HILIC column

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

FIGURE 6.7 A HILIC column (Tosoh Amide-80, 25 cm x 4.6 mm, 5 lm particles) at different flow rates with a solvent of ACN/water/0.1%TFA. The gradient is 80% to 15% ACN. Protein standards A = aprotinin, B = cytochrome C, C = carbonic anhydrase. 

Figure 6. scheme for on-line orthogonal HILIC-RP HPLC system and one detector step (A) sample injection into HILIC column (for polar analytes) and RP column (for non-polar analytes), step (B) analysis of HILIC retained analytes, step (C) analysis of RP phase retained analytes. 

An example of a separation carried out in reversed-phase and in HILIC is shown in Figure 16. The analytes are rather polar, morphine and morphine 3- -glucuronide. On the reversed-phase column, the glu-curonide elutes first, since it is more polar than the parent compound. On the HILIC column, it elutes last. In addition, a gradient is used in the HILIC separation to elute both compounds in the same time frame. This demonstrates clearly the difference in retention between HILIC and reversed-phase chromatography. 

Different developed analytical method are discussed in this chapter related to the determination of illicit substances in blood (either whole blood, plasma, or serum), OF, urine, and hair. These methods take into consideration the particular chemical and physical composition of the matrix and applies each time a suitable pretreatment to remove interfering and matrix effect, to maximize recoveries and to achieve a suitable enrichment if necessary. For liquid matrices the applications of the most common techniques are considered from simple PPT to SPE and LLE the results of recent works from literature are reported and new trends as online SPE, pSPE, automated LLE (SLE) or MAE are examined. Several stationary phases have been shown to be suitable for determination of illicit drugs Cl8, pentafluorophenyl, strong cation-exchange, and HILIC columns. The trend toward fast chromatography is investigated, both UHPLC and HPLC with appropriate arrangements moreover, results obtained with different ion sources, ESI, A PCI, and APPI are compared. 

Conventional LC with CIS-modified silica stationary-phase or HILIC columns has been widely used in antibiotic residue analysis as discussed above. Since the 1980s, the need for faster and more powerful separations for the analyses of constituents, such as protein digests, in complex matrices led to the development of highly mechanically stable (to high back-pressures) columns and... 

Mess, J.N., Cote, C., Bergeron, A., et al. (2009) Slection of HILIC columns to handle matrix effect due to phospholipids. Bioanalysis, 1,57-62. 

For reversed-phase column, buffer A is highly aqueous and for an HILIC column, buffer A is highly organic. 

Since DTC require alkaline conditions during chromatography to guarantee stability, silica materials cannot be used. Therefore, the polymeric ZIC-pHILIC column offered the great chance for an LC/MS approach, when a simple and salt-free gradient of acetonitrile and ammonium hydroxide solution is applied. The active sites of the ZIC-/ HILIC column consist of sulfobetaine (zwitterionic) groups (Figure 26.3), which also comprise... 

During the operations, the HILIC column may loose its performance. In contrast to reversed phase columns, this almost does not happen gradually, but suddenly from one injection to another, and also does not influence all DTC simultaneously, but only one or two of them. As exemplarily shown in Figure 26.14, the PBD peak is broadened or even completely distorted. In the left... 

Due to the absence of hydrogen donor capabilities [31], cyanopropyl silica phases are less retentive in normal-phase liquid chromatography than under-ivatized silica or other NP packing materials. Therefore, very few applications have been reported that utilize cyanopropyl-bonded silica in the HILIC mode [32,33]. The limited number of applications may also be attributed to the mechanical instabiUty of cyanopropyl-bonded silica when operated with solvents of intermediate polarity. This instabihty is caused by a decrease in the adhesion of particles to each other that maintain the integrity of the column bed in either nonpolar or highly polar solvents [25]. Dinh et al. [34] performed a multivariate modeling of column selectivity by principal component analysis of chromatographic data from polar compounds of various structures on 20 commercially available HILIC columns and verified the low potential of cyanopropyl-bonded silica columns due to insufficient hydrophilicity. 

One of the most popular types of HILIC columns today is based on a grafted polymeric layer with sulfoalkylbetaine zwitterionic moieties on wide-pore silica, containing both strongly acidic sulfonate groups and strongly basic quaternary ammonium groups separated by a short alkyl spacer. 

The four Acclaim Mixed-Mode columns mentioned in Section 6.7.2 - Acclaim Mixed-Mode WAX-1, Acclaim Mixed-Mode WCX-1, Acclaim Trinity PI, and Acclaim Trinity P2 - can also be used under HILIC conditions. Acclaim Mixed-Mode WAX-1 and Acclaim Mixed-Mode WCX-1 are based on an alkyl chain with terminal ion-exchange functional groups. Because of this column chemistry, both columns offer multiple retention mechanisms, including ion exchange, reversed phase, and HILIC. As an example. Figure 7.7 demonstrates that in highly organic mobile phase. Acclaim Mixed-Mode WCX-1 can serve as a HILIC column. Hydrophilic analytes such as cytosine elute later than naphthalene as a hydrophobic neutral analyte. 

Acclaim HILIC-10 is similar to Acclaim Mixed-Mode WAX-1. This relatively new column is also based on high-purity spherical silica covalently modified with a proprietary hydrophilic layer. It is suited for use in a broad range of applications including the separation of hydrophilic drugs and their metabolites. Acclaim Mixed-Mode HILIC-1 is a stationary phase that combines both RP and HILIC properties. Unlike traditional RP or HILIC columns, this packing material features a long alkyl chain with a diol functionality at the terminal end. 

Because traditional HILIC columns such as underivatized silica, cyano-, amino-, and diol-bonded phases have hydrophilic surfaces, they are incapable of separating small molecules via hydrophobic interaction. By comparison. 

The ZIC-HILIC column also fails to provide adequate retention for hydrophobic acidic and basic APIs. A typical representative of the class of hydrophobic acidic APIs is naproxen, a nonsteroidal anti-inflammatory drug... 

Figure 7.15 UHPLC separation of common carbohydrates under HILIC conditions on Waters BEH HILIC. Column dimensions ...

Table 7.7 HILIC columns and dimensions used for the separation of nucleobases and nucleosides in Figure 7.26.

Figure 131 Separation of adenosine and its 100 mmol/L ammonium acetate, pH 53 flow nucleotides on Nucleodur HILIC. Column rate 1.3mL/min detection UV (259nm) ...

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