What are the disadvantages of using simple calibration curve (i.e. plotting standard concentrations against peak area for each batch of sample run) compared to external or internal standard methods of quantitative analysis of VOC by GC/FID?
Source of error is going to be much higher if you do not pay attention to the linear range of your detector. Make sure you use at least 5 points in your calibration and that the response is linear throughout. One of the problems associated with using an IS with GC/FID is that sample matrix interferences could enhance the signal of the IS thus contributing a significant error to your quantitative results. This matrix effect will not be present in your calibration material or in your blanks. If you do use IS make sure to carefully monitor the IS responses in the samples and compare to the IS responses in your standards and blanks. Qualify any samle results that have IS areas that are out of range 70% - 130% is a good starting point. Spiking your samples with a surrogate standard (SS) compound may be an alternative or additional QC practice that would show recoveries for the analytical procedure. The devil is in choosing an appropriate IS/SS for your procedure.
Internal standard (IS) based calibration method is generally used in different chromatographic experiment for improving the accuracy and precision of the method , there are many reason for that but mainly because these techniques involved uncontrollable parameters which can affect the response significantly, IS is used to compensate the loss of analyte during the preparation and injection step in other word, because IS whit known conc and response is gone through exactly the same procedure which analyte has been gone, dividing the related responses will eliminate the errors.note that the IS should be as similar as possible to analyte but also has distinguishable response.
Response of analytes varies sometimes significantly due to loss of ions generated when GC-FID technique is used for quantitative analysis, suitable Internal standard is used to minimize this error.
Good afternoon. What you are talking about is the method called "external calibration", which are many disadvantages, in some cases, and advantages, in other cases. In GC/FID, most people, including myself, uses internal calibration, with an internal standard, i.e., a molecule that is not among your analytes (i.e., is absent in your sample), byt appears in the GC/FID chromatograms, and has peak with identical characteristics of the peacs of your sample, except the retention time.
The method of the internal standard is more accurate than external calibration, and the accumulation of errors during the whole processus of analysis is smaller.
I do not have experience in VOC and GC-FID but I agree with others, the use of internal standard is of benefit. However, if this method cant be applied for various reasons, I would suggest to run standards few times during any batch run to check for loses (i.e. if its a short run of let say 10 samples, one standard run before and after the batch of samples).
According to my experience in PAH analysis by GC/MS, I can tell you that IS method is the alternative for calibrating, since error in cuantification by losses of analites are compensed.
What is a simple calibration curve? There is no such thing as simple or complicated. It depends on the underlying statistical assumptions. Most GC relies on one point calibration for automation provided that the curve is linear between 0 and the target response (y value), and there is no significant suppression and enhancement effect. Multivariate regression is more complicated. HPLC using DAD can make full use of this because calculation is based on more than 1 wavelength. Internal standard is for controlling analytical errors mostly coming from the instrument as well as other steps involved. External calibration is more accurate but fasditious for routine. Standard addition is the best approach for complex matrix. It total depends on your purpose. Choose one that is fit for it.
IS is to prefer, not external. GC-MS is also important for identification prior GC-FID.
Source of error is going to be much higher if you do not pay attention to the linear range of your detector. Make sure you use at least 5 points in your calibration and that the response is linear throughout. One of the problems associated with using an IS with GC/FID is that sample matrix interferences could enhance the signal of the IS thus contributing a significant error to your quantitative results. This matrix effect will not be present in your calibration material or in your blanks. If you do use IS make sure to carefully monitor the IS responses in the samples and compare to the IS responses in your standards and blanks. Qualify any samle results that have IS areas that are out of range 70% - 130% is a good starting point. Spiking your samples with a surrogate standard (SS) compound may be an alternative or additional QC practice that would show recoveries for the analytical procedure. The devil is in choosing an appropriate IS/SS for your procedure.
Standard methods are nessesary for determination of unknown amounts of analytes in the sample.
There are many different standard methods, e.g.,
1. External standard method
2. Internal standard method
3. Matrix matched/spike method
External Standard Calibration
Analytes in different concentrations are monitored on the MS.
An external calibration curve is prepared where response in standards is plotted as a function of concentration.
The concentration in samples are determined by measuring the response in samples and comparision to the calibration curve.
Matrix Effect
Even though we remove the matrix during sample preparation, some will always be present in the final sample extract. These may interfere with the GC/MS measurements causing suppression of response.
Internal standard calibration uses three types of standards:
1.Internal standard (ISTD)
2. Quantification standard (QS)
3. Recovery standard (RS)
This calibration method takes losses of analytes during sample preparation into account. Therefore it is a very accurate calibration method.
INTERNAL STANDARD (ISTD)
Internal standard is added to the samples prior to extraction. When using GC-MS, mass labelled compounds are used as ISTD.
