HPLC Column
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This document discusses HPLC columns, including: 1. Silica is commonly used as the surface for HPLC columns, with silanols bonding to the surface. Pore size and surface area impact analyte retention and loading capacity. 2. Column particle sizes have decreased over time from 100 μm to below 2 μm, increasing theoretical plate counts. Column dimensions and particle sizes are selected based on the application. 3. Pore size should be larger than analyte molecules to allow entry without hindrance. Pore sizes of 60-80Å or 95-300Å are recommended for small molecules or proteins, respectively.
![Choose Column Configuration for Application
Column Type I.D.
(mm)
Lengths
(mm)
Particle Size
(µm)
Flow Rate
Ranges
Applications Sensitivity
Increases
Nano 0.1, 0.075 150 3.5 100-600 nL/min Proteomics LC/MS 2000
Capillary 0.3, 0.5 35-250 3.5, 5 1-10µL/min Peptide Mapping LC/MS 100
Micro Bore 1.0 30-150 3.5, 5 30-60µL/min High Sensitivity LC/MS 20
Narrow Bore 2.1 15-150 1.8, 3.5, 5 0.1-0.3 mL/min Sample limited LC/MS 5
Solvent Saver 3.0 150, 250 1.8, 3.5, 5 0.3 – 1.0 mL/min Analytical 2
Analytical 4.6 15-250 1.8, 3.5, 5 1-4 mL/min Analytical 1
Semi-prep 9.4 50-250 5 4-10 mL/min Small Scale Protein
Purification
--
Preparative 21.2 50-250 5, 7 20-60 mL/min CombiChem Purification --
Column dimensions in HPLC: •Analytical [internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm] •Preparative
(i.d. > 4.6 mm; lengths 50 –250 mm) •Capillary (i.d. 0.1 –0.5 mm; various lengths) •Nano (i.d. < 0.1 mm, or sometimes
stated as < 100 μm)
Column Particle Sizes:•7, 5, 3.5 (RR), & 1.8 um (RRHT)
Materials of construction for the tubing •Stainless Steel (the most popular; gives high pressure capabilities)
•Glass(mostly for biomolecules) •PEEK polymer (biocompatible and chemically inert to most solvents)](https://image.slidesharecdn.com/columnhplc-190927174746/85/HPLC-Column-7-320.jpg)
HPLC Column
- 1. HPLC Columns The Basic Chemistry Chandra Prakash Singh Research Scientist
- 2. Silica Column Characteristics Surface Chemistry Silanols Bonding Surface Area Pore Size Distribution Particle Size Distribution HPLC Columns
- 3. Silica Particles for HPLC Columns
- 4. Silica surface showing different types of Silanols. Decreased Acidity
- 5. Pore diameter increases Surface area decreases Phase ratio decreases Retention decreased Retention Increase Phase ratio Increase Surface area Increase Pore diameter decrease The maximum weight of sample that can be injected Proportional to surface area So a greater surface area is usually desirable which suggests the use of the smallest possible pore diameter (and smallest pores). HPLC Columns
- 6. History of HPLC Particle Development Year (s) of Acceptance Particle Size Most Popular Nominal Size Plates / 15 cm 1950 s 100 µm 200 1967 57 µm (pellicular) 1000 1972 10 µm 6000 1985 5 µm 12000 1992 3.5 µm 22000 2003 < 2 µm >30000
- 7. Choose Column Configuration for Application Column Type I.D. (mm) Lengths (mm) Particle Size (µm) Flow Rate Ranges Applications Sensitivity Increases Nano 0.1, 0.075 150 3.5 100-600 nL/min Proteomics LC/MS 2000 Capillary 0.3, 0.5 35-250 3.5, 5 1-10µL/min Peptide Mapping LC/MS 100 Micro Bore 1.0 30-150 3.5, 5 30-60µL/min High Sensitivity LC/MS 20 Narrow Bore 2.1 15-150 1.8, 3.5, 5 0.1-0.3 mL/min Sample limited LC/MS 5 Solvent Saver 3.0 150, 250 1.8, 3.5, 5 0.3 – 1.0 mL/min Analytical 2 Analytical 4.6 15-250 1.8, 3.5, 5 1-4 mL/min Analytical 1 Semi-prep 9.4 50-250 5 4-10 mL/min Small Scale Protein Purification -- Preparative 21.2 50-250 5, 7 20-60 mL/min CombiChem Purification -- Column dimensions in HPLC: •Analytical [internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm] •Preparative (i.d. > 4.6 mm; lengths 50 –250 mm) •Capillary (i.d. 0.1 –0.5 mm; various lengths) •Nano (i.d. < 0.1 mm, or sometimes stated as < 100 μm) Column Particle Sizes:•7, 5, 3.5 (RR), & 1.8 um (RRHT) Materials of construction for the tubing •Stainless Steel (the most popular; gives high pressure capabilities) •Glass(mostly for biomolecules) •PEEK polymer (biocompatible and chemically inert to most solvents)
- 8. However, solute molecules must be able to enter the pores without hindrance, and this requires pores that are larger than the solute molecule. For compounds with molecular weights <500 Da, the average pore diameter should preferably be about 9 nm or larger. The interstitial volume of the column is the space between particles; it is usually about 40% of the total column volume. Larger molecules require larger pores; for example, proteins are usually separated with 30-nm-pore particles. Use 60 -80Åpore size column packings to separate small molecules equal to or less than 4000MW to maximize loading capacity and retention. Use 95 or 300Åpore size columns for larger molecules like polypeptides ad proteins (from 4000 to 500,000 MW) to maintain high efficiency. Increase column diameter to increase loading capacity. HPLC Columns - Pore Size Recommendations
- 9. Potential Ion Exchange and Hydrogen Bonding Secondary Interactions Some mobile phase additives can be added to the mobile phase to reduce these interactions and this will be discussed in the mobile phase section. Hydrogen Bonding -SiOH + RCOO- -Sio-……..H+ ……-OOCR Unprotonated acid can compete for H+ with protonated silanols. This can occur at low pH. Ion-exchange SiO- Na+ + R3NH+ SiO- N+R3 + Na+ Ionized silanols (SiO-) will ion-exchange with protonated bases (R3NH+) which can cause tailing and method variability. This occurs most often at mid pH where silanols are ionized.
- 10. The functional group X is often –Cl or –OEt, and/or –CH3 HPLC Columns Carbon Load Carbon Load refers to the % carbon content of the silica bonded stationary phase. Generally speaking, a high carbon load (example 18-25%) results in a more hydrophobic surface. The surface is also more resistant to high pH. A high carbon load does not necessarily provide the best resolution. Protection of the—Si–O–bond
- 11. Synthesis of various bonded-phase column packings by the reaction of a silane with silica. HPLC Columns (a, d), Monomeric packings; (b, c), potentially polymeric packings.
- 12. Some alternative bonded phases based on different reaction conditions HPLC Columns
- 13. Protection of the—Si–O–bond by a steric-protected bonded phase (for low-pH conditions only) HPLC Columns Options for increasing the stability of Alkylsilica columns Protection of the bonded phase by end-capping.
- 14. Type-C silica - The resulting bonded phase (bidentate C18) HPLC Columns Type-C silica
- 15. Thanks