Standardization curve

The concentration range over which the analyte will be determined must be defined in the method, on the basis of actual standard samples over the range (standard curve). 

Determination of accuracy and precision should be made by analysis of repHcate sets of analyte samples of known concentration from equivalent matrix. At least three concentrations representing the entire range of the caUbration should be studied one near the minimum (MAQ), one near the middle, and one near the upper limit of the standard curve. 

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

The phase curve passes through —90° at a = Wn- Its shape depends upon ( and is obtained from the standard curves given in Figure 6.11. 

When plotting the standard curve it is customary to assign a transmission of 100 per cent to the blank solution (reagent solution plus water) this represents zero concentration of the constituent. It may be mentioned that some coloured solutions have an appreciable temperature coefficient of transmission, and the temperature of the determination should not differ appreciably from that at which the calibration curve was prepared. 

Standard curves in spectrophotometry, 674 Standard deviation I 34 Standard potentials 62, 63, 66 Standard series method 652, 654 Standard solutions 107, 257, 259 for pH, 569, 831 prepn. of, 107, 260, 285, 802 storage of, 108 Standard substances for acidimetry and alkalimetry ... 

Fig. 10.2. Standard curve for cell density define based on growth of Saccharomyces cerevisiae.

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

Figure 15. Standard curves for norepinephrine and dopamine vs. dihydroxybenzylamine as an internal standard (Kawasaki, T. Wong, 0. Imai, K. Higuchi, T., in preparation).

Standard curves performed under our defined radioimmunoassay conditions ([ H]PbTx-3 = 1 nM, antiserum dilution = 1 2000, assay volume = 1 ml) demonstrated the ability of this antiserum to bind equally to PbTx-2 and PbTx-3, suggesting specificity for the cyclic polyether backbone region of the molecule (Figure 8). The linear portion of the curve indicated a lower detection limit of 0.2-0.5 ng in saline buffer under these conditions. Evaluation of this assay for use with biological fluids and tissue extracts is underway. 

Figure 8. Standard curves for PbTx-2 ( ) and PbTx-3 ( ) in the brevetoxin radioimmunoassay. Lower detection limits are 0.2 — 0.5 ng in phosphate-buffered saline (PBS). Standard RIA conditions [ H]PbTx-3 = 1 nM antiserum dilution = 1 2000 sample vol. = 1 ml buffer = 0.1 M PBS, pH = 7.4.

Furthermore, from equation (4) it can be seen that when the amount of P and Q are fixed, the more P added the smaller will be the quantity P Q. Therefore, if various known amounts of P are added and P Q is measured in a suitable counter, the relationship between radioactivity and concentration of P can be established. From this a standard curve can be drawn and used to determine the amount of P in an unknown solution. 

The concentration of the unknown is then read off the standard curve opposite its B/Bq value. This sigmoid shaped standard curve, because of its linear portion, simplifies data handling. A mathematical transform of the B/Bq vs log dose is shown in Figure 2. This logit of B/Bq vs log dose is a widely used method of standard curve presentation (5,6,7). Logit B/B is defined as follows ... 

After counting the samples and obtaining a printout from the counter the data must be arranged into a standard curve of some type from which patient results can be determined. 

In order to do this, a simple calculator may be used to aid in the mathematical manipulations. A desktop computer into which the data is entered may be used to generate a standard curve automatically along with the unknown values which are automatically read from the curve. If a paper tape print out... 

Finally9 tubes for the standard curve are followed by the quality control tubes and the unknown tubes. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

Typical standard curves for a technical grade of parathion are shown in Figure 3. Beer s law is followed within the range 10 to 400 micrograms of parathion per milliliter of dyed solution. Deviation becomes apparent outside this range, under the conditions of test. 

A stock solution of the triazole in hexane was made up and diluted to various strengths, and 1.0-ml. aliquots of the diluted solutions were carried through the procedure described below. The transmittance of the colored solutions obtained from 10 to 50 micrograms of the 118-phenyldihydrotriazole was plotted against concentration to make a standard curve. In subsequent analyses, the amount of Compound 118 is readily calculated from the amount of dihydrotriazole formed. 

Reagents and apparatus are the same as those used in the preparation of the standard curve. 

Procedure. A hexane solution of Compound 118 is diluted or concentrated so as to bring the 118 content within a range of 15 to 150 micrograms per ml. In cases where the hexane solution requires concentration, the evaporation is carried out in a beaker on a steam bath with a gentle stream of air passing over the surface. The concentrated or diluted solution of 118 is washed with hexane into a volumetric flask and made up to volume with the hexane washings. One milliliter of the adjusted Compound 118 solution is precisely measured into a spectrophotometer cell, 2 drops of phenyl azide are added, and the dihydrotriazole is quantitatively formed and then treated with diazotized dinitroaniline to produce the red color as in the preparation of the standard curve. A blank, starting with 1.0 ml. of hexane and 2 drops of azide, is run at the same time. 

The final blank solution is set at 100% transmittance and the transmittance of the test solution is then measured. Reading from the standard curve, one obtains the number of micrograms of triazole. 

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