RP-HPLC chromatograph interpretation

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charity
charity's picture
RP-HPLC chromatograph interpretation

dear all,

from an RP-HPLC peak report, is it possible to determine the actual concentration of the compund being tested? how? what is the formula? the peak report contains the following information: retention time, area, height, and width.

thanks!:)

mbicking
mbicking's picture
The raw peak information from

The raw peak information from a chromatogram (area, height) is just that; the raw data to be used for other calculations.

You cannot determine the amount of a substance present without first analyzing one or more standards of known concentration. Once you know the relationship between peak area and amount analyzed (called a "response factor"), then you can calculate the quantity in an unknown.

Many data systems will give you an "Area Percent" report, which is just the percentage of the total that each peak represents. This also has limited meaning, since different compounds may have much different response factors.

To summarize, to get an amount value, you will need a standard. More questions? Just write.

charity
charity's picture
Thank you for taking time to

Thank you for taking time to answer my query. And yes i do have more questions. :)

Actually I have a standard and the retention time of my "unknown" is within the peak range of that standard. With this, I can say that I have more or less the same substance, right?

The thing is, I'm not satisfied with just saying that I have the same substance in my unknown (qualitative). What I am after now is to report the actual amount of the substance in my sample. You said that I can calculate the quantity in an unknown once I know the response factor. So now, my more important question is how to determine such response factor? What is the formula, if there is any? And how exactly do I use this response factor to compute the quantity of the substance in an unknown?

Again, thanks so much! :)

mbicking
mbicking's picture
Well, we could spend days

Well, we could spend days talking about this, but I will try to keep it a little shorter. :-)

The response factor equation for the standard is:

RF = (Peak Area Std)/(Amount Injected Std)

You can use either concentration or mass, but be consistent in all calculations. It would be best to do several injections and get an average RF.

Now, for your unknown, assume the RF is the same (it's the same compound), and you know the peak area, so you just rearrange the equation for Amount Injected.

Amount Injected (Sample) = (Peak Area Sample)/RF

Again, multiple injections will get a better value.

This is called "Single Point Calibration" because you are only using one standard. It is a good approximation if standard and sample are close in concentration. If not, the accuracy will suffer and you will have to take another approach.

Now, as to whether or not your sample has the same compound: well, that is always something we struggle with. If the retention times are the same (or very close) and the peak shape is the same, we usually ASSUME that it is the same compound. But we really can't say with complete certainty. It is best to have some other information, like an absorbance spectrum or mass spectrum, or some other knowledge of the sample.

This should get you started.

charity
charity's picture
Okay, got it! Your

Okay, got it! Your instructions were very clear and concise :) But let me just clarify, in the formula for RF, you were referring to the Peak Area and not the Area %, right?

As to the compound in my sample, I'm pretty confident that I have the same compound as the standard because I only have a single prominent peak in my sample very close to the retention time of the standard. But then again, I really need to firm up my results with further characterization. I intend to do ESI-MS, CD, and FTIR in the next few months. And I'm sure more questions will arise as I am very new to these.

Thanks v. much, Sir! I really appreciate your help. :)

mbicking
mbicking's picture
You are correct, that you use

You are correct, that you use peak area (or height), not the percent value. The area percent results really have no meaning except to tell you the percentage of the total. There are some specific examples in pharmaceutical analysis where you might be able to get some useful information, but in the general case, no.

Also, I would strongly urge you to get some training in HPLC and analytical chemistry. There are many things to learn; more than I can teach you in this forum. You need to learn how the instrument works, how to use the software, how to troubleshoot problems, and how to set up calibration systems and review results.

There are many resources for training available, including some that I offer. But it is very important that you get it from someone.

efoconnor
efoconnor's picture
charity wrote:Thank you for

charity wrote:

Thank you for taking time to answer my query. And yes i do have more questions. :)

Actually I have a standard and the retention time of my "unknown" is within the peak range of that standard. With this, I can say that I have more or less the same substance, right?

The thing is, I'm not satisfied with just saying that I have the same substance in my unknown (qualitative). What I am after now is to report the actual amount of the substance in my sample. You said that I can calculate the quantity in an unknown once I know the response factor. So now, my more important question is how to determine such response factor? What is the formula, if there is any? And how exactly do I use this response factor to compute the quantity of the substance in an unknown?

