are deviated means not at the standered time. as a run solution i am using acetic acid(0.2%) and acetonitril (100%).
can any one help me regarding my problem.
thanks in advance
-Nafees-
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
-swanny-
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
-circlepoint-
dear i am using the same solvent for both standerd and for my extraction and that is methanol.
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
QUOTE (circlepoint @ Apr 19 2007, 09:58 AM)
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
-Nafees-
QUOTE (Nafees @ Apr 19 2007, 12:20 AM)
dear i am using the same solvent for both standerd and for my extraction and that is methanol.
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
QUOTE (circlepoint @ Apr 19 2007, 09:58 AM)
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
-circlepoint-
type of column is Prontosil 120-5-C18-SH 5.0µm
with dimension : Length x ID= 150x 4.6 mmas mobiole phase i am already using 0,2 % AA 100% AcCNl, i am using 80µl of injection volume of my sample.
QUOTE (circlepoint @ Apr 19 2007, 10:50 AM)
QUOTE (Nafees @ Apr 19 2007, 12:20 AM)
dear i am using the same solvent for both standerd and for my extraction and that is methanol.
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
QUOTE (circlepoint @ Apr 19 2007, 09:58 AM)
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
-Nafees-
QUOTE (Nafees @ Apr 19 2007, 01:10 AM)
type of column is Prontosil 120-5-C18-SH 5.0µm
with dimension : Length x ID= 150x 4.6 mmas mobiole phase i am already using 0,2 % AA 100% AcCNl, i am using 80µl of injection volume of my sample.
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
with dimension : Length x ID= 150x 4.6 mmas mobiole phase i am already using 0,2 % AA 100% AcCNl, i am using 80µl of injection volume of my sample.
QUOTE (circlepoint @ Apr 19 2007, 10:50 AM)
QUOTE (Nafees @ Apr 19 2007, 12:20 AM)
dear i am using the same solvent for both standerd and for my extraction and that is methanol.
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
QUOTE (circlepoint @ Apr 19 2007, 09:58 AM)
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
My colleges did similar exp with ochratoxin . Sample was in 70% AcCN 30% H2O Program was isocratic 70% AcCN 30% H20 They use RP8 column
-circlepoint-
as i have mention that i am using a column with C18 which is apolar, tha is y i am using AcCNI 100%(NON POLAR) and acidic water that in0,2% acitic acid(polar). i dont know much about biochemistory and the condition y we chose diferent solvent for different columns and different extracts. i will be thak ful if you could explain how you friends is extracting that toxine from the source and which programe and column he is using for analysing it?
QUOTE (circlepoint @ Apr 19 2007, 01:02 PM)
QUOTE (Nafees @ Apr 19 2007, 01:10 AM)
type of column is Prontosil 120-5-C18-SH 5.0µm
with dimension : Length x ID= 150x 4.6 mmas mobiole phase i am already using 0,2 % AA 100% AcCNl, i am using 80µl of injection volume of my sample.
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
with dimension : Length x ID= 150x 4.6 mmas mobiole phase i am already using 0,2 % AA 100% AcCNl, i am using 80µl of injection volume of my sample.
QUOTE (circlepoint @ Apr 19 2007, 10:50 AM)
QUOTE (Nafees @ Apr 19 2007, 12:20 AM)
dear i am using the same solvent for both standerd and for my extraction and that is methanol.
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
i want to detect actually pinicilic acid and ochratocin A in my sample. what do you suggest as solvent for my HPLC and wich progrmme should i use like i am using a long programe of about 45 mints at the moment.
QUOTE (circlepoint @ Apr 19 2007, 09:58 AM)
QUOTE (swanny @ Apr 18 2007, 03:29 PM)
Do you make up your standards in the same solutions that you use to prepare your extracts (or at least, the final stage solutions)?
Are your shifting is the same proportional for all your detected peaks?
If you substances of interest in extract are in concentrations far from concentrations used for standarts shift in retention will occure because of non-linear adsorbtion. But the most possible cause of shifting as Swanny said, is difference between solvent what you use for preparation extract and for dissolving standarts. The main rule is for standarts and samle use the same ( mean solvent srength) solvent.
As I understand you use RP HPLC but isocratic or gradient regime?
To futher thinking it will be good to see Chrom of your standarts and samples!
Why did you choose your solvent as 0,2 % AA 100% AcCNl?
What kind of column do you use? dimensions , stationary phase ?? Your injection volume?
Try to make gradient 0( water) -100% methanol with your standarts ( better make a mixture of standarts) for 45 min with 1ml\min
Then we will try to optimize ( time, gradient volume ) your programm after obtained results ( It will be better to see Chrom image after your exp!)
My colleges did similar exp with ochratoxin . Sample was in 70% AcCN 30% H2O Program was isocratic 70% AcCN 30% H20 They use RP8 column
-Nafees-