Are compounds in your sample ghosting your chromatography detection method?

You know there are plenty of compounds hidden in your sample. You know your chromatography run is set up for optimal separations. But have you thought about your detection method? Are you sure you can recognize all that is important for you in your fractions? Here, I introduce five different detectors, UV, ELSD, MS, RI and fluorescence, discuss their benefits and limitations and give you suggestions for what compounds each detector is most suitable.

My sister was over with her family last Saturday and we spent the evening building a crime puzzle. Once we put it together, we had to find clues and solve the crime. It took us about three hours, but we managed to find the guilty culprit. One of the guilty culprits for us taking so long was myself, as I was distracted by the UV lamp that came with the puzzle for finding secret messages in the picture. It never ceases to amaze me that something can be present, but just invisible to the naked eye.

Where can this be any more plainly obvious in the lab than in chromatography with its various detectors? I mean, you have a sample full of so many different compounds. So, when you try to fractionate, you are painfully aware that perhaps you are seeing only half the picture!

I have touched upon the topic of detection methods before by mainly focusing on benefits and limitations of UV detection, evaporative light scattering detectors (ELSD) or UV and ELSD used in combination. I’d like to refresh your memory on those devices, but also introduce you to three other commonly used detection methods in liquid chromatography.

Let’s start with the detection methods we should already be familiar with.

UV Detectors

These are the most frequently used detectors in preparative chromatography. This detection method is selective, as it can be only used to measure substances that absorb light in the ultraviolet range (200 to 400 nm) or visible range (400 to 800 nm). Substances you can successfully observe with a UV detector contain a chromophoric group, such as:

  • Aromatic ring
  • Two conjugated double bonds
  • Double bond adjacent to an atom with one electron pair
  • Carbonyl group
  • Bromine, iodine or sulfur

The UV detector in your chromatography system measures the change in intensity of a UV light beam passing through a solution. The absorption of the light is related to the concentration of the solution which the light beam is traveling through. The relationship is described by the Lambert-Beer Law:

flash chromatography; prep HPLC; UV detection; Beer-Lambart Law equation


E = Extinction [dimensionless]
ε = Extinction coefficient [M–1· cm–1]
c = Solution concentration [mol/l]
d = Path length of the light beam through the solution [cm]

Every solvent you use, has a characteristic UV absorbance cutoff wavelength. At wavelengths that are below this value, the solvent itself absorbs all the light.

When using a UV detector, you should select a solvent which has no significant UV absorption at the wavelength at which measurements are to be taken. If not, the signal of the substance and the solvent will overlap, resulting in incorrect fractionation.

If you don’t know the absorption spectrum of your compound, I would recommend that you use multiple wavelengths simultaneously or even a diode array detector (DAD), which can record the whole UV spectrum. The resulting graph would offer more information for the user:

UV absorption; chromatography; liquid chromatography; flash chromatography; UV detection

To wrap up the UV detection method, I would like to go over a few advantages of the method. UV detectors are easy to use, reliable, relatively inexpensive, compatible with solvent gradients, non-destructive to sample and relatively sensitive and specific. The disadvantages of the UV detection method are that compounds lacking a good chromophoric group are difficult to detect and solvents are limited by UV cutoff, especially at low UV wavelengths.

ELS detectors work by measuring the amount of light scattered by particles of solvent which have been dried through evaporation. The process consists of three steps: nebulization, solvent evaporation and detection. During nebulization, a nebulizer combines a gas flow of air or nitrogen with the column or cartridge effluent to produce an aerosol of tiny droplets. In the second step, the droplets enter a drift tube, where the mobile phase evaporates and leaves behind a particulate form of the target compound. In the final step, light strikes the dried particles that are exiting the drift tube. The light is scattered and the resulting photons are detected by a photodiode.

The mathematical equation describing ELSD is governed by particle size:

A = amb

Where A is the peak area
m is solute mass
a and b are constants which depend on variety of factors, such as particle size, concentration and type of target substances, gas flow rate, mobile phase flow rate and temperature of the drift tube.

The ELS detection method is ideal if you want to purify compounds without a chromophoric group. That’s right, exactly the compounds that cannot be easily detected with a UV detector. These types of compounds include carbohydrates, lipids, fats and polymers.

