Packing method for coupled macrobore liquid chromatography

Simple test for determination of the degree of distortion of the liquid-phase flow profile in columns for preparative liquid chromatography ... R. Gil...
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Anal. Chem. 1983, 55, 1842-1847

Packing Method for Coupled Macrobore Liquid Chromatography Columns Istvln HalPsz* a n d G e r h a r d Maldener Lehrstuhl f u r Angewandte Physikalische Chemie der Universitat des Saarlandes, 6600 Saarbrucken, West Germany A versatlle new method is descrlbed for packing “macrobore” columns (1.d. = 3-11 mm, L = 25-100 cm) with silica. The method Is reproducible, the columns are efficient, have long Ilfetimes, and yleld symmetrical peaks. I f these columns are coupled with caplllarles up to a few meters in length, the plate number Increases linearly with the column length, uOp,does not decrease, and the mass transfer term does not increase. The reduced H,,, for an Inert peak Is less than two and Is independent of the column length. With partlcle slres around 8 pm, 100 plates/s are generated with 1000 plateslbar pressure drop over the long columns and the holdup time Is around 10 m l n h of column length.

There is a growing need and interest in life science studies, petrochemical and clinical analyses, etc., to separate complex mixtures sometimes containing more than 100 compounds. More than half a million plates were generated in packed microbore columns (1-17), i.e., in columns with an internal diameter of about 1 mm. Such columns are advantageous if the mass of sample is extremely small or if the price of the eluent is high. The preparation of microbore columns is not simple and sophisticated equipment ( I , 2,10,17) is requir,ed. At present, ,manufacturers are able to produce excellent 3-7 mm i.d. columns, 10 to 30 cm long, packed with 5-10-pm particles. Such columns will be referred to as “conventional” or “macrobore” columns. Direct “conventional” column butt to butt coupling, however, introduces a significant increase of the relative band broadening, as described by Scott and Kucera: “If widebore columns are joined together, they soon reach a limiting efficiency above which further addition of column length does not increase the overall efficiency” ( 4 ) . Furthermore, “Experimental data presented in this paper indicate, that coupling larger diameter columns results in about 60% loss of column efficiency for each coupling step, whereas coupling microbore columns actually produces efficiencies of 100% for each concatenation” (13). In this paper the reason for the excess peak broadening as a function of the column coupling length will be discussed and a new column packing method will be proposed to avoid coupling problem of conventional columns with 4-11 mm i.d. Such columns can be used with commercially available instruments. In the following the particle size of the support, 6, will be defined (18-21) as

i.e., 6 is defined with the pressure drop to be paid for small particle sizes. The following typical parameters for routine LC will be assumed: (a) The particle size, 6, ranges between 3 and 30 pm. (b) The linear velocity varies between uoptand 2OuOpt, where uoptis the velocity at the minimum of the H vs. u curve. (c) The molecular mass of the samples is less than 300 Dalton. (d) “Low viscosity” eluents are used (i.e., n-heptane, di-

chloromethane, acetonitrile, and methanol). (e) The sample capacity ratios, k’, are less than 2. With these parameters the H vs. u curves are approximately independent of the type of the support (silica or chemically bonded phases) and can be described with a tolerable approximation (22, 23) by 6

H = 1.56 + u

+ -U16

where H and 6 are expressed in pm and u in mm/s. The constants in eq 2 have the corresponding dimensions. These special units permit rapid practical calculations for routine LC. In all the other calculations, the usual CGS or SI systems have to be used. Equation 2 is typical for commercially available columns. With short columns ( L < 30 cm) the outlet pressure of the routinely used pumps is seldom a limiting factor. If efficient long columns are required, the number of is of great plates generated per unit pressure drop, N/AP, importance, and it can be shown that