Again, thanks so much! :)

efoconnor
efoconnor's picture
charity wrote:dear all,

charity wrote:

dear all,

from an RP-HPLC peak report, is it possible to determine the actual concentration of the compund being tested? how? what is the formula? the peak report contains the following information: retention time, area, height, and width.

thanks!:)

qualitative certainty is greatest with MS if that mode will work for you. Next is scanning UV or PDA. You can also spike std into your unknown. THis should give a higher peak without shoulders.

charity
charity's picture
mbicking wrote:

mbicking wrote:

Also, I would strongly urge you to get some training in HPLC and analytical chemistry. There are many things to learn; more than I can teach you in this forum. You need to learn how the instrument works, how to use the software, how to troubleshoot problems, and how to set up calibration systems and review results.

There are many resources for training available, including some that I offer. But it is very important that you get it from someone.

Oh yes, I really intend to attend formal trainings with respect to the analytical tools that I will use in the near future. I am just waiting for an opportunity because trainings like this in our country is very seasonal/rare.

Thanks for the advice! :)

mbicking
mbicking's picture
You did not mention which

You did not mention which country you live in, but you should be able to find some good training that is not too far away. The manufacturer of your instrument may offer classes in your country, or a nearby country. And you can look for a Chromatography organization that may offer training also.

Good luck with your learning. We hope to hear from you again.

charity
charity's picture
Ah right! I really appreciate

Ah right! I really appreciate all your suggestions and I will start looking for trainings right away, or as soon as I finish my thesis. I'm an MS Biology student from the Philippines.

Thank you again Sir. :)

charity
charity's picture
efoconnor wrote:

efoconnor wrote:

qualitative certainty is greatest with MS if that mode will work for you. Next is scanning UV or PDA. You can also spike std into your unknown. THis should give a higher peak without shoulders.

Thank you for the above0-mentioned suggestions. I will definitely consider them in my next experiments. :)

NARRA
NARRA's picture
% Purity= (Area of sample

% Purity= (Area of sample/Area of Std)* con.Std/Con.Sample*DF*100

jocelyn
jocelyn's picture
Good day to all!

Good day to all!
Well, Ms. Charity, if i may suggest some info regarding quantitation in the HPLC, if you are using a PC-driven LC, it is sure that the software you are using has the ability to perform the Calibration of your standard solutions. I don't know how many levels of concentrations of the standard you are going to use but through that. you can have the quantitation calculation. Just see if you can ask th supplier of your LC can teach you on how to do it.
I hope this will help.

bankanidhi
bankanidhi's picture
Hi,

Hi,
     I have an important question about the RF(response factor). Whatare the parameter on the the response factors depends. I mean to ask.....Can i theoritically calculate the RF of a substance (for a perticular hplc system) knowing molecular properties like the extinction coefficient. Somewaht i feel that given a hplc system the RF should just be propertional to the extinction coefficient.  Am i right?
Critical suggestions and coments are most welcome and will be appreciated.

Anaa
Anaa's picture
Hello,

Hello,
 
I'm working in RP HPLC. Firstlly i'm working with standards to find out the retation time of interest substances.
For the same standard with different concentration 10, 30 and 50 ppm i'm getting different retantion time. example for 10 and 50 ppm I have the same retation time but not for 30 ppm. That's weird! Can some one help me to solve this problem please. I'm struggling with this for two weeks. Thank you!

Dr. Analytical
Dr. Analytical's picture
Anaa:

Anaa:
Sorry I could not reply earlier.  I have been out of the country. 
Please give us more information about your system: mobile phase, column, compound you are analyzing.  What are the retention time difference that you are observing.  Are they consistent?  That is, do you get the same results each time?
It is common for the retention time of a standard to decrease as the concentration becomes larger.  This means you are overloading the column.  However, the situation that you describe is unusual.  With some more details we may some more ideas.

