Raptor Polar X: Separate a Wide Variety of Polar Analytes with a Novel Hybrid Stationary Phase

• No time-consuming derivatization or complex ion pairing.
• Switch between retention modes with simple mobile phase changes and short equilibration times.
• Ideal for increasing sensitivity and selectivity in LC-MS analyses.

Simplify the Analysis of Polar Compounds

Analyzing polar compounds using liquid chromatography has historically been a challenge. Poor retention and peak shape, complex mobile phases that may not be MS-friendly, long equilibration times, low sensitivity, and sample derivatization are all complications that reduce lab efficiency and productivity. However, the development of a novel column that is specifically designed for the analysis of a broad range of polar compounds allows scientists to avoid these problems by taking advantage of the true power of chromatography.

Providing the True Power of Chromatography

One of the most significant, yet least understood, aspects of method performance is the importance of using the proper stationary phase for a particular separation. For the analysis of polar compounds, reversed-phase columns do not provide adequate retention without the use of complex mobile phases or sample derivatization to compensate for the lack of effective interactions between the analytes and the column. But, by matching analytes to a stationary phase that provides purpose-built resolving power, you can avoid complex sample preparation procedures, save time and money, and reduce opportunities for error.

The Raptor Polar X stationary phase is specifically designed to selectively retain polar analytes through a balance of two retention mechanisms. This unique hybrid phase is ideal for analyzing a wide variety of polar compounds, especially when coupled with mass spectrometry. Simplify the analysis of polar compounds with the resolving power of Raptor Polar X columns from Restek.

Greater Than the Sum of Its Parts

Recognizing that the retention mechanisms most commonly used to retain polar compounds are hydrophilic interaction chromatography (HILIC) and ion-exchange approaches, Restek has developed a novel stationary phase chemistry that combines both modes on a single ligand. Because this unique ligand is bound to superficially porous particles, Raptor Polar X columns both reliably retain and efficiently separate a broad range of polar analytes.

Typical application-specific columns favor one retention mode over another, and gains made in the retention of one type of polar compound come at the expense of the performance for others. In contrast, with the Raptor Polar X column's patent-pending phase chemistry, two independent retention mechanisms are available for use, which provides truly balanced and flexible retention (Figure 1). Simple changes to mobile phase conditions allow analysts to switch between modes and selectively tune retention for the compounds of interest without needing long equilibration times before use or between samples.

How It Works: Switching Retention Modes

When using a mobile phase with a relatively high-percentage of acetonitrile to water, a layer of water is formed at the surface of the silica into which polar compounds can partition. Partitioning into the water layer results in effective interactions between polar analytes and the ligand bound to the silica surface (Figure 2). The novel Raptor Polar X ligand chemistry makes this technique even easier, allowing for faster-than-ever column equilibration and re-equilibration. This means new columns are ready for use quickly, and sample throughput increases because less re-equilibration time is required between samples.

For smaller polar compounds, these high-percentage acetonitrile conditions generate strong interactions between the analytes and the stationary phase, leading to excellent retention of small, highly polar, charged compounds. To fine-tune retention, simply increase the amount of water in the mobile phase. This will draw the charged, polar compounds into the mobile phase, effectively eluting them. When moving to a higher percent water mobile phase, the distinct HILIC partitioning retention will diminish and the stationary phase’s ion-exchange characteristics will become the dominant retention mechanism (Figure 3).

Retention Mode Switching: An Example

To illustrate these principles in action, Figure 4 demonstrates how two different types of polar compounds interact with the stationary phase, and how their responses are affected by simple mobile phase changes. In this example, we analyzed two water-soluble vitamins: vitamin B3 (niacin), which is an organic acid, and vitamin B1 (thiamine), which has a permanent positive charge.

In the top chromatogram, a highly organic mobile phase was used, which is favorable to HILIC separations. Using the Raptor Polar X column under these conditions, a water layer quickly forms on the surface of the silica particles. This allows polar compounds to partition from the acetonitrile layer into the water layer where they can also interact with the silica surface and the stationary phase. In this way, polar compounds are retained through hydrophilic interactions with the stationary phase and are still eluted under highly organic mobile phase conditions. This is very favorable for LC-MS/MS analysis because of the improved desolvation and increased ionization efficiency.