(3) where CT is the total porosity. I t is trivial that the maximum plate number can be generated a t the minimum of the H vs. u curve (uoptand ETmin). Quite unexpectedly it has been shown that the maximum speed of analysis can be achieved a t uopt,if L and 6 can be chosen freely (23). With increasing capacity ratios, k’, of the samples, uoptdecreases. Consequently the optimum velocity will be uoptfor the compound with the minimum retention. Working a t uopthas the additional advantage that H hardly varies as k’ changes. I n the following it will be always assumed, that the compounds are separated at uOpt.Because uOptis inversely proportional to 6, and Hminis linear in 6, it follows that

(4) and (5)

If extremely high plate numbers are required, relatively large particle sizes are necessary because of the pressure limit of the pump, but the analysis time will be long. If the pressure is not a limiting factor, then small particle sizes are optimum for achieving short analysis times. In the following only those columns will be accepted where the asymmetry factors ( 2 4 ) ,especially for peaks with small capacity ratios, are between 0.9 and 1.2. EXPERIMENTAL SECTION Chromatographic Apparatus. Home-built equipment with a UV detector (18) was used. Eluents and Samples. The water content of the n-heptane eluent was controlled on-line (25). Column Material and Stationary Phases. All columns were packed into drilled-out 4.1 mm i.d. stainless steel tubes by the viscosity method (24).

0003-2700/83/0355-1842$0 1.50/0 0 1983 American Chemical Soclety

ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

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-~

Table I. Constants of the van Deemter Equation and the Speed of Analysis A,pm a a a a b

b b

column column + 3 RU column + 15 RU column (“after”) column column + 8 RU column (“after”)

16 18 4.2 22 9.3 8.5 9.7

105~,

cm*/s

C,ms

3.4 3.8 4.8 5.9

5.1 55 5.2

3.7 4.1 3.4

2.4 3.2 2.4

11

Uapt,

mm/s

Hrninl

Hrnin,

pm

0.82 0.59 0.30

6

Nlto, 5-

NIu, bar-’

1.1

24 31 37 33

3.3 4.3 5.1 4.6

36 19 8 33

774 83 3 1376 420

1.26 1.15 1.18

15.1 15.7 15.5

1.68 1.74 1.72

83 73 76

1267 1122 1107

Old column (6 = 7.2 pm) packed with conventional method. Parameters are the same as those given in Figure 1. Column packed by the new method. Parameters are the same as those given in Figure 6. a

The columns were packed with a homemade silica (H-90-7) having an average pore diameter of 90 A, as determined by the chromatographicmethod (26). The corresponding specific surface areas were 350 m2/g. The ]particlesize distribution was determined with a Coulter Counter Model TAII (Coulter Electronics Ltd., Coldharbour Lane, England) as dw = 4.8 Mm, d50= 6.1 Mm, and dlo = 8.2 pm. Constant pressure pump was used (Haskel Comp., Burbank, CA, Type DHST-202). The dispersing agent was 2propanol/tetrachloromethane (2/1, v/v).

EFFICIENCY OF SINGLE A N D COUPLED COLUMNS PACKED B Y CONVENTIONAL PROCEDURES Attempts to pack macroparticular columns having 3 to 11 mm internal diameter and lengths over 50 cm have not been successful. In order to achieve high plate numbers in LC therefore, long columns are generally obtained as a series of “conventional” columns. The H values measured with such coupled “conventional” columns are substantially higher than those of the individual columns. Here a convenient method is described for quantifying such effects (27,28). First the H vs. u curves were measured with the usual arrangement: pump-sampling device-columnsdetector, using a 25-cm column (“reference”). In the second arrangement a squeezed capillary (29) was connected between the sampling system and column. The original diameter of the stainless steel capillaries was 0.25 mm and their length varied between 8 cm and 15 cm. The average hydrodynamic resistance of a squeezed capillary is expressed in restrictor units, RU. The average flow resistance of one RU is equal to the resistance of the packed column. The flow resistance of the capillaries varied between 3 and 15 RU. For a given linear velocity ithe inlet and outlet pressures of the packed column were identical for both of the arrangements. The measured H vs. u curves were very similar with both arrangements, consequently the peak broadening inside the restrictor capillary was negligible. In the next experiment the squeezed capillary was connected between the column outlet and detector inlet and the H vs. u curves were measured. At a given linear velocity the pressure drop over the column was again identical as in the previous arrangements. Consequently, the heat of friction and the heat conductivity of the packed column were also the same. However, the inlet and outlet pressures (and the average pressure) of the packed column were much higher than before. In other words, this setup allows us to monitor the column effluent under conditions as if it were the first in a series of, say, 16 identical columlns, if the flow resistance of the restriction capillary was 15 RU. In the following experiments the restrictor capillary will always be connected in series between the outlet of the column and inlet of the detector. In the last experimental series the conventional arrangement was used again to measure the H vs. u curves again (“after”). The first H vs. u curves for an inert sample were measured with a column used for more than 6 months in routine work.

