Semi-quantitative TLC: my eyes just see spots

Off-topic conversations and chit-chat.

7 posts Page 1 of 1
Hi everyone,

I'm following a semi-quantitative TLC protocol used for in-field screening of anti-malarials (GHPF Minilab) which uses visual comparison of standard and sample spots under UV light.

The problem is my eyes can't "see" any differences in spot intensity but when I coerce others into looking at my TLC plates, they can. It's getting tiresome having to find someone every time I need the plates read.

I've googled for techniques on how the inept might train themselves to recognise intensity differences but with no avail. Plenty of info on optimizing the method and using scanners but nothing for the incompetent.

We are using a CAMAG system with a viewing cabinet and turn off the lab lights. Wavelength is 254 nm and the plates are commercial pre-coated silica gel 60 F254.

Does anyone have any tips? Thanks :)

A long shot but is there any advantage for improving visual comparison by using plain silica gel glass plates without fluorescent indicator and trying charring your plates by spraying with conc sulphuric acid then oven heating, thus allowing better comparison of the spot density in normal light by eye?

Probably only suitable for in lab screening and not suitable for the in-field screening that you describe

Have your eyes been checked recently? No, I'm not being sarcastic; color blindness comes to mind as one thing that may limit your range of detection.

From your description, you *can* see the spots, so maybe try running more standards at lower and lower levels. At *some* point you should get down to where you "don't" see a spot. That will give you some idea of what your LOD (limit of detection) is. If all the spots above that level look the same, then definitely get your eyes checked to find out why you can't discriminate many levels. If you *can* discriminate just above your LOD, then see how far up you can go before they all start to look the same again. That will give you some idea of what your visual range is. It may be that you are just too sensitive.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
Thanks so much for your replies! Ralph, I am unfortunately limited by the need to keep the method applicable for in-field screening.

Tom, I love your suggestion re finding a lower level that I can "see" a difference with. Perhaps then I can train myself, there's hope! Your comment about colour blindness is very interesting tho. In my undergrad days when colourimetric titrations were a huge part of analytical chemistry, picking colour transitions involving yellow or orange were always impossible for me.

Thanks again! I love the depth of knowledge on this forum :)
Tom has made some interesting observations that I hadn't considered.

Purely out of interest and curiosity, could you upload some images of your plates just to get more of an idea of your problem?

This is a very speculative untried thought based on one of Tom's comments.

To overcome colour differentiation problems - would converting your images to b/w help?

For example
I have taken one of the manufacture's images for your particular plate

then inverted the colour


Then converted to black and white


I used to use an overhead camera to take an image of the plate under UV but I am sure that you could now just use a moblle/cell phone camera - just switch off the auto flash

now returning control of your laptop

Also suggest to mount a digi-cam, take pictures and then process the pictures by dedicated software protocols.
That way you can exclude any personal bias and also document your results the same time.

The goal is to generate a pixel intensity profile for each tlc-lane. That will look like a "normal" chromatogram/pseudo-densitogram, where you can integrate the peaks and quantitate against your standards (the calibration functions may not be of a linear model though).

Camag has incorporated such functionality in their visionCats software but had it as an stand alone module in previous versions.

Otherwise guess this could also be done with the OpenSource tool "ImageJ" or a "R".

Some links I came across, that may help for further search:
ImageJ for Western Blot: ... ification/ ... cation.pdf ... hic_plates
Thought I'd post an update since everyone was so helpful. I took Tom's suggestion and dropped the concentration of the lower standard to where I could see a difference in intensity. Once my brain recognised what a difference actually looked like, I've found good success at being able to read the spots and no longer have had to harass others into reading my plates. Guess that means no holidays for me until this project is finished. ha ha

The idea of photographing the plates and using digital software to analyse the spots is certainly something that would work far better than visual observation. At this stage tho we're still trying to see if the simplicity of the method/ equipment can be minimized for in-field use.

Thanks everyone :)
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