Separation of serum lipoproteins by HPLC

SEPARATION OF SERUM LIPOPROTEINS BY HPLC 3.1. Column selection and calibration 

LDL and HDL, G3000SW for that of HDL subfractions, G5000PW or G6000PW for that of chylomicrons, VLDL and LDL. The applicable range of human serum lipoproteins separated by each column is presented in Table 2. 

15 M NaCl solution is usually used as the eluent for lipoprotein separation in this method. However, various aqueous solutions containing appropriate amounts of inorganic salts can be used as an eluent, as long as the pH is less than 8.5. Salt concentration, buffering and pH of the eluent may alter the separation of the lipoproteins, and may improve the resolution of lipoproteins. 

Effects of the eluent properties (buffering agents, salt concentration and pH) on the resolution of lipoproteins were examined. As presented in Fig. 5, their effects are found to be negligibly small on the separation of lipoproteins larger than LDL (12,16). An increase of the buffering agent or salt concentration leads to peak broadening, and in the case of HOL possibly causes the separation of HDL 

A slower flow rate of the eluent gives better resolution of serum lipoproteins. The effect of flow rate of the eluent on the resolution of LDL and HQL for three column systems is shown in Fig. 6. The flow rate of 0.40 ml/min - 1.0 ml/min is usually used according to column systems (see Section 2.5) due to the balance between the increase in resolution and the time required for analysis. 

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