841.

030

Q82,

2

3 u [mm/sec]

u curves of an old column with 4.1 mm internal diameter: column length, 25 cm; stationary phase, H-90-7; 6 = 7.2 prn; total porosity, 0.85; eluent, n-heptane (22 ppm H20); inert sample, tetrachloroethylene (TCE); (1) reference column, (2) column and restrictor capillary (RU = 3), (3)column and restrictor capillary (RU = 15), (4) column (“after”). Flgure 1. H vs.

The results with column 1 are shown in Figure 1. Line 1 describes the results with the ”old” column alone. Lines 2 and 3 were measured with squeezed capillaries with 3 RU and 15 RU, respectively. The pneumatic resistances of these arrangements were equivalent to columns coupled in series 100 cm and 400 cm in length, respectively. The constants of the van Deemter equation (A, B , C) were calculated and are given in Table I. With increasing flow resistance of the capillaries the C terms increase by a factor of up to 10, the uoptand the speed of analysis at uoptdecrease by factors of about 3 and 4, respectively. After the measurements with the restrictors the efficiency of the column alone was not reproducible (“after” in Figure 1 and Table I). Quantitatively similar results were achieved with freshly packed columns of good quality as demonstrated, for example, in Figure 2. Comparing the reference column and that with 9 RU restrictor capillaries, uOptshifted from 1.2 to 0.9 mm/s and the speed of analysis decreased from 58 to 42 plates/s. The H vs. u curve of the reference column before and after the measurement with the restrictor capillaries was, however, reproducible. Five different types of commercial columns packed with silica, which find worldwide use in practice, were employed in this study to exclude the possibility that the results described above were the consequence of our poor packing method. To save time, band broadening5 at low velocities (u < 1mm/s) were not measured and the constants of the simple relationship H = A* -t C*u (6) were calculated for the reference columns without and with the restrictor capillaries (RU = 8). The internal diameters varied between 3.9 and 4.6 mm and the column lengths between 20 and 30 cm. The nominal particle sizes, as given by the manufacturers, were all 10 pm. However, effective particle

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“1

42

1C cm

i

25-100 c m

r

pre-columr

I t

I 47l

10cm 1 2 3 4 5 u[mrrlsec; Flgure 2. H vs. u curves of a new column with 4.1 mm internal diameter. Parameters are the same as in those given in Figure 2 except: (1) reference column, (2) column and restrictor capillary (RU = 5), (3) column and restrictor capillary (RU = 9).

+

pst-co,urnn cap1llar y

I

12 - 2 0 0 c m

1

I pmi 98.