The function of ELS detectors remains undisturbed by mobile phase variations and gradient baseline shifting. Sensitivity of the ELS detection method is independent of the compound’s physical and chemical properties and is only influenced by the absolute quantity of the compound. As ELSD is a mass-dependent detector, a high signal indicates that a large amount of compound is eluting. Because the detector is semi-quantitative, you can obtain valuable information on the ratio of the compounds in the sample.

ELSD can detect nearly all compounds, except for highly volatile analytes, for example ethanol in wine. Generally, the compound of interest or added modifier must be less volatile than the mobile phase. ELSD will also destroy your sample, so you should aim to use smaller sample amounts.

The lower the boiling point of the mobile phase, the easier it is for the solvent to evaporate. Mobile phases with high boiling points, such as DMS, DMF, toluene or water, need to be evaporated at high temperatures. However, this approach carries the risk of destroying the target substances. Alternatively, the solvents can be nebulized into extremely tiny droplets to allow evaporation even at room temperature.

Now let us look into some detection methods that we haven’t explored on the blog before.

Mass Spectrometry (MS)

The mass spectrometer, as a chromatography detector, enables the identification of species according to each chromatography peak based on its unique mass spectrum. A liquid chromatography system coupled with a MS detector has the following workflow. Firstly, the molecules are converted from chromatography eluent to a charged or ionized state. The mass analyzer is the component of the mass spectrometer which takes ionized masses and separates them based on charge-to-mass ratios. The analyzer then outputs them to the detector, where they are recognized and converted to digital output.

The benefits of the MS detection methods include good sensitivity, selectivity and the possibility to obtain structural information. The disadvantages of the MS detector are the purchasing price and the frequent maintenance the device requires . For me, MS has no place in a typically crowded and busy synthesis lab.

Refractive Index (RI)

The refractive index detector measures changes in the refraction of light caused by a medium as it flows through a measuring cell. This detection method is non-selective, as it detects all substances which flow through the cell. RI detectors measure according to the following principle:

refractive index; RI; flash chromatography; prep HPLC; liquid chromatography; detection method

∆n= Difference between the refractive indices
nG = Refractive index of the dissolved sample
nL = Refractive index of the pure solvent
ni = Refractive index of the sample
c = Concentration of the sample

The advantages of the RI detection method include:

  • Universal nature of the detector response
  • Good linear dynamic range – ~ 4 orders of magnitude
  • Easy to operate

The limitations of the RI detection method include:

  • Cannot be used with solvent gradients
  • Low sensitivity
  • Very sensitive to temperature and pressure fluctuations

Fluorescence detector

When compounds with specific functional groups are excited by shorter wavelength energy, the y emit higher wavelength radiation or fluorescence. Fluorescence intensity is governed by both the excitation and emission wavelength, which enables the selective detection of some components over others. About 15% of all compounds have natural fluorescence.

Aliphatic and alicyclic compounds with carbonyl groups and compounds with highly conjugated double bonds have natural fluorescence. Aromatic components with conjugated pi-electrons give off the strongest fluorescence activity.

The main advantages of fluorescent detectors include:

  • High sensitivity – the sensitivity of fluorescence detectors is 10 – 1000 times higher compared to UV detectors
  • High selectivity
  • Generally insensitive to flow and temperature change

Disadvantages of the fluorescence detection method include:

  • Limited linearity
  • Not many compounds are naturally fluorescent
  • Derivatization method is complicated
  • Complicated use of detector – must have solid grasp of both chemical and instrument variables
  • Some chemicals, such as oxygen, can quench fluorescence – must exercise extra care by degassing

So there you have it! Five detection methods that should help identify any compound in your sample. There is always more to discover on the topic, so I invite you to watch a free webinar on detection methods or to read a free Chromapedia guide with more details, including many solvent tables with relevant properties.

Whew, this is as much detection as I can take, unlike my colleagues in the “Shallot Holmes & the Food Detectives” blog where they must solve food analytics cases at least twice a month! But hey, my name is also not Bart Holmes and Birke is no Watson. But she could be! Check out our latest exchange documented for all those interested in my personal and scientific lab in the latest blog post to celebrate the fourth birthday of Bart’s Blog. Happy reading!

Till next time,

The Signature of Bart Denoulet at Bart's Blog