Selecting the Optimal Flash Column: A Guide to Injection Volume in Purification
Flash chromatography is an essential technique widely used in organic synthesis and pharmaceutical chemistry for purifying valuable compounds, especially in early stages of compound screening. When a promising active compound is identified, isolating and purifying it becomes crucial, and the use of flash columns is one of the most common methods for this process.
Flash columns come in various sizes, with packing material quantities ranging from as small as 4g to as large as 330g. Choosing the right column size primarily depends on the injection volume, which is one of the most critical parameters during purification.
Understanding Injection Volume in Purification
Injection volume is typically the maximum amount of sample that can be applied to a column while maintaining efficiency, purity, and yield. It can be divided into two categories: the theoretical maximum injection volume and the effective maximum injection volume.
- Theoretical Maximum Injection Volume: This refers to the maximum amount a column can handle without considering factors like separation efficiency. It is based solely on the capacity of the packing material. Different types of chromatographic media, such as bare silica gel and C18 silica, have different bonding phases and separation mechanisms, leading to variations in their sample-handling abilities. For example, bare silica gel has a theoretical maximum injection volume of 1/10 of the gel mass, while for C18 silica, it’s 1/50 of the media mass.
- Effective Maximum Injection Volume: In practice, separation factors such as resolution and solubility influence the actual amount of sample that can be applied. Therefore, the effective maximum volume is more relevant in real-world applications, and is commonly referred to when optimizing the purification process.
Factors Affecting Injection Volume
The two main factors determining the injection volume are resolution and solubility, which directly influence whether overloading can be applied, and if so, how it should be approached.
- Resolution: If the target compound and impurities are well-separated, you can increase the injection volume. However, as the load increases, resolution may decrease, eventually compromising the yield and purity. Therefore, resolution dictates whether overloading is feasible.
- Solubility: This determines the method of overloading. If the sample has good solubility, concentration overloading is preferred. Here, the injection volume remains constant, but the sample concentration increases, resulting in sharper peaks and better separation. If the sample has poor solubility, volume overloading is used, where the injection volume is increased. However, this method carries a risk of sample precipitation, as the peaks tend to broaden and flatten.
Choosing the Right Flash Column
If you’re unsure how to select the appropriate flash column size or determine the injection volume for your own flash column, guidelines from manufacturers like Welch’s WelFlash can serve as a helpful reference. These recommendations list flash column sizes and their corresponding sample capacities, offering a practical starting point for efficient purification.
Flash chromatography continues to be a reliable and versatile tool in laboratories, and by understanding key factors like injection volume, resolution, and solubility, researchers can optimize their purification processes for higher efficiency and better results.
Recommended injection volume in the WelFlash manual
Specification |
4g |
2g |
25g |
40g |
80g |
120g |
220g |
330g |
Injection volume 1 (g) |
0.01-0.02 |
0.03-0.06 |
0.05-0.1 |
0.1-0.2 |
0.2-0.4 |
0.3-0.6 |
0.5-1.0 |
0.75-1.5 |
Injection volume 2 (g) |
0.02-0.08 |
0.06-0.24 |
0.1-0.4 |
0.2-0.8 |
0.4-1.6 |
0.6-2.4 |
1.0-4.0 |
1.5-6.0 |
Injection volume 3 (g) |
0.08-0.4 |
0.24-1.2 |
0.4-2.0 |
0.8-4.0 |
1.6-8.0 |
2.4-12.0 |
4.0-22.0 |
6.0-33.0 |
Column volume |
8mL |
24mL |
40mL |
80mL |
160mL |
240mL |
400mL |