Flgure 4. Proposed packing arrangement. Internal diameter of the columns is 4-1 1 mm.

i

2

3

4

5

6 ulrnrnlseci

Flgure 3. H vs. u curves of a commercially available coiumn packed with silica: column length, 30 cm (3.9 mm i.d.); 6 = 9 pm; eluent, n-heptane (22 ppm water); (1) reference column, sample, TCE ( k ’ = 0);( l a ) column and restrictor (RU = 8), TCE; (2) reference column, perylene (k’ = 3.8); (2a) column and restrictor (RU = 8), perylene.

sizes, 6, varied between 7.9 and 9.2 pm. The water content of the n-heptane eluent was maintained at 22 ppm by using an appropriate A1203 cartridge as described previously (25). A four-component sample was used to measure the H vs. u curves for each column a t different capacity ratios, k’. The four components and their respective k’values are as follows: tetrachloroethylene (TCE) (k’ = 0); benzene (0.5);diphenyl (1.7); perylene (3.8). The column to column variation in the k’values was less than 10%. A set of H vs. u curves for TCE and perylene with one of the commercial columns is shown in Figure 3. Because of the pressure limit of the pump, peak broadening was measured only up to u = 3 mm/s, if a squeezed capillary (RU = 8) was connected in series between column outlet and detector inlet (lines l a and 2a in Figure 4). The C* term increased from 4.4 to 7.4 ms for the inert TCE sample and from 9.5 to 16.2 ms for perylene (k’ = 3.8). The use of any postcolumn resstrictor caused the mass transfer term C* to increase by 50% to 110% in different columns. Nevertheless, no correlation was found between the magnitude of increase in C* and the corresponding retention ratios. With increasing retentions the mass transfer term increases and uOptdecreases. Consequently with increasing k’the speed of analysis, N / t R ,decreases much more steeply than is usually assumed. To avoid the steep descending branches of the H vs. u curves, the optimum velocity for the separation is uOpt of the inert sample or that of a sample with the smallest retention.

PROPOSED COLUMN PACKING METHOD I t is an old experience that columns packed at relatively low pressure exhibit excellent specific permeability and ef-

ficiency, but poor stability. The inverse is true for columns packed at very high pressures (i.e., greater than 1000 bar). If columns are coupled in series, the average pressure will be high in the first units after the sampling system. To pack such columns two requirements have to be fulfilled: (a) the packing pressure, Le., the pressure at the top layer during the packing procedure, has to be high and constant and (b) the speed of packing, Le., the column length packed per unit time, also has to be constant. Unfortunately, these requirements are not fulfilled with “constant pressure” or “constant flow rate” pumps. It is calculable, that the flow rate of the suspension decreases steeply, if the column is packed with constant inlet pressure, because of the increasing pneumatical resistance of the column. The column packing will also be inhomogeneous if constant flow rate pumps are used. Of course, the packing speed is constant in this case, but the packing pressure increases steeply as a function of time. To pack homogeneous columns the arrangement shown in Figure 4 is proposed. The slurry reservoir, precolumn, column to be packed, postcolumn, and a packed restrictor column are coupled in series. The first three columns have the same internal diameters (4.1 mm) and are butt-to-butt coupled with the help of drilled-out unions. At the outlet of the empty postcolumn a filter paper with pore diameters of 2.2 pm and a stainless steel sieve is inserted. The packed restrictor column is usually an old unit with internal diameter similar to the column to be packed. The sieve fraction of the silica in the slurry and in this column are nearly alike. The packed restrictor column is filled with the dispersing agent of the slurry. Consequently its pneumatic resistance does not change while the empyt column is being packed. The minimum length of this restrictor column is half that of the column to be packed. This length can be increased by up to a factor of 8 depending of the required packing pressure in the first layer of the column to be packed. Changes in the packing pressure and in the slurry flow rate are reduced if the restrictor column is elongated. Precolumns are often proposed in the literature. A postcolumn is used here to achieve a homogeneous packing. For one thing, because of the compressibility of the slurry, the flow rate as a function of the pressure is undefined when the first layers are deposited.

ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

F lmllminl

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I

7-

6-

35

, I

10

20

30

40

-/Fdt

[mlj or L Icr:

Flgure 5. Measured flow rate of the suspension as a function of the packed column length: constant outlet pressure of the pump, 500 bar: internal diameter of the columns, 4.1 mm; silica, H-90-7; 6 = 9 pan; dispersing agent, 2-propanoi/CCI, = 2/1 (v/v); concentration of the silica in the slurry, ca. 50 rng/mL; packing density, 0.35 g of silica/mL column volume. After the packing procedure the columns are disconnected and the inlet of the freshly packed column is covered with a filter paper and metal Bieve. This end is also locked up with the end-fitting. Pre- and postcolumns are unpacked, the tubes and support as well as the restrictor column can be used over again. The dispersing agent was a mixture of 2-propanol/carbon tetrachloride (2/1, v/v). The concentration of the silica (H-90-7) in the slurry was about 50 mg/mL. One centimeter column length was packed with 1 mL of such a suspension, if the internal diameter of the column was 4.1 mm. The slurry was pressurized with n-heptane. The volume of the slurry, Le., the amount of silica, was chosen to fill about half, or more, of the precolumn. The measured flow rate at the outlet as a function of the packed column length is shown in Figure 5. The length of the restrictor column and that of the column were 25 cm and both columns were packed with identical support. As shown in Figure 5 the speed of packing changed only from 4 to 3 cm/min. The calculated and measured flow rates of the slurry in Figure 5 were the same. Consequently the specific permeability of the column was constant at any time during the packing procedure, i.e., the column packing was homogeneous. The steep increase in the flow rate when the precolumn is packed signals the breakthrough of n-heptane. In previous experimlents the pressure was varied between 300 and 800 bar. If the pressure was less than 500 bar, tailing was observed. If the pressure was greater than 500 bar the efficiency of the column and the peak symmetry were unchanged, but the packing became more dense and the permeability decreased (i.e., the 6 values decreased) systematically. Packed restrictor columns with length up to 75 cm (3 RU) were also used, but the quality of the freshly packed column was practically unchanged. The optimum packing pressure and flow rate of the slurry change a little with the quality of the silica. On the basis of our experience, peak tailing can be observed if the packing pressure is too low. High packing pressures resulted in leading and tailing of the peaks. The efficiency of the 25 cm long column packed with the new method and 500 bar is demonstrated in Figure 6 and Table I. As shown in Figure 6, the H vs. u curves vary only moderately if a restrictor capillary (RU = 8) is connected between column and detector. The reproducibility of the H vs. u curve is good "after" the experiments with the restrictor. As can be seen in Table I, the reduced plate height of the column is excellent ((H/&,,, = 1.7). The value of uopt,the

3

2

I

4

5

-

u [rnmisec:

Flgure 6. H vs. u curves of a silica column (L = 25 cm) packed with the new method: packlng pressing, 500 bar; stationary phase, H-90-7 (6 = 9.1 pm); total porosity, 0.84; interstitial porosity, 0.45; eluent, n-heptane (22 ppm H,O); sample, TCE (k' = 0);(1) reference column, (2) column and restrictor capillary (RU = 8), (3) reference column "after".

"t

[pl

j _ I

2

3

4

5 ~[mmisec;

Figure 7. Hvs. u curves on a silica column (L = 50 cm) packed with the new method: packed with a restrictor column (L = 25 cm) and with 800 bar; stationary phase, H-90-7 (6 = 8.8 pm); eluent, n-heptane (22 ppm H,O); samples, (1) TCE (k'= 0);(2)benzene (0.5);(3) diphenyl (1.7); (4) peryiene (3.7). speed of analysis, and N/APchange only modestly. For the inert peak about 80 plates/s and 1200 plates/bar are generated with 6 = 9.0 pm. The calculated particle size 6, the total porosity (eT = 0.84), the interstitial porosity ( E Z = 0.45)), and the packing density (0.35 g/mL) remained unchanged after measurements under high pressure. The efficiency of more than 95 of 100 packed columns was reproducible with deviations of about *lo%. The lifetime of the columns was longer than 1 year in routine work. Packing o f 50 crn Long Columns. The best columns were packed when the length of the packed restrictor column was only 25 cm and the packing pressure was 800 bar. Due to the higher pressure drop over the column the speed of packing (Le., packed length per unit time) was greater here than in the 25-cm columns. This column (without pre- and postcolumn) was packed in ca. 13 min whereas the packing time of the 25-cm column was ca. 8 min. The packing times, including flushing with one column volume of n-heptane, were 25 and 20 min for the 50- and 25-cm columns, respectively. The permeability of the long column was also unchanged during the packing procedure. The characteristic parameters for an inert peak were very similar in the 25- and 50-cm columns (6 = 8.8 pm, ET = 0.84, urnin= 1.29 mm/s, Hmin= 15.6 pm, H,,d = 1.8, N / t o = 83/s, N/AP= 1150/bar). As demonstrated in Figure 7, the increase in the slope of the H vs. u curves with increasing retentions is acceptable,

