Standard, internal

An internal standard Is a different substance from the analyte. 

When the relative response of an instrument to analyte and standard remains constant over a range of concentrations, we say there is a linear response. For critical work, this assumption should be verified because it is not always true.  

If the detector responds equally to standard and analyte. F = 1. If the detector responds twice as much to analyte as to standard F = 2. If the detector responds half as much to analyte as to standard, F = 0.5. 

An internal standard is a known amount of a compound, different from analyte, that is added to the unknown. Signal from analyte is compared with signal from the internal standard to find out how much analyte is present. 

Internal standards are also desirable when sample loss can occur during sample preparation steps prior to analysis. If a known quantity of standard is added to the unknown prior to any manipulations, the ratio of standard to analyte remains constant because the same fraction of each is lost in any operation. 

The successful application of an external standardization or the method of standard additions, depends on the analyst s ability to handle samples and standards repro-ducibly. When a procedure cannot be controlled to the extent that all samples and standards are treated equally, the accuracy and precision of the standardization may suffer. For example, if an analyte is present in a volatile solvent, its concentration will increase if some solvent is lost to evaporation. Suppose that you have a sample and a standard with identical concentrations of analyte and identical signals. If both experience the same loss of solvent their concentrations of analyte and signals will continue to be identical. In effect, we can ignore changes in concentration due to evaporation provided that the samples and standards experience an equivalent loss of solvent. If an identical standard and sample experience different losses of solvent. 

A standard, whose identity is different from the analyte s, that is added to all samples and standards containing the analyte. 

A standardization is still possible if the analyte s signal is referenced to a signal generated by another species that has been added at a fixed concentration to all samples and standards. The added species, which must be different from the analyte, is called an internal standard. 

Since the analyte and internal standard in any sample or standard receive the same treatment, the ratio of their signals will be unaffected by any lack of reproducibility in the procedure. If a solution contains an analyte of concentration Ca, and an internal standard of concentration, Cis, then the signals due to the analyte, Sa, and the internal standard. Sis, are 

Because equation 5.10 is defined in terms of a ratio, K, of the analyte s sensitivity and the internal standard s sensitivity, it is not necessary to independently determine values for either or kjs. 

If we add a known amount of a compound to our solution, we can use it to quantify the material of interest. This is great except that we may not want to contaminate our material with some other compound. A number of people have looked at using standards that are volatile so that they can be got rid of later (TMS is an example that we have seen published). The problem with this approach is that if the sample is volatile then you need to run it quickly before it disappears. TMS disappears really quickly from DMSO so it is probably not a good idea in this case. TMS also suffers from the fact that it has a long relaxation time so you have to be very careful with your experiment to ensure that you do not saturate the signal. The last major problem with TMS is that it comes at the same part of the spectrum as silicon grease which can be present in samples. Choosing a standard so that it has a short relaxation time, is volatile and comes in a part of the spectrum free of interference is really tricky. In fact, we wouldn t recommend it at all. 

The admixture of the standard should be dimensioned so that the strongest lines of sample and standard become about equal. The right ratio can be calculated if the I/I -values of standard (Is/Ic) and sample (Ip/Ic) are known (see above at intensity calibration). The weight ratio Xp ( sample) (standard) can be estimated by  

If the I/Ic-values are not known, one can admix the standard in small portions until the strongest reflections show about equal intensity. 

If fluorphlogopite is used as an internal standard, an alignment of the mica flakes parallel to the sample surface is preferred. This can be obtained if a small amount of the mixture sample/standard is suspended in acetone or isopropanol and is sedimented to a single crystal sample holder. 

For the Guinier-camera a compromise between external and internal standard is possible, as the camera allows the simultaneous recording of several samples onto the same film by dividing the camera by the installation of separating disks. Mostly three individual patterns are taken simultaneously pure sample, mixture sample/standard, and pure standard. If one wants to dispense with the mixture, it will be sufficient to put the sample pattern in the centre and a standard pattern at each side of the Guinier-film. 

Only those standard reflections are suited for a calibration that do not overlap with sample reflections. If unavoidable, one may try to separate standard and sample reflections by profile fitting. But in the plot for the calibration curve the A20-values of these standard reflections should follow 

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Finally, other methods are used to obtain simulated distillation by gas phase chromatography for atmospheric or vacuum residues. For these cases, some of the sample components can not elute and an internal standard is added to the sample in order to obtain this quantity with precision. 

There are two standard procedures for determining the octane numbers Research or FI and the Motor or F2 methods. The corresponding numbers are designated as RON (Research Octane Number) and MON (Motor Octane Number) which have become the international standard. 

The ISO 8681 standard, which treats all the petroleum products, groups lubricants, industrial oils and related products in the L Class. The international standard ISO 6743/0, accepted as the French standard NF T 60-162, subdivides the L Class into 18 families or categories. 

Furthermore, each sub-category given in Table 6.2 can be divided according to product viscosities, which are classified in the international standard ISO 3448 (French standard NF ISO 3448, index T 60-141). 

International standard ISO 8402 1994 defines procedure as determined mode of the activities realization. We consider activities on testing. 

Flow measurements using tracers are performed in all piping systems carrying oil, gas or water including separators, compressors, injector systems, and flares. Calibration of elsewhere difficult accessible flow meters is regularly performed by the tracer methods, which are based on international standards. Tracer flow measurements are also well suited for special purposes... 

The method is based on the international standard ISO 4053/IV. A small amount of the radioactive tracer is injected instantaneously into the flare gas flow through e.g. a valve, representing the only physical interference with the process. Radiation detectors are mounted outside the pipe and the variation of tracer concentration with time is recorded as the tracer moves with the gas stream and passes by the detectors. A control, supply and data registration unit including PC is used for on site data treatment... 

