Why does a spectrophotometer needed to be calibrated

This is comparable to what calibration does. Just as you clear the number on a calculator, calibrating a spectrometer ensures that the spectrometer is set to zero. Potential issues and errors are also corrected during the calibration. Calibration should be conducted at least once a year, regardless of how often the spectrometer is used. Results can be affected by long-term lack of use as well as things like dust being knocked about can adversely affect results.

Ensuring an accurate calibration is essential to getting reliable and accurate results. Calibration will vary based on the spectrometer, but the steps are relatively universal. While calibration typically refers to setting the machine prior to use, but there are more detailed types of calibration that should be done as part of the regular maintenance.

There are a number of services that are included as part of calibration including a number of cleaning services that are part of regular maintenance, which is why maintenance helps ensure that calibrations provide optimal functionality. However, cleaning is not the same as calibration. The following are some of the primary means of calibrating a spectrometer. The intensity of the light is essential to getting an accurate analysis. If the light intensity is affected, the readings can be very inconsistent.

This means you will run the analysis more times than is necessary, and you will still have results that are not reliable. Regularly checking the light intensity is not exactly part of calibration, but it is essential for ensuring that the calibration is also correct so that you have a good starting point.

This is among one of the most common calibration methods. A conventional calibration lamp provides the necessary illumination of the spectrometer and measures its positions. This includes things like pixel indices. The calibration process of a conventional calibration lamp provides a set of lines that are evenly distributed for the range you would like to calibrate.

We recommend annual calibration for every color measurement instrument. Our technicians who perform this service are highly skilled in calibrating instruments as well as in numerous other repair and maintenance tasks on advanced technological devices.

To watch a video of the calibration process using our CM A spectrophotometer , click here. Enabling Paint Shop Industry 4. Same car different color. Blue Light Safety.

Achieving a Standard Color in Cosmetic Foundations. More results Generic filters Hidden label. Hidden label. Apart from the errors mentioned in section IV—B, these are the influences of a lateral displacement of the light beam. An upper limit for this term may be estimated from. It is tilted so that the reflections by the sample do not impair the result, i. The opposite inclination cancels possible beam shifts. For the first measured ratio of the radiant fluxes it follows that.

R a R b can be calculated from this equation. The user of a commercial photometer cannot carry out such tests. He cannot work in the cell compartment without windows because of the danger of damage to optical surfaces by spilled chemicals or vapors.

If the manufacturer has not eliminated the reflections, the user has to take the instrument as it is. Most spectrophotometers, however, are used to measure solutions. A cell with solution is measured relative to another one containing the solvent. Actually the user wants to determine the internal transmittance. If the solvent does not absorb, it follows from the above mentioned definition of true transmittance equation 5 or 6 that. If these terms are entered in equation 24 it follows that.

Due to the multiple reflections in the instrument provided they have not been eliminated by the manufacturer he will experience an apparent nonlinearity according to eq 35 of. Though higher, it is of the same kind as that expressed in eq 32 for a reflection-free photometer with the following apparent nonlinearity. The errors are so small that they are negligible in practice. Only very few cases are known to the author where the reflection within the sample was considered according to eq 32 or 37 for analytical applications.

Consequently also the somewhat larger error due to eq 36 may be tolerable. When designing instruments for standards laboratories, multiple reflections should be avoided. Mielenz achieved this by imaging with off-axis parabolic mirrors. This doubtless reduces multiple reflections to values which are neglegible even for most exacting demands with regard to measuring accuracy. However, such an arrangement should always be checked for multiple reflections.

Not only must the sample be placed exactly perpendicular to the optic axis, but aberrations must also be kept small and apertures must be blackened.

One of these apertures is the monochromator exit slit, and it may be difficult to eliminate reflections from its sharp edges fig. The interreflective term R a. R b has a very high value in the straight beam from left to right because of the high reflectance of the plate in front of detector 1 signal i 1 and a very low one for the beam to detector 2 signal i 2 because there is no reflecting plate and interreflections are attenuated by the low reflectance of the inclined beam splitting plate.

The filter was at first perpendicular to the beam axis a , then tilted b , and finally the small reflecting edges of the slit were carefully blackened. Curve b shows above nm the influence of the slit edge alone. To avoid reflections the instrument designer can equip the instrument with off-axis mirrors see above , tilt lenses and windows, or provide them with antireflection coating which is, however, possible only for a limited spectral range.