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ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 15

2 I’

I

11

1

0

10

20

39

o i

t~minl

Figure 8. Separation of an artificial mixture with coupled silica column 2 X 50 cm in length: stationary phase, H-90-7 (6 = 8.4 pm); eluent, n-heptane (22 ppm H,O); A f = 90 bar; u = 1.9 mm/s; plate number for the inert peak (No),52000; Ho/6 = 2.3; N i t o = IOO/s; N o / A f = 580/bar; samples, (1) TCE, (2) 1,2,4-trichlorobenzene, (3) cyclohexadiene, (4) odichlorobenzene, (5) benzene, (6) tetrahydronaphthalene, (7) naphthalene, (8) 2,6dlmethylnaphthalene, (9) 2,3dimethylnaphthalene, (IO) anthracene, (11) fluoranthene, (12) benzo[alpyrene, (13) perylene, (14) m-terphenyl, (15) o-terphenyl, (16) benzo [ b] fluoranthene.

and probably better than in commercially available 25 cm columns. The quality and lifetime of these 50 cm long columns were very similar to the shorter ones. Coupled Columns. When six 25-cm columns were coupled, the parameters as given in the first line in Table I were reproduced. Coupling three 50-cm columns yielded results that were about 10% worse than those shown in the first line of Figure 7 . The columns were coupled with stainless steel capillaries 7 cm in length and with internal diameters of 0.25 mm. The separation of an artificial mixture in a column with 2 X 50 cm length is shown in Figure 8. The linear velocity was 1.6 times higher than urnin.About 100 plates were generated per second for the inert sample and N / A P = 580/bar was achieved.

RESULTS AND DISCUSSION If identical “macrobore” columns are coupled in series, the average pressure in a column decreases from top to bottom. If a restrictor is placed after the column, the properties of the first column in series can be measured, provided the peak broadening inside the restrictor is negligible to a first approximation. The experiments described above demonstrate that the mass transfer terms increase with increasing average pressure if all other parameters are kept constant. This effect cannot be caused by the irreproducible column packing, as is usually argued ( 4 , 1 3 ) ,because in our experiments only one column was used. Temperature effects are also excluded, because in our experiments the column was always the “first column” and at a given linear velocity of the eluent the heat produced and conducted is identical whether a restrictor is behind the column or not. We assumed that the reason for the increasing mass transfer term with increasing average pressure is the conventional column packing method. This technology is now excellent, if the length of the column, or that of columns coupled in series, does not exceed about 50 cm. This hypothesis was reinforced by the experience that some of the columns were disturbed after the experiments with the restrictor capillaries. When “macrobore” columns (>3 mm i.d.), packed by conventional methods are coupled, uOptdecreases and the C term of the van Deemter equation increases. Consequently, to describe the efficiency of a column in plates/meter units is