The bottom chromatogram highlights the chromatographic changes that are seen when the retention mechanism is switched by moving to a mobile phase with a higher percentage of water. As we move further away from ideal HILIC conditions, both vitamin B1 and vitamin B3 are less retained. This is because as the percentage of water in the mobile phase increases, conditions shift away from HILIC retention and toward the ion-exchange retention characteristics of the phase. The elution order also changes because vitamin B1 is less retained than vitamin B3 under ion-exchange conditions.

	Peaks
1.	Vitamin B3
2.	Vitamin B1

Column	Raptor Polar X (cat.# 9311A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	0.1% Formic acid in acetonitrile
Mobile Phase
Flow:	0.5 mL/min

Detector	MS/MS
Ion Source:	Electrospray
Ion Mode:	ESI+
Instrument	HPLC
Notes	Mobile phase A: Water, 5 mM ammonium formate, 0.1% formic acid Mobile phase B: Acetonitrile, 0.1% formic acid Top chromatogram: 95% B, 10 min run, 2 μL injection (100 ppm B3, 0.01 ppm B1) Bottom chromatogram: 60% B, 5 min run, 0.5 μL injection (100 ppm B3, 0.1 ppm B1)

Download PDF

Well-Balanced Hybrid Retention Lets You Analyze More Compounds with a Single Method

It is common for columns to specialize, to focus on a single type of interaction to the exclusion of others. This approach works well when the target analytes all share the same characteristics; however, polar compounds vary widely in their chemical traits. For the analysis of polar compounds, a specialized approach to column selection often means having to use multiple methods with different columns or conditions for each type of compound. As shown in the following examples, the hybrid phase chemistry of Raptor Polar X columns is a better alternative because the multimode retention profile allows a wide variety of target analytes to be analyzed in the same method.

QuPPe-Inspired List of Polar Contaminants

The European Quick Polar Pesticides (QuPPe) method includes a range of polar analytes from anionic polar herbicides, such as glyphosate and related compounds, to a variety of oxychloro contaminants, including chlorate and perchlorate. As shown in Figure 5, the Raptor Polar X column retains and quickly separates this diverse mix with the last compound eluting around 10.5 minutes and a total run time of only 13 minutes. This separation is performed with simple unbuffered mobile phases, acidified with 0.5% formic acid to reduce peak broadening and tailing. The method demonstrated in Figure 5 was also able to separate compounds that have similar mass fragments, like AMPA from N-acetyl AMPA and fosetyl aluminum from phosphonic acid and phosphoric acid (a commonly observed matrix component and source of interference).

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion 1	Product Ion 2	Product Ion 3	Precursor 2	Product Ion 2
1.	Aminomethylphosphonic acid (AMPA)	0.805	200	110.1	79.1	63.1	81.1	-	-
2.	Bialophos	0.847	100	322.2	88.2	216.1	134.2	-	-
3.	Perchlorate	2.593	5	101.0	84.95	-	-	98.9	83
4.	Glufosinate	3.376	200	180.2	85.2	95.1	-	-	-
5.	3-(Methylphophinico) propionic acid (MPPA)	4.076	100	151.0	63.0	107.1	133.2	-	-
6.	Trifluoracetic acid (TFA)	4.423	20	113.0	69.1	19.1	-	-	-
7.	2-Hydroxyethane phosphonic acid (HEPA)	4.969	100	125.1	79.0	95.0	63	-	-
8.	Difluoroacetic acid (DFA)	5.018	200	95.0	51.1	-	-	-	-
9.	Chlorate	5.542	100	85.0	69.0	-	-	83.0	67.1
10.	Ethephon	5.564	200	143.1	107.2	-	-	-
11.	Glyphosate	6.113	200	168.1	63.1	79.1	-	-	-
12.	Bromide	6.423	2000	80.9	80.9	-	-	79.0	79.0
13.	Bromate	6.771	600	129.0	113	-	-	127	111.1
14.	N-acetyl AMPA	6.932	200	152.1	110.1	62.9	-	-	-
15.	Fosetyl aluminum	7.775	80	109.1	81.0	63.0	78.9	-	-
16.	Phosphonic acid	8.275	500	81.1	62.9	-	-	-	-
17.	N-acetyl glufosinate	9.980	200	222.2	136.1	134.1	59.0	-	-

