Detection

Methods
Chromapedia
Volume I
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Detection methods in flash and prep HPLC

When performing chromatography separation, it is essential to determine the precise point

when a substance is eluted from the column or cartridge to achieve optimal fractionation. A
chromatography detector is a device used to detect the presence of individual substances as
they elute from the column or cartridge. The detector converts a change in effluent levels into
an electric signal which is recorded by a data system.
In preparative liquid chromatography, the detectors used must accommodate high flow rates
and high sample concentrations associated with the process. Various detector types exist
for this purpose and each type of detector has its specific advantages and limitations. In the
following, five commonly used detector types in liquid chromatography are discussed in more
detail.

UV detector

UV detectors are the most frequently used detectors in preparative chromatography. A UV de-
tector is a selective detector, meaning it only measures substances which absorb light of a se-
lected wavelength in the ultraviolet range (200-400nm) or visible range (400-800nm). Substanc-
es which are suitable for UV detection include compounds with 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 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. This relationship is described by the Lambert-Beer Law:

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 has a UV absorbance cutoff wavelength. At wavelengths below this value the solvent
itself absorbs all the light. When using a UV detector, it is necessary to select a solvent which has
no significant UV absorption at the wavelength at which measurements are to be taken. Other-
wise, the signal of the substance and the solvent will overlap, resulting in incorrect fractionation.

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Typical critical solvents used are acetone, toluene and benzene, as indicated in the following
table:

UV limit/cutoff UV limit/cutoff
Solvent Solvent
(nm) (nm)

Acetone 330 Dipropylether 220

Toluene 285 Hexane 210

Benzene 285 Cyclohexane 210

N,N- Ethanol 210

275
Dimethylformamide
Methanol 210
Dimethyl sulfoxide 275
Propanol 210
Ethyl acetate 260
Heptane 200
Dichloromethane 245
Acetonitrile 200
Chloroform 245
Water 190
Dichloroethane 230

Tetrahydrofurane 220

Table 1: Critical solvents used in flash and prep HPLC

If the absorption spectrum of a compound is not known, it is useful to use multiple wavelengths
simultaneously or even a diode array detector (DAD), which can record the whole UV spectrum.
The DAD can also confirm purity and compound identity by showing the absorption spectrum of
each peak, eliminating post fraction thin layer chromatography (TLC).
For example, the DAD analysis of caffeine, vanillin and piperine is shown below:

Figure 1: Typical chromatogram with average UV signal Figure 2: 3D dataset of UV signals (200-400nm) of
(200- 400nm) of the three compounds caffeine, vanillin caffeine, vanille and piperine indicating the absorption
and piperine maxima

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 Scan at 6.95 min

Figure 3: Detailed UV absorption spectrum of the vanillin peak

Advantages of UV detection Limitations of UV detection

• Easy to use • Poor response if compound doesn’t

• Reliable have a good chromophoric group
• Relatively inexpensive • Solvents limited by UV cutoff especially
• Solvent gradient compatible at low UV wavelengths
• Non-destructive to sample
• Relatively sensitive (depending on
compound activity)
• Specific

Table 2: Advantages and limitations of UV detection

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Evaporative light scattering (ELS) detector

The general mechanism of ELSD involves measuring the amount of light scattered by particles of
solvent which have been dried through evaporation. The process consists of three steps: neb-
ulization, solvent evaporation and detection (see picture below). 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 next step, the droplets are introduced into a drift tube where the mobile
phase evaporates and leaves behind a particulate form of the target compound. In the last step,
light strikes the dried particles which exit the drift tube, the light is scattered, and the resulting
photons are detected by a photodiode.

