Spectrophotometer Guide

General work of spectrophotometer

The basis of the spectrophotometer’s work is to measure the intensity of light in a spectrum of wavelengths created by the greeting.

How to use the spectrophotometer

Based on the contents of the manufacturer’s manual.

How to store the spectrophotometer

The annual service is provided by the device’s backup company.

Spectrophotometer quality control

The quality control of the spectrophotometer includes the evaluation of the wavelength accuracy, the lienearity, photometric accuracy, lamp replacement control, photometric drift (time stability test), identical cuvettes and spectral bandwidth control (SBW).

Correct wavelength

The wavelength accuracy assessment is to prove the manufacturer’s claim that the system has been calibrated for wavelengths.

Verification of wavelengths by replacing a typical optical source of a spectrophotometer with a maximum radiation source (e.g. Mercury or Deuterium lamp) or using glass filters or colored solutions are as follows:

Potassium dichromate solution mg / lit 50 in 0.01 normal sulfuric acid with a maximum absorbance of 257 and 350 nm.
Paranitrophenol solution mmol / lit04 in 0.01 Nm with a maximum absorbance of 401 nm.
Ammonium sulfate solution of 0735 mmol / lit sulfuric acid in sulfuric acid M 0.18 which has an optical absorption maximum of 562 nm.
Cyan methemoglobin solution (20 ml of blood and 5 ml of daphnia) with an absorbance peak of 540 nm.
Oxyglychelin solution (V / V 5% ammonia solution in water + blood) with optical absorption of 540 and 576 nm.

The wavelength control is carried out every three to six months or after each change. It is also recommended to use one or more of the above solutions.

Linearity

Linearity is the power of a spectrophotometer to record a signal proportional to the amount of light. Linearity must be determined by using different dilutions such as potassium dichromate at 350 nm, paranitrofenol (0.04 mmol / lit solution) at 405 nm, copper sulfate solution at 650 nm, cobalt ammonium sulfate solution The length of 512 nm was determined by a methanoglobin cyan solution at 540 nm and an oral green solution at 630 nm and nickel sulfate at 550 nm and plotted in contrast to light absorption, and then the linear spacing or gradient We calculate the slope for each dilution. The non-linearity is a sign of a device failure or a mistake in dilution. The spectrophotometer should be linear at regular intervals and after each change or repair of the device.

Photometric accuracy

The photometric accuracy is whether or not the maximum optical absorption is at a specific wavelength? The photometric accuracy depends on the ability of the lamp to provide maximum radiation, SBW, the type and quality of the chromatograph menu.

You can use one of the following:

The solution of potassium dichromate (mg / lit 50) in 0.01 normal sulfuric acid should have a light absorption of 0.336 ± 0.35 at a wavelength of 350 nm, compared with 0.01 normal sulfuric acid.
Ready-made commercial solutions, including preciset BM at 4.5 nm, can also be used.

Switching lamp control

Due to the luminosity of the lamp in the event of instability, the light absorption should be replaced.

After changing the bulb for any reason, the optical system of the device must be calibrated in such a way that the maximum light reaches the photocell after passing through the cuvette, which is usually accomplished by filling the cuvette from distilled water and changing the position of the lamp and other optical components at a location Maximum T is the best position for the lamp to be installed.

Photometric drift control

The photometric drift is controlled by inserting a cuvette in a package with methamphetamine solution containing methymoglobin solution in the device and optical absorption readings at intervals of every 15-5 minutes for one hour. Of course, this test can also be done with an empty cuvette or distilled water. Existence of a thrust indicates an instability of light absorption or light transmission, which can be due to the burnout of the light source. If there is a thrust, the machine must be repaired, otherwise, at intervals, every 20 to 10 times the specimen should be read, with zero blanks. The maximum acceptable absorbance difference is 0.005 ± 1 / hour.

Stray light

The unwanted lights are light-emitting lights that pass through the light transmitted from the chromatogram menu to the sample. To do this, a solution that completely absorbs the light (such as acetone or sodium nitrite at certain wavelengths) is placed in the light path. In this case, the trans is a zero-percent (infinite and non-readable) absorption because the light does not pass from the space and does not reach the detector.

To investigate unwanted evaporation, aqueous solution of 50 g / l sodium nitrite is prepared and distilled water is read at 300-385 nm. The transient peaks should be zero percent.

Evaluate the same cuvettes

It is necessary that the cuvettes have the same optical absorption rate so that they can not be observed in repeating the measurement of quantities when using multiple cues.

With the help of distilled water (reading optical absorption at 550 nm), cuvettes that have absorption differences of more than 0.01 are discarded.

Using a methanoglobin cyan solution at 540 nm, cuvettes that have more than 0.015 absorbance differences are eliminated.

It is recommended that you do this with a cuvette, Blank Measurement, Standard and Tests to prevent this error.

Spectrophotometer safety device

Before