Spiral Disk Assembly For High-speed Countercurrent Chrom

用于分离蛋白质和其他生物聚合物的螺旋盘组件的改进。 在过去测试的各种螺旋盘中，以前报道的混合-澄清螺旋盘显示了聚合物相系统对蛋白质的最佳拆分。以12.5%(w/w)聚乙二醇1000(PEG1000)和12.5%(w/w)磷酸二氢钾和肌红蛋白(马骨骼肌)和溶菌酶(蛋清)混合物组成的聚合物相体系为实验样品，继续测试螺旋盘室的尺寸、玻璃微珠在通道中的分布、两侧路障的开度、转速、流速、洗脱方式等各种参数。 1.室的尺寸：制作和测试两个单元：2.8 mm×2.8 mm，1460个凹坑(有障碍性的珠链螺旋盘)和2.8 mm×6.0 mm的730个凹坑(有障碍性的环形螺旋盘)，均与路障上下0.4 mm的管道相连。初步试验表明，环形螺旋盘的效率要高得多。 2.玻璃微珠的分布：用开口1.2 mm的环形螺旋盘测试几种不同分布的玻璃微珠。每隔一个坑或每隔三个坑放置一次珠子，效果最好。 3.房室盘内障碍物两侧开口：分别测试1.2 mm、0.8 mm、0.4 mm三种不同的开口。研究发现，开孔越小，效率越高。带有0.2 mm开口的栅栏圆盘现在正在NIH机械车间制造。 4.圆盘转速的影响：测试了600、800和1000三个转速范围。结果表明，当rpm增加到1000时，以Rs(峰值分辨率)表示的效率几乎是线性提高的。例如，以下相为流动相，流速为0.25ml/min时，Rs值在600rpm时为0.91~0.97,800rpm时为1.29~1.31,1000rpm时为1.51~1.56。 5.洗脱模式：试验了以下4种洗脱模式：从内端泵送的下相，即头或尾；以及从外端泵送的上相，即头或尾。所有这些洗脱模式都利用通过螺旋通道产生的离心力梯度来促进固定相的保留(反向洗脱模式导致固定相损失很大，峰分辨率较差)。研究发现，洗脱剂的洗脱 下相具有较高的峰分离度，固定相保留率约为总柱容的50%，而上流动相的峰分离度较低，固定相的保留率极高，可达80%以上。 简而言之，上述研究的结果表明，使用一组串联的有障碍物的螺旋圆盘可以实现蛋白质的有效分离。

Improvement of the spiral disk assembly for separation of proteins and other biopolymers. Among various spiral disks tested in the past, the mixer-settler spiral disk previously reported showed the best resolution of proteins with polymer phase systems. Using a polymer phase system composed of 12.5%(w/w) PEG1000 and 12.5%(w/w) dibasic potassium phosphate and a mixture of myoglobin (horse skeletal muscle) and lysozyme (egg white) as test samples, the experiments were continued to test the following various parameters : dimensions of the locule of the spiral disk; distribution of the glass beads in the channel, opening of the barricade on both sides, effects of revolution speed, flow rate, elution modes, etc. 1. Dimensions of the locule: two units were made and tested: 2.8 mm x 2.8 mm with 1460 pits (barricaded bead chain spiral disk) and 2.8 mm x 6.0 mm 730 pits (barricaded locular spiral disk) both connected with 0.4 mm ducts above and below the barricades. The preliminary test showed that the locular spiral disk yields much higher efficiency. 2. Distribution of the glass beads: Using the locular spiral disk of 1.2 mm opening, several different distributions of the beads were tested. The best results were obtained with placing the beads in every other pit or every third pit. 3. Opening on both sides of the barricaded in the locular disk: three different openings were tested, i.e., 1.2 mm, 0.8 mm and 0.4 mm. It was found that the smaller the opening the higher the efficiency. The barricaded disk with 0.2 mm opening is now under fabrication in the NIH Machine shop. 4. Effect of revolution speeds of the locular disk: Following three rpm ranges were tested: 600, 800 and 1000. The results clearly showed that the efficiency expressed by Rs (peak resolution) is almost linearly improved with an increased rpm up to 1000. For example, using the lower phase as a mobile phase at a flow rate of 0.25 ml/min, the Rs value of is 0.91-0.97 at 600 rpm; 1.29-1.31 at 800 rpm; and 1.51-1.56 at 1000 rpm. 5. Elution mode: The following 4 elution modes were tested: the lower phase pumped from the inner terminal which is either the head or the tail; and the upper phase from the outer terminal which is again either the head or the tail. All these elution modes use the centrifugal force gradient generated through the spiral channel to facilitate the retention of the stationary phase (the reversed elution modes resulted in a great loss of the stationary phase and poor peak resolution). It was found that the elution of the lower phase showed a high peak resolution with satisfactory levels of the stationary phase retention around 50% of the total column capacity, whereas the upper mobile phase showed much lower peak resolution with an extremely high retention of the stationary phase some over 80%. In short the results of the above studies promise that the efficient separation of proteins would be achieved using a set of barricaded spiral disks connected in series.