dangerous, if the plate number was determined in a shorter column. A new packing method that uses a packed restrictor column is proposed. In this method the flow rate of the suspension and the packing pressure change only moderately. Both constant pressure or constant flow rate pumps can be used. At a given outlet pressure (or flow rate) of the pump the speed of packing and the packing pressure can be varied by changing the pneumatic resistance of the restrictor column. Based on experiments (27), only partially described in this paper, columns of unvarying good quality can be packed by the new method, if the internal diameter is varied between 3 and 11 mm with lengths between 15 cm and 100 cm. The minimum reduced plate heights for an inert peak are less than 2 and the asymmetry factors are between 0.9 and 1.2. During the packing procedure the permeability of the column remains constant and the packing density is independent of the column length. Columns packed by the new method were reproducible, had a long lifetime, and yielded symmetrical peaks. The reproducibility of the surface properties of silica is limited. Consequently the required composition of the slurry has to be adjusted from batch to batch if conventional packing methods are used. Such modifications are not necessary if the columns are packed by the new method. Our columns were coupled with capillaries (0.25 mm i.d.) up to a few meters in length. This way of coupling is simple and the capillaries are excellent heat exchangers, especially if the columns are immersed in a water thermostat. This can be important because the heat of friction is a linear function of the pressure drop over the column (18) and is high if long columns are used. Therefore, we recommend the use of short units (L = 25 cm) to increase the heat exchange in the system. In these long coupled columns the shift of uOptand the increase in the mass transfer term were minimal. Furthervalues were excellent. The increase of the C more, the Hmin term with increasing k’was also tolerable. High-speed analyses can be achieved with coupled columns packed by the new method. Because of the compromise between the speed of analysis and the plate number produced by unit pressure, a particle size between 7 and 10 pm seems to be a good choice for coupled long columns. For these particle sizes the following are typical parameters for the inert sample, if “low viscosity” eluents are used: N/AF’ N 1000/bar

N / t , = 1oo/s to N 10 m i n / m column length

It is assumed, that the linear velocity of the eluent is uoptof the inert peak. With 400 bar pressure about 400000 plates will be produced and the hold up time of the column will be around 1 h. The column length will be about 6.7 m and H 31 17 pm will be achieved for the inert peak. Of course these parameters can be improved by increasing the column temperature (Le., lower viscosity of the eluent and higher interdiffusion coefficients of the samples). As mentioned earlier, the experience of most chromatographers with connecting conventional (“macrobore”) columns in series has been disappointing because the resulting column efficiency fell far short of the plate number one would have expected on the basis of the plate number of the column measured individually. The results presented in this paper suggest that the packing structure of the columns described in the literature is responsible for the departure from the theoretically expected additivity of plate numbers. On the other hand, it has been reported that this problem can be solved by long “microbore” columns, i.e., the need for a long

ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

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In a following paper we want to describe the new method for “macrobore” columns packed with reversed phase and to discuss advantages and disadvantages of the use of “microbore” and “macrobore” columns in routine chromatography.

ACKNOWLEDGMENT We wish to acknowledge the help of G. Gutnikov, California State Polytechnic University (Pomona, CA) for correcting the English in this manuscript.

LITERATURE CITED

25

5

75

10

125

15

9L-1

Figure 9. Reduced H vs. ~d curves for inert peaks from stainless steel columns (1 mm i.d.) packed with silica: (-) results of Scott and Kucera Figure 7 In ref 4; eluent, 2-propanol-n-heptane (5:95);“microbore” columns (1 m X 1 mm), columns packed with 1800 bar, particle sizes are given in the figure; (---) own results; eluent, n-heptane with 22 ppm water; coupled “m1c:robore” column, 4 mm and 4 X 25 cm columns packed with 500 bar.

