Inertsil Hybrid-Z HILIC

HILIC vs Reversed Phase

Polar compounds can be retained in HILIC conditions while reversed-phase conditions may provide limited retention.

1. Uracil

2. Pyridine

3. Phenol

Inertsil Hybrid-Z HILIC

Conditions

Column	5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4, A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm

Inertsil Hybrid-C18

Conditions

Column	5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3OH B) H2O, A/B = 30/70, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm

Alkaline Durability of Inertsil Hybrid-Z HILIC (pH 12)

Relative Theoretical Plate Count (%)

Condition

Column	5 µm, 150 x 2.1 mm I.D.
Eluent	2% (0.2 M) Triethylamine in A/B, A) CH3CN B) H2O A/B = 80/20, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C

Retention Time Relative to Initial Value (%)

Condition

Eluent	A) CH3CN B) 10mM HCOONH4 in H2O A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 220 nm
Analyte	Allantoin

Cyanuric Acid / Melamine Example

Hybrid-Z HILIC provides clear retention and separation for highly polar analytes compared with a high-aqueous reversed-phase approach.

Inertsil Hybrid-Z HILIC

Conditions

Column	5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4, A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 220 nm

InertSustain AQ-C18

Conditions

Column	5 µm, 150 x 4.6 mm I.D.
Eluent	A) 20 mM NaH2PO4 B) CH3CN, A/B = 98:2
Flow Rate	1.0 mL/min
Col. Temp.	40 °C
Detection	UV 215 nm

Comparison of Zwitterionic HILIC Columns for Nucleobases and Nucleosides

Inertsil Hybrid-Z HILIC, 5 µm

 

Brand A, 5 µm

 

Brand S, 5 µm

 

Brand P, 1.7 µm

 

Conditions

Column	150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Analytes	1. Thymine 2. Uracil 3. Adenosine 4. Uridine 5. Cytosine 6. Cytidine 7. Guanosine

Application Examples

20 application examples

Antiviral Drugs (Guanosine Analogs)

+

Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	0.5 µL
Analytes	1. Aciclovir 2. Penciclovir 3. Ganciclovir (100 mg/L, each)

Nucleobases, Nucleosides, and Related Compounds

+

Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 20 mM NH4HCO3 in H2O A/B = 90/10-15min-50/50-15.1min-90/10 (v/v)
Flow Rate	0.2 mL/min
Col. Temp.	60 °C
Detection	MS/MS (ESI, Positive, MRM)
Inj. Vol.	1 µL

Analytes	Q1/Q3
1. Caffeine	195.1/138.1
2. Adenine	136.1/119.1
3. Cytosine	112.1/95.1
4. Inosine	269.1/137.1
5. Guanine	152.1/135.1
(10 µmol/L, each)

Nucleic Acid Synthesis Inhibitors

+

Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	0.5 µL
Analytes	1. 5-Fluorouracil 2. 5-Fluorocytosine (100 mg/L, each)

Sugar Alcohols

+

Conditions

Column	Inertsil Hybrid-Z HILIC 5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	RI
Inj. Vol.	4 µL
Analytes	1. Erythritol 2. Xylitol 3. Sorbitol 4. Mannitol (5000 mg/L, each)

Sugars (Monosaccharides and Disaccharides)

+

Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D.
Eluent	A) 50 mM NH3 in (CH3CN/H2O = 95/5) B) 50 mM NH3 in H2O A/B = 90/10 (v/v)
Flow Rate	0.3 mL/min
Col. Temp.	60 °C
Detection	MS/MS (ESI, Negative, MRM)
Inj. Vol.	1 µL

Analytes
1. Fructose	179.1/89.1	250 mg/L
2. Glucose	179.1/89.1	250 mg/L
3. Sucrose	341.0/179.1	2.5 mg/L
4. Maltose	341.0/179.1	250 mg/L
5. Lactose	341.0/179.1	250 mg/L

Urea and Allantoin

+

Conditions

Column	Inertsil Hybrid-Z HILIC 5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10, v/v
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	UV 220 nm
Inj. Vol.	1 µL
Analytes	1. Urea 1000 mg/L 2. Allantoin 500 mg/L

Water-Soluble Vitamins and Related Compounds

+

Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 90/10-10min-40/60-0.1min-90/10, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 265 nm
Inj. Vol.	1 µL
Analytes	1. Nicotinamide 2. Riboflavin 3. Pyridoxamine 4. Nicotinic acid 5. Cyanocobalamin 6. Erythorbic acid 7. Ascorbic acid (50 mg/L, each)

Thiamine and Thiamine Pyrophosphate

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Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 20 mM NH4HCO3 in H2O A/B = 80/20-15 min-40/60-0.1 min-80/20
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	0.5 µL
Analytes	1. Thiamine 2. Thiamine Pyrophosphate (100 mg/L, each)

Amino Acids

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Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) 0.1% HCOOH in CH3CN B) 50 mM HCOONH4 in H2O A/B = 90/10 - 15 min-50/50 - 3 min - 20/80, 3 min hold - 0.1 min - 90/10, (v/v)
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Positive, MRM)
Inj. Vol.	2 µL