Step 1. Nebulization Step 2. Mobile Phase Evaporation Step 3. Detection

Figure 4: Process of ELSD

ELSD is described mathematically by equations which are governed by particle size. The peak
area (A) is related to the quantity of analyte in the column (m):
A = amb

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

ELS detectors are a preferred choice for purification of compounds without a chromophor-
ic group. Such compounds include carbohydrates, lipids, fats and polymers. The function of
ELS detectors remains undisturbed by mobile phase variations and gradient baseline shifting.
Sensitivity in ELSD is independent of the compound’s physical and chemical properties and is
therefore governed only by the absolute quantity of the compound. ELSD is a mass-dependent
detector, meaning a high signal indicates that a large amount of compound is eluting. As a
semi-quantitative technique, it provides valuable information on the ratio of the compounds in
the sample.

ELSD can detect nearly all compounds, except for highly volatile analytes, such as ethanol in
wine. In general, the compound of interest must be less volatile than the mobile phase. Any
modifier in the mobile phase needs to be also volatile. Since ELSD is a destructive detector,
sample amount transferred to the ELSD should be as minimal as possible.
The lower the boiling point of the mobile phase, the easier it is for the solvent to evaporate. Mobile
phases with high boiling points (e.g. DMS, DMF, toluene or water), either need to be evaporated
at high temperatures, with the risk of destroying the target substances, or nebulized into extreme-
ly tiny droplets, which allow evaporation even at room temperature.

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 Solvent Boiling point °C

Dimethyl sulfoxide 189

N,N-Dimethylformamide 153

Toluene 111

Water 100

Propanol 97

Dipropylether 90

Dichloroethane 84

Acetonitrile 82

Cyclohexane 81

Benzene 80

Ethyl acetate 77

Ethanol 78

Hexane 69

Tetrahydrofuran 66

Methanol 64

Chloroform 62

Acetone 56

Dichloromethane 40

Table 3: Boiling points of typical solvents used in flash and prep HPLC

Advantages of ELSD Limitations of ELSD

• Detects any compound less volatile • Only volatile mobile phases can be used
than the mobile phase • The sample must be less volatile than
• Good sensitivity the mobile phase
• Straightforward operation • ELSD is a destructive technique
• Gradient compatible – maintains stable
baselines during gradients

Table 4: Advantages and limitations of ELSD

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Mass spectrometry (MS) detector

Liquid chromatography separates compounds according to their physio-chemical properties,

whereas mass spectrometry (MS) differentiates compounds by mass. The mass spectrometer,
as a chromatography detector, enables the identification of species corresponding to each chro-
matographic peak based on its unique mass spectrum.
The basic components of a liquid chromatography system coupled with a MS detector are shown
below:

Figure 5: Components of a liquid chromatography system with MS detection

The workflow involves converting molecules from the 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.

Advantages of MS detection Limitations of MS detection

• Good sensitivity • Purchase price is very high

• Very selective • Frequent maintenance is needed
• Provides structural information

Table 5: Advantages and limitations of MS detection

Refractive index (RI) detector

The refractive index detector measures changes in the refraction of light caused by a medium
as it flows through a measuring cell. The detector is non-selective as it registers all substances
which flow through the cell. Refractive index detectors measure according to the following prin-
ciple:
= 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

Since the refractive index detector is not specific, it is universally applicable. This type of detector
is however not well suited to applications with gradient elution, since the eluate is always com-
pared with the pure solvent in a reference cell. The system is therefore sensitive to changes in
the composition of the mobile phase. In addition, the detector is highly sensitive to temperature
changes.

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 Advantages of RI detection Limitations of RI detection

• Universal nature of the detector • Cannot be used with solvent

response gradients
• Good linear dynamic range - ~4 orders • Low sensitivity
of magnitude • Very sensitive to temperature
• Easy to operate and pressure fluctuations

Table 6: Advantages and limitations of RI detection

Fluorescence detector

When compounds with specific functional groups are excited by shorter wavelength energy, they
emit higher wavelength radiation or fluorescence. Fluorescence intensity is dictated by both the
excitation and emission wavelength, which enables the selective detection of some components
over others. Approximately 15% of all compounds have natural fluorescence. Some of these
compounds include aliphatic and alicyclic compounds with carbonyl groups and compounds
with highly conjugated double bonds. Aromatic components with conjugated pi-electrons give
off the strongest fluorescence activity.
Fluorescence detectors offer one of the highest sensitivity levels among existing detectors. The
sensitivity of fluorescence detectors is 10 to 1000 times higher compared to UV detectors.

