Solvents for HPLC

As the solvents used will affect both the capacity factors and the selectivity of the system, the mobile phase is selected on its ability to elute the solutes in an acceptably short time with a reasonable resolution between each one. The choice of mobile phase components may also be influenced by the desire to suppress the ionisation of one or more of the analytes or to reduce peak tailing. These considerations, whilst very important are generally viewed separately from the factors which affect the k and a values. 

In both normal phase and reversed phase HPLC, the eluting power or solvent strength of the mobile phase is mainly determined by its polarity. Although most analysts have a good idea of what the term polarity implies and could rank most common solvents in order of their polarity, a more quantitative description would be very useful in chromatography. 

One measure of polarity is the solubility parameter (8) which was suggested by Hildebrand. This is defined as the square root of a solvent s vaporisation energy (AE) divided by its molar volume (V)  

As the data in Table 4.3 show, the solubility parameter reflects how a chemist might rank these solvents in terms of polarity, e.g. water as the most polar (highest 5) and hexane as the least polar (lowest 8) but also one of the difficulties with this measurement of polarity is highlighted. The solubility parameter suggests that tetrahydrofuran (THE) and carbon tetrachloride are very similar even though carbon tetrachloride is immiscible with water whilst THE is miscible with water in all proportions. A similar comparison may be made between chloroform (8 = 19.1, water-immiscible) and acetone (8 = 20.2, water-miscible). 

The reason for this apparent contradiction is that the vaporisation energy of a solvent is affected by all of the interactive forces in which the solvent can take part and hence the total solubility parameter may be 

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Another parameter often measured is the adjusted retention time, Ur. This is the difference between the retention time of a given component and the retention time of an unretained substance, tM, which is often air for GC and the sample solvent for HPLC. Thus, the adjusted retention time is a measure of the exact time a mixture component spends in the stationary phase. Figure 11.17 shows how this measurement is made. The most important use of this retention time information is in peak identification, or qualitative analysis. This subject will be discussed in more detail in Chapter 12. 

Reagents. Organic solvents for HPLC separations—methylene chloride, methanol, isopropyl alcohol, hexane, and acetonitrile—were obtained as HPLC grade from Fisher Scientific. Type I water for HPLC and for the preparation of other aqueous solutions was purified as described previously (7). All HPLC solvents were filtered through a 0.45-/zm Millipore membrane filter (Millipore Corporation) and degassed... 

Method 2. The steroid residue is dissolved in methanol and then acidified by 3-4 drops of concentrated hydrochloric acid. 2,4-Dinitrophenylhydrazine is dissolved in methanol as a stock solution (0.2%). This is added to the residue in a slight molar excess, taking into account the number of carbonyl groups contained in the steroid. The mixture is then stirred and warmed for 5 min at 50 °C. An aliquot portion of this solution may be used for chromatography. If methanol is not suitable as a solvent for HPLC, then the reaction mixture may be evaporated and dissolved in an appropriate solvent such as chloroform or ethyl acetate. 

In principle, it would be possible to use fully deuterated solvents for HPLC-NMR coupling, and then solvent suppression techniques would be unnecessary however, due to the high costs of these solvents, only the use of D2O is economically acceptable. Solutions to this problem can be seen in the development of hyphenating capillary HPLC to NMR spectroscopy, and there, because of the reduced solvent consumption, the use of fully deuterated solvents would be reasonable. However, this approach is difficult and the development is still an on-going process (see Chapter 7.3 below). From the viewpoint of NMR spectroscopy, there could be a completely new situation, when separation techniques could be found which use proton-free solvents as the mobile phases. 

Organic solvents for HPLC are generally very good. There are three rules of thumb to remember always use HPLC grade solvents, buy from a reliable supplier, and filter your solvents and check them periodically with your HPLC. Most manufacturers do both GLC and HPLC quality control on their solvents some do a better job than others. The best way to find good solvents is to talk to other chromatographers. 

