Agilent DE04095141 Infinity Lab Poroshell 120 Instructions

Overview

How do I choose?

• Efficiency
• Particle size
• Column length
• Selectivity
• Bonded phase
• Mobile phase
• Retention
• Polar bonded phase
• HILIC

Resolution Equation

Improve resolution by improving any of these parameters:

• Efficiency describes the separation power of the column.
• Selectivity has the highest influence on the resolution. Small changes in selectivity can lead to big changes in resolution.
• Retention has only a significant influence at small k values.

Selectivity

Selectivity impacts the resolution most Selectivity impacts resolution

• Stationary and mobile phase
• Temperature
•  is strongly influenced by alpha

Efficiency and Retention Factor

Parameters influencing column efficiency:

• Column length (increasing column length increases efficiency)
• Particle size (decreasing particle size increases efficiency)

???? = retention time for sample peak
??0 = retention time for unretained peak
The retention factor measures the period of time that the sample component resides in the stationary phase relative to the time it resides in the mobile phase. It is calculated from the  retention time divided by the time for an unretained peak.

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Agilent’s Small Molecule LC Columns

When to choose which product family

Scalability

Method Transferability Across Product Families
Traditional ZORBAX chemistries are aligned with InfinityLab Poroshell chemistries to offer simplified method transfer from fully porous particles to superficially porous particle columns.

Making a Poroshell Particle

Strict monitoring of every production step ensures column performance and quality

Step 1: Make the solid core
Poroshell 120 column cores have a very smooth surface and a uniform particle size which contributes to a tight overall particle size distribution. As a result, you get a more tightly packed column bed and therefore a better lifetime. Step 2: Apply the porous shell
In contrast to other manufacturers, Agilent applies the porous shell in one single step. This unique single-step process delivers better column-to-column reproducibility.
Step 3: Apply the bonded phase
Most of the Poroshell chemistries are bonded in a single step. This further increases batch-to-batch reproducibility and method scalability from 1.9 to 2.7 to 4 µm.

Poroshell Particles

Designed along with your LC instruments for highest performance

SPP particle| For| Maximum pressure| Typical pressure| Efficiency| Target system
---|---|---|---|---|---
1.9 µm| Highest UHPLC
performance| 1300 bar| Similar to sub-2 µm totally porous| ~120% of sub-2 µm totally porous| 1290 Infinity II
2.7 µm| UHPLC performance at lower pressures| 600 bar /1000 bar| 50% of sub-2 µm totally porous| ~90% of sub-2 µm  totally porous| 1290 Infinity II
1260 Infinity II
4 µm| Improved HPLC
performance| 600 bar| Typically < 200 bar| ~200% of 5 µm totally porous| 1260 Infinity II VL
1220 Infinity II (VL)

Poroshell 120 portfolio: Chemistries with Unique Selectivity

InfinityLab Poroshell 120 offers a broad portfolio to suit your needs * – can be operated at 100% aqueous mobile phase conditions

Chemistries with Unique Selectivity

The influence of stationary phase on selectivity and resolution

Compounds:
1. APAP, 2. Phenacetin, 3. Piroxicam, 4. Tolmetin, 5. Ketoprofen, 6. Naproxen, 7. Sulindac, 8. Diclofenac, 9. Diflunisal

Stationary Phase Selection Guide

Starting Recommendation
Poroshell 120 EC-C18

Change Selectivity Slightly| Change Selectivity  Significantly| For Many Early Eluters
---|---|---
1. Poroshell PhenylHexyl
2. Polaris C18-A
3. Poroshell EC-C8| 1. Poroshell BonusRP
2. Poroshell PFP
3. Pursuit XRs Diphenyl| 1. Poroshell SB-Aq
2. Poroshell PFP
3. Poroshell HILIC-Z
No retention at 98+% Aqueous in RP| Acidic Solvents (pH < 2)| Basic Solvents (pH >6)
1. Poroshell HILIC-Z
2. Poroshell PFP
3. Poroshell HILICOH5| 1. Poroshell SB-C18
2. Poroshell SB-Aq
3. PLRP-S
4. Poroshell SB-C8| 1. Poroshell HPHC18
2. PLRP-S
3. Poroshell CS-C18

Top 3 to keep around (covers 95% of analyses)