Anaa
Anaa's picture
Dr. Analytical

Dr. Analytical
Thank you very much for your reply.
I'm working with phenol and (by-products such as Hydroquinone, catechol, benzoquinone).
The mobile phase is methanol/water 60:40 (I'm working with low pressure). The column is Discovery C18.
The differences of retention time between the same compounds are 2-3 min. And they are consistent. The deviation happened only with 30 ppm concentration.
For information I have tried with different mobile phase composition methanol / water such as : 40:60; 60: 40; 90; 10 but the same problem.
 

Dr. Analytical
Dr. Analytical's picture
There is a problem with your

There is a problem with your 30 standard.  Is it possible that it is contaminated or the analytes have decomposed.  A 2 - 3 minute difference is significant, and usually means that you are not looking at the same compounds.
Has anything else changed?
 

Dr. Analytical
Dr. Analytical's picture
Bankanidhi:

Bankanidhi:
Sorry I did not answer sooner. I have been away.
You are correct that the response factor is proportional to the extinction coefficient.  To calculate the response factor, you would have to calibrate your system with known standards, and use that information.  This step is necessary because of differences in detector optics, chromatography, as well as data system procedures.
This is essentially the same procedures as a normal calibraition, so there would be no real advantage.
 

fresher
fresher's picture
i'm  new in dealing with HPLC

i'm  new in dealing with HPLC, in my first RUN I prepared a standard solution of my substance of intrest for the method development. the first step was to find out the most suitable wavelength. I ran the experement twice  under the same conditions but at different wafelength. however, I found that the chromatogram base line is very linear and constant ( no sloping), but at the another wavelength the base line  sloped up !! I couldn't interpret that , since nothing was different only the wavelength.
 
can any body help me?

Dr. Analytical
Dr. Analytical's picture
Fresher:

Fresher:
Please provide some more information about your system - instrument, column mobile phase, etc.
If you are using a gradient program (changing mobile phase composition with time), then a change in the baseline is normal.  This happens when one of the components absorbs more at a wavelength than the other component (e.g., acetonitrile absorbs more than water at 210 nm), so when you increase the amount of acetonitrile, then the absorbance will increase.  This happens more often at shorter wavelengths.  At longer wavelengths, you do not see this problem.
If this is not your situation, then write back with more details.
 

fresher
fresher's picture
thank you for your reply. in

thank you for your reply. in fact I used an isocratic elution of ( ACN:water, 80:20). the stationary phase was C18, the two wavelengths were 235 and 275 nm.  the sloping occured with 270 nm.  another interisting thing is that when I scanned the absorbance wavelengths using a spectrophotometer to determend the Lambda-max of my subsance, I found that it is 275nm but not 235 nm.
if necessary, I can send you a copy of the chromatograms I got.
thank you again

Dr. Analytical
Dr. Analytical's picture
If you have a changing

If you have a changing baseline in isocratic analysis, it usually means that you have an external source of contamination.  However, the baseline will become stable at some point.
Here is another test:
While the baseline is changing, stop the pump flow, and then observe what happens to the baseline.  Does it continue to rise, or does it now remain flat?
Please post your answer here. 

tariqakbari
tariqakbari's picture
Hi all,

Hi all,
can any 1 help me in getting the exact concentration of a component in the computer generated report, instead of doing it manually by calculating response factor and etc.
 

Dr. Analytical
Dr. Analytical's picture
tariqakbari :

tariqakbari :
Please start a "new" topic in this forum, so that more people can see your question.  In the Chromatography sub-forum page (http://www.scientistsolutions.com/f65-chromatography.html), look for a button that says "Create Topic."
Please post some additional details about your question.  What LC or GC system are you using?  What software are you using, and what version of that software do you have?  Then, please describe what you are doing now and what you would like to do.  (Your original description is OK.  Please repeat it with the additional information.)
Thank you for using scientistsolutions.com
 

efoconnor
efoconnor's picture
Each run should have its own

Each run should have its own calibration curve.   That info will display on the individual chromatograms sowing not only peak parameters but also converting the peak parameter HT or area or Ht or area ratio to a concentration as established with your curve.   You can also add min/max area/min/max ht/min/max ratio as well as peak shape, resolution and retention time windows to flag and reject or accept peaks.

efoconnor
efoconnor's picture
Do all the standards have the

Do all the standards have the same material-water, methanol in them ?

efoconnor
efoconnor's picture
1) what system are you using?