600mL |
Min. flow rate |
5mL/min |
8mL/min |
10mL/min |
20mL/min |
25mL/min |
35mL/min |
45mL/min |
50mL/min |
Max.flow rate |
l8mL/min |
20mL/min |
25mL/min |
40mL/min |
50mL/min |
80mL/min |
90mL/min |
100mL/min |
Pressure |
Max. Pressure: 200psi |
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Note:
injection volume formula: ΔCV=l/Rf1-1/Rf2;
Matrix：silica gel 40-60μm
Injection volume 1: injection volume ΔCV = 1
Injection volume 2: injection volume ΔCV = 2
Injection volume 3: injection volume ΔCV = 6
Appendix: WelFlash Low to Medium Pressure Preparative Liquid Chromatography Columns
Spherical 20-40μm silica gel
P/N |
Specification |
00001-03011 |
WelFlash® SiO2 -1, 4g, 20 pcs/pk |
00001-03021 |
WelFlash® SiO2 -1, 12g, 18 pcs/pk |
00001-03031 |
WelFlash® SiO2 -1, 25g, 12 pcs/pk |
00001-03042 |
WelFlash® SiO2 -1, 40g, 10 pcs/pk |
00001-03053 |
WelFlash® SiO2 -1, 80g, 10 pcs/pk |
00001-03063 |
WelFlash® SiO2 -1, 120g, 10 pcs/pk |
00001-03075 |
WelFlash® SiO2 -1, 220g, 6 pcs/pk |
00001-03084 |
WelFlash® SiO2 -1, 330g, 4 pcs/pk |
Spherical 20-40μm C18
P/N |
Specification |
00002-03011 |
WelFlash® C18-1, 4g, 20 pcs/pk |
00002-03021 |
WelFlash® C18-1, 12g, 18 pcs/pk |
00002-03031 |
WelFlash® C18-1, 25g, 12 pcs/pk |
00002-03042 |
WelFlash® C18-1, 40g, 10 pcs/pk |
00002-03053 |
WelFlash® C18-1, 80g, 10 pcs/pk |
00002-03063 |
WelFlash® C18-1, 120g, 10 pcs/pk |
00002-03075 |
WelFlash® C18-1, 220g, 6 pcs/pk |
00002-03084 |
WelFlash® C18-1, 330g, 4 pcs/pk |
Spherical 20-40μm Phenyl
P/N |
Specification |
00003-03011 |
WelFlash® Phenyl, 4g, 20 pcs/pk |
00003-03021 |
WelFlash® Phenyl, 12g, 18 pcs/pk |
00003-03031 |
WelFlash® Phenyl, 25g, 12 pcs/pk |
00003-03042 |
WelFlash® Phenyl, 40g, 10 pcs/pk |
00003-03053 |
WelFlash® Phenyl, 80g, 10 pcs/pk |
00003-03063 |
WelFlash® Phenyl, 120g, 10 pcs/pk |
00003-03075 |
WelFlash® Phenyl, 220g, 6 pcs/pk |
00003-03084 |
WelFlash® Phenyl, 330g, 4 pcs/pk |
Spherical 20-40μm NH2
P/N |
Specification |
00005-03011 |
WelFlash® NH2 , 4g, 20 pcs/pk |
00005-03021 |
WelFlash® NH2 , 12g, 18 pcs/pk |
00005-03031 |
WelFlash® NH2 , 25g, 12 pcs/pk |
00005-03042 |
WelFlash® NH2 , 40g, 10 pcs/pk |
00005-03053 |
WelFlash® NH2 , 80g, 10 pcs/pk |
00005-03063 |
WelFlash® NH2 , 120g, 10 pcs/pk |
00005-03075 |
WelFlash® NH2 , 220g, 6 pcs/pk |
00005-03084 |
WelFlash® NH2 , 330g, 4 pcs/pk |
Spherical 40-70μm silica gel
P/N |
Specification |
00001-04011 |
WelFlash® SiO2 -1, 4g, 20 pcs/pk |
00001-04021 |
WelFlash® SiO2 -1, 12g, 18 pcs/pk |
00001-04031 |
WelFlash® SiO2 -1, 25g, 12 pcs/pk |
00001-04042 |
WelFlash® SiO2 -l, 40g, 10 pcs/pk |
00001-04053 |
WelFlash® SiO2 -1, 80g, 10 pcs/pk |
00001-04063 |
WelFlash® SiO2 -1, 120g, 10 pcs/pk |
00001-04075 |
WelFlash® SiO2 -1, 220g, 6 pcs/pk |
00001-04084 |
WelFlash® SiO2 -1, 330g, 4 pcs/pk |
Spherical 40-70μm C18
P/N |
Specification |
00002-04011 |
WelFlash® C18-1, 4g, 20 pcs/pk |
00002-04021 |
WelFlash® C18-1, 12g, 18 pcs/pk |
00002-04031 |
WelFlash® C18-1, 25g, 12 pcs/pk |
00002-04042 |
WelFlash® C18-1, 40g, 10 pcs/pk |
00002-04053 |
WelFlash® C18-1, 80g, 10 pcs/pk |
00002-04063 |
WelFlash® C18-1, 120g, 10 pcs/pk |
00002-04075 |
WelFlash® C18-1, 220g, 6 pcs/pk |
00002-04084 |
WelFlash® C18-1, 330g, 4 pcs/pk |
Spherical 20-40μm CN
P/N |
Specification |
00004-03011 |
WelFlash® CN, 4g, 20 pcs/pk |
00004-03021 |
WelFlash® CN, 12g, 18 pcs/pk |
00004-03031 |
WelFlash® CN, 25g, 12 pcs/pk |
00004-03042 |
WelFlash® CN, 40g, 10 pcs/pk |
00004-03053 |
WelFlash® CN, 80g, 10 pcs/pk |
00004-03063 |
WelFlash® CN, 120g, 10 pcs/pk |
00004-03075 |
WelFlash® CN, 220g, 6 pcs/pk |
00004-03084 |
WelFlash® CN, 330g, 4 pcs/pk |
Spherical 20-40μm SAX
P/N |
Specification |
00007-03011 |
WelFlash® SAX, 4g, 20 pcs/pk |
00007-03021 |
WelFlash® SAX, 12g, 18 pcs/pk |
00007-03031 |
WelFlash® SAX, 25g, 12 pcs/pk |
00007-03042 |
WelFlash® SAX, 40g, 10 pcs/pk |
00007-03053 |
WelFlash® SAX, 80g, 10 pcs/pk |
00007-03063 |
WelFlash® SAX, 120g, 10 pcs/pk |
00007-03075 |
WelFlash® SAX, 220g, 6± pcs/pk |
00007-03084 |
WelFlash® SAX, 330g, 4 pcs/pk |