As far as the linear function of the photometer can be influenced, he can correct the apparent linearity error resulting from eq They were eliminated by so-called correctors described by Hansen [ 24 ] figs. The following numerical value was determined for correction:. Correction is positive because of the negative error.

Corrector 1 is rotatable around an axis A perpendicular to the diaphragm plane, corrector 2 around an axis B parallel to the diaphragm plane. Also shown as curves are the linearity changes brought about by the correctors. The above considerations reveal a danger in testing photometers with multiple reflections by standards which are calibrated against air.

Multiple reflections do not only supply higher absolute values than actually available of the linearity error, but compared with the error involved in the measurement of solutions against solvent this error has the opposite sign compare eqs 25 and If standards are used which are pairs of equally reflecting, yet differently absorbing substances, the errors caused by multiple reflections when measuring the standards and the samples themselves will be at least approximately equal, and the error caused by a multiple reflection will be eliminated at least approximately by the linearity correction.

The calculation of multiple reflection given in section IV—D is based on the reflection coefficient of the radiant flux. With strictly collimated and monochromatic light, there will be interferences at the parallel surfaces. The transmittance of such a sample will show periodic maxima and minima as a function of the wavelength. This is well known from IR instrument applications, but is also observed in the visible under special conditions. Mielenz has stated corresponding formulae [ 22 ].

These interferences are generally regarded as disturbances in spectrophotometric measurements and are eliminated, if possible. There are three ways to achieve this: sufficiently large bandwidth, varying thickness of the sample, and sufficiently large aperture angles.

To get from one inference maximum to the next by changing the thickness, the change of thickness must be. If again 10 interference maxima Fizeau fringes are to be averaged [ 28 ], a thickness change of 3. Interferences can thus not be compensated by intentional variation of the sample thickness over the measuring area unless the accuracy requirements are low.

The same applies to the compensation of interference by the use of larger aperture cones. The relation of oblique beam passage and change in path length has been derived in section IV-B. The same limits as mentioned above are true because, although with oblique rays Haidinger rings the change of the transmitted layer is desirable to compensate the interference, it is undesirable for the constancy of absorption. Interferences cannot be effectively compensated by the aperture cone in collimated-beam photometers of standards laboratories, however, the larger cone in commercial photometers causes freedom from interferences.

To calibrate standards of high accuracy, several measurements must be made at intervals of approx. The small increase in transmittance of gray glasses over the years may also be considered as an interference phenomenon. It is due to the formation of surface layers by a kind of aging, which have a reflection-reducing effect. For this reason, gray glasses should not be used alone but in combination with other glass types.

It has up to now not been possible to obtain glass of equal refractive index and chemical composition which changes with time in the same manner as gray glasses. As a rule, the light in commercial photometers is partially polarized. The horizontal and vertical cross sections of the light beams being of different shape, this polarization affects the reflection at oblique incidence. If such a photometer is tested with standards having surfaces similar to those of the cells used for measurement, the error caused by polarization is corrected together with the linearity error.

An instrument with rotational symmetry of the beam cross section must be used to calibrate the standards. If this proves impossible, measurements must be made in the two preferred polarization directions. To avoid systemmatic errors in partially polarized light, the standards should be free from birefringence, strain or optical activity. As mentioned before, the sensitivity of all known photomultiplier tubes depends considerably on the position on the cathode and on the direction of incidence.

If the beam is shifted when the sample is brought into the beam path, errors occur which differ even with instruments of the same type. Shifts of the light beam on the cathode may be due to wedge errors, tilt errors or focusing errors. This changes the cross section of the light beam at the cathode. If collimated light passes through the sample, the tilt and focusing errors will not shift the beam cross section in the focal plane of the collimator but the direction of the beams.

If the light beam falls directly on the detector the cross section on the detector should not be too small. This is the reason for producing an image of the pupil on the detector. However, even with collimated light the sample may cause changes of the pupil image. The best solution is to eliminate the dependency of the sensitivity on place and direction. This can be achieved with an averaging sphere, which because of its low efficiency has so far only been used in special equipment.

Whether or not the progress made in designing averaging spheres [ 25 ] will make them suitable for commercial spectrophotometers remains to be settled. Whoever wants to improve spectrophotometry must know the inherent sources of error. According to the author there are enough means to test the spectral characteristics.

