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The Effect Measurement of Chromatographic Column

The column effect is the separation effect of chromatographic column . The higher the number of effective plates or the lower the height of effective plates, the better the column effect.


The Number Of Theoretical Plate (HETP)

According to the plate theory, the number of theoretical plate reached by a chromatographic column can be calculated, or expressed as theoretical plates per meter(n/m).The column length equivalent to a theoretical plate is called the theoretical plate height H, and the column effect is usually expressed by the effective plate number N and the effective plate height L,W is the peak width, and W1/2 is the half-peak width:

N=16(TR/W)2

=5.54(TR/W1/2)2

=L/H

Obviously , the larger the value of N or the smaller the value of H, the higher the column effect.


The column effect mainly depends on the packing (i.e., the stationary phase), however in the process we often find what The same batch of packing, the same size of root chromatographic column, the same appearance, the column effect is completely different; The same batch of packing, the same chromatographic column before and after two packing, its column effect is still different. Finally, the reason is the packing column.

The packing quality of chromatographic column is very important to ensure the stability of purification process and the safety of final product. The ACC series automatic axial compression column of hanbon science&technology is matched with an automatic packed column workstation. The fully automatic and intelligent column packing method can ensure the consistency of each packing effect and the repeatability of chromatography results .Scientific distributor design can uniformly distribute the feed and improve the column effect.

Even with such a good chromatography column, it takes a suitable method to measure the high column effect.


The Measurement of Chromatography Column Effect

This method is suitable for the medium&low pressure liquid chromatography pre-packed column and self-packed chromatography column.

The introduction is divided into five parts, pre-test preparation, reagent selection, test column effect operation, test result integral, and frequent problem.

1.    Pre-test preparation:

(1) Chromatography system .

The type of the system is required to match the size of the chromatography column, and the volume between the injection valve and the UV and conductivity detector should be kept to a minimum (the pipe inner diameter should be as thin as possible, the pipe should be as short as possible), which is very important for accurate measurement of the column effect of the lab-scale small chromatography column.

(2)The test of balance solution and sample.

(3)The packed chromatography column .

Using a balance buffer solution to balance at least 1.5cv (column volume) at the flow rate of measuring column. The balance direction must be consistent with the column effect direction.

(4)Injection ring.  

Volume is larger than 1% of column volume.

2. Reagent selection

The two main test methods for measuring column effect: NaCl test and Acetone test. As long as the operation is correct, the results of the two methods are basically the same. However, it should be noted that the concentration of each reagent cannot be changed at will , otherwise the test results will be affected.

(1) NaCl test

Sodium chloride can be used to measure column effect for all types of columns and packing

Balance solution: 0.4 M NaCl aqueous solution

Sample: 0.8 M NaCl aqueous solution

(2)Acetone test

For the chromatography column and packing which are affinity, ion exchange, and gel filtration technologies

Balance solution: water

Sample: 1% acetone in aqueous solution

For reversed and hydrophobic column and packing

Balance solution: 20% ethanol

Sample: 1% acetone dissolved in 20% ethanol

3. The Measurement Operation Of Column Effect

Linear flow rate of 30 cm/h is used throughout the column effect test. An balance solution of at least 1.5 times the column volume (CV) is required to balance the chromatographic column. 1% CV sample (NaCl or Acetone) is injected into the column and flushed with the balance solution until a response peak appears at 280nm of conductance or UV.

4. Test Result

Software workstation automatically integrates the response peak and reads the integral: theoretical plate number (HETP) and symmetry (Asymmetry).

Note: the NaCl test integral curve is the conductance curve, and the acetone test integral curve is the UV curve at 280nm.

In the Peak data area, the co-selected analysis results will show that the standard for judging whether the chromatography column effect is qualified is Plates per meter and Asymmetry. The Plates per meter should be larger than the minimum limit required by the instruction (e.g. >3000 or 20000) or than 1000000/ average particle size /3 (calculated as the value on the instructions). Asymmetry requires a range between 0.8 and 1.8.If these two points are met, the column can be packed.


Cause And Solution Of Column Effect

If you are unlucky to find out the column effect is not qualified, it may be caused by the following reasons, and herein solution is attached:

Column packing non-uniform --- re-pack

Fluid flow instable in pipeline --- NaOH cleaning system; Balance column above 1.5CV

Bubbles in pipeline --- solution degasification ; Backpressure valve on-line, flushing enough

Agglomeration phenomenon of gel --- down chromatography column, re-pack column after cleaning gel with NaOH ,renew the gel

Bad flow distribution of column --- change distributor or chromatography  column

Big system dead volume --- change fine line; Short-connected post valve

Too big sample volume --- 1% column volume

Too fast flow rate --- measure according to the use of flow rate