HPLC internal standard [May 4, 2004]
Posted: Wed Aug 25, 2004 4:36 am
By Anonymous on Tuesday, May 4, 2004 - 02:58 am:
Hello all,
I'd like to use an internal standard in HPLC.
But I have no experience in this kind of standardisation.
Could someone recomend me a good IS to use in these conditions.
Column C18
CH3CN/H2O isocratic
Sample Temp 35°C
Thx,
Laurent
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 06:55 am:
what is this internal standard for? To make sure the HPLC works fine or something else?
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 07:59 am:
To calculate the factor response of my compounds.
We got very small amounts of pure compounds to make external standardisation.
I know that in GC we could use "Alcane" to perform internal calibration but what could I use in HPLC ?
Ah yes I work at 200nm :/
-------------------------------------------------------------------------------------------------------
By tom jupille on Tuesday, May 4, 2004 - 10:43 am:
I'm not sure why you would want to use an internal standard. You will still have to consume some of your pure compounds to prepare the calibrator solutions.
Internal standards are useful in LC primarily to correct for precision problems caused by injection volume variations or sample workup (i.e., anything that will affect all the peaks in the same proportion). If your precision is OK using external standardization, then the use of an internal standard will provide no advantage (and may, in fact, contribute addtional error).
That said, what you want to find is a compound that is *never* present in your sample, that elutes near your analytes, and is completely resolved from all of them. The alkyl phenones represent a good set of choices (pick the chain length that gives appropriate retention for your purposes).
Final comment: my recollection is that the "alkanes" are used in GC not so much as internal standards as to provide the basis for determining retention indices. If that's what you're looking for, there is no equivalent in LC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 10:45 am:
Does that mean you want to use the internal standard to calculate the concentration of your compound? I never did this for HPLC. Since every compound has its own UV absorbance. The only thing you can do may be choosing something with similar UV performance based on the structure and should not be eluted out at the same time. However, I could not guarantee the result will be accurate.
-------------------------------------------------------------------------------------------------------
By Andreas Neumaier on Wednesday, May 5, 2004 - 07:05 am:
You can choose a compound with similar UV spectrum for external calibration and calculate sample amount by using the relative response factor of the compound you want to quantitate.
But you have to make sure that RRF stay the same after changing the lamp and/or column. Which means you have to use up some small amount of your rare compound every time you check the RRF.
You have a higher rsd than without using RRF.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, May 5, 2004 - 07:42 am:
One should not forget that the internal standard method is an absolute "must" in many biological analyses, where the recovery varies more than the final measurement. Often one can get a synthetic variant of the analyte to use as an internal standard.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 11, 2004 - 03:22 am:
I agree with Tom, that internal standard is useful in HPLC, if you have some problem with injection. If you have very small amount of analyte, you can use other substance to calculate a relative response factor for your analyte, and use this factor for quantitative analysis.
In case of analysis of biological samples the use of internal standard is a bad habit, it can result in faulty data because of the different behaviour of analyte and internal standard during the sample preparation steps. You can determine all necessary data for validation (precision, accuracy, linearity) using samples spiked with the analyte. This way you get more correct data.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, May 11, 2004 - 07:03 am:
Last anon.,
what do you do when you have to analyze plasma/serum of patients with various illnesses, states of metabolism, etc., with wildly differing matricese? You do your stuff with a plasma/serum pool and then pray that all the patient´s sample will behave like your pool? One can usually choose internal standards so close in chemistry, etc., to the analytes that discrimination is negligible (this parallel behavior can be much more robust than the determination of precision, etc. with a pool, or whatever). Anyway, if you want to be sure in chromatography you should do two different chrom methods.... See J Chrom B, 678, 137(1996). One HPLC method without an internal standard can be a fast road to licentiousness.
Also, a problem with injection can usually be controled, whereas the sort of patient´s samples you get is out of your control.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, May 12, 2004 - 06:12 am:
Just a quick note here, not meant to stir things up. When I worked in an environmental lab we used two different compounds during analysis, an "internal standard" and a "surrogate". The "surrogate" compound was added to the sample prior to preparation and went through all the same manipulations as the sample. Then the "internal standard" was added to the final extract before analysis. Obviously the "surrogate" was meant to evaluate the extraction/preparation efficiency and the "internal standard" meant to compensate for any variations during injection. I think it would be clearer here to make some distinction between the uses of the added compounds.
Regards,
Mark
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, May 12, 2004 - 07:25 am:
Also, there is no rule which prevents one from using the external standard method in conjunction (parallel to) the internal Standard method. One has some nice monitoring available that way. The argument that the internal st. method deteriorates accuracy...... is then completely diffused.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, May 16, 2004 - 08:45 am:
We have somewhat departed from the initial question. There was a publication in Analytical Chemistry in July last year on the handling of quantitative assays from biological samples by Matuszewski (sp?) in the case of LC with MS detection. It is supposed to get around the problems of ion suppression common to MS detectors.
-------------------------------------------------------------------------------------------------------
By and on Friday, June 11, 2004 - 06:24 am:
I think you do need the internal standards for the quantitation LC especially LC/MS for the complex samples. There is no one internal standard for all samples. Usually people will choose those non interfering compounds or internal isotopically labled analogue of the analyte.Isotopically labled analogue normally will give you better accuracy.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 17, 2004 - 08:27 pm:
Why internal standard use in hplc?what is mean by % RsD/
Hello all,
I'd like to use an internal standard in HPLC.