Column	Raptor Polar X (cat.# 9311A32)
Dimensions:	30 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	35 °C
Standard/Sample
Diluent:	Water
Inj. Vol.:	1 µL
Mobile Phase
A:	Water, 0.5% formic acid
B:	Acetonitrile, 0.5% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 35 65 5.0 0.5 90 10 11.5 0.5 90 10 11.51 0.5 35 65 13 0.5 35 65

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC

Download PDF

In addition to the Restek-developed method shown in Figure 5, Raptor Polar X analytical and guard columns were independently evaluated and included in the QuPPe method itself [1]. They are used in a method for the detection of a wide variety of polar pesticide by LC-MS/MS in negative ESI mode.

Underivatized Amino Acids

Amino acids are a diverse group of very polar compounds that are commonly analyzed with reversed-phase or ion-exchange chromatography using pre- or post-column derivatization. Direct analysis of underivatized amino acids is challenging due to limited retention and poor chromatographic performance. However, underivatized amino acids with nonpolar, polar, positively charged, and negatively charged side chains are all retained and easily separated in one method on a Raptor Polar X column. Figure 6 shows the analysis of 21 amino acids, including a taurine supplement, in a liquid baby formula matrix using a simple protein precipitation and direct analysis of the resulting extract.

	Peaks	tR (min)	Precursor Ion	Product Ion
1.	Tryptophan	1.17	205.07	146.08
2.	Phenylalanine	1.26	166.13	120.10
3.	Leucine	1.41	132.13	86.10
4.	Isoleucine	1.55	132.13	86.10
5.	Methionine	1.62	150.07	104.10
6.	Tyrosine	1.69	182.10	136.08
7.	Taurine	1.91	126.07	108.07
8.	Valine	1.98	118.13	72.11
9.	Proline	2.29	116.13	70.09
10.	Alanine	3.00	90.03	44.10

	Peaks	tR (min)	Precursor Ion	Product Ion
11.	Threonine	3.12	120.13	74.08
12.	Glycine	3.62	76.10	30.11
13.	Glutamine	3.87	147.13	84.07
14.	Serine	3.93	106.07	60.09
15.	Asparagine	4.08	133.13	74.07
16.	Arginine	4.47	175.17	70.09
17.	Histidine	4.66	156.07	110.16
18.	Lysine	4.97	147.13	84.13
19.	Glutamic acid	5.89	148.10	84.10
20.	Cystine	6.10	241.13	152.00
21.	Aspartic acid	7.12	134.07	74.06

Column	Raptor Polar X (cat.# 9311A12)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	20:80 Water:acetonitrile, 0.01 N HCl
Conc.:	Endogenous amino acids
Inj. Vol.:	5 µL
Mobile Phase
A:	Water, 0.5% formic acid
B:	Mobile Phase B: 9:1 Acetonitrile:20 mM ammonium formate in water (pH 3.0) (The ammonium formate concentration is 20 mM relative to the total volume of mobile phase B. See preparation notes for instructions on diluting a 200 mM aqueous starting solution.)
	Time (min) Flow (mL/min) %A %B 0.00 0.5 12 88 3.50 0.5 12 88 8.00 0.5 70 30 8.01 0.5 12 88 10.0 0.5 12 88

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	A 200 µL aliquot of protein hydrolysate formula (Similac ALIMENTUM) was mixed with 800 µL of acetonitrile and 10 µL of 1 N HCl. After centrifugation at 4000 rpm for 5 minutes, the supernatant was diluted 20-fold with 20:80 water:acetonitrile (0.01 N HCl) and injected for analysis.
Notes	Mobile Phase B Preparation: To make 500 mL of mobile phase B, measure ~45 mL of water into a small beaker and add 1 mL of 10 M ammonium formate solution. Adjust pH to 3.0 by adding formic acid and then bring the volume to 50 mL with water. Combine this 50 mL ammonium formate solution (pH 3.0) with 450 mL of acetonitrile to complete the preparation.

