Cuvette cleaning

Cuvette material Optically clean glass Electrode cell material Organic glass... 

Cuvettes used with the spectrometer are not simply test tubes. They are specially made tubes or cuvettes and are often matched such that a set of tubes or cuvettes will all have the same absorbance characteristics. Cuvettes should never be used as test tubes they must be kept clean at all times, and care must be taken not to scratch them. When using cuvettes that have not been used before, they should be tested to make sure they are all the same. This is accomplished by inserting them into the spectrophotometer and noting their absorbance. All should be the same. Keep in mind that empty cuvettes will have a higher absorbance than when filled with water. This is because light is refracted at each surface, and when filled with water or solvent, there is less refraction at the surfaces. 

Fill a cuvette about three-fourths full with distilled water and dry its outside with a tissue. To calibrate the colorimeter, place the cuvette in the colorimeter and close the lid. Turn the wavelength knob to 0%T. Press TRIGGER on the CBL and enter 0 into the calculator. Turn the wavelength knob to Red (635 nm). Press TRIGGER on the CBL and enter 100 into the calculator. Leave the colorimeter set on Red for the rest of the lab. Remove the cuvette from the colorimeter. Empty the distilled water from the cuvette. Dry the inside of the cuvette with a clean cotton swab. 

Remove the cuvette and pour out the solution. Rinse the inside of the cuvette with distilled water and dry it with a clean cotton swab. Repeat this step for test tubes 2 through 5. 

Clean the cuvette with a cotton swab and fill it about three-fourths full with the unknown dye solution. Place the cuvette in the colorimeter and close the lid. From the MAIN MENU, select COLLECT DATA (do not select SET UP PROBES as this will erase your data lists). Select MONITOR INPUT from the DATA COLLECTION MENU. Press ENTER to monitor the absorbance value of the colorimeter. After about 10-15 seconds, record the absorbance value and record it in your data table. 

Turn off the colorimeter. Clean and dry the cuvette. Return all equipment to its proper place. 

Remove the sample, clean the cuvette and fill with another sample for readings. 

Practical Considerations. Typical absorption assay methods utilize ultraviolet (UV) or visible (vis) wavelengths. With most spectrophotometers, the measured absorbance should be less than 1.2 to obtain a strictly linear relationship (/.c., to obey the Beer-Lambert Law). Nonlinear A versus c plots can result from micelle formation, sample turbidity, the presence of stray light (see below), bubble formation, stacking of aromatic chromophores, and even the presence of fine cotton strands from tissue used to clean the faces of cuvettes. One is well advised to confirm the linearity of absorbance with respect to product (or substrate) concentration under the exact assay conditions to be employed in... 

Never clean cuvettes or any optically polished glassware with ethanolic KOH or other strong base, as this will cause etching. All cuvettes should be cleaned carefully with 0.5% detergent solution, in a sonicator bath, or in a cuvette washer. 

In order to determine the specific activity of the enzyme, the exact concentration of the enzyme must be known. The concentration of the solution of tyrosinase may be determined as a class project by the following procedure. Turn on the spectrophotometer and the UV lamp. Adjust the wavelength to 280 nm. Allow the instrument and lamp to warm up for 15 to 20 minutes. Transfer 1.0 or 3.0 mL of the phosphate buffer to a 1- or 3-mL quartz cuvette. Place it in the sample position of the spectrophotometer and adjust the balance to zero absorbance. Discard the buffer, and clean and dry the cuvette. Transfer 1.0 or 3.0 mL of the tyrosinase solution into the quartz cuvette. Place in the sample position and record the absorbance at 280 nm. Calculate the tyrosinase concentration as described in the Analysis of Results section. 

Pour the contents of the cuvette into the waste ethidium container provided by your instructor. Clean the cuvette carefully with warm water, rinse several times with distilled water, and dry. 

Standardize the spectrofluorimeter in the following way. Pipet 2.0 mL of the pH 7.5, ethidium bromide-Tris buffer into a cuvette. Add 1.0 mL of Tris buffer I and 20 fiL of standard DNA solution. Mix and place in the fluorimeter. Adjust the fluorescence intensity to 100. Clean the cuvette as described in part A and repeat the assay using various concentrations of spermine. Prepare a table displaying the amount of each component to be added. Four reagents must be in the table pH 7.5, ethidium bromide-Tris buffer, DNA solution, spermine, and Tris buffer I. Maintain the volume of DNA at 20 fiL and ethidium bromide solution at 2.0 mL for all assays. Use 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of spermine in the assays. Remember that the total volume of all constituents in the cuvette must remain constant at 3.02 mL for all the assays. Therefore, the amount of Tris buffer I must change with the amount of spermine added. Prepare each assay separately by adding the proper amount of each component to the cuvette. Mix well and record the fluorescence intensity of each cuvette. 

Disposable polystyrene cuvettes eliminate thejob of cleaning dye-stained quartz or glass cuvettes. 

A single spectrum, with associated baseline, will require -1 hr, including the time needed to fill and clean the cuvette. Up to an additional hour may be needed for optimizing instrument parameters if the sample is unfamiliar. For a number of samples, economies of time can be effected (see Basic Protocol). 

Calibrated fluorescence spectrometer, buffer solution, and cleaned cuvettes (see Strategic Planning)... 

Fill a cleaned cuvette with a filtered sample from the actual batch of buffer used to dissolve or dialyze the protein, then place the cuvette in the cell holder of the spectrometer in a reproducible orientation (see Strategic Planning, discussion of Cells). Scan this buffer blank using the same instrument settings as are appropriate for the sample, store the spectrum, and check for any unexpected fluorescence bands. 

Make sure that cuvette surfaces are clean. 

Clean the cuvette, syringe, and in particular the syringe tip (made of stainless steel) of a drop volume tensiometer with diluted chromic acid ( 1 mM). Rinse extensively with ultrapure water to completely remove any remaining acid. 

For a liquid/gas interface, the primary fluid" is simply air and the cuvette will therefore remain empty. In this case, extensive cleaning of the cuvette is not required. 

Click only once to adjust to zero when using the reagent blank, because spectrophotometers usually adjust themselves again after a second click. Newer spectrophotometers should adjust to zero automatically. Use only one clean 1-ml quartz cuvette with two opaque walls. If the spectrophotometer has a flow-through cell, make sure that it is cleaned regularly. For further details, see Critical Parameters and Troubleshooting. 

---