Cuvettes volume determination

Various concentrations of ascorbic acid (0.2, 2.0, and 20 fxM) were incubated in a cuvette with the various buffers listed in Table II and monitored at 254 nm. The concentration of ascorbic acid was selected to approximate the sample ascorbic acid coming into contact with a buffer volume determined by its time in transit through the column. No detectable losses were observed over the 5-min incubation time for any concentration of ascorbic acid under any concentration of citrate buffer or pH. 

The estimation of the exact working volume of the cuvette is made individually for each cuvette by determining the dilution of a colored... 

Figure 5. Determination of the cuvette volume, the time for mixing the reactants, and the time for emptying the cuvette. The cuvette used in this experiment (Fig. 2A) has a path of 6 mm. By injecting 8 pi of 2.16mAf Hb02 solution (first arrow) the extinction increases by 0.672 units, and from this the calculated cuvette volume is 2.16 x 34 x 0.6 x 8/0.672 = 525 pi. The second arrow indicates the start of the cuvette emptying, at a rate of the pump of 4 ml/min. (Inset) Calculation of the first-order rate constant and tU2 for the cuvette emptying.

The initial mixture and each time point are then assayed for doxorubicin and lipid. Lipid concentrations can be quantified by the phosphate assay (see above) or by liquid scintillation counting of an appropriate radiolabel. Doxorubicin is quantified by an absorbance assay (see below). The percent uptake at any time point (e.g., t = 30 minutes) is determined by %-uptake = [(D/L), =30minutes] x 100/[(D/L) inuiai]. Doxorubicin can be assayed by both a fluorescence assay and an absorbance assay, but we find the latter to be more accurate. The standard curve consists of four to five cuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL samples to be assayed are of the same volume. To each tube is added 0.9 mL of 1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems such as DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to 15 seconds, until the detergent turns cloudy. Samples are allowed to cool, and absorbance is read at 480 nm on a UV/Visible spectrophotometer. 

Make up the volume with distilled water up to 1.6 ml. Then, add 0.4 ml of Coomassie Blue. Mix slowly, and measure the OD at 595 nm. Concentrations of the different enzyme dilutions can be determined using the standard plot. The concentration of -galactosidase stock solution is obtained by multiplying the enzyme concentration in the cuvette by the dilution factor. 

Using the data shown below, answer questions a and b The GOT assay was performed in a total volume of 3 ml. The value in parentheses indicates how much the fraction was diluted before using 0.1 ml in the assay. The protein assay was performed by diluting the samples as indicated in parentheses and determining an A2so value in a 1-cm pathlength cuvette. Assume... 

These are often multi-parameter analysers and enable several determinations to be carried out on the same sample furthermore, the number of determinations can be programmed for each individual sample. An aliquot of the sample is placed in a transparent measurement cuvette following addition of the reagents, a colorimetric measurement is then carried out directly on the cuvette. The analysis rate varies from 50 to 1000 determinations per hour, and the principal applications are carried out either by chemical or enzymatic analysis. The volumes of reagent required for sequential analysis are small by comparison with FTA which substantially reduces the cost per analysis, particularly for enzymatic determinations. 

A pre-determined volume of sample is removed by the robotic arm from the vial in the sampling tray and placed in the reaction cuvette. 

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