GE公司培训讲义—凝胶过滤层析技术

-pores filled with eluent 孔内充满洗脱液

-carefully controlled size range 孔径精密控制

-chemically /physically inert (lack of adsorptive properties)

1. Spherical particles packed into a column.

2. Sample applied.

3. Buffer (mobile phase) and sample

move through column.Molecules

diffuse in and out of matrix

4. Large molecules leave the column

first followed by smaller molecules in

Molecules larger than the

pass straight through.

V = void volume V = retention volume or elution volume V = total liquid volume of the bed V = inner pore volume = V V = total geometric volume of the column V = volume of gel matrix V o

V t

V c

V R1V R2 V R3 V R4

V o

V R

volume

c o n c e n t r a t i o n

非常大的分子在外水体积洗出, V o

volume

c o n c e n t r a t i o n

V t

V c

V o

V R

V = V 0+V 柱床的几何体积K d

V t

V R

volume

c o n c e n t r a t i o n

elution volume when K = 1

elution with K Adsorption has occurred.

elution with K Serious problem!

流动相中需要加入150mM NaCl av K av versus av 1

log (M r )

Exclusion limit

All molecules bigger than this elute in the void volume.均在外水体积洗出

10

log (M r )

0.7

0.1fractionation range 填料分级范围

av av Rs Rs Rs V 2 -V 1(W 1 + W 2) / 2

=

两峰相对于其峰宽而言，分开的有多远

high efficiency low efficiency

高柱效需要良好的柱填装技术高柱效有时可弥补选择性不足

lower selectivity

high selectivity

high selectivity

low selectivity

high efficiency

low efficiency

1k 10k 100k

1000k

Sephacryl S-100

40 80 120 ml

Sephacryl S-300

Sephacryl S-200

BSA

Cyt C IgG

b-L

Cytidine

AU A gel has different

fractionation ranges for native, globular proteins and random coil.

对于形状不同的分子，某填料的分级范围不同

denatured proteins 变性蛋白

1

0.7

0.1

log (M r )

native proteins 天然蛋白

Molecules with different shapes have different selectivity curves.

对于形状不同的分子，有不同的选择性曲线。

K av 1

globular proteins 球蛋白

1

log (M r )

linear polysaccharides 线性多糖

relative viscosity: 1.0relative viscosity: 4.2relative viscosity: 11.8relative viscosity: 1.0

relative viscosity: 4.2

relative viscosity: 11.8

Principles of gel filtration Optimize : How to get the expected results Content 内容

Column:Sample:Load:Buffer:组分离: 柱高< 60cm, 进样体积较大16% -44% CV

V c

Measure elution position

计算分子量大小V

R

log (M

r

)

V

R

Log(M)

V R~ Log (M r)

Superdex™200 HR 10/30

快速脱盐，置换缓冲液，去除试剂常用于对分辨率要求比较高的精细纯化!

cross-linked agarose

dextran

cross-section from Superdex™particle

葡聚糖链Tricorn Superdex10/30GL_24ml Hiload Superdex16/26 120ml /320ml

13um 34um