But, to test the linearity of the transmittance scale, standards are required which must be issued by a standards laboratory. As far as the author knows there are being offered only two types of standards which are calibrated according to independent and published procedures: the gray glasses and solutions issued by NBS and the gray glasses of ZEISS see note [ 26 ].

A standards laboratory will be responsible for the increase in accuracy up to a technically feasible limit. Important progress has recently been made in this respect.

Yet the errors mentioned at the beginning are about 3 orders of magnitude above the accuracies obtained in standards laboratories. It would be an important step forward if an accuracy of a few tenths of a percent were achieved for routine applications. Standards with transmittances guaranteed to within approx. They must be easy to handle and to clean and must, of course, be stable. They should also be neutral. Gray glasses meet these specifications for the visible spectral range, but the formation of a surface layer impairs the stability of the values with time.

Changes of up to 1 percent of actual transmittance have been observed by us within ten years. If a material of higher stability is not found, it should be tried to calibrate these glasses with reference to a similar absorption-free glass, which would eliminate most of the time-dependency [ 27 ].

This would best meet practical requirements, and would ensure the smallest influence by multiple reflections. Gray glasses cannot be used in the UV. Blackened quartz glass being commercially available, attempts should be made to produce quartz glass which absorbs in the UV almost independent of the wavelength. Vaccum-deposited, neutrally transmissive metal coatings can be used within a much wider spectral range than glasses.

In spite of this the author doubts their usefulness even for moderate accuracy requirements, because they reflect too much light. Even if such filters are tilted by means of a suitable mount, for instance to eliminate part of the errors due to multiple reflection, this may cause errors in commercial spectrophotometers, because the reflected light is much stronger than the reflection at glass surfaces; even a reflection on to a black surface may cause measuring errors.

Furthermore, these coatings are very sensitive, but in spite of this the author would not recommend cementing with a coverglass, because all cementing agents are known to increase their UV absorption with time. Solutions, even if they are transported in sealed ampouls, are still problematic with regard to durability, contamination and the cells required for their use. Photometers with fixed cells to measure liquids continuously or in cycles should be tested with reference to gray glasses. These usually can be inserted, because the cells must be removable for cleaning and replacement.

Liquids are needed in the visible spectral range to test such photometers only if the cell cuts off the beam path and an additional gray glass cannot be provided. The improvement of routine spectrophotometry is more a problem of instruction of the user and provision of suitable equipment than of improving the accuracy in the standards laboratories. The author thankfully acknowledges the assistance of K.

Mielenz and R. Mavrodineanu in revising the English text. His participation at the workshop seminar held Nov. In no instances does such identification imply endorsement by the National Bureau of Standards. National Center for Biotechnology Information , U. Published online Aug 1. Author information Copyright and License information Disclaimer. Carl Zeiss, Oberkochen, Germany. Copyright notice.

The papers are in the public domain and are not subject to copyright in the United States. Articles from J Res may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Abstract Based on simple principles, spectrophotometry nevertheless demands a lot of precautions to avoid errors.

The following properties of spectrophotometers will be discussed together with methods to test them: Spectral properties—wavelength accuracy, bandwidth, stray light; photometric linearity; interactions between sample and instrument—multiple reflections, polarization, divergence, sample wedge, sample tilt, optical path length refractive index , interferences.

Keywords: Bandwidth, calibration, errors in spectrophotometry, interferences, multiple reflections, photometric linearity, polarization, sample characteristics, stray light, wavelength accuracy.

Introduction The comparison of measured results of different optical parameters reveals considerable differences in accuracy. Solution p. Chromate 40 Chromate 40 9. Open in a separate window. Testing the Spectral Characteristics A. Accuracy of the Wavelength Scale The wavelengths of a great number of emission lines within the ultraviolet and visible spectral regions are known exactly.

Two facts deserve special mention: Even in regions without absorption the dispersion of prism materials is not as homogeneous as may be expected. Figure 1. Irregularities in the dispersion of a prism made of F—2 glass Schott. Figure 2. Different wavelength display systems.

Figure 3. Periodic error of a sine bar mechanism in this case due to an unsuitable ball bearing. Figure 4.

Line emission of a commercial deuterium source showing D and H lines. Table II Emission lines of hydrogen and deuterium. Figure 5. Spectral transmittance of Holmium in aqueous solution 1. Figure 6.