Poroshell EC-C18
Poroshell HILIC-Z
Poroshell PFP

USP L1

1. Poroshell 120 EC-C18
2. Polaris C18-A
3. Polaris C18-Ether
4. Pursuit XRs C18

USP L3

1. Poroshell 120 HILIC
2. ZORBAX Rx-Sil
3. Pursuit XRs Si

USP L7

1. Poroshell 120 EC-C8
2. Polaris C8-A

USP L8

1. Polaris NH2
2. ZORBAX NH2

USP L11

1. Poroshell Phe-Hex
2. Pursuit XRs Diphenyl

Recommended Solvent A (Weak)

1. 0.1% Formic Acid (pH ~2.7)
2. 10 mmol Ammonium Acetate (adj. pH 5)
3. 0.1% Ammonium Hydroxide (pH ~10)
4. 0.1% Trifluoroacetic acid (pH ~1.5, no MS)
5. 150 mmol Sodium Phosphate (adj. pH 3, no MS)

Solvent B (Strong)

1. Acetonitrile
2. Methanol
3. Isopropanol
4. THF
5. Acetone

Sugars (RI or ELSD)

1. Poroshell HILIC-Z
2. Hi-Plex H
3. Hi-Plex Ca
4. Polaris NH2

Normal Phase

1. Poroshell HILIC
2. Poroshell EC-CN
3. Polaris NH2

Chiral

1. Poroshell Chiral-V
2. Poroshell Chiral-T
3. Poroshell Chiral-CD
4. Poroshell Chiral-CF

HPLC Chemistry Selection: Poroshell 120

1. C8 phases have nearly identical selectivity compared to C18.
   Suitable for faster analysis times when sufficient resolution and retention is achieved.

2. Bare silica column. Shipped in RP solvents. For NP use, please properly flush out shipping solvent before.
   RP = reversed phase
   NP = normal phase

Particle Size and Dimension: P120

1. ULD kit recommended (p/n 5067-5963)
2. not possible with regulated gradient methods, not recommended lab technicians that lack experience with UHPLC
3. Delay and dispersion volume. E.g., 0.17 mm ID tubing or bigger + 10 mm classic flow cell, valves, long tubing connections, old mixer design

400 bar = 6000 psi
600 bar = 9000 psi
1200 bar =17000 psi
1300 bar = 19000 psi

Limitations
V – System volume (dispersion/delay)
P – Pressure limits
V+P – System volume and pressure
H/I – if instrument is used for HPLC methods / ISET emulation
Recommended
Acceptable
Limited Configurations
Not Recommended

Scalability

Scaling Water-Soluble Vitamins on InfinityLab Poroshell 120 SB-Aq

An example of scalability between particle sizes

Increase Throughput with Ultrafast Separations

• ZORBAX Eclipse Plus C18, 5 µm
• 36 min runtime

• Poroshell 120 EC-C18, 1.9 µm

• 3 min runtime (12 x faster)

• 96% less solvents used

• 95% less sample injected

• Poroshell 120 EC-C18, 1.9 µm

• 0.3 min runtime (120 x faster)

• 98% less solvent

Modern columns help to increase the number of samples measured per day

High throughput UHPLC at 1150 bar and 60 °C

Agilent InfinityLab Poroshell 120 EC-C18, 2.1 x 50 mm. 1.9 pm column 2.3 mL/min, 50-100 %B in 0.3 minutes, 60 °C, 254 nm
Minimum resolution = 2.2
Compared to 250 mm. 5 jm analysis:

• 120x faster
• 98 % less mobile phase used

A pH Change Can Strongly Affect Selectivity

Mobile phase pH is a powerful method development tool for separating ionizable compounds

• In RPLC mode, ionizable analytes are more retained in their neutral state
• Acids are more retained at low pH
• Bases are more retained at high pH
• Neutrals are not impacted by mobile phase pH

Selectivity Can be Controlled by Changing pH

Agilent InfinityLab Poroshell HPH-C18 4.6 x 50 mm, 2.7 µm

1. Procainamide
2. Caffeine
3. Acetyl Salicylic Acid
4. Hexanophenone Deg.
5. Dipyrimadole
6. Diltiazem
7. Diflunisal
8. Hexanophenone

A pH Change Can Strongly Affect Selectivity

CS-C18 is another high-pH compatible L1 stationary phases Read more: Application Note 5994-2274EN

5-95% CH3CN in 10 min, 4 min post run, mobile phase A varies, 0.4 mL/min, 2.1 x 100 mm, 2.7 µm Agilent InfinityLab Poroshell 120 CS-C18, 30 o C, DAD: 254 nm, 80 Hz; Sample: uracil, amitriptyline, butyl paraben, dipropyl phthalate, acenaphthene

