SepaFlash HILIC ARG Cartridge and Its Application in the Purification of Amino Acid Derivatives

Introduction
Reversed phase liquid chromatography (RPLC) is by far the most widely used liquid chromatography separation technology RPLC plays a leading role in the separation and purification of moderately polar or some highly polar compounds Alkyl bonded phase LC columns, such as C18, C8, C4, etc are usually the first choice for separating these compounds With the rapid development of biomedicine, scientific researchers are p resenting
increasing  demand on the separation of highly polar compounds, including small molecule organic acids, amino acids and their derivatives, nucleic acids, peptides and etc   However, conventional LC columns with alkyl bonded phase have poor retention for these highly polar samples Considering this situation, numerous brands of manufacturers have introduced hydrophilic interaction chromatography (HILIC) columns that can improve the retention of highly polar compounds For instance, there are plenty of LC columns used for analytical area, including InfinityLab Poroshell 120 HILIC series and Zorbax HILIC series from Agilent, Atlantis HILIC series and XBridge HILIC series from Waters, Accucor HILIC series and Syncronis HILIC series from Thermo Fisher, etc
In Flash chromatography, SepaFlash HILIC ARG cartridge is specifically developed by Santai Technologies to improve the retention for compounds of high polarity With the bonding of arginine molecules to the surface of silica, the interaction between the stationary phase surface and the molecules of high polarity is enhanced, thereby the retention of such molecules on the cartridge is improved The retention mechanism in HILIC is a multi-mode one mixed with liquid-liquid distribution, ion exchange and hydrogen bonding The schematic diagram of the separation mechanism in HILIC is shown in Figure 1 Same as conventional RPLC, the mobile phase used in HILIC is usually water/acetonitrile system or water/methanol system, except that the elution order in HILIC is exactly the opposite of RPLC In RPLC, organic phases such as acetonitrile or methanol are solvents with higher elutropic strength In contrast, organic phase becomes the weaker one in HILIC, meanwhile water becomes the solvent with the highest elutropic strength For example, when setting the eluting gradient profile in HILIC mode, we usually start with acetonitrile/water (V : V = 95 : 5) and water ratio is increased during the separating procedure to elute out the highly polar samples gradually
Figure 1 The schematic diagram of the separation mechanism in HILIC mode
In this post, the sample is an amino acid derivative kindly provided by a synthetic lab from a pharmaceutical R&D company The sample has high polarity and good solubility in water However, the sample had poor retention on the hydrophilic C18AQ cartridge For the analysis of raw sample, analytical amino column combined with buffer salt solution as the mobile phase was used Considering the challenge of buffer salt removal in post separation sample process, HILIC ARG cartridge was used for the sample purification The application engineers from Santai Technologies utilized SepaFlash HILIC ARG cartridge in combination with a Flash chromatography system SepaBean machine T to purify the sample As a result, the target product meeting the purity requirement were successfully obtained, suggesting an efficient and cost effective solution for the purification of these highly polar compounds
Experimental Section
1   Sample information
The sample used in this application was kindly provided by a pharmaceutical R&D company The sample was an amino acid derivative that was highly polar and has a good solubility in water The chemical structure cannot be obtained due to confidential reasons
2   Sample purification by Flash chromatography
The sample was purified by a Flash chromatography system SepaBean machine T according to the parameters as shown in Table 1
Instrument
SepaBean machine T
Flash cartridge
12 g SepaFlash  C18AQ  reversed phase cartridge
(spherical silica, 20 - 45 μm, 100 Å, Order number ： SW-5222-120-SP(AQ))
12 g SepaFlash HILIC ARG  cartridge
(spherical silica, 20 - 45 μm, 100 Å, Order number ： SW-5 6 22- 012 -SP)
Wavelength
220 nm ; 254 nm
Mobile phase
Solvent A : Water ;
Solvent B :  Acetonitrile
Flow rate
15  m L /min
Sample loading
0 5 mL ( 50  mg)
Gradient
Time (min)
Solvent B (%)
Time (min)
Solvent B (%)
0
0
0
95
8.5
0
2.0
95
12.5
17
14.0
78
1 2.6
37
17.5
78
17.3
80
20.0
62
21.0
80
25.0
62
Table 1 The experimental setup for Flash purification
Results and Discussion
According to previous experience of the purification of these highly polar samples, we used an aqueous C18AQ cartridge as a start to purify the sample The Flash chromatogram of the sample was shown in Figure 2