Download PDF

Ultrashort- to Long-Chain PFAS

The last illustration of the Raptor Polar X column’s ability to maximize method productivity is an application that paves a path for future testing. Current LC-MS/MS methods for per- and polyfluoroalkyl substances (PFAS) analysis are focused on short-chain (C4-C6), long-chain (C8 and up), and alternative compounds and do not include emerging ultrashort-chain (C2 and C3) compounds. Typical reversed-phase methods for PFAS analysis do not have sufficient retention for ultrashort-chain PFAS; whereas other methods based on anionic exchange often have too much retention, resulting in poor chromatographic performance. Because of the balanced retention mechanisms found in the Raptor Polar X column, ultrashort-chain PFAS and long chain PFAS can all be analyzed in a single 5-minute isocratic run (Figure 7).

	Peaks	tR (min)	Conc. (ng/L)	Precursor Ion	Product Ion
1.	11-Chloroeicosafluoro-3-oxanonane-1-sulfonate (11CL-PF3OUdS)	1.25	400	630.78	450.80
2.	9-Chlorohexadecafluoro-3-oxanonane-1-sulfonate (9Cl-PF3ONS)	1.34	400	530.78	350.85
3.	Perfluorooctanesulfonic acid (PFOS)	1.38	400	498.84	79.97
4.	Perfluorohexanesulfonic acid (PFHxS)	1.49	400	398.90	79.97
5.	Perfluorobutanesulfonic acid (PFBS)	1.64	400	298.97	79.97
6.	Perfluoropropanesulfonic acid (PFPrS)	1.73	400	248.97	79.98
7.	Perfluoroethanesulfonic acid (PFEtS)	1.86	400	198.98	79.92

	Peaks	tR (min)	Conc. (ng/L)	Precursor Ion	Product Ion
8.	Hexafluoropropylene oxide dimer acid (HFPO-DA)	2.06	400	284.97	168.92
9.	Perfluorooctanoic acid (PFOA)	2.11	400	412.90	368.91
10.	Ammonium 4,8-dioxa-3H-perfluorononanoate (ADONA)	2.15	400	376.90	250.93
11.	Perfluorohexanoic acid (PFHxA)	2.36	400	312.97	268.90
12.	Perfluorobutanoic acid (PFBA)	2.76	400	212.97	168.97
13.	Perfluoropropionic acid (PFPrA)	3.06	400	163.03	119.01
14.	Trifluoroacetic acid (TFA)	3.77	400	113.03	69.01

Column	Raptor Polar X (cat.# 9311A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Temp.:	40 °C
Standard/Sample
Diluent:	50:50 Water:methanol
Conc.:	400 ng/L
Inj. Vol.:	10 µL
Mobile Phase
A:	Water, 10 mM ammonium formate, 0.05% formic acid
B:	60:40 Acetonitrile:methanol, 0.05% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 15 85 8.00 0.5 15 85

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC

Download PDF

To Passivate or Not to Passivate?

Glyphosate can be difficult to analyze at trace levels because it chelates with active metal sites in an LC system. Even though the Raptor Polar X column contains a treated sample flow path and arrives in your lab ready for initial use, metal components along the sample flow path through the LC instrument may benefit from passivation. Whether or not passivation is required will depend on the application and on your particular instrument setup, but for the analysis of polar compounds that are known to chelate, such as glyphosate, Restek recommends treating your system with LC passivation solution (cat.# 32475) prior to running samples.

