I have been using benzophenone for other reasons, but it should do the trick in your case, cheap, easy to buy great grade, stongly UV positiv.

Thanks! I will see if I can get this. However, please, can you tell me if it is strongly coloured when in solution? It is essential that the dye be conspicuous when coming out from the fraction collector.

Sorry Eduardo but benzophenone will not have a strong color, just a great UV response.

Whats detector are you using with you HPLC?

I am not familiar with dyes, but I bought some indigo some time ago, great blue dye and cheap.

a fluorescent dye, such as Thioflavin-T could be used. You may need a UV lamp to see the fraction coming (a lamp used for TLC is sufficient.

I suggest betacarotene, u should have no problem eluting. Normally we use it at 100ppm and a flow rate of 1.5 or 2.0 ml/min using a mixtute of solvents (ACN,IPA,ethyl acetate) plus methanol to elute it.

Did you think about calculating the volume of the tubing connection instead?

I don't understand why you want to test the delay time between the UV detector and your fraction collector with the column on line??? The column is before the detector so you don't care about this volume. So remove the column from your system put an union and inject one colored solution (red soudan if you want more concentrated) and run an injection. Set up flow rate at 1ml/min and inject and collecte when you get the signal on the detector, calculate the time that the color appear in the first but and you get the delay time to have the right peak in the right tube. That's done .-)

Sorry, you should read tube not but !!! "calculate the time that the color appear in the first TUBE and you get the delay time"

As proposed by Luisa, a quick solution is to perform fractionnation by very small volumes (the smallest volume you fractionnate, the more accurate you will be), and then to analyse each fraction by analytical HPLC. The shift you see between the peak detected by your prep-detector and the fraction containinf the highest concentration of the compound, corresponds to the shift you will have to take into account for next purifications.

And this can be done with every UV-detectable compound.

Otherwise, if you want to inject a colourized product, no need to use a column since you want to measure the delay between the detector and the collector.

If you can change your mobile phase and use a methanol/water mixture, then maybe you can try with K2Cr2O7. All you need is a small amount (1mM) and you will be able to see the color. That been said, I also believe that it would be more useful if you calculate the tubing volume between the detector and the fraction collector.

Good luck!

I am very happy, as everyone provided excellent tips. I will clarify few aspects mentioned: i) indeed the best would be remove the column completely but I have been told in my course that with the Shimadzu system there should always be something connected (in fact ends do not fit directly) thus the dye will have to be compatible with the C18 from the guard column. ii) I would prefer seeing the color coming out from the collector since it is unambiguous and instantaneous. iii) indeed there is a manual telling of the volume from standard tubing and collector head, however the technician gave me a standard volume which clearly is not right, thus I decided to measure directly. iv) I can use a UV transilluminator if the compound is will shine strong enough. I found that a much stronger % of Sudan 3 is workable but still rather weak after elution -- thus I will try to get some of the other good options suggested. Will post what happens here! Thanks to everyone!

oh, btw, the tubing between collector and detector is all rolled up and tied with a plastic strap, thus measuring it to get the volume seems more complicated than just measuring the delay directly... Sorry, I am new to HPLC yet...

you do not need the column or precolumn in place to determine lag time so disconnect them and a good dye to use would be a pH indicator such as meta-cresol purple it is soluble in your mobile phase and under acidic conditions (0.2% TFA) is red and under alkaline pH (0.2% ammonia) is very dark purple. you could try either and see how you go.

God Bless you,

Peter Mansour

I suggest you use a sub-2-μm particle columns for fast

HPLC analysis and you will get excellent results. A

increase in pressure for the pump works well give it a try too.

I agree with M. Mansour (see Principles in preparative HPLC Agilent handbook by U. Hubner and R. E. Majors). But if you would like to see the separation and colour of fraction, use 4-nitrophenol or 2-chloro-4-nitrophenol. Both have intensive yellow colour (can measure at 405 nm) and the elution from C18 phase is excelent with MeCN-water = 1:9 or 4:6 (shorter or longer retention time as you need)

You can downlood the Agilent handbook from the Agilent home page. Search for it by Google.

If you want a very cheap colorant you can try to buy some dye in the agro alimentary industry as tartrazine. You can find on any supermarket as several names and usually is an orange-yellow powder (0,1 ppm is enought)

On the other hand regarding the speed of the analysis I am totally agree with Paul regarding the sub-2-μm particle for fasta analysis, I am currently using in my lab in order to improve our productivity.

If you are afraid of ruining your column when using a concentrated dye, you could always use an ionized dye, which should not have any or minimal interactions with the stationary phase. I cannot recommend such a dye at the moment but I am sure they exist. Or, as someone already mentioned, you can exclude your column from your experiment altogether since you are intersted only in the dead volume from your detector to the fraction collector.

Best of luck...

A colorant that can also be used is a DNPH derivative of aldehyde or cetone. It is a easily prepared derivative obtained quickly by mixing equimolar quantity of DNPH and an aldehyde/cetone, and isolated just by filtration since this derivative precipitates.

As for the HPLC analysis, the here above cetones/acetaldehydes are eluted within 10 minutes on a C18 column with a mobile phase composed of Water/ACN/THF with the following gradient : in 15min : 50>30% of water, 30>65 of ACN, 20>5 of THF ; flow rate of 1.5 mL/min, UV detection at 365 nm ; standards prepared at 5 mg/L (but you may have to prepared higher concentration to more easily view the dye yourself).

The aldehydes/cetones you can used and commonly found in a lab are : acetaldehyde (TR=4min), acetone (TR=4.5min), propionaldehyde (TR=5.5min), benzaldehyde (TR10min). All the DNPH derivatives have intensive colours from yellow to red.

Dear colleagues,

Again, many thanks for the many suggestions provided. I have stuck to Sudan III however increased concentration to 10mg\ml, which gives me enough colour at the exit. I am amazed to find that my delay volume is a bit over 500 ul, yielding a delay of 1 min at flux of 0.5 ml/min (and 30 sec at 1mL/min). I have asked friends using older Shimadzu systems and they reported theirs returned a delay of 1-2 seconds when tested by similar methods. Sorry to raise yet another question, but could any more experienced colleague tell me if a 500 ul dead volume is normal? It seems to me there might be an issue in the system.

Dear Leonardo, I absolutely LOVED solution B1, using the bubbles. Since I am new to HPLC at first I was afraid of unplugging the exit of the detector, but upon looking at it and trying it out, it proved very easy. Tracking the air in tubing was realy really useful. I have determined my dead volume thus without dyes to be 566 ul until collector and 2.3mL (!!) until discard exit. It takes over 1 min at 0.5mL/min to reach the collector -- this is not very practical. Is this normal to other readers here, please? I will probably turn this into another question.

Thank you, again, your tips were truly clarifying. I think tubes fitted after the detector exit have a wider gauge than would be desirable, thus causing this unwanted delay plus dissolving sample just after successful separation. These tubes surely seem wider in the inside than those before the detector. Maybe changing them is the best/easiest solution, I will discuss this with the technical support. Thanks!

The following text was quite useful too:

http://www.modernmedicine.com/modernmedicine/data/articlestandard/lcgc/502003/78758/article.pdf

Thought readers here would get interested.

Soluble dyes should not affect your stationary phase. Most dyes I know are charged molecules, and because thy are deeply colored, are used very diluted. Perhaps the concentration of your dye es below the LOD/LOQ or it is eluting with you front at t= t0.