Standard chromatogram

Figure 13.4 shows the standard chromatogram obtained by using this method. In this sample, isoprene and major interference compounds, i.e. 2-methyl pentane and hexane, were spiked at 1 p.g 1 , plus DMS at lOpig 1 . ... 

To characterize the difference between two chromatograms, an overall mismatch index and a list of the areas, helglits, and positions of the main peaks in the difference chromatogram are provided. The difference chromatogram is the result of subtracting the standard chromatogram for that class of specimen from the chromatogram of the specimen. 

Figure 2 Example of a standard chromatogram (in pure solvent without matrix components) with the analyte peak (11) eluting at 18.23 min, solvent peaks (1, 3), and instrumental noise (5,6, 13)...

Where Cone, is concentration, s is sample chromatogram, IS is the internal standard and std. is the standard chromatogram. 

The concentrations of each analyte can be calculated by relating the peak area in the sample chromatogram to the peak area in the standard chromatogram as follows peak area in sample... 

Figure 4.11—Standard chromatogram producedhy a recording integrator showing retention time (min), area of the peak (arbitrary units) and corresponding % of area. The % area of the peaks must not be confused with the % quantity of the sample constituents since the response factors are not identical for all compounds (non-normalised report).

Reference Solutions. Prepare a chloroform solution containing 10 Lig/ml of each of barbitone, butobar bitone,amylobarbitone,pentobarbitone,qumalbarb-itone. and tetraphenylethylene (internal standard), and inject 3 Lil on to both columns to obtain standard chromatograms (e.g. Fig. 1). 

Identify any peaks which appear on the chromatograms by reference to the standard chromatogram and 10 the retention data listed in Table 9 the index of Gas Chromatographic Data in Part 3 may also be consulted. Corroborative evidence must be obtained from both systems. For quantification, prepare standard solutions of the identified drug containing 0., 10, 20, 50. and 100 Lig/ml in plasma. Examine at least three standards by the same method as used for the sample. Duplicate analyses of samples and standards are essential. 

Identify any peaks which appear on the chromatograms by reference to the standard chromatogram and to the retention data listed in Table 11 the indexes of Gas Chromatographic Data in Part 3... 

Reference Solution. Prepare a solution in chloroform containing 30 g/ml of edichlorvynol, 50 jig/ml of each of chlormethiazole and trichloroeOmiol, Mid 25 Lig/ml of 2-methylnaphtiialene (internal stMidM-d). Inject 3 )Lil of this solution on to the gas chromatographic column to obtain the standard chromatogram. 

Method. Add 50 li1 of whole blood or urine to 0.5 ml of distilled water in a stoppered test-tube, mix in a vortex mixer, and inject 1 illI on to the column. Identify any peaks which appear by reference to the standard chromatogram. 

Inject aliquots from 0.01 to 1.0 p.1 of this reference mixtiu-e on to the coliunn to produce the standard chromatogram. 

Identity is a general requirement for dosage forms. When determining specificity for identity, the assay and related substances or the content uniformity methods can be used. Assay and content uniformity methods are quantitated by external reference standard. This identity test confirms that the correct active ingredient (s) is present and is present in correct ratio if multiple variants are available. The method could also be used for post-packaging analysis. The general requirements are that the sample and standard chromatograms should correspond in retention time and normalized peak area within 10%. 

Figure 1. Thiohydantoin-amino acid standard chromatogram (about 50 pmoles).

Fig. 2 Typical polystyrene narrow molecular-weight range standard chromatograms and calibration curve.

Detection and identification Comparison of sample and standard chromatograms scanned at six different wavelengths with a densitometer. 

Column Partisil SCX-10 (250x4.6 nm ID), protected with a precolumn (30x2.8 mm ID) packed with 30 pm pellicular beads, mobile phase methanol - water (1 4) containing 0.2% ammonium phosphate and 0.1% potassium chloride (pH 4.5), flow rate 1 ml/min, temperature 50° C. Peaks 1, psilocybin 2, psilocin 3, dimethyltryptamine (internal standard). Chromatogram a standard mixture, detection UV 267 nm chromatogram b mushroom extract, detection UV 267 nm chromatogram c standard mixture, fluorescence detection ( excitation 267 nm, emission 335 nm) chromatogram d mushroom extract, fluorescence detection. 

Other important information that has to be derived from the standard chromatogram are the column porosities and the void fraction. These parameters have to be carefully determined as they will be the basis for detailed modeling and simulation of the purification process. As illustrated in Fig. 2.4, the total volume of a packed column (Vc) is divided into two sub-volumes the interstitial volume of the fluid phase (Vint) as well as the volume of the stationary phase (Vads) (Eq. 2.4)... 

Fig. 3-14. Standard chromatogram of an AS2 separator column. - Eluent 0.003 moI/L Na2C03 + 0.002 mol/L NaOH other chromatographic conditions see Fig. 3-13.

The first successful polyamine separations were accomplished in the mid 70s after the introduction of surface-sulfonated cation exchangers. Fig. 3-164 displays a standard chromatogram with the separation of putrescine (1,4-diaminobutane) and cadaverine (1,5-diaminopentane) as well as spermidine (A-(3-aminopropyl-1,4-diaminobutane) and... 

Standard addition is elegant if the sample amount is not limited. It allows calibration of the analysis under realistic conditions, i.e. not with a standard chromatogram which is free from interferences. The standard addition and internal standard methods can be combined. 

The retention volume of an analyte represents the volume of mobile phase necessary to enable its migration throughout the column from the moment of entrance to the moment in which it leaves. To estimate this volume, different methods (direct or indirect) may be used, that depend of the physical state of the mobile phase. On a standard chromatogram with time in abscissa, Tg is calculated from expression 1.16, if the flow rate F is constant. 

When unusual spots are found on these two standard chromatograms, it is of course necessary to run more samples of the urine through the process to provide supplementary chromatograms for other location techniques (N2) or to make specific tests on the eluate of such unknown spots. 

IsocUric Acid. Isocitric acid is not separated from citric acid on standard chromatograms of organic acids. Special techniques have been described for the paper chromatographic separation of these two acids (CIO, P7), but they do not seem to have been applied to biological fluids. 

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