Advantages of Fluoresence detection Limitations of Fluoresence detection

• Very sensitive • Limited linearity

• Very selective • Not many compounds naturally
• Generally insensitive to flow and fluorescent
temperature changes • Derivatization complicated method
• Complicated to use – must
have firm grasp on both chemical and
instrument variables
• Some chemicals, such as oxygen, may
quench fluorescence - must degas well

Table 7: Advantages and limitations of fluorescence detection

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Combination of UV and ELS detectors

In general, users in preparative chromatography require detectors which are universal and easy
to use, with high sensitivity, with non-destructive technology and without mobile phase effects.
However, none of the currently available detectors fulfill all these criteria as shown in the following
table.

Detector Compounds Gradient Easy to

Sensitivity Destructive
name detected compatible use

UV Chromophoric Dependent Yes No Yes

compounds on compound
activity

ELSD Non volatile Medium Yes Yes Yes

compounds

MS Ionizable Medium Yes Yes No

compounds

RI Universal Low No No Yes

Fluorescence Fluorescent High Yes No No

compounds

Table 8: Summary of detection methods in flash and prep HPLC

UV detection is the default method in preparative chromatography, since it is an established

technique and a lot of molecules absorb UV light. However, for some applications, UV detec-
tion alone may not provide the needed purity levels. Especially in synthesis, the detection of all
by-products in the reaction mixture is crucial for an efficient separation and sometimes the user
is not certain if all compounds are UV active. Therefore, to be on the safe side, a combination
with another detector, preferably ELSD, is often used to meet the needs of the user.
Both UV and ELS detectors are easy to operate, gradient compatible and feature good levels
of sensitivity. The fact ELSD is destructive can be overcome by choosing a detector with lowest
sample loss in the µl range. Besides this, the combination of UV and ELSD allows the detection of
almost any compound: chromophoric and non-chromophoric, as well as volatile and non–volatile
(see figures below).

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 Solvent % ELSD UV1 UV2

Time (min)

CH3 O O
CH3
H3C H
CH3

O
H 3C H
OH OCH3
H
H

H3C O H2N HO

Cholesteryl acetate Methyl Paraben 4- Aminobenzoic

(non-chromophoric) (chromophoric) acid (chromophoric)

Figure 6: Separation of chromophoric and non-chromophoric compounds in a mixture

Time (min)

O O O

N N H O
HO
N HO
O N
OCH3 O

Caffeine (non-volatile) Vanillin (high volatile) Piperine (non-volatile)

Figure 7: Separation of volatile and non-volatile compounds in a mixture

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References

Aced G. and Möckel HJ. (1991). Liquid-chromatographie Apparative, theoretische und metho-
dische Grundlagen der HPLC. Weinheim, Germany: VCH.

Hostettmann K. et al. (2010). Handbook of Strategies for the Isolation of Bioactive Natural
Products. Beijing, China: Science Press.

Pitt JJ. (2009). Principles and Applications of Liquid Chromatography-Mass Spectrometry in

Clinical Biochemistry. 30(1):19-34.

Schmidt-Traub H. et al. (2012). Preparative Chromatography. (2nd ed.). Weinheim, Germany:

Wiley-VCH Verlag.

Talamona A. (2005). Laboratory Chromatography Guide. Flawil, Switzerland: BÜCHI

Labortechnik AG.

Young C.S. and Dolan J.W. (2003). Success with Evaporative Light-Scattering Detection.
LCGC Europe.
https://pdfs.semanticscholar.org/86fe/6906062968f3e659798669428c1a119f063a.pdf

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