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HPLC Solvent Properties
 

Introduction for non-chemists

Organic solvents fall into classes, such as alkanes, alcohols, acids, aldehydes, etc where each member of the class has the same functional group, e.g. OH (-alcohol)

Each member has a different alkyl chain length (=the number of carbon atoms in the molecule joined is a chain.). This then has a standard naming system (nomenclature).

 

No Functional Group

Functional Group

1 Carbon

Methane, CH4

Methyl, CH3-

2 Carbons

Ethane, CH3CH3

Ethyl, CH3CH2-

3 Carbons

Propane, CH3CH2CH3

Propyl, CH3CH2CH2-

4 Carbons

Butane

 

5 Carbons

Pentane

 

6 Carbons

Hexane

 

7 Carbons

Heptane

 

8 Carbons

Octane

 

9 Carbons

Nonane

 

10 Carbons

Decane

 

(NB 18 carbons – octadecane = ODS)

When the carbons are all in a straight line, it is prefixed by ‘n’ e.g. n-pentane, n-hexane etc. Some solvents have more than one name because different naming systems have been adopted. For example acetic acid, CH3COOH, is also known as Ethanoic Acid (2 carbons).  Isopropyl alcohol is also Propan-2-ol, iso-octane is also 2,2,4 trimethyl pentane and methyl ethyl ketone is also known as butan-2-one.

Function groups determine the rest of the name:

  • -OH                     adds –ol (ie alcohol, e.g. methanol, ethanol etc)
  • -CN                     nitrile e.g. Acetonitrile
  • -NH2                   amino
  • -C6H5                 Phenyl

Solvent Parameters relating to HPLC

UV Transparency and UV cut-off

Most solvents are more transparent to UV down to a certain wavelength and below that they totally absorbs UV so the spectrum looks as follows:

                              

To be useful with UV detection, the solvent has to have a lower UV cut off then the absorption of any of the sample components. In general, reverse phase eluents have much lower UV cut-off’s than normal phase eluents.

Solvent Miscibility

Some solvents such as alkanes (e.g. hexane, pentane etc) are very non-polar and will not mix with others (such as water) which are very polar.  Since solvents are mixed in HPLC to fine tune the polarity, thereby controlling their eluent strength, it is essential that solvents chosen are totally miscible. Also when changing from one solvent to another, the new solvent must be miscible with the old one. If this is not the case, a stepwise change must be made.  See table for miscibility.

Viscosity

HPLC operates in dynamic equilibrium.  Almost 90% of the surface area a 5u packing material is inside the pores. So the lower the viscosity of a solvent, the lower the back pressure and the better the mass transfer in and out of the pores. This in turn gives better separation efficiency, ie sharper peaks.  See the table for viscosity of most HPLC solvents.

Purity

In general lab use we are familiar with AR, LR and technical grades. For HPLC we require a higher level of purity. However, just as different manufacturers spec for Analytical Reagent (=AR, Analar) and Laboratory Reagent (=SLR, GPR etc) varies, so does ‘HPLC Grade’.  For the purpose of this, our HPLC grade starts out as AR grade raw material, following which it is distilled at least once and filtered.  Some manufacturers test only the optical purity (ie UV transparency) and hence it does not even meet AR spec.

The importance of solvent purity is that when analysing 20ul of sample, looking for possible sub picogramme levels of sample, impurities in the 20-30ml of solvent used during a run can be quite significant.

Some solvents (such as heptane) are very expensive in pure form and 95 –97% may have to be used. However, impurities are very similar alkanes and normally have little effect on the separations.

Stability

Most solvents are stable for long periods.  This is indicated by their shelf-life unopened. Some solvents, especially THF, have very limited shelf life, so it is important only to order as many as can be used within this period and to ascertain the confidence level that can be placed in the integrity of a bottle of solvent retrieved from a solvent cupboard.  Look for Date of Manufacture, and shelf life.

Eluent Strength

For Reverse Phase HPLC, water is the weakest eluent.  It’s eluent strength is then modified by adding a less polar but miscible solvent such as methanol. The less polar, the greater the eluent strength.

For Normal Phase HPLC, hexane (or heptane) is the weakest eluent and a more polar solvent is added to modify eluent strength.  These include Chloroform, Dichloromethane, Ethyl Acetate, Acetone, Ether etc

If a change in eluent composition is made for a selectivity reasons e.g. from Methanol to Acetonitrile in RP-HPLC, the ratios must be changed to maintain the same eluent strength.

COSHH

Some solvents are more hazardous than others.  Look for toxicity, flammability , carcenogenicity etc.  Some have a very unpleasant odour.  Some have a low flash point.  It is important to be aware of the hazards.

Compressibility

Compared to gases, solvents are not compressible.  But compared to each other, some are more compressible than others.  Some pumps have the facility to correct for this, thereby achieving a more accurate flow rate (See table.)

Refractive Index

Light is refracted (a deviation from the angle of incidence, ‘bent’ to the geography teachers) when it passes from one medium to another, e.g. from air into a liquid, or into glass. The refractive index of a material is an index of the extent of this deviation. Taking air as 1.0, water is 1.3 and glass is 1.5.

A refractive index detector operates by measuring a change in refractive index when a sample component passes through the flow cell. It is therefore important that the RI of the sample and of the eluent are as different as possible.  Otherwise no peaks! See table.