Choosing between C18s

• – can be operated at 100% aqueous mobile phase conditions

Applications with Poroshell 120 Aq-C18 Columns

• Organic acids
• Water-soluble vitamins
• Characteristic chromatogram on TCM formula granules

Screening C18 columns within Poroshell 120 family under 0.1% H3PO4

Aq-C18 performs better than conventional C18 for polar organic acids MP: 97.5% 0.1%H3PO4/2.5% methanol; 1 ml/min; 210 nm; 2 uL; 25°C

Comparison with existing products under phosphate buffer

Aq-C18 performs better than conventional C18 for polar organic acids MP: 99% 50mM NaH2PO4/1% methanol; 1 ml/min; 210 nm; 1 uL; 25°C

Water soluble vitamins separation with Aq-C18
A gradient start with 100% aqueous phosphate buffer

1. Thiamine
2. Ascorbic acid
3. Nicotinic acid
4. Nicotinamide
5. Pyridoxine
6. Pantothenic acid
7. Aminobenzoic acid
8. Folic acid
9. Caffeine
10. Cyanocobalamin
11. Riboflavin
12. Biotin

• 1290 Infinity II Binary pump;
• MP: A, 10mM NaH2PO4 buffer with pH 2.5; B, ACN
• 0-2min, 0-6% B; 2-6 min, 625%B; 6-8min 25-70%B, 8-10 min 70%B, stop time 10min; post run: 2 min
• Flow rate: 1.5 ml/min;
• 30C
• 1 uL
• 260nm/210 nm;

Excellent Reproducibility from consecutive 6 injections

Reproducibility on Poroshell 120 Aq-C18, 2.1×50 mm

HPLC Conditions:
Column: 2.1 x 50 mm,
Poroshell 120 Aq-C18;
Mobile phase: 50mM sodium
acetate in HPLC-grade
deionized water at pH 4.6
Flow rate: 0.3 ml/min;
Detector: 254 nm;
Sample: (1) nicotinic acid, (2)
pyridoxine, (3) niacinamide,
and (4) thiamine. 0.25 mg/ml in water.
Injection volume: 0.1 uL Excellent reproducibility and almost no retention loss under 100% aqueous mobile phase.

Good stability under 100% aqueous phase

Less retention loss with Aq-C18 than conventional C18.

1. Thiourea
2. 5-Fluorocytosine
3. Adenine
4. Nicotinamide
5. Resorcinol
6. Guanosine

What is HILIC and When Should I Consider it?
HILIC Complements RPLC

HILIC Method Development

InfinityLab Poroshell 120 HILIC column options

Starting mobile phases
*Mobile Phase A (Strong phase, H2O): Note:** Phosphates have low solubility in high % ACN (1-30 mM). Always test solubility before running. Never run in

80% ACN to avoid precipitation.

Mobile Phase B (Weak phase, CH3CN):

• Buffer concentration should match Mobile Phase A for improved reproducibility
• Adding 10% water in ACN generally recommended for improved solubility and faster re-equilibration
• Pure MeOH is too strong a solvent for most HILIC separations. Mixed with ACN in small quantities (<15%), it can be used to change selectivity slightly.

Example of mobile phase preparation:
Stock: 200 mM ammonium formate adjusted to pH 3 with formic acid
A: 900 mL water + 100 mL stock
B: 900 mL acetonitrile + 100 mL stock

Effect of pH on Retention of Acidic Compounds with HILIC

Starting Mobile Phases

In HILIC mode, ionizable compounds are better retained when they are ionized

• Acids at high pH
• Bases at low pH

Once the analyte is fully ionized, retention should stabilize

• Note: if other retention mechanisms are occurring, this may not be true

Biotin pKa = 4.5
Nicotinic acid pKa = 4.8
Pantothenic acid pKa = 4.3
Mobile Phase A: H2O, B: CH3CN, D: varies, 200 mM Ammonium Formate or Acetate; Flow Rate: 0.5 mL/min; Gradient: 95% B for 1 min, 95–65% B in 9 min, hold 5% D constant throughout analysis, 5 min post run; Injection: 0.5 µL of 13.3 µg/mL each in CH3CN/H2O 19:1; Column: 25 , 2.1 x 100 mm, 2.7 µm
Agilent InfinityLab Poroshell 120 HILIC-Z; Detection: Ultivo TQ/MS ESI+ dMRM °C

Analysis of Amino Acids (and Isobars) in Plant Tissue with LC-MS/MS

1. Aspartic acid
2. Phenylalanine
3. Leucine
4. Isoleucine
5. Methionine
6. Valine
7. Proline
8. Tyrosine
9. Cysteine
10. Alanine| 11. Homoserine
12. Threonine
13. Glycine
14. Glutamine
15. Aspargine
16. Glutamic acid
17. Citrulline
18. Histidine
19. Lysine
20. Ornithine
---|---