In the fiber mode, the sorbent coated fiber is housed in a microsyringelike protective holder. With the fiber retracted inside the syringe needle, the needle is used to pierce the septum of the sample vial. The plunger is depressed to expose the sorbent-coated fiber to the sample. After equilibrium is reached or at a specified time prior to reaching equilibrium, the fiber is retracted into the protection of the microsyringe needle and the needle is withdrawn from the sample. The sorbent is then interfaced with an analytical instrument where the analyte is desorbed thermally for GC or by solvents for HPLC or capillary electrophoresis. For the in-tube mode, a sample aliquot is repeatedly aspirated and dispensed into an internally coated capillary. An organic solvent desorbs the analyte and sweeps it into the injector [68,130,133]. An SPME autosampler has been introduced by Varian, Inc., that automates the entire process for GC analyses. 

The standard assay contained 100 vaM Tris-HCl (pH 9), 100 mAf KG, 20 mM threonine and/or serine, and the enzyme. After incubation at 37°C for various times, the reaction was terminated by addition of 3 N HQ. The reaction mixture was centrifuged and 250 / L of the supernate was mixed with 250 /iL of o-phenylenediamine solution (10 mg/mL in 3 N HQ). The tubes were tightly capped and heated in a boiling water bath for 30 minutes. The derivatized keto acids were extracted with ethyl acetate. An aliquot of the ethyl acetate layer was dried under nitrogen. The residue was taken up in 1 mL of the running solvent for HPLC Assays were linear for 30 minutes and with amount of protein added. 

Solvents used for chromatography should always be fractionally distilled to ensure that non-volatile impurities are removed. Solvents for hplc should be of high purity and again many suppliers provide special hplc grade solvents, which have been purified and filtered to remove contaminants which might degrade hplc columns. 

TLC and HPLC. To determine the best solvents for HPLC, 5pl of the C18 concentrated PVP fraction were separated on analytical TLC plates coated with either C18 reversed phase adsorbant or with silica. One cm sections of the plates were removed, placed in buffer, hydrolyzed, extracted and the p-damascenone charm determined. Standards containing 5 mg each of 1 -O-n-octyl- p-D-glucopyranoside and a-methyl-D-mannoside were run simultaneously on the same plate and visualized by spraying with aniline-diphenylamine-acetone-phosphoric acid 80% (4 mL 4 g 200 mL 30 mL) (10). HPLC separations were performed on a 0.46 mm x 25 cm column packed with 5 im C18, eluted with 60/40 methanol water at a flow of 0.8 mL/min. The refractive index of samples was recorded with a Knauer differential refractometer. Fractions were collected every minute and assayed for p-damascenone charm. 

Monoacyl CGAs are more polar than the diacyl CGA, so they eluted first and appeared early in the chromatogram [6-8,10,11,13,15-20]. Generally, water, acetic acid, methanol, and acetonitrile have been used as solvents for HPLC with reverse phase stationary phases, such as C18, C8, phenyl hexyl, and diphenyl. 

SPME uses a fused sihca fiber that is coated on the outside (occasionally internally if a capillary is used) with an appropriate stationary phase, typically an immobilized polymer, a solid adsorbent, or a combination of the two. A wide range of coatings is available, but probably the most widely used is polydimethylsiloxane (PDMS), weU known as a GC stationary phase that is thermally stable (can be used in a temperature range 20—320 ° C). The anal54es in the sample (liquid or gas) are directly extracted to the fibre coating, and are then thermally desorbed directly into the injection port of a GC or are extracted from the fiber using a suitable solvent for HPLC analysis this process provides high sensitivity because the complete extract can be analyzed. 

The flexibility of TLC relative to HPLC is enhanced by the greater choice of solvents available for preparing TLC mobile phases. The choice of solvents for HPLC is limited by the requirements for their chemical and physical properties imposed by the nature of the method. HPLC is a closed system operated under high pressure with on-line detection, most often using a UV monitor, and the column is continually reused. Solvent components with high vapor pressure (e.g., ethyl ether) or UV absorbance (benzene) or those that might degrade the column (NaOH) are difficult to use in HPLC but are readily applicable to TLC. 

Although not specific2Jly considered below, the preparation of aqueous-organic solvents for HPLC can cause the introduction of impurities. Mixing acetonitrile and water is endothermic and can sometimes exacerbate air dissolution. Buffers are also a special case in that the pH can change due to exposure to air and... 

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