UPLCS suggests that chromatographic operators pay attention to the importance of sample filtration before performing liquid chromatographic analysis. It is mainly to prevent impurities such as proteins from being deposited on the chromatographic column, and the biological samples need to be pretreated for deproteinization before loading the column. This can extend column life and reduce maintenance costs, thereby helping to increase laboratory throughput.
Deproteinization also plays a very important role in the analysis of antibiotics and residual pesticide content in biological fluids (such as plasma, serum, urine and cerebrospinal fluid). And if the precipitated protein is not removed from the sample, it will cause the performance of the chromatographic column to decline, and ultimately affect the accuracy of the chromatographic analysis results.
There are 3 processing methods for removing protein from biological samples:
Precipitate by protein dehydration. Use organic solvents such as acetonitrile, methanol, etc., or neutral salts such as ammonium sulfate, sodium sulfate, etc. to dehydrate and precipitate the protein. When acetonitrile is used, the resulting precipitate is easy to separate. When 1 to 3 times the volume of organic solvent is used, more than 90% of the protein can be precipitated. After mixing and centrifuging the plasma sample with acetonitrile, about 95% of the protein will precipitate. Non-polar proteins (such as albumin) remain in the liquid phase. If a guard column is used to remove the remaining 5% of the protein, the supernatant can be injected directly. For long-term use, the guard column needs to be replaced or inverted and washed away to prevent protein from entering the main column.
We suggest that add a preparation guard column before the HPLC column. The sample and mobile phase can be filtered, which can maintain the efficiency of the chromatographic column for a longer time, effectively protect the chromatographic column, and extend the service life of the chromatographic column. UHPLCS preparation guard column after design optimization, the influence on the chromatogram under various separation conditions is extremely low. Its low dead volume design can greatly reduce peak diffusion and peak tailing, thereby maintaining resolution and analysis efficiency. The upper limit of withstanding pressure can reach 15,000 psi, which protects HPLC, UHPLC column and preparative column products cost-effectively and exerts good performance.
There are various types of HPLC columns, such as direct-connection guard column, rotation type guard column, analytical guard column, preparative columns and Semi-prep columns. They combine the strength of stainless steel with the convenience and efficiency of PEEK pre-packed cartridges. To better meet the various needs of customers.
Use protein to form insoluble salt precipitation. Commonly used are acidic precipitation agents, such as trichloroacetic acid, perchloric acid, etc., or use heavy metal ions to precipitate proteins. Acidification with trichloroacetic acid, centrifugation and neutralization with sodium hydroxide can remove 99% of the protein. Perchloroacetic acid can also be used.
Other methods.Heating makes the protein precipitate, which is suitable for samples that are stable to heat.The sample can be placed in a boiling water bath to directly denature the protein to form a precipitate.Or use ultrafiltration and dialysis methods to remove the protein in the body fluid sample. During the processing, the protein in the sample is not denatured, and the combined component to be tested cannot be liberated and cannot be detected. This method can be measured in serum or plasma. The concentration of the free component to be tested.However, it is time-consuming and subject to certain limitations when analyzing a large number of samples.
Post time: May-29-2021