HILIC for Classes of Sticky Analytes
Interact with stainless steel in the system

Phosphorylated Metabolites
(higher degree of phosphorylation = worse sticking) Organophosphates

Chelating Organic Acids

Improvements in Signal and Peak Shape

Repeatability after Wash Step

HILIC/MS Sensitivity Can Be Improved with a PEEK Lined Column and Inert LC
Nucleotide Phosphates on a PEEK Lined Agilent InfinityLab Poroshell 120 HILIC-Z Column used was 2.1 x 100 mm, 2.7 μm Agilent InfinityLab Poroshell 120 HILIC-Z (PEEK lined stainless steel); A: 10 mM Ammonium Formate pH 6.8 in water, B: acetonitrile + 10 mM Ammonium Formate pH 6.8, 95-30% B in 10 minutes, 0.25 mL/min, 0.2 μL injection (5 ng each on column), MS Source: ESI-, m/z 191.02, 346.06, 426.02, 505.99, 521.99, 742.067, 743.067, 259.03

Separation of water-soluble vitamins

Separation of water soluble vitamins on competitor C18 2.1 x 100mm 1.7µm column

Separation of Sugars

Separation of Organic Acids

1. Oxalic acid
2. cis-Aconitic acid
3. Tartaric acid
4. Malic acid
5. Lactic acid
6. Formic acid
7. Fumaric acid
8. Propionic acid
9. Butyric acid

Columns Selection Quick Reference

Agilent offers a wide range of analyticalscale columns from up to 10 mm ID

Agilent InfinityLab Preparative LC
Columns are optimized to meet customer needs and available in the most common prep dimensions
Agilent offers a selection of 50 mm ID prepacked and load and lock columns for those who require larger yield

Agilent’s InfinityLab Preparative LC Columns Portfolio

Agilent InfinityLab Poroshell 120 preparative LC columns

Made with 4 µm Poroshell particles

• Symmetrical particles with smooth surfaces (↓ A term)
• Narrow particle size distribution (↓ A term)
• Short analyte diffusion path in/out of the porous layer (↓ C term)

What makes Poroshell Prep Columns so Special for High Throughput?

• Higher performance at high flow rates offers shorter run times
• 4 µm particles mitigate concerns of excessive pressure and a large system volume that could negate chromatographic benefits. 4 µm is large enough to operate well within pressure range of traditional prep instruments without significant band broadening while still performing better than TPP prep columns| Deliver purified samples faster and
increase throughput
Available phases from A to P| Seamless Scalability
• Fast analytical screening with seamless scalability to prep
Optimized Proprietary
Packing Process| Robust and Reliable
• Excellent bed stability with tried and true hardware
• Delivering robust and reproducible performance even when tested to 1000 injections
4 µm superficially porousparticle Columns| Excellent Loadability for Drug Discovery Needs
• 4 µm SPP particle column efficiencies drive higher resolution and sharper peaks than traditional prep columns of comparable surface areas

Full Compatibility

Designed to seamlessly integrate into the InfinityLab family
Agilent InfinityLab products are designed to provide you the best efficiency in your liquid chromatography workflow regardless of application area. When relying on Agilent InfinityLab instruments, columns, and supplies be assured that every part works together seamlessly.

Agilent Resources for Support

• Resource page www.agilent.com/chem/agilentresources
  – Quick reference guides, product catalogs
  – Online selection tools, “How-to” videos
  – Column user guides – www.agilent.com/en- us/support/liquidchromatography/kb005965
  – Biocolumn user guides – www.agilent.com/en/support/liquidchromatography/kb005960

• Tech support: www.agilent.com/chem/techsupport

• InfinityLab LC Supplies catalog (5991-8031EN)

• Agilent University www.agilent.com/crosslab/university

• YouTube – Agilent Channel

• Subscribe to Agilent Peak Tales podcasts at peaktales.libsyn.com

15 November 2023
How Do I Choose?
DE04095141

Documents / Resources

| Agilent DE04095141 Infinity Lab Poroshell 120 [pdf] Instructions
DE04095141 Infinity Lab Poroshell 120, DE04095141, Infinity Lab Poroshell 120, Lab Poroshell 120, Poroshell 120
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References

• Agilent Collection of Columns, Supplies, and Standards Resources - Wiki - Consumables - Agilent Community
• Technical Support | Agilent
• Agilent University | Agilent

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