A Novel Stationary Phase Built on the Time-Tested Foundation of Raptor Quality

The Raptor Polar X has been designed, manufactured, and tested to the same exacting standards that have made Raptor columns synonymous with quality. Whether it’s robust injection-to-injection performance when analyzing real-world samples in challenging matrices like spinach (Figure 8) or reliable column-to-column performance when it is eventually time to replace the column (Figure 9), you can count on Raptor brand quality. In addition, the single ligand approach to the Raptor Polar X column’s hybrid retention functionality helps ensure that each column is made uniformly and can withstand even aggressive conditions such as large volume water injections without loss of stationary phase.

	Peaks	Conc. (ng/mL)	Precursor Ion	Product Ion 1	Product Ion 2	Product Ion 3
1.	AMPA	100	110.10	79.05	63.10	81.10
2.	Glufosinate	100	180.20	85.15	95.10	-
3.	Glyphosate	100	168.10	63.05	79.05	-

Column	Raptor Polar X (cat.# 9311A32)
Dimensions:	30 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Guard Column:	Raptor Polar X guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9311A0252)
Temp.:	35 °C
Standard/Sample	Spinach extract
Inj. Vol.:	2 µL
Mobile Phase
A:	Water, 0.5% formic acid
B:	Acetonitrile, 0.5% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 35 65 5.0 0.5 90 10 6.5 0.5 90 10 6.51 0.5 35 65 8 0.5 35 65

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC
Sample Preparation	Frozen spinach was added to a Blixer processor with dry ice (3:1-4:1 ratio) and then ground into a very fine powder. The homogenate was placed into the freezer immediately. A 5.0 gram sample of the spinach powder was weighed into a 50 mL centrifuge tube (cat.# 25846). According to the QuPPe method (Quick Polar Pesticides Method), 5.0 mL of methanol with 1.0% formic acid was added into the centrifuge tube. The tube was shaken by hand for 1 min and then by a mechanical shaker vigorously for 5 min. After centrifuging for 10 min at 4200 rpm, the supernatant was filtered through a 0.22 μm filter (cat.# 23984). The final extract was fortified with the AMPA, glufosinate, and glyphosate at a final concentration of 100 ng/mL.

Download PDF

	Peaks	Conc. (ng/mL)	Precursor Ion	Product Ion 1	Product Ion 2	Product Ion 3
1.	AMPA	100	110.10	79.05	63.10	81.10
2.	Glufosinate	100	180.20	85.15	95.10	-
3.	Glyphosate	100	168.10	63.05	79.05	-

Column	Raptor Polar X (cat.# 9311A32)
Dimensions:	30 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	35 °C
Standard/Sample
Diluent:	Water
Inj. Vol.:	5 µL
Mobile Phase
A:	Water, 0.5% formic acid
B:	Acetonitrile, 0.5% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 35 65 5.0 0.5 90 10 6.5 0.5 90 10 6.51 0.5 35 65 8 0.5 35 65

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC

Download PDF

Raptor Polar X Columns: Revolutionizing the LC Analysis of Polar Compounds

Poor retention, low response, complex sample preparation procedures…the analysis of polar compounds historically has posed challenges to both data quality and lab productivity. Now, Raptor Polar X columns—with their novel phase chemistry that blends HILIC and ion-exchange retention on a single ligand—significantly simplify the analysis of these difficult and varied compounds. And, by bonding that unique ligand to superficially porous particles (SPP), Restek has created a phase that retains and efficiently separates a wide variety of polar compounds. The synergy created by these features results in an unparalleled ability to analyze a wide variety of polar compounds, providing a powerful, productive, and versatile tool for LC scientists across many industries.

References

1. M. Anastassiades, A.-K. Wachtler, D. I. Kolberg, E. Eichhorn, H. Marks, A. Benkenstein, S. Zechmann; D. Mack, C. Wildgrube, A. Barth, I. Sigalov, S. Görlich, D. Dörk, G. Cerchia, Quick method for the analysis of highly polar pesticides in food involving extraction with acidified methanol and LC - or ICMS/MS Measurement - I. Food of Plant Origin (QuPPe-PO-Method)–Version 12 (published on EURL-SRM website on July 23, 2021). https://www.eurl-pesticides.eu/docs/public/tmplt_article.asp?LabID=200&CntID=1115&Theme_ID=1&Pdf=False&Lang=EN