Your Question -

 What protocol do you use to clean the RP C18 HPLC column daily?

I am running 30-50 samples/day I wonder if I need to do regular cleaning after use. My mobile phase 5% acetic acid-methanol-acetonitrile 70:15:15. What solvent do you suggest?

Answer - I usually make morning wash and night wash for column C18 RP, befor and after working in the end of the day.

The morning wash is consist of four stages; each stage about 10 minutes

1) 10% acetonitrile+90%water

2) 100% Methanol

3) 100% Acetonitrile

4) 100% mobile phase

The night wash is consist of four stagese too.

1) 10% acetonitrile+ 90% water

2) 100% Methanol

3) 100% Acetonitrile

4) 80% Acetonitrile+20% Water

This will longer the age of column what ever you work  on the column.

 YOUR QUESTION :-

                  How can you know that a sample is UV active?

       Please update me- is there any specification on structure basis, that a compound is UV active?As a large molecule, can we make a decision to look into whether the structure of that molecule is UV active or inactive?

Answer - UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.

• Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the colour and changes the wavelength of maximum absorption (λmax).

• Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.

• While charge transfer complexes also give rise to colours, the colours are often too intense to be used for quantitative measurement.

almost all aromatic compounds are UV active and also compounds with double bonds with extended conjugation. Any basic book on spectroscopy can help you to know whether your molecule will be uv active or not. 

 Your Question :-

How to analyse UV inactive compound on HPLC using UV Detector?

In my research field I got UV inactive compound having one Chiral Center I want to separate the enantiomers on HPLC having UV detector So what can i do to get UV response on HPLC. ?

Answer - 

I suggest to complex the compound with some kind of regents to give a color so you can detect in visible range. For the enantiomer you can modify a mobile phase containing 5% beta-cyclodextrine to enhance selectivity towards enentiomers.Derivatization is one way, but more laborious and expensive. You can try the more straightforward approach of indirect UV detection.  The detection of a UV-Vis-transparent analyte is accomplished by adding light-absorbing species into the mobile phase. The presence of the analyte is monitored by measuring a decrease or The only way you could analyze a UV inactive compound on a UV detector is to derivatize it to add a UV active functionality, such as a phenyl ring.  Otherwise you can't use UV on the compounds in their native state since there is nothing for the UV detector to detect. If you are doing this as a preparative LC than derivatization wouldn't be an option.  Your only recourse would be to use another type of detector.

 Your Question :-

    Can Chloroform degrade C18 reverse phase Silica?

Answer - Chloroform will not damage your C18 RP column. I was hesitant before using it the first time, so I used a really old column that had been used with a lot of lipid-containing samples. The improvement after a {20% Ethanol or Methanol:water(ultra-pure) > 100% Methanol > 100% Acetonitrile > 100% Chloroform > 100% Acetonitrile > 100% Methanol > 20% Methanol:ultra-pure water} progression was very dramatic, restoring the column's theoretical plate number to close to that when it was new. This suggested that a lot of lipid had been accumulating on my column, impairing its performance and maybe introducing a 'lipid partition' into my separations. I have repeated this 'column-stripping' procedure several times since, on old columns that were losing resolution, and it does usually help, depending on the history of the column, and whether it has been used and stored within the limits set by the manufacturer.

I agree, however, that chloroform would not be appropriate in a mobile phase solvent for a Reverse-Phase column.

                                                                                                                                Thank you

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