CN bonded phase

The first toxic peak(s) from the Bio-gel P-2 run (volume reduced to 1 ml) is passed through a preparative mini column (Sep-pak 18 to separate the toxin(s) from yellowish pigments. The eluant is then subjected to HPLC using a semipreparative column (CN bonded phase 9.4 mm x 25 cm). Fig. 3 illustrates the presence of neosaxitoxin (second last peak) and saxitoxin (last peak). A total of 150 mu (mouse units) was loaded. The profile in Fig. 4 shows only neosaxitoxin (500 mu) is present because only a portion of the first toxic peak(s) from the P-2 run was injected. The bottom profile is that of a standard neosaxitoxin (200 mu). 

FIGURE 4-7. Comparison of relative retention due to the polarity (hydrophobicity) of Cj8, phenyl, and cyano (CN) bonded phases. Column Nova Pak family (4/a) 3.9 mm ID x 15 cm. (a) C, phase, (b) Phenyl phase, (c) CN phase. Mobile phase— acetonitrile water, 35 65 flowrate 2 mL/min detection 254 nm sample 1. benzyl alcohol, 2. acetophenone, 3. p-tolualdehyde, 4. anisole. Nova-Pak CN is the least hydrophobic and therefore the least retentive, followed by the more hydrophobic Nova-Pak Phenyl, and Nova-Pak C18 with the most hydrophobicity and the longest retention times. 

Partition (solubility) Polar interaction Non-polar hydrophobic interactions NH2 CN Bonded phase -normal Bonded phase -reverse... 

My approach in this chapter has been to present you with the options and then discuss the alternatives. I placed emphasis on reliability and reproducibility, if the techniques look otherwise similar. Therefore I recommended, for example, size-exclusion techniques over retention chromatography, if the analytical problem can be solved by both techniques. Also, for reasons of lepr ucibility, I recommended reversed-phase methods over normal-phase methods. Among the options for reversed-phase packings, I recommended Cjg bonded phases over CN bonded phases. Most of my recommendations are paralleled by the popularity of a particular technique. We can see this as a confirmation of the recommendations thousands of chromatographers cannot be wrong. 

Figure 3.3 Retention factor of the cationic solute benzyltrimeth>iammonium bromide (BTAB) versus the adsorbed amount of SDS on a CN bonded phase. Column 10 cm x 4.6 mm i.d., CPS Hypersil 5 pm. Inset shows log k vs. the SDS concentration in the aqueous mobile phase. Reprinted from Ref 11 with permission of the American Chemical Society.

To answer to the introduction question, a study of the retention behavior of a set of solutes of various polarities was done with different stationary phases and identical micellar mobile phases [26]. SDS and CTAB micellar phases were used with five different HypersiKD phases. The retention of the polar, apolar as well as ionic solutes studied obeyed Armstrong s three phase model (eq. 3. 4, Chapter 3). Table 4.3 lists the P m solute-micelle partition coefficient and the Pws solute-stationary phase affinity coefficient for different solutes on four bonded phases. The Pwm values are similar within experimental errors (see Chapter 5). The Pws values are similar for the C8 and C18 bonded phases. These two phases do behave similarly in classical RPLC. The more polar Cl and CN bonded phases do show significantly lower affinity coefficients. The surfactant layer is there cationic solutes are not retained by the four bonded phases and hydro-organic mobile phases. Their high affinity for the stationary phases with SDS mobile phases is due... 

The pH of a buffered mobile phase is significant not only because retention of ionizable compounds is dependent upon pH but because bonded phase columns are made via an acid catalyzed reaction. Manufacturers recommendations are to avoid any system with a pH lower than 2 or accept the risk of reversing the bonding reaction. However, the effective pH experienced by a column in a water organic mixture is difficult to predict. We have operated at a pH of 1.5 in this laboratory with no adverse effects to the column, but exercise the simple precaution of not allowing the acidic mobile phase to remain on the column overnight or static for long periods of time (1 hr). We have particularly noticed that the CN column is stable at low pH s. 

Strength. The only other possibility was to change the polarity of the bonded phase therefore, a CN column with equivalent polarity as silica itself was chosen and tailing eliminated as demonstrated by the peak shapes in Figure 4. Using the CN column and an eluent of 25 75 acetonitrilerwater with 0.035% sulfuric acid (v v v), good peak shapes were obtained with no change in retention time with variation in sample concentration. 

The bonded phases carrying polar groups such as -NH2, -OH, -CN, etc., which are separated from the packing surface by a spacer (usually the n-propyl groups) behave in an intermediate manner between the solid surfaces and the nonpolar bonded phases though the effect of polar end groups usually dominates (Sections 16.3.5 and 16.3.6). 

While there are no thermochemical data available for the (ZnCN)+ ion or related neutral, the latter species, in its gas phase, was found to have a ground state with an atypically short CN bond length ". Valence isoelectronic to the latter species is the (ZnCS)+ ion for which a 0 K gas phase bond energy of 149 23 kJmoR was recently determined. ... 

PNIPAM containing pendant triphenylmethane leuconitrile groups also showed photostimulated phase transition from the phase separated state to the homogeneous state [13]. The triphenylmethane leuconitrile group is known to change the polarity more pronouncedly than the azobenzene chromophore by the ionic photodissociation of C-CN bond [14]. 

Acidic solutes can localize on cyano groups of the bonded phase. The cyano phase is very versatile and has been proposed as the unique stationary phase in an expert system (22). In re-versed-phase mode, CN-bonded silica exhibits better selectivities than alkyl-bonded silica toward proteins and peptides. 

Fluorescence and Ultraviolet Absorbance of Pesticides and Naturally Occurring Chemicals in Agricultural Products After HPLC Separation on a Bonded-CN Polar Phase... 

Pesticides Separated on a CN-Bonded Polar Phase. Figures 3 and 4 contain the chromatograms obtained for several of the pesticides listed in Table 2. Various mobile phases were used to facilitate separation on the CN-bonded polar phase. Detection is shown in both absorbance and fluorescence modes. It would appear that fluorescence detection is more sensitive for some pesticides, while absorbance detection is more sensitive for others. However, comparisons of one manufacturer s absorbance monitor with another manufacturer s fluorescence monitor could be misleading. (I will refer to this under Instrumental Parameters.) Furthermore, the ultimate useful comparison is obtained when practical samples are chromatographed since these... 

Figure 3. HPLC of several pesticides (100 ng) on a CN-bonded polar phase with absorbance and fluorescence detection (A PM = attenuation setting of photomultiplier signal)...

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