column having high plate numbers can be met. That this is not necessarily the case, is illustrated by the reduced H vs. u plots in Figure 9. The three upper curves were replotted from the data of Scott and Kucera (4) who used 1 m long, 1 mm i.d. columns packed with 5, 10, and 20 pm silica. The lower curve is a plot of data obtained in our laboratory with a 4 X 25 cm long, 4.1 mm i.d. column packed with 9.3-~msilica by using the packing procedure described above. Otherwise the eluents and operating conditions were essentially the same in the two sets of experiments. The results depicted in Figure 9 demonstrate that the long column prepared by connecting regular size columns packed by our procedure yields efficiencies superior to those obtained with long “microbore” columns by leading authorities. Unfortunately it is not possible to establish whether instrumental factors or the column packing property is responsible for the poor efficiencies exhibited by the “microbore” columns. Nevertheless, we believe that the results shown in Figure 9 are sufficient to invalidate the claims frequently advocated in the literature that long “microbore” columns are superior to long “macrobore” columns and that when regular size microparticulate columns are connected in series an inevitable loss of efficiency occurs.

(1) HorvBth, C.; Lipsky, S. R. Anal. Chem. 1989, 47, 1227-1234. (2) Ishll, D.; Asal, K.; Hibl, K.; Jonokuchi, T.; Nagay, M. J . Chromatogr. 1977, 744, 157-168. (3) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1978, 725, 251-263. (4) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 169, 51-72. (5) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 785, 27-41. (6) Scott, R. P. W.; Kucera, P.; Munroe, M. J . Chromatogr. 1979, 786, 475-87. (7) Kucera, P. J . Chromatogr. 1980, 798, 93-109. (8) Scott, R. P. W. J . Chromatogr. Scl. 1980, 78,49-54. (9) Knox, J. H. J . Chromatogr. Scl. 1980, 78,453-461. (10) Reese, C. E.; Scott, R. P. W. J . Chromatogr. Scl. 1980, 18, 479-486. (11) Gulochon, G. Anal. Chem. 1981, 5 3 , 1318-1325. (12) Kucera, P.; Moros, S. A.; Mlodozeniec, A. R.; J . Chromatogr. 1981, 270,373-388. (13) Kucera, P.; Manius, G. J . Chromatogr. 1981, 276,9-21. (14) Novotny, M. Anal. Chem. 1981, 53, 1294A-1308A. (15) Yang, F. J. J . Chromatogr. 1982, 236, 265-277. (16) Liao, J. C.; Ponz6, J. L. J . Chromatogr. Sci. 1982, 20, 14-18. (17) Scott, R. P. W.; Slmpson, C. F. J . Chromatogr. Sci. 1982, 20, 62-66. (18) HalBsz, I.; Endele, R.; Asshauer, J. J . Chromatogr. 1975, 772, 37-60. (19) Endele, R.; Halisz, I.; Unger, K. J . Chromatogr. 1974, 9 9 , 377-393. (20) Ohmacht, R.; Hal% I. Chrohatographia 1981, 14, 155-162. (21) Ohmacht, R.; HalBsz, I. Chromatographla 1981, 14, 216-228. (22) HalBsz, I.; Schmldt, H.; Vogtel, P.J . Chromatogr. 1978, 126, 19-33. (23) HalBsz, I.; Gorlltz, G. Angew. Chem., Int. Ed. Engl. 1982, 27, 50-61. (24) Asshauer, J.; Halgsz, I. J. Chromatogr. Sci. 1974, 72, 139-147. (25) Bohme, W.; Engelhardt, H. J . Chromatogr. 1977, 733, 67-81. (26) Hallsz, I.; Martln, K. Angew. Chem., Int. Ed. Engl. 1978, 17, 901-908. (27) Maldener, G. Diplomarbeit, Universitat des Saarlandes, Saarbrucken, 1980. (28) Steuer, W. Diplomarbeit, Universltat des Saarlandes, Saarbrucken, 1980. (29) HalBsz, I.; Gerlach, H. 0.;Kroneisen, A.; Walkllng. P. 2.Anal. Chem. 1988, 234, 97-108.

RECEIVED for review September 21,1982. Resubmitted May 2, 1983. Accepted July 5, 1983. Our thanks are due to the Deutsche Forschungsgemeinschaft for financial assistance. This paper was presented at the “NI. International Symposium on Column Liquid Chromatography”, Avignon, France, 11-15 May 1981.