7
difficult. Since guanidine is such a polar compound, it also requires a 100% aqueous mobile phase to be retained in reversed- phase on the TYPE-C C18 column. Normally this analysis would require a specialty column such as an ion-exchange phase to enhance retention of this polar molecule but there are other issues using this approach [8] including long run and re- equilibration times. Since the Cogent Bidentate C18™ (TYPE-C with C18 bonded moiety) does not suffer any loss of retention from run to run which is commonly known to be due to dewetting or ‘phase collapse’ and it is not a ‘specialty column’, it can be used as an L1 or reversed phase C18 column for USP methods. In Figure 2A the reproducibility of the method is shown where after 100 injections the retention time of guanidine (second peak) is essentially the same as the first injection. In Figure 2B is the analysis of the residual guanidine removed from a protein preparation and the retention is compared to a standard sample. Such a procedure is common if the protein is to be used for therapeutic applications. One of the advantages of this column is that it is hydrolytically stable under aggressive, acidic mobile phase conditions as shown in this application. Also, since it is still considered an L1 column, it could be interchanged with a C18 method column without revalidation of a validated GMP method.
Figure 2. Analysis of guanidine in a 100% aqueous mobile phase on a Cogent Bidentate C18 column.
A. Repeatability of analysis using guanidine Standard.
B. Comparison of retention times of standard and real sample. Mobile Phase: 100% DI water + 0.5% phosphoric acid + 1.5g/L pentane sulfonic acid (isocratic). Column: 4.6 x 150mm. Flow rate: 1.0mL/min.
Detection at 200 nm. Standard Sample: 100 ppm. Injection Volume: 20µL.
Figure 3. Separation of melamine and cyanuric acid on the Cogent Diamond Hydride column using an acetic acid mobile phase and a gradient from high to low concentration of acetonitrile in the mobile phase. Mobile Phase: A: DI water + 0.1% acetic acid; B: Acetonitrile + 0.1% acetic acid. Gradient: 100% B to 50% B in 15 min. Column: 2.1 x 150mm. Flow rate: 0.4mL/min. Sample Concentration: Cyanuric acid 1.5µg/ml and melamine 3ug/mL. Detection: m/z 128 in the negative ion mode for cyanuric acid and m/z 127 in the positive ion mode on an Agilent 6210 MSD TOF spectrometer.
An improvement in peak shape can be achieved by adding a small amount of ammonia to the sample. The amount used here (5 µL of 12% ammonia/mL) does not appreciably affect the retention times.
Aqueous Normal Phase
Every TYPE-C stationary phase displays ANP properties, for example, increased retention of hydrophilic species as the amount of the least polar component in the mobile phase increases. The mobile phase consists of water and typically either acetonitrile or acetone. Thus it is very easy to transition (no hysteresis) from RP to ANP since water is the common mobile phase constituent in each mechanism and the attraction of water to the particle surface is weak and desorbs very easily. In general, the extent of ANP retention is dependent on the amount and type of modification of the particle surface. Maximum retention of hydrophilic species under ANP conditions is obtained for the totally unmodified or minimally modified surface while lesser, but still appreciable, polar retention is obtained even when C18 or C8 bonded groups are present.
The retention of polar compounds in the ANP mode is best illustrated by some examples that utilise the Cogent Diamond Hydride™ (DH) stationary phase. This phase, which has a small amount of carbon on the surface, has both high hydrophilic retention as well as excellent peak shape over a wide range of polar compounds. An analytical problem that seems to continually make the news is the determination of melamine and its degradation product, cyanuric acid, which have been found to be contaminants in both human and animal food sources. Figure 3 shows the analysis of these two compounds using LCMS with the DH stationary phase. The detection of cyanuric acid is done in the negative ion mode while melamine is detected as a positive ion. Using a TOF MS system the sensitivity is in the nanogram range while with an MS/MS system the detection limit is sub- nanogram. As can be seen in Figure 3, the separation of these two compounds is substantial with both peaks having excellent efficiency and peak shape.
An important metabolic determination is the analysis of adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Nucleotides are important phosphate containing compounds that are found in living cells and are associated with a broad array of metabolic and biological processes. They have significant roles in the synthesis of DNA and RNA, are involved in signal transduction pathways, function as coenzymes in biosynthetic pathways and serve as energy reservoirs in biological systems. Figure 4 shows an example of the separation of the two adenosine analytes using an ANP gradient method. The gradient starts at a high percent of acetonitrile in the mobile phase and goes to 70% (v/v) over 10 min. The last component, adenosine 5′-triphosphate (ATP) has some tailing in comparison to the first components.
Finally, two examples of pharmaceutical analyses will be described to document the retention of polar compounds on TYPE-C-based columns having reasonably high surface coverages of larger bonded molecules. Metformin, a drug used in the treatment of diabetes, was analysed on a Bidentate C18 column [9]. A mobile phase of acetonitrile and DI water with 0.1% formic acid is used for the analysis as is the case for many basic compounds. Retention begins to increase at 60% acetontrile, becomes substantial at 70% and has a k (retention factor) greater than 5 when the amount of acetonitrile reaches 80%. An even more basic drug, Tobramycin, an antibiotic has been analysed on a TYPE-C-based UDC- Cholesterol™ column [10]. The additive again is formic acid but an even more unusual aspect of this analysis is that methanol as well as acetonitrile can be used as the organic component of the mobile phase. This situation is relatively rare since in most cases methanol is generally too strong of a solvent to produce significant retention in the ANP mode. But in the case of some highly polar compounds, methanol becomes a viable solvent for ANP. Naturally retention is considerably less than with acetonitrile or acetone.
Figure 4. Gradient separation of AMP and ATP on a Cogent Diamnond Hydride column. Mobile Phase: A, DI water + 0.1% ammonium formate; B, 90:10 acetonitrile/DI water + 0.1% ammonium formate. Gradient: 95% B to 70% in 10 min. Column: 2.1 x 100mm. Flow rate: 0.3mL/min. Sample concentration: 0.3mg/mL of each. Detection @ 254nm.
References
1. J.J. Pesek, M.T. Matyska, A. Sharma,
J.Liq. Chromatogr. & Rel. Technologies, 31 (2008) 134.
2. J.J. Pesek, M.T. Matyska in ‘Advances in Chromatography, Vol. 48’, E. Grushka, N. Grinberg, eds, CRC Press, Boca Raton, FL, 2010, pp. 255.
3. J.J. Pesek, M.T. Matyska, S.M. Fischer,
T.R.Sana, J. Chromatogr. A, 1204 (2008) 48.
4. J.J. Pesek, M.T. Matyska, M.T.W. Hearn, R.I. Boysen, J. Chromatogr. A, 1216, (2009) 1140.
5. J.J. Pesek, M.T. Matyska, J.A. Loo, S.M. Fischer, T.R. Sana, J. Sep. Sci, 32 (2009) 2200.
6. J.J. Pesek, M.T. Matyska, J. Sep. Sci., 32 (2009) 3999.
7. J.J. Pesek, M.T. Matyska, J. Duley, M. Zamzami, S.M. Fischer, J. Sep. Sci, 33 (2010) 930.
8. J. Qui, H. Lee, C. Zhou, J. Chromatogr. A, 1073 (2005) 263. 9. J.J. Pesek, M.T. Matyska, LCGC, 24 (2006) 296. 10.
http://www.microsolvtech.com/hplc/
app_udc_amgly.asp
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