Gradient makers

The analysis of pharmaceutical agents in preparations has been one of the most important applications of modem RPC. A simple gradient maker to be used on the low pressure side of the pump for use in gradient elution of pharmaceuticals has been described (560). For the detection and identification of pharmaceuticals, micro internal reflection infrared spectroscopy (561) and ultraviolet scanning spectroscopy with stopped flow (562) were also employed. 

Side-outlet gradient maker for linear gradients (e.g., Hoefer SG50 for standard gels and Hoefer SGI5 for mini-format gels Amersham Pharmacia Biotech) Peristaltic pump capable of delivering 1 to 6 ml/min... 

Connect a piece of pump tubing to the outlet tubing connector of a side-outlet gradient maker. 

Pour heavy gradient solution into the chamber closest to the outlet (mixing chamber) of the gradient maker and add a small stir-bar. Use 9.3 ml per chamber for 1-mm-thick standard (14 x 16-cm) gels. Use 2.3 ml per chamber for 1-mm-thick mini-format (8... 

Place the gradient maker on a magnetic stirrer and begin stirring. 

Gradient makers are available commercially, but instructions for making a simple but effective device are given by Flurkey (13). 

Since Lucite is not wetted by water, 0.02% Pluronic L92 surfactant (BASF Wyandotte) was added to the aqueous liquids in the gradient maker. 

From a linear gradient maker, pump 5 ml of 5% and 5 ml of 20% NaCl solution for each gradient. Note that these volumes are appropriate when using a Beckman (Palo Alto, CA) SW-41 rotor as each tube holds a total volume of 11 ml. Form the gradient by pumping in the less dense NaCl solution into the bottom of the tube followed by the more dense NaCl solution. The less dense solution will rise above as the more dense solution is added. 

Using a linear gradient maker, form a 5 to 20% NaG gradient in... 

Assembly of the column and gradient maker are shown in Figure 4-13. 

Connect the glass tube protruding from the stopper to a type 1 gradient maker (see Figure 4-9 for the various types) by means of a piece... 

Prior to addition of the components of the gradient into the gradient maker, fill one side of the maker with water. Open the valve between the vessels and allow the water to run from one side to the other for about 10 seconds. While the water is still flowing, close the valve between the two vessels of the gradient maker. This procedure removes any bubbles trapped in the valve. 

Open the valve between the stirred side of the gradient maker and the column and allow the tube leading to the column to fill. The distance... 

Gently empty the water from both sides of the gradient maker. 4-37. The gradient to be used is a 500 ml, 0-5JV linear formic acid gradient. Place 250 ml water in the stirred side (side connected to the column) of the gradient maker and 250 ml 5M formic acid in the unstirred side. Be sure that the gradient maker is perfectly level. 

After a few seconds unclamp the rubber tube connecting the column and gradient maker. 

Gently open the valve between the stirred side of the gradient maker and the tubing. 

When it is certain that everything is flowing well, gently open the valve between the two sides of the gradient maker. Why is it advisable to do this last ... 

Check to see that the solution is flowing between the two sides of the gradient maker. Do this by looking for the schlieren patterns that exist at the entry of the formic acid into the water. 

Recheck the speed of the stirring motor during the run. The speed must be gradually and gently decreased as the level of the liquid in the gradient maker decreases. 

Figure 9-12. Gradient maker used to form linear density gradients over a broad range of volumes. A vibrator type of stirring motor is usually positioned above the unit. (Courtesy of Buchler Instruments, Fort Lee, N.J.)...

Sophisticated gradient makers capable of generating complex gradients are now on the market (e.g. LKB). The simplest ones to operate generate the gradient using two variable speed pumps and two buffer reservoirs. The pump speeds are automatically controlled by the device so that the gradient follows any pre-determined curve. One other type, which has been used in this field and can be fairly simply constructed is shown in Fig. 4.4. A number, maybe 9, of... 

SDS was obtained from Fisher Scientific. The gradient maker, acrylamide and bis-acrylamide of the highest purity were obtained from Bio-Rad (Hercules, CA). The gel equipment was a Hoefer SE-660 18 x 24 cm apparatus from Amersham-Phar-macia. The 0.2 pm filters were from Nalgene (Rochester, NY)... 

Pour the heavy solution in front chamber of gradient maker add stirbar. 

Pour light solution in back chamber of gradient maker, add stirbar balancer. 

Such a gradient maker is used in the Perkin-Elmer Model 604 liquid chro-... 

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