The most important feature of editing software is the option to save the structure in standard file formats which contain information about the structure (e,g., Mol-filc. PDB-filc). Most of these file formats arc ASCII text files (which can be viewed in simple text editors) and cover international standardized and normalized specifications of the molecule, such as atom and bond types or connectivities (CT) (see Section 2,4). Thus, with these files, the structure can be exchanged between different programs. Furthermore, they can seiwe as input files to other chemical software, e.g, to calculate 3D structures or molecular properties. 

In a single-point internal standardization, a single standard is prepared, and K is determined by solving equation 5.10... 

A sixth spectrophotometric method for the quantitative determination of Pb + levels in blood uses CQ+ as an internal standard. A standard containing 1.75 ppb Pb + and 2.25 ppb CQ+ yields a ratio of Sa/Sis of 2.37. A sample of blood is spiked with the same concentration of Cu +, giving a signal ratio of 1.80. Determine the concentration of Pb + in the sample of blood. 

A single-point internal standardization has the same limitations as a singlepoint normal calibration. To construct an internal standard calibration curve, it is necessary to prepare several standards containing different concentrations of analyte. These standards are usually prepared such that the internal standard s concentration is constant. Under these conditions a calibration curve of (SA/Sis)stand versus Ca is linear with a slope of K/Cis-... 

A seventh spectrophotometric method for the quantitative determination of Pb + levels in blood gives a linear internal standards calibration curve for which... 

When the internal standard s concentration cannot be held constant the data must be plotted as (SA/Sis)stand versus Ca/Cis, giving a linear calibration curve with a slope of K. 

Using the Regression Equation Once the regression equation is known, we can use it to determine the concentration of analyte in a sample. When using a normal calibration curve with external standards or an internal standards calibration curve, we measure an average signal for our sample, Yx, and use it to calculate the value of X... 

Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample s matrix is to explore the effect of pH on the quantitative analysis of an acid-base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 buffer and used to analyze samples buffered to different pHs in the range of 6-10. Results can be compared with those obtained using a standard addition. 

A standard sample was prepared containing 10.0 ppm of an analyte and 15.0 ppm of an internal standard. Analysis of the sample gave signals for the analyte and internal standard of 0.155 and 0.233 (arbitrary units), respectively. Sufficient internal standard was added to a sample to make it 15.0 ppm in the internal standard. Analysis of the sample yielded signals for the analyte and internal standard of 0.274 and 0.198, respectively. Report the concentration of analyte in the sample. 

Troost and Olavesen investigated the application of an internal standardization to the quantitative analysis of polynuclear aromatic hydrocarbons. The following results were obtained for the analysis of the analyte phenanthrene using isotopically labeled phenanthrene as an internal standard... 

Caffeine is extracted from beverages by a solid-phase microextraction using an uncoated fused silica fiber. The fiber is suspended in the sample for 5 min and the sample stirred to assist the mass transfer of analyte to the fiber. Immediately after removing the fiber from the sample it is transferred to the gas chromatograph s injection port where the analyte is thermally desorbed. Quantitation is accomplished by using a C3 caffeine solution as an internal standard. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

Precision For samples and standards in which the concentration of analyte exceeds the detection limit by at least a factor of 50, the relative standard deviation for both flame and plasma emission is about 1-5%. Perhaps the most important factor affecting precision is the stability of the flame s or plasma s temperature. For example, in a 2500 K flame a temperature fluctuation of +2.5 K gives a relative standard deviation of 1% in emission intensity. Significant improvements in precision may be realized when using internal standards. 

Samples of analyte are dissolved in a suitable solvent and placed on the IR card. After the solvent evaporates, the sample s spectrum is obtained. Because the thickness of the PE or PTEE film is not uniform, the primary use for IR cards has been for qualitative analysis. Zhao and Malinowski showed how a quantitative analysis for polystyrene could be performed by adding an internal standard of KSCN to the sample. Polystyrene was monitored at 1494 cm- and KSCN at 2064 cm-. Standard solutions were prepared by placing weighed portions of polystyrene in a 10-mL volumetric flask and diluting to volume with a solution of 10 g/L KSCN in... 

What is the %w/w Pb in a sample of brass that gives an emission intensity of 9.25 X lO" The analysis for Ni uses an internal standard. Results for a typical calibration are shown in the following table. ... 

Zinc can be used as an internal standard in the analysis of thallium by differential pulse polarography. A standard... 

Assume that p-xylene is the analyte and that methylisobutylketone is the internal standard. Determine the 95% confidence interval for a single-point standardization, with and without using the internal standard. 

For a single-point external standard (omitting the internal standard) the relationship between peak area, A2, and the concentration, C2, ofp-xylene is... 

For an internal standardization, the relationship between the peak areas for the analyte, A2, and the internal standard, Ai, and their respective concentrations, Ci and C2, is... 

As this example clearly shows, the variation in individual peak areas between injections is substantial. The use of an internal standard, however, corrects for these variations, providing a means for accurate and precise calibration. 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

One advantage of an HPLC analysis is that a loop injector often eliminates the need for an internal standard. Why is an internal standard used in this analysis What assumption(s) must we make about the internal standard ... 

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

A quantitative analysis for vitamin Bi was carried out using this procedure. When a solution of 100.0 ppm Bi and 100.0 ppm o-ethoxybenzamide was analyzed, the peak area for vitamin Bi was 71 % of that for the internal standard. The analysis of a 0.125-g vitamin B complex tablet gave a peak area for vitamin Bi that was 1.82 times as great as that for the internal standard. How many milligrams of vitamin Bi are in the tablet ... 

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