IS is used for quantification of analytes. By comparing the response in the sample from the ISTD to the response from the analytes, it is possible to determine the amount of analyte in the sample.
The internal standard behave similar to the analytes, therefore one can assume that a loss of ISTD during sample preparation implies the same loss of analyte. Therefore, there is no need for recovery corrections.
QUANTIFICATION STANDARD (QS)
Contains known amounts of the analytes, the internal standards and the recovery standard.
Preferable, QS should contain the SAME internal standard as the samples.
QS is analyzed on the GC-MS at the same time as the samples. Every 10-20 injection should be the quantification standard.
RECOVERY STANDARD (RS)
Recovery standard is added to the samples in the end of sample preparation.
RS is used to calculate the recovery of internal standards during sample preparation. Since we assume the loss of ISTD to be equal to the loss of analyte, the recovery will be a measure on the performance during sample preparation.
Matrix Matched Calibration
Built on the same principles as internal standard calibration with IS, QS and RS.
QS is added to the matrix. The standard sample is extracted and cleaned-up at the same time as the samples.
The method corrects for matrix effects but is very time consuming!
Make sure you use at least 3 points in your calibration and that the response is linear throughout. One of the problems associated with using an IS with GC/FID is that sample matrix?. This matrix effect will not be present in your calibration material or in your blanks.
External Standard Calibration
An external calibration curve is prepared where response in standards is plotted as a function of concentration.The concentration in samples are determined by measuring the response in samples and comparision to the calibration curve.
In my experience with PAHs (16) first, I think that you need to know the range of your probable levels. At least you can use 5 points in your calibration curve. In the extraction I add a different compound (analyte) among the 16 PAHs of my study in the duplicate samples, it’s increase the number of total samples but you have quality in your analysis and always I use blanks.
You have to wisely select between internal standard calibration and external standard calibration, however IS have certain more advantages. The issue has already been explained well by various scientist above. Before adding an IS you have to consider the matrix of the sample.
Actually for highest accuracy you should use both internal standards to correct for error in the instrument and or extraction method and external calibration.
If your analysis is based on manual injection you need to add one or more (for complex mix) IS to your sample. The addition of IS guarantees the reproducibility of ratio between analytes and IS and calibration curve reports this ratio versus the concentration of each VOC.
If your GC is equipped with an autosampler you will obtain a good reproducibility and so even an external calibration will give good results.
Sadegh,
Also - You can access EPA Method 8000B, Determinative Chromatographic Separations here http://www.epa.gov/waste/hazard/testmethods/sw846/pdfs/8000b.pdf this has accepted practices for the use of GC as an analytical quantitative tool. A very good reference, and the gold standard in the U.S.
use 3 point calibration curve for quantitative analysis. And for this you must use Response factor (RF) method for analysis.
I agree wholeheartedly with Peter Philbrook in relation to the need to take note of linear range and the possibility of matrix interferences affecting the measured peak areas of the IS peak. I also agree with the need to check the expected IS peak areas as a routine QA process. To this excellent advice I would add that, particularly if the chromatographic run is a long one, it is advisable to use several internal standards to take into account any drift in detector conditions during the run. The presence of several internal standards within a run also allows an additional means of routinely checking for interferences at one or more IS peaks. Even if two IS peaks are interfered with it is unlikely that the amount of interference will be identical for both. Thus if the analyte concentrations measured using one internal standard match with the analyte concentrations measured using another then the IS peaks are unlikely to be subject to interference. Careful scrutiny of the chromatogram for any sample runs where, by whatever means, potential problems are identified should be a matter of course. I would also add that if the analysis is of a well established and understood matrix the chromatography should already have been optimised such that the IS peaks are separable from all expected non-analyte matrix peaks as well as all of the analyte peaks. Finally, except for the simplest of analytical problems (of which I would not include a VOCs determination from most samples), I would caution against reliance on “FID only” peak identification unless it is backed up with a determination carried out on a column with different polarity. Of course this would also depend to some extent on the use to which one proposes to put the data but with the availability of relatively low cost GC-MS these days I am surprised that this determination is being proposed with an FID.
Assuming there is no matrix effects, the main disadvantage of direct calibration is the poor reproducibility due to variation in injection volume and it does not compensate for sample losses during sample preparation.
If preparing sample that same condition u should prepare standard. Atleast u should take more then 3 standard curves can get good result. internal method get good result for ur spike recovery then compare sample.
Hello Jai Ghosh! To avoid that limitations, is common practice in most reference labs, to calculate the "response factor" of each FID detector, with certain periodicity. The response factor, for each molecule you want to analyse, can be calculated by this way: first, you determine the area under the Gas-chromatography peak, of your molecule, with a known amount injected, comparing with the area under the peak of the internal standard.
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