Cost

Some solvents are very much more expensive than others.  The range is from about £7 per 2.5 litre Winchester to over £100 per bottle.  Disposal may be expensive

A few specific solvents

  • Chloroform – Should be stabilised, 0.5% Ethanol.  Unstabilised is dangerous.  Especially with Aluminium.
  • THF – Unstabilised has 6 months.  Stabilised 2 years. Do not distil (contains free radicals). Stabiliser is BHT.  (Not miscible with water, strong UV absorption).
  • Water -  De-ionised to 18MW. Distilled with KMnO4 to oxidise and remove organics.  Filtered.  Very viscous.
  • Hexanes – n-hexane, iso-hexane, hexane fraction. 95%, 97%, 99%
  • Pet Spirit (also called Pet Ether.) Boiling factions 30-40, 40-60, 60-80, 80-100, 100-120oC
  • Ethanol – Not a good eluent. £49 duty per Winchester (2.5litres)
  • IPA – Excellent air solubility. Mixes with almost everything.

Acids – Use acetic, formic or phosphoric.

HPLC Solvent Properties Mol Wt. Density  Viscosity Eluent Solvent R I UV Cut-off Miscibility Dipole  Freezing  Boiling  Flash Pt
     (20oC) (cP) Strength Polarity (20oC) (nm) Number Moment  Point 'C Point (oC) (oC)
        (NP)       D<15=Misc        
1,2-Dichloroethane 98.96 1.2521 0.78 0.44 3.5 1.442 230   - -35.7 83 13
2,2,4-Trimethylpentane (= iso octane) 114.23 0.6919 0.47 0.01 0.1 1.389 200 29 - -107.4 99 -12
Acetic Acid     1.10   6 1.370 235     118  
Acetone  58.08 0.7900 0.32 0.56 5.1 1.359 330 15, 17 2.88 -94.7 56 -18
Acetonitrile  41.05 0.7822 0.34 0.65 5.8 1.344 210 11,17 3.92 -43.8 82 6
Carbon Disulphide     0.34 0.15 0.3 1.626 380     46  
Carbon Tetrachloride     0.90 0.18 1.6 1.457 265     77  
Chlorobenzene     0.80 0.3 2.7 1.525   21     132  
Chloroform 119.38 1.4891 0.57 0.4 4.1 1.443 245 19 1.04 -63.5 61 N/A
Cyclohexane  84.16 0.7786 0.90 0.04 -0.2 1.427 200 28 - 6.7 81 -17
Cyclopentane     0.42 0.05 -0.2 1.406 200     -103.5 49 -20
Dichloromethane (=Methylene Chloride) 84.93 1.3256 0.41 0.42 3.1 1.424 240 20 1.60 -94.9 40 N/A
Diethyl ether 74.12 0.7136 0.24 0.38 2.8 1.352 220 23 1.15 -116.3 34 -45
Dimethylformamide  73.09 0.9487 0.80 0.64 6.4 1.430 275 12 3.82 -60.4 153 58
Dimethylsulfoxide 78.13 1.1004 2.00 0.75 7.2 1.479 275 9 3.96 18.5 189 95
Dioxan              
Ethanol  46.07 0.7892 1.08 0.88 4.3 1.361 220 12 1.69 -114.5 78 12
Ethyl acetate  88.11 0.9006 0.45 0.58 4.4 1.372 260 19 1.78 -83.6 77 -4
Fluoroalkanes     -0.25 <-2 1.250      
Heptane 100.20 0.6838 0.40 0.01 0.2 1.388 195 29 0 -90.6 98 -4
Hexane 86.18 0.6593 0.29 0.01 0.1 1.375 190 29 - -95.3 69 -23
Methanol  32.04 0.7910 0.54 0.95 5.1 1.328 210 12 1.7 -97.7 65 10
Methyl ethyl ketone, MEK 72.11 0.8049 0.38 0.51 4.7 1.376 330 17 2.78 -86.7 80 -7
m-Xylene 106.17 0.8644 0.62 0.26 2.5 1.497 290   - 139.1 139 25
n-Butyl Acetate     0.73 0.4 4 1.394 254 22   126  
n-Butyl Ether     0.64 0.25 2.1 1.397 220          
Nitromethane     0.67 0.64 6 1.380 380     101  
N-Methyl Pyrollidone     1.67 0.7 6.7 1.468 285     202  
Pentane 72.15 0.6262 0.22 0 0 1.355 195   - -129.7 36 -48
Petroleum Ether     0.30 0.01 0.1 1.390 210 29    
Propan-1-ol 60.10 0.8036 1.90 0.8 4.0 1.386 240   1.55 -126.2 97 15
Propan-2-ol (=IPA) 60.10 0.7855 1.90 0.82 3.9 1.384 210 15 1.56 -88.0 82 12
Pyridine 79.10 0.9832 0.88 0.71 5.3 1.510 305 16 2.21 -41.6 115 20
Tetrahydrofuran,THF 72.11 0.8892 0.46 0.6 4.0 1.407 215 17 1.75 -108.4 66 -17
Toluene  92.14 0.8668 0.55 0.29 2.4 1.497 285 23 0.37 -95.0 111 4.4
Triethylamine     0.36 0.54 1.9 1.398 275     89  
Water 18.02 0.9982 1.00 High 10.2 1.333 <190   1.85 0.0 100 N/A