1) what system are you using?
2) have you prepared a standard curve
3) are your injection volumes fixed
4) do you use an internal standard
 
You software program should come with several options for quantitation including standard curve fitting.   try the simplest first the change as needed to get your curve values to return measurementss within 15-20% of the expected value.   You can push to tighter tolerance 5-10% if you need that.
Response factors vary and will change from the low end to the high end of the curve.
 
 

jaypee
jaypee's picture
hi there, i'm a student from

hi there, i'm a student from a state university here at Philippines. and im doing my research for HPLC... i need to know limitations of it....i need your answers as soon as possible... i want to know what HPLC can't to.. i need it please..... reply to this msg. or e-mail your answers on this e-maail

jaypeeitsme@gmail.com

again this is my question:

What are/is the limitations of HPLC??

pls reply as soon as possible...

thankz

Dr. Analytical
Dr. Analytical's picture
This is really a new topic.

This is really a new topic.  In the future please start a new topic so it is easier to find.

Your question is difficult to answer without having more information about what you are trying to do.  (In fact, this question sounds like an examination question.  )

But here are some general rules:

  1. The molecules that you want to determine must be soluble in the mobile phase.  If they are not soluble, then they will never reach the column.
  2. The molecules must produce some response from the detector.  This may mean that you have to adjust detector settings or use a different detector.
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?

Dr. Analytical
Dr. Analytical's picture
abhigyansaha:

abhigyansaha:

Thanks for posting your question.  In the future, please start a new topic so that it is easier to find.

Regarding your problem with the monograph, you did not specify which column you are using.  Is it exactly the same as specified in the monograph?  It is common for different C18 columns to provide different retention times for the same conditions.  So, if you are using a different column, the results may be normal.  Also, be aware that the pH adjustment must be made before adding the methanol.  And sonication does nothing to help your chromatography.  You can skip that step.

If your chromatography is good, and everything else is stable, I would just use the conditions you have.  Some differences are to be expected (although this is a bit larger than usual).  If your calibration information is acceptable, you may not have a problem.

If you want to try adjusting conditions, do not adjust the flow.  That does not really change anything.  You can change the methanol concentration.

tariqakbari
tariqakbari's picture
Efoconnor,

Efoconnor,
I am using Perkin Elmer Series 200, with Total Chrom Software.
The Injection volumes are fixed that is 100 microlitre and the system loop take only 20 micro litre. I am using Retinyl Acetate as internal standard. I have not prepared any standard curve.
Let me tell you about the whole procedure I take for retinol:
I take 100 microlitre serum in a test tube and 100 microlitre of Retinyl Acetate as Internal Standard and 200 microlitre of n-Hexane as deproteinizer. then I centrifuge them all and got the 100 micrlitre of supernatant and injected this 100 microlitre in the chromatograph. The chromatograph take only 20microlire of it and discard the rest.
Now My question is: how to get the exact concentration of retinol in the total chrom  pre formatted report,  instead of manually calculating the response areas of retinol and retinyl acetate. I want to get the exact concentration that may be either in 1 ml or 100 microlitre on the total chrom report.
my second question is: any value I get , either from manual calculation or the report, how much volume this concentration will represent?
Thanks.

lakeduck
lakeduck's picture
 Hello.

 Hello.

I have HPLC/MS chromatograms from my standard and my substance. I am trying to get close to the concentration of my substance, but i don´t know what are the units of the peak areas. I know this is not an accurate method, but it is what i have for the moment. Later i will quantify by HPLC-UV. 

I also need to know what "intensity" of the Mass Spectrum refers to. What are the units?

Can you help me? Thank you very much. 

Dr. Analytical
Dr. Analytical's picture
The units for the

The units for the chromatogram are not important if both the standard and sample were analyzed using the same system and same conditions.  You can use either area or height measurements.  WIth this information you can calculate the response factor for the standard:

Response Factoer (RF) = Area (or Height)/Amount Injected

For the sample, you know the RF and Area (or Height), so you can calculate the Amount.

"Intensity" means the same as response, or peak height.  Higher intensity means more response (a taller peak).

Thank you for posting on scientistsolutions.com.  Next time  please start a new topic so that more people will see the discussion.