UV spectrophotometer calibration


UV-1800 UV-VIS spectrophotometer has been designed in accordance with the Japanese and European Pharmacopoeia, UV-1800 is the only UV-VIS spectrophotometer which achieves a resolution of 1 nm, that is the highest in its class, in such a compact design. UV spectrophotometer calibration is very easy in this model.

UV-1800 UV-VIS spectrophotometer has designed in accordance with the United States, Japanese and European Pharmacopoeia is very user-friendly in features, the UV-1800 can be used either as a stand-alone instrument or as a PC-controlled instrument.

UV calibration procedure:-

General Cleaning:

Ensure that power to the instrument is switched off. Clean the outer surface of the instrument with a dry cloth. If required, wipe with a cloth soaked in a good quality detergent. Subsequently, wipe with a piece of dry cloth.


(1) Switch on the instrument from the mains and wait for Initialization.

(2) After completion of Initialization. instrument show different (mode MENU) on the screen.

(3) Log in –User Name – administration

(4) Input password – Enter (If any)

(5) Select number 8 (Utilities) from MODE MENU, details will appear on the screen, Select F3 key and press number 2 (instrument baseline correction) and wait for 15 minutes till baseline correction is over and press again numerical key 1 for select photometric.

(6) After baseline correction is over press (Return) key and then the (Mode MENU) key. Now press key number 1, (Photometric) details will appear on the screen.

(7) Press (gotoWL) for required wavelength using the numerical key by pressing key (F1), %T/Abs as per requirement %T stands for percentage transmittance and Abs stands for absorbance.

(8) Place the cells filled with diluents into the cell holder and press (Auto zero) key. Fill the front cell with the test solution and measure the %T/Abs as per requirement

(9) Clean the cuvette in between the sample or standards by washing it with the diluents and rinsing it with the corresponding standards and samples.

(10) After the analysis, clean the cuvette thoroughly, press the (Start/Stop) key and switch off the instrument. Subsequently, switch off the respective main supply.


(i) To check the correction of the absorbance readings generated by the spectrophotometer, the spectrophotometer needs to be calibrated. In a UV-Visible spectrophotometer, calibration is required for wavelength and absorbance.

(ii) Control of Wavelength:

(iii) Check with Holmium Filter: Verify scale using the absorption maxima of the holmium filter. Peaks should be seen at 240.15nm, 287.15nm 361.5nm and 536.3nm. The permitted tolerance is ± 1nm for the ultraviolet range and ± 3nm for the visible range.

(iv) Control of Absorbance Value: this can be checked by measuring the absorbance of potassium dichromate solution at the wavelengths indicated in the following table, which gives for each wavelength the exact value of A (1%, 1cm) and the permissible limits. The tolerance for the absorbance is ±0.01.

235124.5122.9 TO 126.2
257144.5142.8 TO 146.2
31348.647.0 TO 50.3
350107.3105.6 TO 109
45015.915.7 TO 16.1

(v) Preparation of 0.005M Sulphuric acid: Dilute 2.8ml of the sulphuric acid to 100ml with diluted water and mix. Pipette out 10ml and makeup the volume 1000ml.

(vi) Potassium Dichromate solution: Dry a quantity of potassium dichromate to constant weight at 1300C. Weigh accurately 57.0 to 63.0 mg and dissolve it in sufficient 0.005M sulphuric acid to produce 1000ml.

(vii) Potassium Dichromate solution (430nm): Dry a quantity of potassium dichromate to constant weight at 1300C. Weigh accurately 57.0 to 63.0 mg and dissolve it in sufficient 0.005M sulphuric acid to produce 100ml.

(viii) Calculation of A (1%, 1cm)

A (1%, 1cm) at a particular wavelength is calculated as follows;

                          Absorbance of the solution at that wavelength

A (1%, 1cm) =       ____________________________________

                              Percentage concentration of the solution

(ix) Limit of Stray Light (1.2%w/v potassium chloride solution): transfer an accurately 1.2g of potassium chloride in 100ml volumetric flask, add about 80 ml distilled water, shake to dissolve, and make up the volume up to the mark with distilled water, and mix. The absorbance of the solution at a 200nm of path length 1cm should be greater than 2.0.

(x) Resolution (0.02% v/v toluene in n-hexane): Transfer an accurately 2.0ml of toluene in 100ml volumetric flask, add about 80mlof n-hexane, mix properly and make up the volume up to the mark with n-hexane. Dilute 1ml of this solution to 100ml with n-hexane, check the ratio of absorbance of the solution at maximum at about 269nm to that at the minimum at about 266nm it should not be less than 1.5.

(xi) Record the observation in calibration report.

Calibration:Frequency: Every three month.


(1) Switch OFF the mains of the Instrument (Unplug the switch).

(2) Allow the oven to cool down at room temp if hot.


User manual of UV- spectrophotometer UV-1800.   

Safety Precautions

(1) Cuvette should be keep dry and clean place.

(2) Silica should be kept in cabinet to absorb the moisture.

(3) Silica gel should be blue and dry.

(4) Ensure that electricity should not interrupt during analysis.


1             SOP          :           Standard Operating Procedure

2             QC             :           Quality Control

3             min            :           Minute

4              cm             :           Centimeter

5             ml              :           Milliliter

6             Sec           :           Second.

7             %               :          Percentage

8            nm              :          Nanometer

9            mg              :          millimeter

10          0C               :         degree centigrade


UV spectrophotometer calibration is a very essential practice for every laboratory to ensure testing results are valid. UV-VIS spectrophotometer calibration should perform in every 3 months to avoid any testing error by the machine. These days very high-end technology apparatus have come in the market, they are also very sensitive in operation so that their frequent calibration check is very necessary.

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Darshan Singh

The author has 20 years of experience in the Pharmaceutical Manufacturing Companies. Have exposure to all audit documents for WHO-GMP, PIC/S, USFDA, Export documents, US-FDA documents, Validation of Pharma machinery, and Quality Control Instruments. Expert in Pharma Plant Setup and sale and acquisition of running pharma company.

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