Analytes	Q1/Q3
1. Phenylalanine	166.1/120.1
2. Leucine	132.1/86.1
3. Isoleucine	132.1/86.1
4. Tryptophan	205.1/188.1
5. Methionine	150.1/104.1
6. Valine	118.1/72.1
7. GABA	104.1/87.1
8. Tyrosine	182.1/136.1
9. Proline	116.1/70.1
10. Alanine	90.1/44.1
11. Threonine	120.1/74.1
12. Glutamine	147.1/84.1
13. Asparagine	133.1/74.1
14. Serine	106.1/60.1
15. Aspartic acid	134.1/74.1
16. Histidine	156.1/110.1
17. Lysine	147.1/84.1
18. Ornithine	133.1/70.1
(10 µmol/L)

Imidazole Dipeptides

+

Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D.
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 80/20 (v/v)
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	UV 220 nm
Inj. Vol.	1 µL
Analytes	1. Carnosine 2. Anserine (100 mg/L, each)

Dipeptides

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) 0.1% HCOOH in CH3CN B) 20 mM HCOONH4 in H2O A/B = 80/20-10 min-50/50-0.1 min-80/20
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Positive, MRM)
Inj. Vol.	1 µL

Analytes	Q1/Q3
1. Ala-Phe	237/166
2. Ala-Val	189/118
3. Gly-Phe	223/120
4. Ala-Tyr	253/182
5. Ala-Ala	161/44
6. Ala-Gly	147/44
7. Ala-Gln	218/147
8. Gly-Lys	204/84
9. Ala-His	227/110
10. Gly-His	213/110
(1 µg/mL, each)

Glutathione (GSH and GSSG)

+

Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 100 mM HCOONH4 A/B = 70/30-10 min-50/50-0.1 min-70/30
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	UV 245 nm
Inj. Vol.	1 µL
Analytes	1. Glutathione reduced 2. Glutathione oxidized (100 mg/L, each)

Cyclic Nucleotides and cGAMP

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 10 mM HCOONH4 in H2O A/B = 85/15-10 min-50/50-0.1 min-85/15
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	1 µL
Analytes	1. cAMP 2. cGMP 3. 2’3’-cGAMP (100 mg/L, each)

NAD, NADH, NADP, and NADPH

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Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 20 mM NH4HCO3 in H2O A/B = 80/20-10 min-50/50-0.1 min-80/20
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	0.5 µL
Analytes	1. NAD+ 2. NADH 3. NADP 4. NADPH (100 mg/L, each)

Nucleotide Monophosphates

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Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 50 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 100 mM HCOONH4 in H2O A/B = 70/30, v/v
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 254 nm
Inj. Vol.	0.5 µL
Analytes	1. AMP 2. UMP 3. CMP 4. GMP (100 mg/L, each)

Sugar Phosphates

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Conditions

Column	Inertsil Hybrid-Z HILIC 1.7 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 20 mM NH4HCO3 (pH 10 NH3 adjusted) in H2O A/B = 85/15-10 min-40/60-2 min-hold-0.1 min-85/15
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Negative, MRM)
Inj. Vol.	5 µL

Analytes	Q1/Q3
1. Glucose-1-phosphate (G1P)	259.0/79.0
2. Glucose-6-phosphate (G6P)	259.0/97.0
(1 µg/mL)

Oligonucleotides

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) CH3CN B) 200 mM HCOONH4 in H2O A/B = 70/30-30 min-45/55-0.1 min-70/30 (v/v)
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	UV 260 nm
Inj. Vol.	1 µL

Analytes
1. 5’-TATGACGTTCCTGATGCT-3’	18 mer
2. 5’-CATGACGTTCCTGATGCT-3’	18 mer
3. 5’-TCTATGACGTTCCTGATGCT-3’	20 mer
4. 5’-GCTATGACGTTCCTGATGCT-3’	20 mer
(20 nmol/mL, each)

Glufosinate and Glyphosate

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) 50 mM NH3 in (CH3CN/H2O=95/5, v/v) B) 50 mM NH3 + 10 mM HCOONH4 in H2O A/B = 85/15-10 min-60/40, 2 min hold, -0.1 min-85/15
Flow Rate	0.2 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Negative, MRM)
Inj. Vol.	10 µL

Analytes	Q1/Q3
1. Glufosinate	180.0/136.0
2. Glyphosate	168.0/63.0
(1 µg/mL, each)

Quaternary Ammonium Pesticides

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) 0.1% HCOOH in CH3CN B) 100 mM HCOONH4 in H2O A/B = 85/15-2 min, hold-8 min-50/50-0.1 min-85/15
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Positive, MRM)
Inj. Vol.	1 µL

Analytes	Q1/Q3
1. Chlormequat	122.0/58.2
2. Mepiquat	114.0/98.2
3. Paraquat	186.0/171.1
4. Diquat	183.1/157.1
(1 ng/mL, each)

Aminoglycosides – Spectinomycin and Streptomycin

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Conditions

Column	Inertsil Hybrid-Z HILIC 3.5 µm, 150 x 2.1 mm I.D. (PEEK Hardware)
Eluent	A) 0.1% HCOOH in CH3CN B) 20 mM HCOONH4 in H2O A/B = 80/20-6 min-20/80-5min, hold-0.1 min-80/20
Flow Rate	0.3 mL/min
Col. Temp.	40 °C
Detection	MS/MS (ESI, Positive, MRM)
Inj. Vol.	1 µL

Analytes	Q1/Q3
1. Spectinomycin	333.2/189.1
2. Streptomycin	582.3/246.1
(1 µg/mL, each)

No application example lists this compound.