Figure 2 The Flash chromatogram of the sample on a C18AQ cartridge
As shown in Figure 2, the sample could not be well retained on the C18AQ cartridge The sample was eluted out from the cartridge by the mobile phase in about one column volume (CV), resulting poor separation between the target product and the impurities in raw sample The reason behind this might be the existence of a large number of polar groups in the sample structure, such as Serine, Threonine,
Asparagine, Glutamine, etc
(Figure 3) On the other hand, there might be plenty of electrically charged groups in the sample structure, including Aspartate, Glutamate, Lysine, Arginine, Histidine, etc These polar or electrically charged groups cannot have enough hydrophobic interactions with the hydrophobic C18 carbon chains, resulting poor retention of the sample on the C18AQ cartridge
Figure 3 The chemical structure of twenty-one amino acids (reproduced from Wikipedia)
Based on the previous experimental results, we tried with a SepaFlash HILIC ARG cartridge to purify the sample The Flash chromatogram was shown in Figure 4
Figure 4 The Flash chromatogram of the sample on a HILIC ARG cartridge
As shown in Figure 4, the sample could be well retained on the SepaFlash HILIC ARG cartridge With the help of mixed separation mode in HILIC, the components in the sample have different retention time on the cartridge due to their different polarity or electric charge state The resolution for the target product and the impurities in the raw sample were good After post-separation process of the collected fractions, the target product meeting the purity requirement was obtained
Conclusion
For the separation and purification of the highly polar amino acid derivative samples, HILIC ARG cartridge is a good choice due to its multi-mode separation mechanism, including liquid-liquid partitioning, ion exchange and hydrogen bonding
User Guide for the SepaFlash HILIC ARG Cartridges
1   The Choice of Mobile Phase
As a start, water/acetonitrile is recommended as the mobile phase in HILIC mode To keep the stationary phase in wet condition, at least 5% of the water phase should be maintained in the mobile phase Furthermore, the water phase should not exceed 50% in the mobile phase when setting the elution gradient profile As a replacement for acetonitrile in order to gain more selectivity for the analytes, other organic solvents including methanol, acetone, isopropanol, etc could be considered
2   The Choice of Buffer Salt
When buffer salt is needed as an additive to the mobile phase, it is suggested that avoid using phosphate buffer salts since these salts have poor solubility in organic solvents and might induce salt precipitation during separation, thus resulting over pressure in chromatography system As a recommendation, the ammonium salts including ammonium acetate and ammonium formate have good performance in reproducibility and maintaining good peak shape Moreover, ammonium salts are easy to remove when purified sample needs to be further processed The suggested concentration for the ammonium salts as an additive to the mobile phase is 10 mM
3   The Choice of Sample Dissolution Solvents
Avoid using pure water to dissolve the sample when using a HILIC cartridge It is recommended to use 100% organic solvents as the sample dissolution solvent, including acetonitrile, methanol, isopropanol, etc If the sample cannot be completely dissolved in the above mentioned organic solvents, add water or DMSO appropriately to dissolve the sample and then dilute with those organic solvents
4   Cleaning and Storage of HILIC Cartridges
Cleaning : Use acetonitrile/water (V:V=50:50) as the cleaning solvent If there are some components strongly retained on the cartridge, use acetonitrile/water (V:V=5:95) to thoroughly clean the cartridge and then replace with acetonitrile/water (V:V=95:5) for cartridge storage
Storage : Use acetonitrile/water (V:V=95:5) as the cartridge storage solvent Do not store the cartridge in solvents containing buffer salts Store the cartridge well-capped and wet in proper storage solvent Never dry out the HILIC cartridge since it will induce channeling due to expansion and contraction of the stationary phase
About SepaFlash HILIC ARG Cartridges
There are a series of the SepaFlash HILIC ARG c artridges with different specifications from Santai Technologies  (as shown in Table 2)
Item Number
Column Size
Sample Size
Max.Pressure
(psi/bar)
SW-5 6 22-004-SP
5.4 g
5.4 mg –  108 mg
400/27.5
SW-5 6 22-012-SP
20 g
2 0  mg –  0.40 g
400/27.5
SW-5 6 22-025-SP
33 g
33g –  0.66 g
400/27.5
SW-5 6 22-040-SP
48 g
48 mg –  0.96 g
400/27.5
SW-5 6 22-080-SP
105 g
105 mg –  2.1 g
350/24.0
SW-5 6 22-120-SP
155 g
15