But I have no experience in this kind of standardisation.
Could someone recomend me a good IS to use in these conditions.
Column C18
CH3CN/H2O isocratic
Sample Temp 35°C
Thx,
Laurent
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 06:55 am:
what is this internal standard for? To make sure the HPLC works fine or something else?
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 07:59 am:
To calculate the factor response of my compounds.
We got very small amounts of pure compounds to make external standardisation.
I know that in GC we could use "Alcane" to perform internal calibration but what could I use in HPLC ?
Ah yes I work at 200nm :/
-------------------------------------------------------------------------------------------------------
By tom jupille on Tuesday, May 4, 2004 - 10:43 am:
I'm not sure why you would want to use an internal standard. You will still have to consume some of your pure compounds to prepare the calibrator solutions.
Internal standards are useful in LC primarily to correct for precision problems caused by injection volume variations or sample workup (i.e., anything that will affect all the peaks in the same proportion). If your precision is OK using external standardization, then the use of an internal standard will provide no advantage (and may, in fact, contribute addtional error).
That said, what you want to find is a compound that is *never* present in your sample, that elutes near your analytes, and is completely resolved from all of them. The alkyl phenones represent a good set of choices (pick the chain length that gives appropriate retention for your purposes).
Final comment: my recollection is that the "alkanes" are used in GC not so much as internal standards as to provide the basis for determining retention indices. If that's what you're looking for, there is no equivalent in LC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 10:45 am:
Does that mean you want to use the internal standard to calculate the concentration of your compound? I never did this for HPLC. Since every compound has its own UV absorbance. The only thing you can do may be choosing something with similar UV performance based on the structure and should not be eluted out at the same time. However, I could not guarantee the result will be accurate.
-------------------------------------------------------------------------------------------------------
By Andreas Neumaier on Wednesday, May 5, 2004 - 07:05 am:
You can choose a compound with similar UV spectrum for external calibration and calculate sample amount by using the relative response factor of the compound you want to quantitate.
But you have to make sure that RRF stay the same after changing the lamp and/or column. Which means you have to use up some small amount of your rare compound every time you check the RRF.
You have a higher rsd than without using RRF.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, May 5, 2004 - 07:42 am:
One should not forget that the internal standard method is an absolute "must" in many biological analyses, where the recovery varies more than the final measurement. Often one can get a synthetic variant of the analyte to use as an internal standard.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 11, 2004 - 03:22 am:
I agree with Tom, that internal standard is useful in HPLC, if you have some problem with injection. If you have very small amount of analyte, you can use other substance to calculate a relative response factor for your analyte, and use this factor for quantitative analysis.
In case of analysis of biological samples the use of internal standard is a bad habit, it can result in faulty data because of the different behaviour of analyte and internal standard during the sample preparation steps. You can determine all necessary data for validation (precision, accuracy, linearity) using samples spiked with the analyte. This way you get more correct data.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, May 11, 2004 - 07:03 am:
Last anon.,
what do you do when you have to analyze plasma/serum of patients with various illnesses, states of metabolism, etc., with wildly differing matricese? You do your stuff with a plasma/serum pool and then pray that all the patient´s sample will behave like your pool? One can usually choose internal standards so close in chemistry, etc., to the analytes that discrimination is negligible (this parallel behavior can be much more robust than the determination of precision, etc. with a pool, or whatever). Anyway, if you want to be sure in chromatography you should do two different chrom methods.... See J Chrom B, 678, 137(1996). One HPLC method without an internal standard can be a fast road to licentiousness.
Also, a problem with injection can usually be controled, whereas the sort of patient´s samples you get is out of your control.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, May 12, 2004 - 06:12 am:
Just a quick note here, not meant to stir things up. When I worked in an environmental lab we used two different compounds during analysis, an "internal standard" and a "surrogate". The "surrogate" compound was added to the sample prior to preparation and went through all the same manipulations as the sample. Then the "internal standard" was added to the final extract before analysis. Obviously the "surrogate" was meant to evaluate the extraction/preparation efficiency and the "internal standard" meant to compensate for any variations during injection. I think it would be clearer here to make some distinction between the uses of the added compounds.
Regards,
Mark
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, May 12, 2004 - 07:25 am:
Also, there is no rule which prevents one from using the external standard method in conjunction (parallel to) the internal Standard method. One has some nice monitoring available that way. The argument that the internal st. method deteriorates accuracy...... is then completely diffused.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, May 16, 2004 - 08:45 am:
We have somewhat departed from the initial question. There was a publication in Analytical Chemistry in July last year on the handling of quantitative assays from biological samples by Matuszewski (sp?) in the case of LC with MS detection. It is supposed to get around the problems of ion suppression common to MS detectors.
-------------------------------------------------------------------------------------------------------
By and on Friday, June 11, 2004 - 06:24 am:
I think you do need the internal standards for the quantitation LC especially LC/MS for the complex samples. There is no one internal standard for all samples. Usually people will choose those non interfering compounds or internal isotopically labled analogue of the analyte.Isotopically labled analogue normally will give you better accuracy.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 17, 2004 - 08:27 pm:
Why internal standard use in hplc?what is mean by % RsD/