As I understand, you use several cuvettes.

To identify the errors I recommend to

1. Scan a broader range of wavenumbers. Though you need only one specific absorption, this can help to detect contamination (and also bubbles)

2. Compare spectra of different cuvettes.

3. What about temperature? (I would not expect such big impact)

Are you using QUARTZ cuvettes for all readings?

To troubleshoot the problem, start at the beginning. Establish that the spectrophotometer is stable. Set up several cuvettes with high purity n-Hexane (from the same bottle) in the spectrophotometer. Measure and record the absorbance of each solution. 20 minutes later, repeat the measurements, one-at-a-time. Repeat at 40 minutes (or 1 hour). Is significant variation observed ? If the readings are stable and consistent, repeat the test using "washed" cuvettes, being sure to hold at least one back as a control (that has not been washed).

See this link

https://www.researchgate.net/figure/UV-vis-absorption-and-emission-spectra-of-1N-in-n-hexane-and-1-2-

Attila Kovács ,

It´s not clear to me if you have the same measure with the sample after a time... The sample maintain the same value along the time? If so, then the problem it´s not about equipment. If not, I sugest you follow the William Letter´s sugestion

I hope this helps

Be careful with acetone; it decreases transmission even in small amounts!

Thank you for all the answers.

I followed the method what was was described by William Letter. Apparently the quartz cuvettes I used worked correctly, even after washing. The problem was with the n-hexane I used. Althoug I'm using HPLC quality n-hexane, the transmittance of different bottles varies a lot. Earlier I didn't noticed that, because the two bottle I checked first had quite similar absorbance, but after I excluded all other reasons, I checked additional bottles: these are the same type of product from the same manufacturer, but still, for four different bottles I got absorbances of 1.251, 1.073, 1,350 and 0,873 at 200nm (based on the average of 3 measurements with small deviation between the measurements). Is that normal to have this big difference in absorbance between different bottles of the same product?

What brand and exact grade did you use (Vendor and P/N) ? Did you compare your results to the vendor's UV spectral data supplied with their certifications for each grade? High quality solvents supplied by the leading manufacturers will include a lot analysis sheet often showing the full UV spectral data along with other relevant purity info. Any company can apply an "HPLC grade" label to their bottle, but the best companies can back it up with data from each bottle, batch or lot. How does their info compare to what you measured?

William Letter :

I am using the product of Acros Organics (Product number: 44572).

However, the specification does not give too detailed information on the UV absorption (against water), see below the related part:

200 nm A: =

If you have stored the product per the vendor's requirements, taken measurements at the same temperature each time AND your instrument is working properly, then any readings of > 0.7 AU (The Spec is < 0.7) are certainly higher than the specification. Did you obtain those four readings from the SAME BOTTLE (*if so, that would imply your instrument or technique is faulty) or from 4 different bottles? If from different bottles (on the same day/time), then the vendor's general specification indicates it is unreliable. *Let them know about the issue, but also select a different, higher quality grade and/or vendor with better HPLC quality too (e.g. Burdick Jackson).

• BTW: I could not find the specific Acros product # 44572 (?) in Fisher's catalog, but the closest solvent matching your description appeared to be one for n-hexane, 95+% "HPLC". Just because a company puts a label on the bottle which says "HPLC", it does not mean that you should use it for this application. Your own needs and requirements should determine if their product is suitable for your application. Use their advert only as a guide. If the product is found to vary considerably from bottle to bottle and not meet their suggested specification, then I would certainly advise not using it or purchasing anything that does not include a spec sheet (or at least something that you can verify is reliable).

William Letter : The four different values came from four different bottles; repeated measurements of the same bottle gave acceptable variance. That is why I thought the problem is with the solvent not with spectrophotometer.

Thank you for the detailed instructions, I will switch to a more appropriate product for my measurements.

It sounds like you should certainly avoid that brand or grade of solvent in the future. Generally, at 200nm, only the highest quality (often the most expensive) grades will offer the lower UV abs values. Asking for bottle-to-bottle reliability at 200 nm for n-Hexane is going to be difficult to achieve in general. Higher grades should be better, but at a cost.

One other option may be available to you for your project. If you need a known volume of n-Hexanes to complete your project, then you could just mix all of the needed bottles together to make one final solution for use. At the end of the day the only thing that matters is the relative difference. If the solutions used are the same, and enough dynamic headroom still exists at the wavelength needed, then it should be fine.